Atomistic insights into regulatory mechanisms of the HER2 tyrosine kinase domain: a molecular dynamics study.

PubMed

Telesco, Shannon E; Radhakrishnan, Ravi

2009-03-18

HER2 (ErbB2/Neu) is a receptor tyrosine kinase belonging to the epidermal growth factor receptor (EGFR)/ErbB family and is overexpressed in 20-30% of human breast cancers. Although several crystal structures of ErbB kinases have been solved, the precise mechanism of HER2 activation remains unknown, and it has been suggested that HER2 is unique in its requirement for phosphorylation of Y877, a key tyrosine residue located in the activation loop. To elucidate mechanistic details of kinase domain regulation, we performed molecular dynamics simulations of a homology-modeled HER2 kinase structure in active and inactive conformations. Principal component analysis of the atomistic fluctuations reveals a tight coupling between the activation loop and catalytic loop that may contribute to alignment of residues required for catalysis in the active kinase. The free energy perturbation method is also employed to predict a role for phosphorylated Y877 in stabilizing the kinase conformations. Finally, simulation results are presented for a HER2/EGFR heterodimer and reveal that the dimeric interface induces a rearrangement of the alphaC helix toward the active conformation. Elucidation of the molecular regulatory mechanisms in HER2 will help establish structure-function relationships in the wild-type kinase, as well as predict mutations with a propensity for constitutive activation in HER2-mediated cancers.

Lack of Csk-mediated negative regulation in a unicellular SRC kinase.

PubMed

Schultheiss, Kira P; Suga, Hiroshi; Ruiz-Trillo, Iñaki; Miller, W Todd

2012-10-16

Phosphotyrosine-based signaling plays a vital role in cellular communication in multicellular organisms. Unexpectedly, unicellular choanoflagellates (the closest phylogenetic group to metazoans) possess numbers of tyrosine kinases that are comparable to those in complex metazoans. Here, we have characterized tyrosine kinases from the filasterean Capsaspora owczarzaki, a unicellular protist representing the sister group to choanoflagellates and metazoans. Two Src-like tyrosine kinases have been identified in C. owczarzaki (CoSrc1 and CoSrc2), both of which have the arrangement of SH3, SH2, and catalytic domains seen in mammalian Src kinases. In Capsaspora cells, CoSrc1 and CoSrc2 localize to punctate structures in filopodia that may represent primordial focal adhesions. We have cloned, expressed, and purified both enzymes. CoSrc1 and CoSrc2 are active tyrosine kinases. Mammalian Src kinases are normally regulated in a reciprocal fashion by autophosphorylation in the activation loop (which increases activity) and by Csk-mediated phosphorylation of the C-terminal tail (which inhibits activity). Similar to mammalian Src kinases, the enzymatic activities of CoSrc1 and CoSrc2 are increased by autophosphorylation in the activation loop. We have identified a Csk-like kinase (CoCsk) in the genome of C. owczarzaki. We cloned, expressed, and purified CoCsk and found that it has no measurable tyrosine kinase activity. Furthermore, CoCsk does not phosphorylate or regulate CoSrc1 or CoSrc2 in cells or in vitro, and CoSrc1 and CoSrc2 are active in Capsaspora cell lysates. Thus, the function of Csk as a negative regulator of Src family kinases appears to have arisen with the emergence of metazoans.

Functional and structural analysis of the sialic acid-binding domain of rotaviruses.

PubMed Central

Isa, P; López, S; Segovia, L; Arias, C F

1997-01-01

The infectivity of most animal rotaviruses is dependent on the interaction of the virus spike protein VP4 with a sialic acid (SA)-containing cell receptor, and the SA-binding domain of this protein has been mapped between amino acids 93 and 208 of its trypsin cleavage fragment VP8. To identify which residues in this region are essential for the SA-binding activity, we performed alanine mutagenesis of the rotavirus RRV VP8 expressed in bacteria as a fusion polypeptide with glutathione S-transferase. Tyrosines were primarily targeted since tyrosine has been involved in the interaction of other viral hemagglutinins with SA. Of the 15 substitutions carried out, 10 abolished the SA-dependent hemagglutination activity of the protein, as well as its ability to bind to glycophorin A in a solid-phase assay. However, only alanine substitutions for tyrosines 155 and 188 and for serine 190 did not affect the overall conformation of the protein, as judged by their interaction with a panel of conformationally sensitive neutralizing VP8 monoclonal antibodies (MAbs). These findings suggest that these three amino acids play an essential role in the SA-binding activity of the protein, presumably by interacting directly with the SA molecule. The predicted secondary structure of VP8 suggests that it is organized as 11 beta-strands separated by loops; in this model, Tyr-155 maps to loop 7 while Tyr-188 and Ser-190 map to loop 9. The close proximity of these two loops is also supported by previous results from competition experiments with neutralizing MAbs directed at RRV VP8. PMID:9261399

The cyclolignan PPP induces activation loop-specific inhibition of tyrosine phosphorylation of the insulin-like growth factor-1 receptor. Link to the phosphatidyl inositol-3 kinase/Akt apoptotic pathway.

PubMed

Vasilcanu, Daiana; Girnita, Ada; Girnita, Leonard; Vasilcanu, Radu; Axelson, Magnus; Larsson, Olle

2004-10-14

The insulin-like growth factor-1 receptor (IGF-1R) is crucial for many functions in neoplastic cells, for example, antiapoptosis. Recently, we demonstrated that the cyclolignan PPP efficiently inhibited phosphorylation of IGF-1R without interfering with insulin receptor activity. PPP preferentially reduced phosphorylated Akt, as compared to phosphorylated Erk1/2, and caused apoptosis. Now, we aimed to investigate how PPP inhibits the IGF-1R tyrosine kinase (IGF-1RTK) and the PI3K/Akt apoptotic pathway. Using a baculovirus driven IGF-1RTK we found that PPP interfered with tyrosine phosphorylation in the activation loop of the kinase domain. Specifically, it blocked phosphorylation of tyrosine (Y) 1136, while sparing the two others (Y1131 and Y1135). To explore the impact of inhibition of Y1136 on Akt phosphorylation we transfected P6 cells (overexpressing IGF-1R) and malignant melanoma cells with different IGF-1R mutants, including Y1136F (tyrosine replaced by phenylalanine). Y1136F was found to strongly decrease IGF-1 stimulated phosphorylation of Akt. Conversely, Akt phosphorylation was weakly affected in the Y1131F transfectant. Taken together, our data suggest that the preferential inhibition of phosphorylated Akt, after PPP treatment, may be due to specific inhibition of Y1136. PPP was proven not to interfere directly with Akt or any of its downstream molecules in the apoptotic pathway.

Crystal structure of the FLT3 kinase domain bound to the inhibitor quizartinib (AC220)

DOE PAGES

Zorn, Julie A.; Wang, Qi; Fujimura, Eric; ...

2015-04-02

More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain ofmore » FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.« less

In silico analysis of surface structure variation of PCV2 capsid resulting from loop mutations of its capsid protein (Cap)

PubMed Central

Wang, Aibing; Zhang, Lijie; Khayat, Reza

2016-01-01

Outbreaks of porcine circovirus (PCV) type 2 (PCV2)-associated diseases have caused substantial economic losses worldwide in the last 20 years. The PCV capsid protein (Cap) is the sole structural protein and main antigenic determinant of this virus. In this study, not only were phylogenetic trees reconstructed, but variations of surface structure of the PCV capsid were analysed in the course of evolution. Unique surface patterns of the icosahedral fivefold axes of the PCV2 capsid were identified and characterized, all of which were absent in PCV type 1 (PCV1). Icosahedral fivefold axes, decorated with Loops BC, HI and DE, were distinctly different between PCV2 and PCV1. Loops BC, determining the outermost surface around the fivefold axes of PCV capsids, had limited homology between Caps of PCV1 and PCV2. A conserved tyrosine phosphorylation motif in Loop HI that might be recognized by non-receptor tyrosine kinase(s) in vivo was present only in PCV2. Particularly, the concurrent presence of 60 pairs of the conserved tyrosine and a canonical PXXP motif on the PCV2 capsid surface could be a mechanism for PXXP motif binding to and activation of an SH3-domain-containing tyrosine kinase in host cells. Additionally, a conserved cysteine in Loop DE of the PCV2 Cap was substituted by an arginine in PCV1, indicating potentially distinct assembly mechanisms of the capsid in vitro between PCV1 and PCV2. Therefore, these unique patterns on the PCV2 capsid surface, absent in PCV1 isolates, might be related to cell entry, virus function and pathogenesis. PMID:27902320

In silico analysis of surface structure variation of PCV2 capsid resulting from loop mutations of its capsid protein (Cap).

PubMed

Wang, Naidong; Zhan, Yang; Wang, Aibing; Zhang, Lijie; Khayat, Reza; Yang, Yi

2016-12-01

Outbreaks of porcine circovirus (PCV) type 2 (PCV2)-associated diseases have caused substantial economic losses worldwide in the last 20 years. The PCV capsid protein (Cap) is the sole structural protein and main antigenic determinant of this virus. In this study, not only were phylogenetic trees reconstructed, but variations of surface structure of the PCV capsid were analysed in the course of evolution. Unique surface patterns of the icosahedral fivefold axes of the PCV2 capsid were identified and characterized, all of which were absent in PCV type 1 (PCV1). Icosahedral fivefold axes, decorated with Loops BC, HI and DE, were distinctly different between PCV2 and PCV1. Loops BC, determining the outermost surface around the fivefold axes of PCV capsids, had limited homology between Caps of PCV1 and PCV2. A conserved tyrosine phosphorylation motif in Loop HI that might be recognized by non-receptor tyrosine kinase(s) in vivo was present only in PCV2. Particularly, the concurrent presence of 60 pairs of the conserved tyrosine and a canonical PXXP motif on the PCV2 capsid surface could be a mechanism for PXXP motif binding to and activation of an SH3-domain-containing tyrosine kinase in host cells. Additionally, a conserved cysteine in Loop DE of the PCV2 Cap was substituted by an arginine in PCV1, indicating potentially distinct assembly mechanisms of the capsid in vitro between PCV1 and PCV2. Therefore, these unique patterns on the PCV2 capsid surface, absent in PCV1 isolates, might be related to cell entry, virus function and pathogenesis.

Phosphorylation of Mutationally Introduced Tyrosine in the Activation Loop of HER2 Confers Gain-of-Function Activity

PubMed Central

Hu, Zexi; Wan, Xiaobo; Hao, Rui; Zhang, Heng; Li, Li; Li, Lin; Xie, Qiang; Wang, Peng; Gao, Yibo; Chen, She; Wei, Min; Luan, Zhidong; Zhang, Aiqun; Huang, Niu; Chen, Liang

2015-01-01

Amplification, overexpression, and somatic mutation of the HER2 gene have been reported to play a critical role in tumorigenesis of various cancers. The HER2 H878Y mutation was recently reported in 11% of hepatocellular carcinoma (HCC) patients. However, its functional impact on the HER2 protein and its role in tumorigenesis has not been determined. Here, we show that HER2 H878Y is a gain-of-function mutation. Y878 represents a phosphorylation site, and phospho-Y878 interacts with R898 residue to stabilize the active conformation of HER2, thereby enhancing its kinase activity. H878Y mutant is transforming and the transformed cells are sensitive to HER2 kinase inhibitors. Thus, our study reveals the following novel mechanism underlying the tumorigenic function of the HER2 H878Y mutation: the introduction of a tyrosine residue into the kinase activation loop via mutagenesis modulates the conformation of the kinase, thereby enhancing its activity. PMID:25853726

A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in Mycobacterium smegmatis.

PubMed

Bansal, Ankita; Kar, Debasish; Pandey, Satya Deo; Matcha, Ashok; Kumar, N Ganesh; Nathan, Soshina; Ghosh, Anindya S

2017-06-01

Mycobacterial beta-lactamases are involved in exerting beta-lactam resistance, though many of these proteins remain uncharacterized. Here, we have characterized MSMEG_4455 of Mycobacterium smegmatis as a beta-lactamase using molecular, biochemical and mutational techniques. To elucidate its nature in vivo and in vitro, and to predict its structure-function relationship in silico analysis is done. The MSMEG_4455 is cloned and expressed ectopically in a beta-lactamase deficient Escherichia coli mutant to establish the in vivo beta-lactamase like nature via minimum inhibitory concentration (MIC) determination. Likewise the in vivo results, purified soluble form of MSMEG_4455 showed beta-lactam hydrolysis pattern similar to group 2a penicillinase. In silico analyses of MSMEG_4455 reveal glutamic acid (E)193 and tyrosine (Y)194 of omega-like loop might have importance in strengthening hydrogen bond network around the active-site, though involvement of tyrosine is rare for beta-lactamase activity. Accordingly, these residues are mutated to alanine (A) and phenylalanine (F), respectively. The mutated proteins have partially lost their ability to exert beta-lactamase activity both in vivo and in vitro. The Y194F mutation had more prominent effect on the enzymatic activity. Therefore, we infer that Y194 is the key for beta-lactamase activity of MSMEG_4455.

Herpes simplex virus requires VP11/12 to induce phosphorylation of the activation loop tyrosine (Y394) of the Src family kinase Lck in T lymphocytes.

PubMed

Wagner, Melany J; Smiley, James R

2009-12-01

Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses, the function of some, like virion protein 11/12 (VP11/12), have not been clearly defined. Previously, we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis, M. J. Wagner, R. C. Doepker, J. M. Maciejko, C. M. Crider, K. R. Jerome, and J. R. Smiley, J. Virol. 82:6098-6108, 2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here, we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394), a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However, we were unable to detect an interaction between ICP0 and Lck in T lymphocytes, and we show that ICP0, US3, and UL13 are not strictly required for Lck activation. In contrast, VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus, VP11/12 likely modulates host cell signaling pathways for the benefit of the virus.

Herpes Simplex Virus Requires VP11/12 To Induce Phosphorylation of the Activation Loop Tyrosine (Y394) of the Src Family Kinase Lck in T Lymphocytes ▿

PubMed Central

Wagner, Melany J.; Smiley, James R.

2009-01-01

Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses, the function of some, like virion protein 11/12 (VP11/12), have not been clearly defined. Previously, we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis, M. J. Wagner, R. C. Doepker, J. M. Maciejko, C. M. Crider, K. R. Jerome, and J. R. Smiley, J. Virol. 82:6098-6108, 2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here, we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394), a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However, we were unable to detect an interaction between ICP0 and Lck in T lymphocytes, and we show that ICP0, US3, and UL13 are not strictly required for Lck activation. In contrast, VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus, VP11/12 likely modulates host cell signaling pathways for the benefit of the virus. PMID:19776125

Structural Characterization of Proline-rich Tyrosine Kinase 2 (PYK2) Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Seungil; Mistry, Anil; Chang, Jeanne S.

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptormore » tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.« less

An EGFR wild type-EGFRvIII-HB-EGF feed forward loop regulates the activation of EGFRvIII

PubMed Central

Li, Li; Chakraborty, Sharmistha; Yang, Chin-Rang; Hatanpaa, Kimmo J.; Cipher, Daisha J.; Puliyappadamba, Vineshkumar Thidil; Rehman, Alizeh; Jiwani, Ameena J.; Mickey, Bruce; Madden, Christopher; Raisanen, Jack; Burma, Sandeep; Saha, Debabrata; Wang, Zhixiang; Pingle, Sandeep C.; Kesari, Santosh; Boothman, David A.; Habib, Amyn A.

2014-01-01

EGFRvIII is a key oncogene in glioblastoma (GBM). EGFRvIII results from an in frame deletion in the extracellular domain of EGFR, does not bind ligand, and is thought to be constitutively active. While EGFRvIII dimerization is known to activate EGFRvIII, the factors that drive EGFRvIII dimerization and activation are not well understood. Here we present a new model of EGFRvIII activation and propose that oncogenic activation of EGFRvIII in glioma cells is driven by co-expressed activated EGFR wild type (EGFRwt). Increasing EGFRwt leads to a striking increase in EGFRvIII tyrosine phosphorylation and activation while silencing EGFRwt inhibits EGFRvIII activation. Both the dimerization arm and the kinase activity of EGFRwt are required for EGFRvIII activation. EGFRwt activates EGFRvIII by facilitating EGFRvIII dimerization. We have previously identified HB-EGF, a ligand for EGFRwt, as a gene induced specifically by EGFRvIII. In this study we show that HB-EGF, is induced by EGFRvIII only when EGFRwt is present. Remarkably, altering HB-EGF recapitulates the effect of EGFRwt on EGFRvIII activation. Thus, increasing HB-EGF leads to a striking increase in EGFRvIII tyrosine phosphorylation while silencing HB-EGF attenuates EGFRvIII phosphorylation, suggesting that an EGFRvIII-HB-EGF-EGFRwt feed forward loop regulates EGFRvIII activation. Silencing EGFRwt or HB-EGF leads to a striking inhibition of EGFRvIII induced tumorigenicity, while increasing EGFRwt or HB-EGF levels resulted in accelerated EGFRvIII mediated oncogenicity in an orthotopic mouse model. Furthermore, we demonstrate the existence of this loop in human GBM. Thus, our data demonstrate that oncogenic activation of EGFRvIII in GBM is likely maintained by a continuous EGFRwt-EGFRvIII-HBEGF loop, potentially an attractive target for therapeutic intervention. PMID:24077285

Phosphatidylinositol-3-kinase-dependent phosphorylation of SLP-76 by the lymphoma-associated ITK-SYK fusion-protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hussain, Alamdar, E-mail: alamdar.hussain@ki.se; Department of Biosciences, COMSATS Institute of Information Technology, Chak Shazad Campus, Islamabad; Faryal, Rani

Recurrent chromosomal translocations have long been implicated in various types of lymphomas and other malignancies. Novel recurrent t(5;9)(q33;q22) has been recently discovered in un-specified peripheral T-cell lymphoma. To elucidate the role of this translocation, the corresponding fusion construct encoding the N-terminal portion of the ITK kinase and the C-terminal catalytic region of the SYK kinase was generated. We herein show that the ITK-SYK fusion-protein is constitutively active. Moreover, we demonstrate that ITK-SYK is phosphorylated on key tyrosine residues and is capable of potently phosphorylating the related adapter proteins BLNK and SLP-76. In transiently transfected cells, SYK was phosphorylated at Y352more » but not detectably at the activation-loop tyrosines Y525/Y526. In contrast, ITK-SYK was phosphorylated both at Y212 and the activation-loop tyrosines Y385/Y386, corresponding to Y352 and Y525/Y526 in SYK, respectively. In resting primary lymphocytes, ITK-SYK predominantly localizes to the cell surface. In addition, we demonstrate that following stimulation, the ITK-SYK fusion-protein in cell lines translocates to the cell membrane and, moreover, that this phenomenon as well as SLP-76 phosphorylation are blocked upon phosphatidylinositol-3-kinase (PI3-kinase) inhibition.« less

Skin peptide tyrosine-tyrosine, a member of the pancreatic polypeptide family: isolation, structure, synthesis, and endocrine activity.

PubMed

Mor, A; Chartrel, N; Vaudry, H; Nicolas, P

1994-10-25

Pancreatic polypeptide, peptide tyrosine-tyrosine (PYY), and neuropeptide tyrosine (NPY), three members of a family of structurally related peptides, are mainly expressed in the endocrine pancreas, in endocrine cells of the gut, and in the brain, respectively. In the present study, we have isolated a peptide of the pancreatic polypeptide family from the skin of the South American arboreal frog Phyllomedusa bicolor. The primary structure of the peptide was established as Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly-Glu10-Asp-Ala-Ser-Pro-Glu-Glu- Met-Asn- Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Val-Thr- Arg-Gln-Arg-Tyr-NH2 . This unusual peptide, named skin peptide tyrosine-tyrosine (SPYY), exhibits 94% similarity with PYY from the frog Rana ridibunda. A synthetic replicate of SPYY inhibits melanotropin release from perifused frog neurointermediate lobes in very much the same way as NPY. These results demonstrate the occurrence of a PYY-like peptide in frog skin. Our data also suggest the existence of a pituitary-skin regulatory loop in amphibians.

Skin peptide tyrosine-tyrosine, a member of the pancreatic polypeptide family: isolation, structure, synthesis, and endocrine activity.

PubMed Central

Mor, A; Chartrel, N; Vaudry, H; Nicolas, P

1994-01-01

Pancreatic polypeptide, peptide tyrosine-tyrosine (PYY), and neuropeptide tyrosine (NPY), three members of a family of structurally related peptides, are mainly expressed in the endocrine pancreas, in endocrine cells of the gut, and in the brain, respectively. In the present study, we have isolated a peptide of the pancreatic polypeptide family from the skin of the South American arboreal frog Phyllomedusa bicolor. The primary structure of the peptide was established as Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly-Glu10-Asp-Ala-Ser-Pro-Glu-Glu- Met-Asn- Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Val-Thr- Arg-Gln-Arg-Tyr-NH2 . This unusual peptide, named skin peptide tyrosine-tyrosine (SPYY), exhibits 94% similarity with PYY from the frog Rana ridibunda. A synthetic replicate of SPYY inhibits melanotropin release from perifused frog neurointermediate lobes in very much the same way as NPY. These results demonstrate the occurrence of a PYY-like peptide in frog skin. Our data also suggest the existence of a pituitary-skin regulatory loop in amphibians. PMID:7937944

Multiple myeloma phosphotyrosine proteomic profile associated with FGFR3 expression, ligand activation, and drug inhibition

PubMed Central

St-Germain, Jonathan R.; Taylor, Paul; Tong, Jiefei; Jin, Lily L.; Nikolic, Ana; Stewart, Ian I.; Ewing, Robert M.; Dharsee, Moyez; Li, Zhihua; Trudel, Suzanne; Moran, Michael F.

2009-01-01

Signaling by growth factor receptor tyrosine kinases is manifest through networks of proteins that are substrates and/or bind to the activated receptors. FGF receptor-3 (FGFR3) is a drug target in a subset of human multiple myelomas (MM) and is mutationally activated in some cervical and colon and many bladder cancers and in certain skeletal dysplasias. To define the FGFR3 network in multiple myeloma, mass spectrometry was used to identify and quantify phosphotyrosine (pY) sites modulated by FGFR3 activation and inhibition in myeloma-derived KMS11 cells. Label-free quantification of peptide ion currents indicated the activation of FGFR3 by phosphorylation of tandem tyrosines in the kinase domain activation loop when cellular pY phosphatases were inhibited by pervanadate. Among the 175 proteins that accumulated pY in response to pervanadate was a subset of 52 including FGFR3 that contained a total of 61 pY sites that were sensitive to inhibition by the FGFR3 inhibitor PD173074. The FGFR3 isoform containing the tandem pY motif in its activation loop was targeted by PD173074. Forty of the drug-sensitive pY sites, including two located within the 35-residue cytoplasmic domain of the transmembrane growth factor binding proteoglycan (and multiple myeloma biomarker) Syndecan-1/CD138, were also stimulated in cells treated with the ligand FGF1, providing additional validation of their link to FGFR3. The identification of these overlapping sets of co-modulated tyrosine phosphorylations presents an outline of an FGFR3 network in the MM model and demonstrates the potential for pharmacodynamic monitoring by label-free quantitative phospho-proteomics. PMID:19901323

The Cytoplasmic Adaptor Protein Dok7 Activates the Receptor Tyrosine Kinase MuSK via Dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bergamin, E.; Hallock, P; Burden, S

Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK.more » The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.« less

A peptide representing the carboxyl-terminal tail of the met receptor inhibits kinase activity and invasive growth.

PubMed

Bardelli, A; Longati, P; Williams, T A; Benvenuti, S; Comoglio, P M

1999-10-08

Interaction of the hepatocyte growth factor (HGF) with its receptor, the Met tyrosine kinase, results in invasive growth, a genetic program essential to embryonic development and implicated in tumor metastasis. Met-mediated invasive growth requires autophosphorylation of the receptor on tyrosines located in the kinase activation loop (Tyr(1234)-Tyr(1235)) and in the carboxyl-terminal tail (Tyr(1349)-Tyr(1356)). We report that peptides derived from the Met receptor tail, but not from the activation loop, bind the receptor and inhibit the kinase activity in vitro. Cell delivery of the tail receptor peptide impairs HGF-dependent Met phosphorylation and downstream signaling. In normal and transformed epithelial cells, the tail receptor peptide inhibits HGF-mediated invasive growth, as measured by cell migration, invasiveness, and branched morphogenesis. The Met tail peptide inhibits the closely related Ron receptor but does not significantly affect the epidermal growth factor, platelet-derived growth factor, or vascular endothelial growth factor receptor activities. These experiments show that carboxyl-terminal sequences impair the catalytic properties of the Met receptor, thus suggesting that in the resting state the nonphosphorylated tail acts as an intramolecular modulator. Furthermore, they provide a strategy to selectively target the MET proto-oncogene by using small, cell-permeable, peptide derivatives.

Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4.

PubMed

Klaus-Heisen, Dörte; Nurisso, Alessandra; Pietraszewska-Bogiel, Anna; Mbengue, Malick; Camut, Sylvie; Timmers, Ton; Pichereaux, Carole; Rossignol, Michel; Gadella, Theodorus W J; Imberty, Anne; Lefebvre, Benoit; Cullimore, Julie V

2011-04-01

Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.

Visualizing Active-Site Dynamics in Single Crystals of HePTP: Opening of the WPD Loop Involves Coordinated Movement of the E Loop

DOE Office of Scientific and Technical Information (OSTI.GOV)

D Critton; L Tautz; R Page

2011-12-31

Phosphotyrosine hydrolysis by protein tyrosine phosphatases (PTPs) involves substrate binding by the PTP loop and closure over the active site by the WPD loop. The E loop, located immediately adjacent to the PTP and WPD loops, is conserved among human PTPs in both sequence and structure, yet the role of this loop in substrate binding and catalysis is comparatively unexplored. Hematopoietic PTP (HePTP) is a member of the kinase interaction motif (KIM) PTP family. Compared to other PTPs, KIM-PTPs have E loops that are unique in both sequence and structure. In order to understand the role of the E loopmore » in the transition between the closed state and the open state of HePTP, we identified a novel crystal form of HePTP that allowed the closed-state-to-open-state transition to be observed within a single crystal form. These structures, which include the first structure of the HePTP open state, show that the WPD loop adopts an 'atypically open' conformation and, importantly, that ligands can be exchanged at the active site, which is critical for HePTP inhibitor development. These structures also show that tetrahedral oxyanions bind at a novel secondary site and function to coordinate the PTP, WPD, and E loops. Finally, using both structural and kinetic data, we reveal a novel role for E-loop residue Lys182 in enhancing HePTP catalytic activity through its interaction with Asp236 of the WPD loop, providing the first evidence for the coordinated dynamics of the WPD and E loops in the catalytic cycle, which, as we show, is relevant to multiple PTP families.« less

The SH2 domain interaction landscape.

PubMed

Tinti, Michele; Kiemer, Lars; Costa, Stefano; Miller, Martin L; Sacco, Francesca; Olsen, Jesper V; Carducci, Martina; Paoluzi, Serena; Langone, Francesca; Workman, Christopher T; Blom, Nikolaj; Machida, Kazuya; Thompson, Christopher M; Schutkowski, Mike; Brunak, Søren; Mann, Matthias; Mayer, Bruce J; Castagnoli, Luisa; Cesareni, Gianni

2013-04-25

Members of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a high-density peptide chip technology that allows for probing of the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique, we have experimentally identified thousands of putative SH2-peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2-mediated probabilistic interaction network, which we make available as a community resource in the PepspotDB database. A predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the extracellular signal-regulated kinase activation loop was validated by experiments in living cells. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Signaling of the ITK (interleukin 2-inducible T cell kinase)-SYK (spleen tyrosine kinase) fusion kinase is dependent on adapter SLP-76 and on the adapter function of the kinases SYK and ZAP70.

PubMed

Hussain, Alamdar; Mohammad, Dara K; Gustafsson, Manuela O; Uslu, Merve; Hamasy, Abdulrahman; Nore, Beston F; Mohamed, Abdalla J; Smith, C I Edvard

2013-03-08

The inducible T cell kinase-spleen tyrosine kinase (ITK-SYK) oncogene consists of the Tec homology-pleckstrin homology domain of ITK and the kinase domain of SYK, and it is believed to be the cause of peripheral T cell lymphoma. We and others have recently demonstrated that this fusion protein is constitutively tyrosine-phosphorylated and is transforming both in vitro and in vivo. To gain a deeper insight into the molecular mechanism(s) underlying its activation and signaling, we mutated a total of eight tyrosines located in the SYK portion of the chimera into either phenylalanine or to the negatively charged glutamic acid. Although mutations in the interdomain-B region affected ITK-SYK kinase activity, they only modestly altered downstream signaling events. In contrast, mutations that were introduced in the kinase domain triggered severe impairment of downstream signaling. Moreover, we show here that SLP-76 is critical for ITK-SYK activation and is particularly required for the ITK-SYK-dependent phosphorylation of SYK activation loop tyrosines. In Jurkat cell lines, we demonstrate that expression of ITK-SYK fusion requires an intact SLP-76 function and significantly induces IL-2 secretion and CD69 expression. Furthermore, the SLP-76-mediated induction of IL-2 and CD69 could be further enhanced by SYK or ZAP-70, but it was independent of their kinase activity. Notably, ITK-SYK expression in SYF cells phosphorylates SLP-76 in the absence of SRC family kinases. Altogether, our data suggest that ITK-SYK exists in the active conformation state and is therefore capable of signaling without SRC family kinases or stimulation of the T cell receptor.

Signaling of the ITK (Interleukin 2-inducible T Cell Kinase)-SYK (Spleen Tyrosine Kinase) Fusion Kinase Is Dependent on Adapter SLP-76 and on the Adapter Function of the Kinases SYK and ZAP70*

PubMed Central

Hussain, Alamdar; Mohammad, Dara K.; Gustafsson, Manuela O.; Uslu, Merve; Hamasy, Abdulrahman; Nore, Beston F.; Mohamed, Abdalla J.; Smith, C. I. Edvard

2013-01-01

The inducible T cell kinase-spleen tyrosine kinase (ITK-SYK) oncogene consists of the Tec homology-pleckstrin homology domain of ITK and the kinase domain of SYK, and it is believed to be the cause of peripheral T cell lymphoma. We and others have recently demonstrated that this fusion protein is constitutively tyrosine-phosphorylated and is transforming both in vitro and in vivo. To gain a deeper insight into the molecular mechanism(s) underlying its activation and signaling, we mutated a total of eight tyrosines located in the SYK portion of the chimera into either phenylalanine or to the negatively charged glutamic acid. Although mutations in the interdomain-B region affected ITK-SYK kinase activity, they only modestly altered downstream signaling events. In contrast, mutations that were introduced in the kinase domain triggered severe impairment of downstream signaling. Moreover, we show here that SLP-76 is critical for ITK-SYK activation and is particularly required for the ITK-SYK-dependent phosphorylation of SYK activation loop tyrosines. In Jurkat cell lines, we demonstrate that expression of ITK-SYK fusion requires an intact SLP-76 function and significantly induces IL-2 secretion and CD69 expression. Furthermore, the SLP-76-mediated induction of IL-2 and CD69 could be further enhanced by SYK or ZAP-70, but it was independent of their kinase activity. Notably, ITK-SYK expression in SYF cells phosphorylates SLP-76 in the absence of SRC family kinases. Altogether, our data suggest that ITK-SYK exists in the active conformation state and is therefore capable of signaling without SRC family kinases or stimulation of the T cell receptor. PMID:23293025

Role of B61, the Ligand for the Eck Receptor Tyrosine Kinase, in TNF- α-Induced Angiogenesis

NASA Astrophysics Data System (ADS)

Pandey, Akhilesh; Shao, Haining; Marks, Rory M.; Polverini, Peter J.; Dixit, Vishva M.

1995-04-01

B61, a cytokine-inducible endothelial gene product, is the ligand for the Eck receptor protein tyrosine kinase (RPTK). Expression of a B61-immunoglobulin chimera showed that B61 could act as an angiogenic factor in vivo and a chemoattractant for endothelial cells in vitro. The Eck RPTK was activated by tumor necrosis factor-α (TNF-α) through induction of B61, and an antibody to B61 attenuated angiogenesis induced by TNF-α but not by basic fibroblast growth factor. This finding suggests the existence of an autocrine or paracrine loop involving activation of the Eck RPTK by its inducible ligand B61 after an inflammatory stimulus, the net effect of which would be to promote angiogenesis, a hallmark of chronic inflammation.

Role of B61, the ligand for the Eck receptor tyrosine kinase, in TNF-alpha-induced angiogenesis.

PubMed

Pandey, A; Shao, H; Marks, R M; Polverini, P J; Dixit, V M

1995-04-28

B61, a cytokine-inducible endothelial gene product, is the ligand for the Eck receptor protein tyrosine kinase (RPTK). Expression of a B61-immunoglobulin chimera showed that B61 could act as an angiogenic factor in vivo and a chemoattractant for endothelial cells in vitro. The Eck RPTK was activated by tumor necrosis factor-alpha (TNF-alpha) through induction of B61, and an antibody to B61 attenuated angiogenesis induced by TNF-alpha but not by basic fibroblast growth factor. This finding suggests the existence of an autocrine or paracrine loop involving activation of the Eck RPTK by its inducible ligand B61 after an inflammatory stimulus, the net effect of which would be to promote angiogenesis, a hallmark of chronic inflammation.

Interleukin 6 inhibits proliferation and, in cooperation with an epidermal growth factor receptor autocrine loop, increases migration of T47D breast cancer cells.

PubMed

Badache, A; Hynes, N E

2001-01-01

Interleukin (IL)-6, a multifunctional regulator of immune response, hematopoiesis, and acute phase reactions, has also been shown to regulate cancer cell proliferation. We have investigated IL-6 signaling pathways and cellular responses in the T47D breast carcinoma cell line. The IL-6-type cytokines, IL-6 and oncostatin M, simultaneously inhibited cell proliferation and increased cell migration. In T47D cells, IL-6 stimulated the activation of Janus-activated kinase 1 tyrosine kinase and signal transducers and activators of transcription (STAT) 1 and STAT3 transcription factors. Expression of dominant negative STAT3 in the cells strongly reduced IL-6-mediated growth inhibition but did not prevent IL-6-induced cell migration. IL-6 treatment led to activation of the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3'-kinase (PI3K) pathways. Inhibition of MAPK or PI3K activity reversed IL-6- and oncostatin M-stimulated migration. Because cross-talk between cytokine receptors and members of the ErbB family of receptor tyrosine kinases has been described previously, we have examined their interaction in T47D cells. Down-regulation of ErbB receptor activity, through the use of specific pharmacological inhibitors or dominant negative receptor constructs, revealed that IL-6-induced MAPK activation was largely dependent on epidermal growth factor (EGF) receptor activity, but not on ErbB-2 activity. Using a monoclonal antibody that interferes with EGF receptor-ligand interaction, we have shown that in T47D cells, IL-6 cooperates with an EGF receptor autocrine activity loop for signaling through the MAPK and PI3K pathways and for cell migration. Both the tyrosine phosphatase SHP-2 and the multisubstrate docking molecule Gab1, which are potential links between IL-6 and the MAPK/PI3K pathways, were constitutively associated with the active EGF receptor. On IL-6 stimulation, SHP-2 and Gab1 were recruited to the gp130 subunit of the IL-6 receptor and tyrosine phosphorylated, allowing downstream signaling to the MAPK and PI3K pathways. Thus, in T47D breast carcinoma cells, IL-6 acts in synergy with EGF receptor autocrine activity to signal through the MAPK/PI3K pathways. Cooperation between IL-6 and the EGF receptor in T47D breast carcinoma cells illustrates how a combination of multiple stimuli, either exogenous or endogenous, may result in synergistic cellular responses.

Computational Study of the “DFG-Flip” Conformational Transition in c-Abl and c-Src Tyrosine Kinases

PubMed Central

2015-01-01

Protein tyrosine kinases are crucial to cellular signaling pathways regulating cell growth, proliferation, metabolism, differentiation, and migration. To maintain normal regulation of cellular signal transductions, the activities of tyrosine kinases are also highly regulated. The conformation of a three-residue motif Asp-Phe-Gly (DFG) near the N-terminus of the long “activation” loop covering the catalytic site is known to have a critical impact on the activity of c-Abl and c-Src tyrosine kinases. A conformational transition of the DFG motif can switch the enzyme from an active (DFG-in) to an inactive (DFG-out) state. In the present study, the string method with swarms-of-trajectories was used to computationally determine the reaction pathway connecting the two end-states, and umbrella sampling calculations were carried out to characterize the thermodynamic factors affecting the conformations of the DFG motif in c-Abl and c-Src kinases. According to the calculated free energy landscapes, the DFG-out conformation is clearly more favorable in the case of c-Abl than that of c-Src. The calculations also show that the protonation state of the aspartate residue in the DFG motif strongly affects the in/out conformational transition in c-Abl, although it has a much smaller impact in the case of c-Src due to local structural differences. PMID:25548962

The tyrosine kinase Stitcher activates Grainy head and epidermal wound healing in Drosophila.

PubMed

Wang, Shenqiu; Tsarouhas, Vasilios; Xylourgidis, Nikos; Sabri, Nafiseh; Tiklová, Katarína; Nautiyal, Naumi; Gallio, Marco; Samakovlis, Christos

2009-07-01

Epidermal injury initiates a cascade of inflammation, epithelial remodelling and integument repair at wound sites. The regeneration of the extracellular barrier and damaged tissue repair rely on the precise orchestration of epithelial responses triggered by the injury. Grainy head (Grh) transcription factors induce gene expression to crosslink the extracellular barrier in wounded flies and mice. However, the activation mechanisms and functions of Grh factors in re-epithelialization remain unknown. Here we identify stitcher (stit), a new Grh target in Drosophila melanogaster. stit encodes a Ret-family receptor tyrosine kinase required for efficient epidermal wound healing. Live imaging analysis reveals that Stit promotes actin cable assembly during wound re-epithelialization. Stit activation also induces extracellular signal-regulated kinase (ERK) phosphorylation along with the Grh-dependent expression of stit and barrier repair genes at the wound sites. The transcriptional stimulation of stit on injury triggers a positive feedback loop increasing the magnitude of epithelial responses. Thus, Stit activation upon wounding coordinates cytoskeletal rearrangements and the level of Grh-mediated transcriptional wound responses.

Autophagy induced by AXL receptor tyrosine kinase alleviates acute liver injury via inhibition of NLRP3 inflammasome activation in mice.

PubMed

Han, Jihye; Bae, Joonbeom; Choi, Chang-Yong; Choi, Sang-Pil; Kang, Hyung-Sik; Jo, Eun-Kyeong; Park, Jongsun; Lee, Young Sik; Moon, Hyun-Seuk; Park, Chung-Gyu; Lee, Myung-Shik; Chun, Taehoon

2016-12-01

Severe hepatic inflammation is a common cause of acute or chronic liver disease. Macrophages are one of the key mediators which regulate the progress of hepatic inflammation. Increasing evidence shows that the TAM (TYRO3, AXL and MERTK) family of RTKs (receptor tyrosine kinases), which is expressed in macrophages, alleviates inflammatory responses through a negative feedback loop. However, the functional contribution of each TAM family member to the progression of hepatic inflammation remains elusive. In this study, we explore the role of individual TAM family proteins during autophagy induction and evaluate their contribution to hepatic inflammation. Among the TAM family of RTKs, AXL (AXL receptor tyrosine kinase) only induces autophagy in macrophages after interaction with its ligand, GAS6 (growth arrest specific 6). Based on our results, autophosphorylation of 2 tyrosine residues (Tyr815 and Tyr860) in the cytoplasmic domain of AXL in mice is required for autophagy induction and AXL-mediated autophagy induction is dependent on MAPK (mitogen-activated protein kinase)14 activity. Furthermore, induction of AXL-mediated autophagy prevents CASP1 (caspase 1)-dependent IL1B (interleukin 1, β) and IL18 (interleukin 18) maturation by inhibiting NLRP3 (NLR family, pyrin domain containing 3) inflammasome activation. In agreement with these observations, axl -/- mice show more severe symptoms than do wild-type (Axl +/+ ) mice following acute hepatic injury induced by administration of lipopolysaccharide (LPS) or carbon tetrachloride (CCl 4 ). Hence, GAS6-AXL signaling-mediated autophagy induction in murine macrophages ameliorates hepatic inflammatory responses by inhibiting NLRP3 inflammasome activation.

HRD Motif as the Central Hub of the Signaling Network for Activation Loop Autophosphorylation in Abl Kinase.

PubMed

La Sala, Giuseppina; Riccardi, Laura; Gaspari, Roberto; Cavalli, Andrea; Hantschel, Oliver; De Vivo, Marco

2016-11-08

A number of structural factors modulate the activity of Abelson (Abl) tyrosine kinase, whose deregulation is often related to oncogenic processes. First, only the open conformation of the Abl kinase domain's activation loop (A-loop) favors ATP binding to the catalytic cleft. In this regard, the trans-autophosphorylation of the Y412 residue, which is located along the A-loop, favors the stability of the open conformation, in turn enhancing Abl activity. Another key factor for full Abl activity is the formation of active conformations of the catalytic DFG motif in the Abl kinase domain. Furthermore, binding of the SH2 domain to the N-lobe of the Abl kinase was recently demonstrated to have a long-range allosteric effect on the stabilization of the A-loop open state. Intriguingly, these distinct structural factors imply a complex signal transmission network for controlling the A-loop's flexibility and conformational preference for optimal Abl function. However, the exact dynamical features of this signal transmission network structure remain unclear. Here, we report on microsecond-long molecular dynamics coupled with enhanced sampling simulations of multiple Abl model systems, in the presence or absence of the SH2 domain and with the DFG motif flipped in two ways (in or out conformation). Through comparative analysis, our simulations augment the interpretation of the existing Abl experimental data, revealing a dynamical network of interactions that interconnect SH2 domain binding with A-loop plasticity and Y412 autophosphorylation in Abl. This signaling network engages the DFG motif and, importantly, other conserved structural elements of the kinase domain, namely, the EPK-ELK H-bond network and the HRD motif. Our results show that the signal propagation for modulating the A-loop spatial localization is highly dependent on the HRD motif conformation, which thus acts as the central hub of this (allosteric) signaling network controlling Abl activation and function.

Hydrophobic interaction between the SH2 domain and the kinase domain is required for the activation of Csk.

PubMed

Mikkola, Esa T; Gahmberg, Carl G

2010-06-18

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the beta3-alphaC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the beta3-alphaC loop. The mutation of the beta3-alphaC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the beta3-alphaC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

P-loop Conformation Governed Crizotinib Resistance in G2032R-Mutated ROS1 Tyrosine Kinase: Clues from Free Energy Landscape

PubMed Central

Sun, Huiyong; Li, Youyong; Tian, Sheng; Wang, Junmei; Hou, Tingjun

2014-01-01

Tyrosine kinases are regarded as excellent targets for chemical drug therapy of carcinomas. However, under strong purifying selection, drug resistance usually occurs in the cancer cells within a short term. Many cases of drug resistance have been found to be associated with secondary mutations in drug target, which lead to the attenuated drug-target interactions. For example, recently, an acquired secondary mutation, G2032R, has been detected in the drug target, ROS1 tyrosine kinase, from a crizotinib-resistant patient, who responded poorly to crizotinib within a very short therapeutic term. It was supposed that the mutation was located at the solvent front and might hinder the drug binding. However, a different fact could be uncovered by the simulations reported in this study. Here, free energy surfaces were characterized by the drug-target distance and the phosphate-binding loop (P-loop) conformational change of the crizotinib-ROS1 complex through advanced molecular dynamics techniques, and it was revealed that the more rigid P-loop region in the G2032R-mutated ROS1 was primarily responsible for the crizotinib resistance, which on one hand, impaired the binding of crizotinib directly, and on the other hand, shortened the residence time induced by the flattened free energy surface. Therefore, both of the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the kinase resistance. PMID:25033171

P-loop conformation governed crizotinib resistance in G2032R-mutated ROS1 tyrosine kinase: clues from free energy landscape.

PubMed

Sun, Huiyong; Li, Youyong; Tian, Sheng; Wang, Junmei; Hou, Tingjun

2014-07-01

Tyrosine kinases are regarded as excellent targets for chemical drug therapy of carcinomas. However, under strong purifying selection, drug resistance usually occurs in the cancer cells within a short term. Many cases of drug resistance have been found to be associated with secondary mutations in drug target, which lead to the attenuated drug-target interactions. For example, recently, an acquired secondary mutation, G2032R, has been detected in the drug target, ROS1 tyrosine kinase, from a crizotinib-resistant patient, who responded poorly to crizotinib within a very short therapeutic term. It was supposed that the mutation was located at the solvent front and might hinder the drug binding. However, a different fact could be uncovered by the simulations reported in this study. Here, free energy surfaces were characterized by the drug-target distance and the phosphate-binding loop (P-loop) conformational change of the crizotinib-ROS1 complex through advanced molecular dynamics techniques, and it was revealed that the more rigid P-loop region in the G2032R-mutated ROS1 was primarily responsible for the crizotinib resistance, which on one hand, impaired the binding of crizotinib directly, and on the other hand, shortened the residence time induced by the flattened free energy surface. Therefore, both of the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the kinase resistance.

Diurnal suppression of EGFR signalling by glucocorticoids and implications for tumour progression and treatment.

PubMed

Lauriola, Mattia; Enuka, Yehoshua; Zeisel, Amit; D'Uva, Gabriele; Roth, Lee; Sharon-Sevilla, Michal; Lindzen, Moshit; Sharma, Kirti; Nevo, Nava; Feldman, Morris; Carvalho, Silvia; Cohen-Dvashi, Hadas; Kedmi, Merav; Ben-Chetrit, Nir; Chen, Alon; Solmi, Rossella; Wiemann, Stefan; Schmitt, Fernando; Domany, Eytan; Yarden, Yosef

2014-10-03

Signal transduction by receptor tyrosine kinases (RTKs) and nuclear receptors for steroid hormones is essential for body homeostasis, but the cross-talk between these receptor families is poorly understood. We observed that glucocorticoids inhibit signalling downstream of EGFR, an RTK. The underlying mechanism entails suppression of EGFR's positive feedback loops and simultaneous triggering of negative feedback loops that normally restrain EGFR. Our studies in mice reveal that the regulation of EGFR's feedback loops by glucocorticoids translates to circadian control of EGFR signalling: EGFR signals are suppressed by high glucocorticoids during the active phase (night-time in rodents), while EGFR signals are enhanced during the resting phase. Consistent with this pattern, treatment of animals bearing EGFR-driven tumours with a specific kinase inhibitor was more effective if administered during the resting phase of the day, when glucocorticoids are low. These findings support a circadian clock-based paradigm in cancer therapy.

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics

PubMed Central

Smirnov, Ivan; Carletti, Eugénie; Kurkova, Inna; Nachon, Florian; Nicolet, Yvain; Mitkevich, Vladimir A.; Débat, Hélène; Avalle, Bérangère; Belogurov, Alexey A.; Kuznetsov, Nikita; Reshetnyak, Andrey; Masson, Patrick; Tonevitsky, Alexander G.; Ponomarenko, Natalia; Makarov, Alexander A.; Friboulet, Alain; Tramontano, Alfonso; Gabibov, Alexander

2011-01-01

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (VL) and variable heavy (VH) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes. PMID:21896761

Transition path theory analysis of c-Src kinase activation

PubMed Central

Meng, Yilin; Shukla, Diwakar; Pande, Vijay S.; Roux, Benoît

2016-01-01

Nonreceptor tyrosine kinases of the Src family are large multidomain allosteric proteins that are crucial to cellular signaling pathways. In a previous study, we generated a Markov state model (MSM) to simulate the activation of c-Src catalytic domain, used as a prototypical tyrosine kinase. The long-time kinetics of transition predicted by the MSM was in agreement with experimental observations. In the present study, we apply the framework of transition path theory (TPT) to the previously constructed MSM to characterize the main features of the activation pathway. The analysis indicates that the activating transition, in which the activation loop first opens up followed by an inward rotation of the αC-helix, takes place via a dense set of intermediate microstates distributed within a fairly broad “transition tube” in a multidimensional conformational subspace connecting the two end-point conformations. Multiple microstates with negligible equilibrium probabilities carry a large transition flux associated with the activating transition, which explains why extensive conformational sampling is necessary to accurately determine the kinetics of activation. Our results suggest that the combination of MSM with TPT provides an effective framework to represent conformational transitions in complex biomolecular systems. PMID:27482115

Emerging Agents for the Treatment of Advanced, Imatinib-Resistant Gastrointestinal Stromal Tumors: Current Status and Future Directions.

PubMed

Bauer, Sebastian; Joensuu, Heikki

2015-08-01

Imatinib is strongly positioned as the recommended first-line agent for most patients with advanced gastrointestinal stromal tumor (GIST) due to its good efficacy and tolerability. Imatinib-resistant advanced GIST continues to pose a therapeutic challenge, likely due to the frequent presence of multiple mutations that confer drug resistance. Sunitinib and regorafenib are approved as second- and third-line agents, respectively, for patients whose GIST does not respond to imatinib or who do not tolerate imatinib, and their use is supported by large randomized trials. ATP-mimetic tyrosine kinase inhibitors provide clinical benefit even in heavily pretreated GIST suggesting that oncogenic dependency on KIT frequently persists. Several potentially useful tyrosine kinase inhibitors with distinct inhibitory profiles against both KIT ATP-binding domain and activation loop mutations have not yet been fully evaluated. Agents that have been found promising in preclinical models and early clinical trials include small molecule KIT and PDGFRA mutation-specific inhibitors, heat shock protein inhibitors, histone deacetylase inhibitors, allosteric KIT inhibitors, KIT and PDGFRA signaling pathway inhibitors, and immunological approaches including antibody-drug conjugates. Concomitant or sequential administration of tyrosine kinase inhibitors with KIT signaling pathway inhibitors require further evaluation, as well as rotation of tyrosine kinase inhibitors as a means to suppress drug-resistant cell clones.

Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B.

PubMed

Ozcan, Ahmet; Olmez, Elif Ozkirimli; Alakent, Burak

2013-05-01

In protein tyrosine phosphatase 1B (PTP1B), the flexible WPD loop adopts a closed conformation (WPDclosed ) in the active state of PTP1B, bringing the catalytic Asp181 close to the active site pocket, while WPD loop is in an open conformation (WPDopen ) in the inactive state. Previous studies showed that Asp181 may be protonated at physiological pH, and ordered water molecules exist in the active site. In the current study, molecular dynamics simulations are employed at different Asp181 protonation states and initial positions of active site water molecules, and compared with the existing crystallographic data of PTP1B. In WPDclosed conformation, the active site is found to maintain its conformation only in the protonated state of Asp181 in both free and liganded states, while Asp181 is likely to be deprotonated in WPDopen conformation. When the active site water molecule network that is a part of the free WPDclosed crystal structure is disrupted, intermediate WPD loop conformations, similar to that in the PTPRR crystal structure, are sampled in the MD simulations. In liganded PTP1B, one active site water molecule is found to be important for facilitating the orientation of Cys215 and the phosphate ion, thus may play a role in the reaction. In conclusion, conformational stability of WPD loop, and possibly catalytic activity of PTP1B, is significantly affected by the protonation state of Asp181 and position of active site water molecules, showing that these aspects should be taken into consideration both in MD simulations and inhibitor design. Copyright © 2013 Wiley Periodicals, Inc.

Protease-activated Receptor-4 Signaling and Trafficking Is Regulated by the Clathrin Adaptor Protein Complex-2 Independent of β-Arrestins*

PubMed Central

Smith, Thomas H.; Coronel, Luisa J.; Li, Julia G.; Dores, Michael R.; Nieman, Marvin T.; Trejo, JoAnn

2016-01-01

Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of β-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of β-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation. PMID:27402844

The adapter protein, Grb10, is a positive regulator of vascular endothelial growth factor signaling.

PubMed

Giorgetti-Peraldi, S; Murdaca, J; Mas, J C; Van Obberghen, E

2001-07-05

Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis and angiogenesis. Activation of VEGF receptors leads to the recruitment of SH2 containing proteins which link the receptors to the activation of signaling pathways. Here we report that Grb10, an adapter protein of which the biological role remains unknown, is tyrosine phosphorylated in response to VEGF in endothelial cells (HUVEC) and in 293 cells expressing the VEGF receptor KDR. An intact SH2 domain is required for Grb10 tyrosine phosphorylation in response to VEGF, and this phosphorylation is mediated in part through the activation of Src. In HUVEC, VEGF increases Grb10 mRNA level. Expression of Grb10 in HUVEC or in KDR expressing 293 cells results in an increase in the amount and in the tyrosine phosphorylation of KDR. In 293 cells, this is correlated with the activation of signaling molecules, such as MAP kinase. By expressing mutants of Grb10, we found that the positive action of Grb10 is independent of its SH2 domain. Moreover, these Grb10 effects on KDR seem to be specific since Grb10 has no effect on the insulin receptor, and Grb2, another adapter protein, does not mimic the effect of Grb10 on KDR. In conclusion, we propose that VEGF up-regulates Grb10 level, which in turn increases KDR molecules, suggesting that Grb10 could be involved in a positive feedback loop in VEGF signaling.

Molecular cloning of a novel receptor tyrosine kinase, tif, highly expressed in human ovary and testis.

PubMed

Dai, W; Pan, H; Hassanain, H; Gupta, S L; Murphy, M J

1994-03-01

Using a combination of polymerase chain reaction and conventional cDNA library screening approaches, we have cloned and characterized a putative receptor tyrosine kinase termed tif. The extracellular domain of tif has an immunoglobulin-like loop and a fibronectin type III structure. The intracellular domain contains a tyrosine kinase domain. Compared with ryk, a ubiquitously expressed receptor tyrosine kinase, tif expression is tissue-specific with human ovary and testis containing the highest amount of tif mRNA. Many other tested human tissues such as heart, liver, pancreas and thymus do not contain detectable levels of tif mRNA. The molecular cloning and characterization of tif cDNA will facilitate the identification of a potential ligand(s) for the putative receptor and the study of its biological role.

Modeling of Receptor Tyrosine Kinase Signaling: Computational and Experimental Protocols.

PubMed

Fey, Dirk; Aksamitiene, Edita; Kiyatkin, Anatoly; Kholodenko, Boris N

2017-01-01

The advent of systems biology has convincingly demonstrated that the integration of experiments and dynamic modelling is a powerful approach to understand the cellular network biology. Here we present experimental and computational protocols that are necessary for applying this integrative approach to the quantitative studies of receptor tyrosine kinase (RTK) signaling networks. Signaling by RTKs controls multiple cellular processes, including the regulation of cell survival, motility, proliferation, differentiation, glucose metabolism, and apoptosis. We describe methods of model building and training on experimentally obtained quantitative datasets, as well as experimental methods of obtaining quantitative dose-response and temporal dependencies of protein phosphorylation and activities. The presented methods make possible (1) both the fine-grained modeling of complex signaling dynamics and identification of salient, course-grained network structures (such as feedback loops) that bring about intricate dynamics, and (2) experimental validation of dynamic models.

Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins.

PubMed

Tanaka, Shiho; Miyamoto, Kazuhisa; Noda, Hiroaki; Endo, Haruka; Kikuta, Shingo; Sato, Ryoichi

2016-04-01

In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+(234)Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+(234)Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins. Copyright © 2016. Published by Elsevier Inc.

Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao

2010-07-20

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalentmore » intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.« less

Dok1 and Dok2 proteins regulate natural killer cell development and function

PubMed Central

Celis-Gutierrez, Javier; Boyron, Marilyn; Walzer, Thierry; Pandolfi, Pier Paolo; Jonjić, Stipan; Olive, Daniel; Dalod, Marc; Vivier, Eric; Nunès, Jacques A

2014-01-01

Natural killer (NK) cells are involved in immune responses against tumors and microbes. NK-cell activation is regulated by intrinsic and extrinsic mechanisms that ensure NK tolerance and efficacy. Here, we show that the cytoplasmic signaling molecules Dok1 and Dok2 are tyrosine phosphorylated upon NK-cell activation. Overexpression of Dok proteins in human NK cells reduces cell activation induced by NK-cell-activating receptors. Dok1 and Dok2 gene ablation in mice induces an NK-cell maturation defect and leads to increased IFN-γ production induced by activating receptors. Taken together, these results reveal that Dok1 and Dok2 proteins are involved in an intrinsic negative feedback loop downstream of NK-cell-activating receptors in mouse and human. PMID:24963146

The Drosophila Receptor Protein Tyrosine Phosphatase LAR Is Required for Development of Circadian Pacemaker Neuron Processes That Support Rhythmic Activity in Constant Darkness But Not during Light/Dark Cycles

PubMed Central

Agrawal, Parul

2016-01-01

In Drosophila, a transcriptional feedback loop that is activated by CLOCK-CYCLE (CLK-CYC) complexes and repressed by PERIOD-TIMELESS (PER-TIM) complexes keeps circadian time. The timing of CLK-CYC activation and PER-TIM repression is regulated post-translationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Although kinases that control PER, TIM, and CLK levels, activity, and/or subcellular localization have been identified, less is known about phosphatases that control clock protein dephosphorylation. To identify clock-relevant phosphatases, clock-cell-specific RNAi knockdowns of Drosophila phosphatases were screened for altered activity rhythms. One phosphatase that was identified, the receptor protein tyrosine phosphatase leukocyte-antigen-related (LAR), abolished activity rhythms in constant darkness (DD) without disrupting the timekeeping mechanism in brain pacemaker neurons. However, expression of the neuropeptide pigment-dispersing factor (PDF), which mediates pacemaker neuron synchrony and output, is eliminated in the dorsal projections from small ventral lateral (sLNv) pacemaker neurons when Lar expression is knocked down during development, but not in adults. Loss of Lar function eliminates sLNv dorsal projections, but PDF expression persists in sLNv and large ventral lateral neuron cell bodies and their remaining projections. In contrast to the defects in lights-on and lights-off anticipatory activity seen in flies that lack PDF, Lar RNAi knockdown flies anticipate the lights-on and lights-off transition normally. Our results demonstrate that Lar is required for sLNv dorsal projection development and suggest that PDF expression in LNv cell bodies and their remaining projections mediate anticipation of the lights-on and lights-off transitions during a light/dark cycle. SIGNIFICANCE STATEMENT In animals, circadian clocks drive daily rhythms in physiology, metabolism, and behavior via transcriptional feedback loops. Because key circadian transcriptional activators and repressors are regulated by phosphorylation, we screened for phosphatases that alter activity rhythms when their expression was reduced. One such phosphatase, leukocyte-antigen-related (LAR), abolishes activity rhythms, but does not disrupt feedback loop function. Rather, Lar disrupts clock output by eliminating axonal processes from clock neurons that release pigment-dispersing factor (PDF) neuropeptide into the dorsal brain, but PDF expression persists in their cell bodies and remaining projections. In contrast to flies that lack PDF, flies that lack Lar anticipate lights-on and lights-off transitions normally, which suggests that the remaining PDF expression mediates activity during light/dark cycles. PMID:27030770

Src is required for migration, phagocytosis, and interferon beta production in Toll-like receptor-engaged macrophages.

PubMed

Maa, Ming-Chei; Leu, Tzeng-Horng

2016-06-01

As an evolutionarily conserved mechanism, innate immunity controls self-nonself discrimination to protect a host from invasive pathogens. Macrophages are major participants of the innate immune system. Through the activation of diverse Toll-like receptors (TLRs), macrophages are triggered to initiate a variety of functions including locomotion, phagocytosis, and secretion of cytokines that requires the participation of tyrosine kinases. Fgr, Hck, and Lyn are myeloid-specific Src family kinases. Despite their constitutively high expression in macrophages, their absence does not impair LPS responsiveness. In contrast, Src, a barely detectable tyrosine kinase in resting macrophages, becomes greatly inducible in response to TLR engagement, implicating its role in macrophage activation. Indeed, silencing Src suppresses the activated TLR-mediated migration, phagocytosis, and interferon-beta (IFN-β) secretion in macrophages. And these physiological defects can be restored by the introduction of siRNA-resistant Src. Notably, the elevated expression and activity of Src is inducible nitric oxide synthase (iNOS)-dependent. Due to (1) iNOS being a NF-κB target, which can be induced by various TLR ligands, (2) Src can mediate NF-κB activation, therefore, there ought to exist a loop of signal amplification that regulates macrophage physiology in response to the engagement of TLRs.

The SH2 Domain Regulates c-Abl Kinase Activation by a Cyclin-Like Mechanism and Remodulation of the Hinge Motion

PubMed Central

Dölker, Nicole; Górna, Maria W.; Sutto, Ludovico; Torralba, Antonio S.; Superti-Furga, Giulio; Gervasio, Francesco L.

2014-01-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors. PMID:25299346

The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

PubMed

Dölker, Nicole; Górna, Maria W; Sutto, Ludovico; Torralba, Antonio S; Superti-Furga, Giulio; Gervasio, Francesco L

2014-10-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

Hotspot Mutations in KIT Receptor Differentially Modulate Its Allosterically Coupled Conformational Dynamics: Impact on Activation and Drug Sensitivity

PubMed Central

Chauvot de Beauchêne, Isaure; Allain, Ariane; Panel, Nicolas; Laine, Elodie; Trouvé, Alain; Dubreuil, Patrice; Tchertanov, Luba

2014-01-01

Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D) localized in crucial regulatory segments, the juxtamembrane region (JMR) and the activation (A-) loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts. PMID:25079768

PTP1B is a negative regulator of interleukin 4–induced STAT6 signaling

PubMed Central

Lu, Xiaoqing; Malumbres, Raquel; Shields, Benjamin; Jiang, Xiaoyu; Sarosiek, Kristopher A.; Natkunam, Yasodha

2008-01-01

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed enzyme shown to negatively regulate multiple tyrosine phosphorylation-dependent signaling pathways. PTP1B can modulate cytokine signaling pathways by dephosphorylating JAK2, TYK2, and STAT5a/b. Herein, we report that phosphorylated STAT6 may serve as a cytoplasmic substrate for PTP1B. Overexpression of PTP1B led to STAT6 dephosphorylation and the suppression of STAT6 transcriptional activity, whereas PTP1B knockdown or deficiency augmented IL-4–induced STAT6 signaling. Pretreatment of these cells with the PTK inhibitor staurosporine led to sustained STAT6 phosphorylation consistent with STAT6 serving as a direct substrate of PTP1B. Furthermore, PTP1B-D181A “substrate-trapping” mutants formed stable complexes with phosphorylated STAT6 in a cellular context and endogenous PTP1B and STAT6 interacted in an interleukin 4 (IL-4)–inducible manner. We delineate a new negative regulatory loop of IL-4–JAK-STAT6 signaling. We demonstrate that IL-4 induces PTP1B mRNA expression in a phosphatidylinositol 3-kinase–dependent manner and enhances PTP1B protein stability to suppress IL-4–induced STAT6 signaling. Finally, we show that PTP1B expression may be preferentially elevated in activated B cell–like diffuse large B-cell lymphomas. These observations identify a novel regulatory loop for the regulation of IL-4–induced STAT6 signaling that may have important implications in both neoplastic and inflammatory processes. PMID:18716132

Geometrical criteria for characterizing open and closed states of WPD-loop in PTP1B

NASA Astrophysics Data System (ADS)

Shinde, Ranajit Nivrutti; Elizabeth Sobhia, M.

2012-06-01

Distinctive movement of WPD-loop occurs during the catalysis of phosphotyrosine by protein tyrosine phosphatase 1B (PTP1B). This loop is in the "open" state in apo-form whereas it is catalytically competent in the "closed" state. During the closure of this loop, unique hydrogen bond interactions are formed between different residues of the PTP1B. Present study examines such interactions from the available 118 crystal structures of PTP1B. It gives insights into the five novel hydrogen bonds essentially formed in the "closed" loop structures. Additionally, the study provides distance ranges between the atoms involved in the hydrogen bonds. This information can be used as a geometrical criterion in the characterization of conformational state of the WPD-loop especially in the molecular dynamics simulations.

Epithelial cell kinase-B61: an autocrine loop modulating intestinal epithelial migration and barrier function.

PubMed

Rosenberg, I M; Göke, M; Kanai, M; Reinecker, H C; Podolsky, D K

1997-10-01

Epithelial cell kinase (Eck) is a member of a large family of receptor tyrosine kinases whose functions remain largely unknown. Expression and regulation of Eck and its cognate ligand B61 were analyzed in the human colonic adenocarcinoma cell line Caco-2. Immunocytochemical staining demonstrated coexpression of Eck and B61 in the same cells, suggestive of an autocrine loop. Eck levels were maximal in preconfluent cells. In contrast, B61 levels were barely detectable in preconfluent cells and increased progressively after the cells reached confluence. Caco-2 cells cultured in the presence of added B61 showed a significant reduction in the levels of dipeptidyl peptidase and sucrase-isomaltase mRNA, markers of Caco-2 cell differentiation. Cytokines interleukin-1beta (IL-1beta), basic fibroblast growth factor, IL-2, epidermal growth factor, and transforming growth factor-beta modulated steady-state levels of Eck and B61 mRNA and regulated Eck activation as assessed by tyrosine phosphorylation. Functionally, stimulation of Eck by B61 resulted in increased proliferation, enhanced barrier function, and enhanced restitution of injured epithelial monolayers. These results suggest that the Eck-B61 interaction, a target of regulatory peptides, plays a role in intestinal epithelial cell development, migration, and barrier function, contributing to homeostasis and preservation of continuity of the epithelial barrier.

Autoantibody-mediated regulation of B cell responses by functional anti-CD22 autoantibodies in patients with systemic sclerosis.

PubMed

Odaka, M; Hasegawa, M; Hamaguchi, Y; Ishiura, N; Kumada, S; Matsushita, T; Komura, K; Sato, S; Takehara, K; Fujimoto, M

2010-02-01

Studies have demonstrated that B cells play important roles in systemic sclerosis (SSc), especially through the CD19/CD22 autoimmune loop. CD22 is a B cell-specific inhibitory receptor that dampens B cell antigen receptor (BCR) signalling via tyrosine phosphorylation-dependent mechanism. In this study, we examined the presence and functional property of circulating autoantibodies reacting with CD22 in systemic sclerosis. Serum samples from 10 tight skin (TSK/+) mice and 50 SSc patients were assessed for anti-CD22 autoantibodies by enzyme-linked immunosorbent assays using recombinant mouse or human CD22. The association between anti-CD22 antibodies and clinical features was also investigated in SSc patients. Furthermore, the influence of SSc serum including anti-CD22 autoantibodies for CD22 tyrosine phosphorylation was examined by Western blotting using phosphotyrosine-specific antibodies reacting with four major tyrosine motifs of CD22 cytoplasmic domain. Anti-CD22 autoantibodies were positive in 80% of TSK/+ mice and in 22% of SSc patients. Patients positive for anti-CD22 antibodies showed significantly higher modified Rodnan skin thickness score compared with patients negative for anti-CD22 antibodies. Furthermore, anti-CD22 antibodies from patients' sera were capable of reducing phosphorylation of all four CD22 tyrosine motifs, while sera negative for anti-CD22 antibodies did not affect CD22 phosphorylation. Thus, a subset of SSc patients possessed autoantibodies reacting with a major inhibitory B cell response regulator, CD22. Because these antibodies can interfere CD22-mediated suppression onto B cell activation in vitro, SSc B cells produce functional autoantibodies that can enhance their own activation. This unique regulation may contribute to the autoimmune aspect of SSc.

Activation and reactivation of the RNA polymerase II trigger loop for intrinsic RNA cleavage and catalysis

PubMed Central

Čabart, Pavel; Jin, Huiyan; Li, Liangtao; Kaplan, Craig D

2014-01-01

In addition to RNA synthesis, multisubunit RNA polymerases (msRNAPs) support enzymatic reactions such as intrinsic transcript cleavage. msRNAP active sites from different species appear to exhibit differential intrinsic transcript cleavage efficiency and have likely evolved to allow fine-tuning of the transcription process. Here we show that a single amino-acid substitution in the trigger loop (TL) of Saccharomyces RNAP II, Rpb1 H1085Y, engenders a gain of intrinsic cleavage activity where the substituted tyrosine appears to participate in acid-base chemistry at alkaline pH for both intrinsic cleavage and nucleotidyl transfer. We extensively characterize this TL substitution for each of these reactions by examining the responses RNAP II enzymes to catalytic metals, altered pH, and factor inputs. We demonstrate that TFIIF stimulation of the first phosphodiester bond formation by RNAP II requires wild type TL function and that H1085Y substitution within the TL compromises or alters RNAP II responsiveness to both TFIIB and TFIIF. Finally, Mn2+ stimulation of H1085Y RNAP II reveals possible allosteric effects of TFIIB on the active center and cooperation between TFIIB and TFIIF. PMID:25764335

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

PubMed Central

Xu, Qifang; Malecka, Kimberly L.; Fink, Lauren; Jordan, E. Joseph; Duffy, Erin; Kolander, Samuel; Peterson, Jeffrey; Dunbrack, Roland L.

2016-01-01

Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets. PMID:26628682

The pituitary-skin connection in amphibians. Reciprocal regulation of melanotrope cells and dermal melanocytes.

PubMed

Vaudry, H; Chartrel, N; Desrues, L; Galas, L; Kikuyama, S; Mor, A; Nicolas, P; Tonon, M C

1999-10-20

In amphibians, alpha-MSH secreted by the pars intermedia of the pituitary plays a pivotal role in the process of skin color adaptation. Reciprocally, the skin of amphibians contains a number of regulatory peptides, some of which have been found to regulate the activity of pituitary melanotrope cells. In particular, the skin of certain species of amphibians harbours considerable amounts of thyrotropin-releasing hormone, a highly potent stimulator of alpha-MSH release. Recently, we have isolated and sequenced from the skin of the frog Phyllomedusa bicolor--a novel peptide named skin peptide tyrosine tyrosine (SPYY), which exhibits 94% similarity with PYY from the frog Rana ridibunda. For concentrations ranging from 5 x 10(-10) to 10(-7) M, SPYY induces a dose-related inhibition of alpha-MSH secretion. At a dose of 10(-7) M, SPYY totally abolished alpha-MSH release. These data strongly suggest the existence of a regulatory loop between the pars intermedia of the pituitary and the skin in amphibians.

Insights into the conformational switching mechanism of the human vascular endothelial growth factor receptor type 2 kinase domain.

PubMed

Chioccioli, Matteo; Marsili, Simone; Bonaccini, Claudia; Procacci, Piero; Gratteri, Paola

2012-02-27

Human vascular endothelial growth factor receptor type 2 (h-VEFGR2) is a receptor tyrosine kinase involved in the angiogenesis process and regarded as an interesting target for the design of anticancer drugs. Its activation/inactivation mechanism is related to conformational changes in its cytoplasmatic kinase domain, involving first among all the αC-helix in N-lobe and the A-loop in C-lobe. Affinity of inhibitors for the active or inactive kinase form could dictate the open or closed conformation of the A-loop, thus making the different conformations of the kinase domain receptor (KDR) domain different drug targets in drug discovery. In this view, a detailed knowledge of the conformational landscape of KDR domain is of central relevance to rationalize the efficiency and selectivity of kinase inhibitors. Here, molecular dynamics simulations were used to gain insight into the conformational switching activity of the KDR domain and to identify intermediate conformations between the two limiting active and inactive conformations. Specific energy barriers have been selectively removed to induce, and hence highlight at the atomistic level, the regulation mechanism of the A-loop opening. The proposed strategy allowed to repeatedly observe the escape of the KDR domain from the DFG-out free energy basin and to identify rare intermediate conformations between the DFG-out and the DFG-in structures to be employed in a structure-based drug discovery process.

Mitochondrial translocation of signal transducer and activator of transcription 5 (STAT5) in leukemic T cells and cytokine-stimulated cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chueh, Fu-Yu; Leong, King-Fu; Yu, Chao-Lan, E-mail: chaolan.yu@rosalindfranklin.edu

2010-11-26

Research highlights: {yields} STAT5 interacts with a mitochondrial protein PDC-E2 in a leukemic T cell line LSTRA. {yields} Tyrosine-phosphorylated STAT5, but not STAT3, is present in LSTRA mitochondria. {yields} Cytokines induce mitochondrial translocation of STAT5, but not STAT1 or STAT3. {yields} Cytokine-induced mitochondrial translocation of tyrosine-phosphorylated STAT5 is transient. {yields} Mitochondrial STAT5 binds to a putative STAT5 site in the mitochondrial DNA in vitro. -- Abstract: Signal transducers and activators of transcription (STATs) were first identified as key signaling molecules in response to cytokines. Constitutive STAT activation also has been widely implicated in oncogenesis. We analyzed STAT5-associated proteins in amore » leukemic T cell line LSTRA, which exhibits constitutive tyrosine phosphorylation and activation of STAT5. A cellular protein was found to specifically interact with STAT5 in LSTRA cells by co-immunoprecipitation. Sequencing analysis and subsequent immunoblotting confirmed the identity of this STAT5-associated protein as the E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2). Consistent with this interaction, both subcellular fractionation and immunofluorescence microscopy revealed mitochondrial localization of STAT5 in LSTRA cells. Mitochondrial localization of tyrosine-phosphorylated STAT5 also occurred in cytokine-stimulated cells. A time course experiment further demonstrated the transient kinetics of STAT5 mitochondrial translocation after cytokine stimulation. In contrast, cytokine-induced STAT1 and STAT3 activation did not result in their translocation into mitochondria. Furthermore, we showed that mitochondrial STAT5 bound to the D-loop regulatory region of mitochondrial DNA in vitro. It suggests a potential role of STAT5 in regulating the mitochondrial genome. Proliferative metabolism toward aerobic glycolysis is well known in cancer cells as the Warburg effect and is also observed in cytokine-stimulated cells. Our novel findings of cytokine-induced STAT5 translocation into mitochondria and its link to oncogenesis provide important insights into the underlying mechanisms of this characteristic metabolic shift.« less

Regulation of Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase (Btk) through a Novel SH3-Dependent Interaction with Ankyrin Repeat Domain 54 (ANKRD54)

PubMed Central

Hussain, Alamdar; Mohammad, Dara K.; Mohamed, Abdalla J.; Nguyen, Vivian; Metalnikov, Pavel; Colwill, Karen; Pawson, Tony; Nore, Beston F.

2012-01-01

Bruton's tyrosine kinase (Btk), belonging to the Tec family of tyrosine kinases (TFKs), is essential for B-lymphocyte development. Abrogation of Btk signaling causes human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). We employed affinity purification of Flag-tagged Btk, combined with tandem mass spectrometry, to capture and identify novel interacting proteins. We here characterize the interaction with ankryin repeat domain 54 protein (ANKRD54), also known as Lyn-interacting ankyrin repeat protein (Liar). While Btk is a nucleocytoplasmic protein, the Liar pool was found to shuttle at a higher rate than Btk. Importantly, our results suggest that Liar mediates nuclear export of both Btk and another TFK, Txk/Rlk. Liar-mediated Btk shuttling was enriched for activation loop, nonphosphorylated Btk and entirely dependent on Btk's SH3 domain. Liar also showed reduced binding to an aspartic acid phosphomimetic SH3 mutant. Three other investigated nucleus-located proteins, Abl, estrogen receptor β (ERβ), and transcription factor T-bet, were all unaffected by Liar. We mapped the interaction site to the C terminus of the Btk SH3 domain. A biotinylated, synthetic Btk peptide, ARDKNGQEGYIPSNYVTEAEDS, was sufficient for this interaction. Liar is the first protein identified that specifically influences the nucleocytoplasmic shuttling of Btk and Txk and belongs to a rare group of known proteins carrying out this activity in a Crm1-dependent manner. PMID:22527282

Structural Basis of a Key Factor Regulating the Affinity between the Zonula Occludens First PDZ Domain and Claudins.

PubMed

Nomme, Julian; Antanasijevic, Aleksandar; Caffrey, Michael; Van Itallie, Christina M; Anderson, James M; Fanning, Alan S; Lavie, Arnon

2015-07-03

The molecular seal between epithelial cells, called the tight junction (TJ), is built by several membrane proteins, with claudins playing the most prominent role. The scaffold proteins of the zonula occludens family are required for the correct localization of claudins and hence formation of the TJ. The intracellular C terminus of claudins binds to the N-terminal PDZ domain of zonula occludens proteins (PDZ1). Of the 23 identified human claudin proteins, nine possess a tyrosine at the -6 position. Here we show that the claudin affinity for PDZ1 is dependent on the presence or absence of this tyrosine and that the affinity is reduced if the tyrosine is modified by phosphorylation. The PDZ1 β2-β3 loop undergoes a significant conformational change to accommodate this tyrosine. Cell culture experiments support a regulatory role for this tyrosine. Plasticity has been recognized as a critical property of TJs that allow cell remodeling and migration. Our work provides a molecular framework for how TJ plasticity may be regulated. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein tyrosine phosphatase 1B inhibitory activity of alkaloids from Rhizoma Coptidis and their molecular docking studies.

PubMed

Choi, Jae Sue; Ali, Md Yousof; Jung, Hyun Ah; Oh, Sang Ho; Choi, Ran Joo; Kim, Eon Ji

2015-08-02

Rhizoma Coptidis (the rhizome of Coptis chinensis Franch) has commonly been used for treatment of diabetes mellitus in traditional Chinese medicine due to its blood sugar-lowering properties and therapeutic benefits which highly related to the alkaloids therein. However, a limited number of studies focused on the Coptis alkaloids other than berberine. In the present study, we investigated the anti-diabetic potential of Coptis alkaloids, including berberine (1), epiberberine (2), magnoflorine (3), and coptisine (4), by evaluating the ability of these compounds to inhibit protein tyrosine phosphatase 1B (PTP1B), and ONOO(-)-mediated protein tyrosine nitration. We scrutinized the potentials of Coptis alkaloids as PTP1B inhibitors via enzyme kinetics and molecular docking simulation. The Coptis alkaloids 1-4 exhibited remarkable inhibitory activities against PTP1B with the IC50 values of 16.43, 24.19, 28.14, and 51.04 μM, respectively, when compared to the positive control ursolic acid. These alkaloids also suppressed ONOO(-)-mediated tyrosine nitration effectively in a dose dependent manner. In addition, our kinetic study using the Lineweaver-Burk and Dixon plots revealed that 1 and 2 showed a mixed-type inhibition against PTP1B, while 3 and 4 noncompetitively inhibited PTP1B. Moreover, molecular docking simulation of these compounds demonstrated negative binding energies (Autodock 4.0=-6.7 to -7.8 kcal/mol; Fred 2.0=-59.4 to -68.2 kcal/mol) and a high proximity to PTP1B residues, including Phe182 and Asp181 in the WPD loop, Cys215 in the active sites and Tyr46, Arg47, Asp48, Val49, Ser216, Ala217, Gly218, Ile219, Gly220, Arg221 and Gln262 in the pocket site, indicating a higher affinity and tighter binding capacity of these alkaloids for the active site of the enzyme. Our results clearly indicate the promising anti-diabetic potential of Coptis alkaloids as inhibitors on PTP1B as well as suppressors of ONOO(-)-mediated protein tyrosine nitration, and thus hold promise as therapeutic agents for the treatment of diabetes and related disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Structural basis of agrin-LRP4-MuSK signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zong, Yinong; Zhang, Bin; Gu, Shenyan

Synapses are the fundamental units of neural circuits that enable complex behaviors. The neuromuscular junction (NMJ), a synapse formed between a motoneuron and a muscle fiber, has contributed greatly to understanding of the general principles of synaptogenesis as well as of neuromuscular disorders. NMJ formation requires neural agrin, a motoneuron-derived protein, which interacts with LRP4 (low-density lipoprotein receptor-related protein 4) to activate the receptor tyrosine kinase MuSK (muscle-specific kinase). However, little is known of how signals are transduced from agrin to MuSK. Here, we present the first crystal structure of an agrin-LRP4 complex, consisting of two agrin-LRP4 heterodimers. Formation ofmore » the initial binary complex requires the z8 loop that is specifically present in neuronal, but not muscle, agrin and that promotes the synergistic formation of the tetramer through two additional interfaces. We show that the tetrameric complex is essential for neuronal agrin-induced acetylcholine receptor (AChR) clustering. Collectively, these results provide new insight into the agrin-LRP4-MuSK signaling cascade and NMJ formation and represent a novel mechanism for activation of receptor tyrosine kinases.« less

Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis.

PubMed

Luthra, Pratibha Mehta; Singh, Satendra

2010-05-01

Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which L: -dopa is formed from the substrate L-tyrosine. L-Dopa concentration and activity of L-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum L-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH(4), and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K (m) value of 808.63 microM for L-tyrosine at 37 degrees C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 microM L-tyrosine. Higher concentrations of the cofactor 6-MPH(4), however, completely inhibited the synthesis of L-dopa. Tyrosine hydroxylase converted specific monophenols such as L-tyrosine (808.63 microM) and tyramine (K (m) 1.1 mM) to diphenols L-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.

What's the FOX Got to Do with the KITten? Regulating the Lineage-Specific Transcriptional Landscape in GIST.

PubMed

Lee, Donna M; Duensing, Anette

2018-02-01

Transcriptional regulation of the KIT receptor tyrosine kinase, a master regulator in gastrointestinal stromal tumors (GIST) and their precursors, the interstitial cells of Cajal (ICC), is part of a positive feedback loop involving the transcription factor ETV1. A new study now shows that the forkhead box (FOX) family transcription factor FOXF1 not only is an upstream regulator of ETV1 and hence ICC/GIST lineage-specific gene transcription, but also functions as lineage-specific pioneer factor with an active role in chromatin rearrangement to facilitate ETV1 binding and transcriptional activity. Cancer Discov; 8(2); 146-9. ©2018 AACR See related article by Ran et al., p. 234 . ©2018 American Association for Cancer Research.

Inhibition of Shp2 suppresses mutant EGFR-induced lung tumors in transgenic mouse model of lung adenocarcinoma

PubMed Central

Schneeberger, Valentina E.; Ren, Yuan; Luetteke, Noreen; Huang, Qingling; Chen, Liwei; Lawrence, Harshani R.; Lawrence, Nicholas J.; Haura, Eric B.; Koomen, John M.; Coppola, Domenico; Wu, Jie

2015-01-01

Epidermal growth factor receptor (EGFR) mutants drive lung tumorigenesis and are targeted for therapy. However, resistance to EGFR inhibitors has been observed, in which the mutant EGFR remains active. Thus, it is important to uncover mediators of EGFR mutant-driven lung tumors to develop new treatment strategies. The protein tyrosine phosphatase (PTP) Shp2 mediates EGF signaling. Nevertheless, it is unclear if Shp2 is activated by oncogenic EGFR mutants in lung carcinoma or if inhibiting the Shp2 PTP activity can suppress EGFR mutant-induced lung adenocarcinoma. Here, we generated transgenic mice containing a doxycycline (Dox)-inducible PTP-defective Shp2 mutant (tetO-Shp2CSDA). Using the rat Clara cell secretory protein (CCSP)-rtTA-directed transgene expression in the type II lung pneumocytes of transgenic mice, we found that the Gab1-Shp2 pathway was activated by EGFRL858R in the lungs of transgenic mice. Consistently, the Gab1-Shp2 pathway was activated in human lung adenocarcinoma cells containing mutant EGFR. Importantly, Shp2CSDA inhibited EGFRL858R-induced lung adenocarcinoma in transgenic animals. Analysis of lung tissues showed that Shp2CSDA suppressed Gab1 tyrosine phosphorylation and Gab1-Shp2 association, suggesting that Shp2 modulates a positive feedback loop to regulate its own activity. These results show that inhibition of the Shp2 PTP activity impairs mutant EGFR signaling and suppresses EGFRL858R-driven lung adenocarcinoma. PMID:25730908

Pleiotrophin suppression of receptor protein tyrosine phosphatase-β/ζ maintains the self-renewal competence of fetal human oligodendrocyte progenitor cells.

PubMed

McClain, Crystal R; Sim, Fraser J; Goldman, Steven A

2012-10-24

Oligodendrocyte progenitor cells (OPCs) persist in human white matter, yet the mechanisms by which they are maintained in an undifferentiated state are unknown. Human OPCs differentially express protein tyrosine phosphatase receptor β/ζ (PTPRZ1) and its inhibitory ligand, pleiotrophin, suggesting the maintenance of an autocrine loop by which PTPRZ1 activity is tonically suppressed. PTPRZ1 constitutively promotes the tyrosine dephosphorylation of β-catenin and, thus, β-catenin participation in T cell factor (TCF)-mediated transcription. Using CD140a/PDGFRα-based fluorescence-activated cell sorting to isolate fetal OPCs from the fetal brain at gestational ages 16-22 weeks, we asked whether pleiotrophin modulated the expansion of OPCs and, if so, whether this was effected through the serial engagement of PTPRZ1 and β-catenin-dependent signals, such as TCF-mediated transcription. Lentiviral shRNAi knockdown of PTPRZ1 induced TCF-mediated transcription and substantially augmented GSK3β inhibition-induced TCF-reporter luciferase expression, suggesting dual regulation of β-catenin and the importance of PTPRZ1 as a tonic brake upon TCF-dependent transcription. Pharmacological inhibition of GSK3β triggered substrate detachment and initiated sphere formation, yet had no effect on either proliferation or net cell number. In contrast, pleiotrophin strongly potentiated the proliferation of CD140a(+)-sorted OPCs, as did PTPRZ1 knockdown, which significantly increased the total number of population doublings exhibited by OPCs before mitotic senescence. These observations suggest that pleiotrophin inhibition of PTPRZ1 contributes to the homeostatic self-renewal of OPCs and that this process is mediated by the tonic activation of β-catenin/TCF-dependent transcription.

Pleiotrophin Suppression of Receptor Protein Tyrosine Phosphatase-β/ζ Maintains the Self-Renewal Competence of Fetal Human Oligodendrocyte Progenitor Cells

PubMed Central

McClain, Crystal R.; Sim, Fraser J.

2012-01-01

Oligodendrocyte progenitor cells (OPCs) persist in human white matter, yet the mechanisms by which they are maintained in an undifferentiated state are unknown. Human OPCs differentially express protein tyrosine phosphatase receptor β/ζ (PTPRZ1) and its inhibitory ligand, pleiotrophin, suggesting the maintenance of an autocrine loop by which PTPRZ1 activity is tonically suppressed. PTPRZ1 constitutively promotes the tyrosine dephosphorylation of β-catenin and, thus, β-catenin participation in T cell factor (TCF)-mediated transcription. Using CD140a/PDGFRα-based fluorescence-activated cell sorting to isolate fetal OPCs from the fetal brain at gestational ages 16–22 weeks, we asked whether pleiotrophin modulated the expansion of OPCs and, if so, whether this was effected through the serial engagement of PTPRZ1 and β-catenin-dependent signals, such as TCF-mediated transcription. Lentiviral shRNAi knockdown of PTPRZ1 induced TCF-mediated transcription and substantially augmented GSK3β inhibition-induced TCF-reporter luciferase expression, suggesting dual regulation of β-catenin and the importance of PTPRZ1 as a tonic brake upon TCF-dependent transcription. Pharmacological inhibition of GSK3β triggered substrate detachment and initiated sphere formation, yet had no effect on either proliferation or net cell number. In contrast, pleiotrophin strongly potentiated the proliferation of CD140a+-sorted OPCs, as did PTPRZ1 knockdown, which significantly increased the total number of population doublings exhibited by OPCs before mitotic senescence. These observations suggest that pleiotrophin inhibition of PTPRZ1 contributes to the homeostatic self-renewal of OPCs and that this process is mediated by the tonic activation of β-catenin/TCF-dependent transcription. PMID:23100427

Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr(215) in Aerococcus viridans lactate oxidase.

PubMed

Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K; Nidetzky, Bernd

2016-06-15

L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr(215) in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr(215), effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr(215) can thus lead to a kinetic bottleneck in product release.

D-tyrosine negatively regulates melanin synthesis by competitively inhibiting tyrosinase activity.

PubMed

Park, Jisu; Jung, Hyejung; Kim, Kyuri; Lim, Kyung-Min; Kim, Ji-Young; Jho, Eek-Hoon; Oh, Eok-Soo

2018-05-01

Although L-tyrosine is well known for its melanogenic effect, the contribution of D-tyrosine to melanin synthesis was previously unexplored. Here, we reveal that, unlike L-tyrosine, D-tyrosine dose-dependently reduced the melanin contents of human MNT-1 melanoma cells and primary human melanocytes. In addition, 500 μM of D-tyrosine completely inhibited 10 μM L-tyrosine-induced melanogenesis, and both in vitro assays and L-DOPA staining MNT-1 cells showed that tyrosinase activity is reduced by D-tyrosine treatment. Thus, D-tyrosine appears to inhibit L-tyrosine-mediated melanogenesis by competitively inhibiting tyrosinase activity. Furthermore, we found that D-tyrosine inhibited melanogenesis induced by α-MSH treatment or UV irradiation, which are the most common environmental factors responsible for melanin synthesis. Finally, we confirmed that D-tyrosine reduced melanin synthesis in the epidermal basal layer of a 3D human skin model. Taken together, these data suggest that D-tyrosine negatively regulates melanin synthesis by inhibiting tyrosinase activity in melanocyte-derived cells. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bacteriophage phi 6 RNA-dependent RNA polymerase: molecular details of initiating nucleic acid synthesis without primer.

PubMed

Laurila, Minni R L; Makeyev, Eugene V; Bamford, Dennis H

2002-05-10

Like most RNA polymerases, the polymerase of double-strand RNA bacteriophage phi6 (phi6pol) is capable of primer-independent initiation. Based on the recently solved phi6pol initiation complex structure, a four-amino acid-long loop (amino acids 630-633) has been suggested to stabilize the first two incoming NTPs through stacking interactions with tyrosine, Tyr(630). A similar loop is also present in the hepatitis C virus polymerase, another enzyme capable of de novo initiation. Here, we use a series of phi6pol mutants to address the role of this element. As predicted, mutants at the Tyr(630) position are inefficient in initiation de novo. Unexpectedly, when the loop is disordered by changing Tyr(630)-Lys(631)-Trp(632) to GSG, phi6pol becomes a primer-dependent enzyme, either extending complementary oligonucleotide or, when the template 3' terminus can adopt a hairpin-like conformation, utilizing a "copy-back" initiation mechanism. In contrast to the wild-type phi6pol, the GSG mutant does not require high GTP concentration for its optimal activity. These findings suggest a general model for the initiation of de novo RNA synthesis.

Carboxyl group footprinting mass spectrometry and molecular dynamics identify key interactions in the HER2-HER3 receptor tyrosine kinase interface.

PubMed

Collier, Timothy S; Diraviyam, Karthikeyan; Monsey, John; Shen, Wei; Sept, David; Bose, Ron

2013-08-30

The HER2 receptor tyrosine kinase is a driver oncogene in many human cancers, including breast and gastric cancer. Under physiologic levels of expression, HER2 heterodimerizes with other members of the EGF receptor/HER/ErbB family, and the HER2-HER3 dimer forms one of the most potent oncogenic receptor pairs. Previous structural biology studies have individually crystallized the kinase domains of HER2 and HER3, but the HER2-HER3 kinase domain heterodimer structure has yet to be solved. Using a reconstituted membrane system to form HER2-HER3 kinase domain heterodimers and carboxyl group footprinting mass spectrometry, we observed that HER2 and HER3 kinase domains preferentially form asymmetric heterodimers with HER3 and HER2 monomers occupying the donor and acceptor kinase positions, respectively. Conformational changes in the HER2 activation loop, as measured by changes in carboxyl group labeling, required both dimerization and nucleotide binding but did not require activation loop phosphorylation at Tyr-877. Molecular dynamics simulations on HER2-HER3 kinase dimers identify specific inter- and intramolecular interactions and were in good agreement with MS measurements. Specifically, several intermolecular ionic interactions between HER2 Lys-716-HER3 Glu-909, HER2 Glu-717-HER3 Lys-907, and HER2 Asp-871-HER3 Arg-948 were identified by molecular dynamics. We also evaluated the effect of the cancer-associated mutations HER2 D769H/D769Y, HER3 E909G, and HER3 R948K (also numbered HER3 E928G and R967K) on kinase activity in the context of this new structural model. This study provides valuable insights into the EGF receptor/HER/ErbB kinase structure and interactions, which can guide the design of future therapies.

HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

PubMed

Nakashima, Kenji; Takeuchi, Kenji; Chihara, Kazuyasu; Horiguchi, Tomoko; Sun, Xuedong; Deng, Lin; Shoji, Ikuo; Hotta, Hak; Sada, Kiyonao

2012-01-01

Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg(176) to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334) was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition.

PubMed

Devkota, Sujan; Joseph, Raji E; Boyken, Scott E; Fulton, D Bruce; Andreotti, Amy H

2017-06-13

Pleckstrin homology (PH) domains are well-known as phospholipid binding modules, yet evidence that PH domain function extends beyond lipid recognition is mounting. In this work, we characterize a protein binding function for the PH domain of interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of nonreceptor tyrosine kinases. Its N-terminal PH domain is a well-characterized lipid binding module that localizes ITK to the membrane via phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) binding. Using a combination of nuclear magnetic resonance spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK, inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP 3 . By clarifying the allosteric role of the ITK PH domain in controlling ITK function, we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.

SH2-dependent autophosphorylation within the Tec family kinase Itk.

PubMed

Joseph, Raji E; Severin, Andrew; Min, Lie; Fulton, D Bruce; Andreotti, Amy H

2009-08-07

The Tec family kinase, Itk (interleukin-2 tyrosine kinase), undergoes an in cis autophosphorylation on Y180 within its Src homology 3 (SH3) domain. Autophosphorylation of the Itk SH3 domain by the Itk kinase domain is strictly dependent on the presence of the intervening Src homology 2 (SH2) domain. A direct docking interaction between the Itk kinase and SH2 domains brings the Itk SH3 domain into the active site where Y180 is then phosphorylated. We now identify the residues on the surface of the Itk SH2 domain responsible for substrate docking and show that this SH2 surface mediates autophosphorylation in the full-length Itk molecule. The canonical phospholigand binding site on the SH2 domain is not involved in substrate docking, instead the docking site consists of side chains from three loop regions (AB, EF and BG) and part of the betaD strand. These results are extended into Btk (Bruton's tyrosine kinase), a Tec family kinase linked to the B-cell deficiency X-linked agammaglobulinemia (XLA). Our results suggest that some XLA-causing mutations might impair Btk phosphorylation.

Conformational changes induced in the protein tyrosine kinase p72syk by tyrosine phosphorylation or by binding of phosphorylated immunoreceptor tyrosine-based activation motif peptides.

PubMed Central

Kimura, T; Sakamoto, H; Appella, E; Siraganian, R P

1996-01-01

A critical event in signaling in immune cells is the interaction of Syk or ZAP-70 protein tyrosine kinases with multisubunit receptors that contain an approximately 18-amino-acid domain called the immunoreceptor tyrosine-based activation motif (ITAM). Tyrosine-phosphorylated Syk from activated cells was in a conformation different from that in nonstimulated cells as demonstrated by changes in immunoreactivity. The addition of tyrosine-diphosphorylated ITAM peptides resulted in a similar conformational change in Syk from nonactivated cells. The peptides based on FcepsilonRIgamma were more active than those based on Fcepsilon RIbeta. In vitro autophosphorylation of Syk was dramatically enhanced by the addition of the diphosphorylated ITAM peptides. The conformational change and the enhanced autophosphorylation required the presence of both phosphorylated tyrosines on the same molecule. These conformational changes in Syk by tyrosine phosphorylation or binding to diphosphorylated ITAM could be critical for Syk activation and downstream propagation of intracellular signals. PMID:8657120

Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity.

PubMed

Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno; Stensballe, Allan; Jensen, Ole N; Carter-Su, Christin

2004-06-01

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2.

Regulation of tyrosine hydroxylase activity and phosphorylation at Ser(19) and Ser(40) via activation of glutamate NMDA receptors in rat striatum.

PubMed

Lindgren, N; Xu, Z Q; Lindskog, M; Herrera-Marschitz, M; Goiny, M; Haycock, J; Goldstein, M; Hökfelt, T; Fisone, G

2000-06-01

The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser(19)-tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser(40) and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser(40) phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser(40)-tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser(40) via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis.

The use of Imatinib resistance mutation analysis to direct therapy in Philadelphia chromosome/BCR-ABL1 positive chronic myeloid leukaemia patients failing Imatinib treatment, in Patan Hospital, Nepal.

PubMed

Kayastha, Gyan K; Ranjitkar, Nora; Gurung, Radha; Kc, Raj Kumar; Karki, Sanjit; Shrestha, Roshan; Rajbhandari, Piyush; Thapa, Raj K; Poudyal, Buddhi; Acharya, Paras; Roberts, David J; Hayes, Bruce; Zimmerman, Mark; Basnyat, Buddha

2017-06-01

Philadelphia chromosome/BCR-ABL1 positive chronic myeloid leukaemia (CML) can be successfully treated with Glivec (Imatinib), which is available free of cost through the Glivec International Patient Assistance programme (GIPAP) to patients with proven CML without means to pay for the drug. We review the acquired mutations in the tyrosine kinase encoded by the BCR-ABL1 gene underlying Glivec failure or resistance in a cohort of 388 imatinib-treated CML patients (149 Female and 239 male) registered between February 2003 and June 2016 in Nepal. Forty-five patients (11 female 34 male) were studied; 18 different BCR-ABL1 mutations were seen in 33 patients. P-loop mutation, Kinase domain and A-loop mutations were seen in 9, 16 and 4 patients respectively. Other mutations were seen in five patients. A T315I mutation was the most common mutation, followed by F359V and M244V. Sixteen mutations showed intermediate activity to complete resistance to Glivec. Among the 45 patients evaluated for BCR-ABL1 mutations, 4 were lost to follow-up, 14 died and 27 are still alive. Among the surviving patients, 16 are receiving Nilotinib, 5 Dasatinib and 3 Ponatinib, while 3 patients were referred to India, one of who received allogenic bone marrow transplantation. Understanding the spectrum of further acquired mutations in BCR-ABL1 may help to choose more specific targeted tyrosine kinase inhibitors that can be provided by GIPAP. © 2017 John Wiley & Sons Ltd.

Tyrosine-Kinase Inhibitors Therapies with Mainly Anti-Angiogenic Activity in Advanced Renal Cell Carcinoma: Value of PET/CT in Response Evaluation

PubMed Central

Ranieri, Girolamo; Marech, Ilaria; Niccoli Asabella, Artor; Di Palo, Alessandra; Porcelli, Mariangela; Lavelli, Valentina; Rubini, Giuseppe; Ferrari, Cristina; Gadaleta, Cosmo Damiano

2017-01-01

Renal cell carcinoma (RCC) is the most frequent renal tumor and the majority of patients are diagnosed with advanced disease. Tumor angiogenesis plays a crucial role in the development and progression of RCC together with hypoxia and glucose metabolism. These three pathways are strictly connected to the cell growth and proliferation, like a loop that is self-feeding. Over the last few years, the ever-deeper knowledge of its contribution in metastatic RCC led to the discovery of numerous tyrosine kinase inhibitors (TKIs) targeting pro-angiogenic receptors at different levels such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, and dovitinib. As anti-angiogenic agents, TKIs interfere the loop, being able to inhibit tumor proliferation. TKIs are now available treatments for advanced RCC, which demonstrated to improve overall survival and/or progression free survival. Their effects can be detectable early on Positron Emission Tomography/Computed Tomography (PET/CT) by change in 18F-fluoro-2-deoxy-2-d-glucose (18F-FDG) uptake, the main radiotracer used to date, as a strong indicator of biological response. 18F-FDG PET/CT demonstrated an ability to predict and monitor disease progression, allowing an early and reliable identification of responders, and could be used for image-guided optimization and “personalization” of anti-angiogenic regimens. New radiotracers for biometabolic imaging are currently under investigation, which exploit the other pathways involved in the cancer process, including cellular proliferation, aerobic metabolism, cell membrane synthesis, hypoxia and amino acid transport, as well as the angiogenic process, but they require further studies. PMID:28891933

Distinct roles of immunoreceptor tyrosine-based motifs in immunosuppressive indoleamine 2,3-dioxygenase 1.

PubMed

Albini, Elisa; Rosini, Verdiana; Gargaro, Marco; Mondanelli, Giada; Belladonna, Maria L; Pallotta, Maria Teresa; Volpi, Claudia; Fallarino, Francesca; Macchiarulo, Antonio; Antognelli, Cinzia; Bianchi, Roberta; Vacca, Carmine; Puccetti, Paolo; Grohmann, Ursula; Orabona, Ciriana

2017-01-01

The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the initial, rate-limiting step in tryptophan (Trp) degradation, resulting in tryptophan starvation and the production of immunoregulatory kynurenines. IDO1's catalytic function has long been considered as the one mechanism responsible for IDO1-dependent immune suppression by dendritic cells (DCs), which are master regulators of the balance between immunity and tolerance. However, IDO1 also harbours immunoreceptor tyrosine-based inhibitory motifs, (ITIM1 and ITIM2), that, once phosphorylated, bind protein tyrosine phosphatases, (SHP-1 and SHP-2), and thus trigger an immunoregulatory signalling in DCs. This mechanism leads to sustained IDO1 expression, in a feedforward loop, which is particularly important in restraining autoimmunity and chronic inflammation. Yet, under specific conditions requiring that early and protective inflammation be unrelieved, tyrosine-phosphorylated ITIMs will instead bind the suppressor of cytokine signalling 3 (SOCS3), which drives IDO1 proteasomal degradation and shortens the enzyme half-life. To dissect any differential roles of the two IDO1's ITIMs, we generated protein mutants by replacing one or both ITIM-associated tyrosines with phospho-mimicking glutamic acid residues. Although all mutants lost their enzymic activity, the ITIM1 - but not ITIM2 mutant - did bind SHPs and conferred immunosuppressive effects on DCs, making cells capable of restraining an antigen-specific response in vivo. Conversely, the ITIM2 mutant would preferentially bind SOCS3, and IDO1's degradation was accelerated. Thus, it is the selective phosphorylation of either ITIM that controls the duration of IDO1 expression and function, in that it dictates whether enhanced tolerogenic signalling or shutdown of IDO1-dependent events will occur in a local microenvironment. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Towards a Molecular Understanding of the Link between Imatinib Resistance and Kinase Conformational Dynamics

PubMed Central

Lovera, Silvia; Morando, Maria; Pucheta-Martinez, Encarna; Martinez-Torrecuadrada, Jorge L.; Saladino, Giorgio; Gervasio, Francesco L.

2015-01-01

Due to its inhibition of the Abl kinase domain in the BCR-ABL fusion protein, imatinib is strikingly effective in the initial stage of chronic myeloid leukemia with more than 90% of the patients showing complete remission. However, as in the case of most targeted anti-cancer therapies, the emergence of drug resistance is a serious concern. Several drug-resistant mutations affecting the catalytic domain of Abl and other tyrosine kinases are now known. But, despite their importance and the adverse effect that they have on the prognosis of the cancer patients harboring them, the molecular mechanism of these mutations is still debated. Here by using long molecular dynamics simulations and large-scale free energy calculations complemented by in vitro mutagenesis and microcalorimetry experiments, we model the effect of several widespread drug-resistant mutations of Abl. By comparing the conformational free energy landscape of the mutants with those of the wild-type tyrosine kinases we clarify their mode of action. It involves significant and complex changes in the inactive-to-active dynamics and entropy/enthalpy balance of two functional elements: the activation-loop and the conserved DFG motif. What is more the T315I gatekeeper mutant has a significant impact on the binding mechanism itself and on the binding kinetics. PMID:26606374

Autophosphorylation of JAK2 on Tyrosines 221 and 570 Regulates Its Activity

PubMed Central

Argetsinger, Lawrence S.; Kouadio, Jean-Louis K.; Steen, Hanno; Stensballe, Allan; Jensen, Ole N.; Carter-Su, Christin

2004-01-01

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2. PMID:15143187

Structure of isocitrate dehydrogenase with alpha-ketoglutarate at 2.7-A resolution: conformational changes induced by decarboxylation of isocitrate.

PubMed

Stoddard, B L; Koshland, D E

1993-09-14

The structure of the isocitrate dehydrogenase (IDH) complex with bound alpha-ketoglutarate, Ca2+, and NADPH was solved at 2.7-A resolution. The alpha-ketoglutarate binds in the active site at the same position and orientation as isocitrate, with a difference between the two bound molecules of about 0.8 A. The Ca2+ metal is coordinated by alpha-ketoglutarate, three conserved aspartate residues, and a pair of water molecules. The largest motion in the active site relative to the isocitrate enzyme complex is observed for tyrosine 160, which originally forms a hydrogen bond to the labile carboxyl group of isocitrate and moves to form a new hydrogen bond to Asp 307 in the complex with alpha-ketoglutarate. This triggers a number of significant movements among several short loops and adjoining secondary structural elements in the enzyme, most of which participate in dimer stabilization and formation of the active-site cleft. These rearrangements are similar to the ligand-binding-induced movements observed in globins and insulin and serve as a model for an enzymatic mechanism which involves local shifts of secondary structural elements during turnover, rather than large-scale domain closures or loop transitions induced by substrate binding such as those observed in hexokinase or triosephosphate isomerase.

Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

PubMed

Catarsi, S; Drapeau, P

1997-08-01

Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

Nonreceptor Protein-Tyrosine Kinases in Neutrophil Activation

PubMed

Welch; Mauran; Maridonneau-Parini

1996-06-01

Nonreceptor protein-tyrosine kinases are involved in the regulation of almost all neutrophil responses such as adhesion, chemotaxis, priming, oxidative burst, and degranulation. Here, we show that phagocytosis is also regulated by protein-tyrosine kinase activity. Using various protein-tyrosine kinase inhibitors, we further demonstrate that opsonized zymosan-induced degranulation of specific and azurophil granules is regulated by protein-tyrosine kinase activity, whereas phorbol ester-induced degranulation is not. Several of the nonreceptor protein-tyrosine kinases involving in neutrophil signal transduction are known, including Fgr, Hck, Lyn, Yes, and Syk. Among these, Hck and Fgr are localized on the azurophil and specific granules, suggesting the involvement of these two protein-tyrosine kinases in the regulation of degranulation. In this report, we characterize some of the molecular properties of Hck and Fgr. We discuss the methods generally used for the measurement of protein-tyrosine kinase activities in neutrophils highlighting precautions against proteolysis. In addition, we show that in subcellular fractions of retinoic acid-differentiated neutrophil-like NB4 cells, the 59- and 61-kDa forms of Hck are attached to the membranes of their respective compartments by different mechanisms. Finally, we discuss the functional roles of protein-tyrosine kinases in the regulation of neutrophil activation and speculate on the importance of their subcellular localization.

Structural Basis for Selective Inhibition of Mycobacterium tuberculosis Protein Tyrosine Phosphatase PtpB

PubMed Central

Grundner, Christoph; Perrin, Dominique; van Huijsduijnen, Rob Hooft; Swinnen, Dominique; Gonzalez, Jérome; Gee, Christine L.; Wells, Timothy N.; Alber, Tom

2007-01-01

Tyrosine kinases and phosphatases establish the crucial balance of tyrosine phosphorylation in cellular signaling, but creating specific inhibitors of protein Tyr phosphatases (PTPs) remains a challenge. Here we report the development of a potent, selective inhibitor of Mycobacterium tuberculosis PtpB, a bacterial PTP that is secreted into host cells where it disrupts unidentified signaling pathways. The inhibitor, (oxalylamino-methylene)-thiophene sulfonamide (OMTS), showed an IC50 of 440 +/− 50 nM and >60-fold specificity for PtpB over six human PTPs. The 2-Å resolution crystal structure of PtpB in complex with OMTS revealed a large rearrangement of the enzyme, with some residues shifting >27 Å relative to the PtpB:PO4 complex. Extensive contacts with the catalytic loop provide a potential basis for inhibitor selectivity. Two OMTS molecules bound adjacent to each other, raising the possibility of a second substrate phosphotyrosine binding site in PtpB. The PtpB:OMTS structure provides an unanticipated framework to guide inhibitor improvement. PMID:17437721

A Novel Positive Feedback Loop Mediated by the Docking Protein Gab1 and Phosphatidylinositol 3-Kinase in Epidermal Growth Factor Receptor Signaling

PubMed Central

Rodrigues, Gerard A.; Falasca, Marco; Zhang, Zhongtao; Ong, Siew Hwa; Schlessinger, Joseph

2000-01-01

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4,5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR. PMID:10648629

Signal transduction through the IL-4 and insulin receptor families.

PubMed

Wang, L M; Keegan, A; Frankel, M; Paul, W E; Pierce, J H

1995-07-01

Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a pre-active conformation of a pentameric channel receptor

PubMed Central

Menny, Anaïs; Lefebvre, Solène N; Schmidpeter, Philipp AM; Drège, Emmanuelle; Fourati, Zaineb; Delarue, Marc; Edelstein, Stuart J; Nimigean, Crina M; Joseph, Delphine; Corringer, Pierre-Jean

2017-01-01

Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical signaling through global allosteric transitions. Despite the existence of several high-resolution structures of pLGICs, their dynamical properties remain elusive. Using the proton-gated channel GLIC, we engineered multiple fluorescent reporters, each incorporating a bimane and a tryptophan/tyrosine, whose close distance causes fluorescence quenching. We show that proton application causes a global compaction of the extracellular subunit interface, coupled to an outward motion of the M2-M3 loop near the channel gate. These movements are highly similar in lipid vesicles and detergent micelles. These reorganizations are essentially completed within 2 ms and occur without channel opening at low proton concentration, indicating that they report a pre-active intermediate state in the transition pathway toward activation. This provides a template to investigate the gating of eukaryotic neurotransmitter receptors, for which intermediate states also participate in activation. DOI: http://dx.doi.org/10.7554/eLife.23955.001 PMID:28294942

Prostate Cancer Cell–Stromal Cell Cross-Talk via FGFR1 Mediates Antitumor Activity of Dovitinib in Bone Metastases

PubMed Central

Wan, Xinhai; Corn, Paul G.; Yang, Jun; Palanisamy, Nallasivam; Starbuck, Michael W.; Efstathiou, Eleni; Li-Ning Tapia, Elsa M.; Zurita, Amado J.; Aparicio, Ana; Ravoori, Murali K.; Vazquez, Elba S.; Robinson, Dan R.; Wu, Yi-Mi; Cao, Xuhong; Iyer, Matthew K.; McKeehan, Wallace; Kundra, Vikas; Wang, Fen; Troncoso, Patricia; Chinnaiyan, Arul M.; Logothetis, Christopher J.; Navone, Nora M.

2015-01-01

Bone is the most common site of prostate cancer (PCa) progression to a therapy-resistant, lethal phenotype. We found that blockade of fibroblast growth factor receptors (FGFRs) with the receptor tyrosine kinase inhibitor dovitinib has clinical activity in a subset of men with castration-resistant PCa and bone metastases. Our integrated analyses suggest that FGF signaling mediates a positive feedback loop between PCa cells and bone cells and that blockade of FGFR1 in osteoblasts partially mediates the antitumor activity of dovitinib by improving bone quality and by blocking PCa cell–bone cell interaction. These findings account for clinical observations such as reductions in lesion size and intensity on bone scans, lymph node size, and tumor-specific symptoms without proportional declines in prostate-specific antigen concentration. Our findings suggest that targeting FGFR has therapeutic activity in advanced PCa and provide direction for the development of therapies with FGFR inhibitors. PMID:25186177

Prostate cancer cell-stromal cell crosstalk via FGFR1 mediates antitumor activity of dovitinib in bone metastases.

PubMed

Wan, Xinhai; Corn, Paul G; Yang, Jun; Palanisamy, Nallasivam; Starbuck, Michael W; Efstathiou, Eleni; Li Ning Tapia, Elsa M; Tapia, Elsa M Li-Ning; Zurita, Amado J; Aparicio, Ana; Ravoori, Murali K; Vazquez, Elba S; Robinson, Dan R; Wu, Yi-Mi; Cao, Xuhong; Iyer, Matthew K; McKeehan, Wallace; Kundra, Vikas; Wang, Fen; Troncoso, Patricia; Chinnaiyan, Arul M; Logothetis, Christopher J; Navone, Nora M

2014-09-03

Bone is the most common site of prostate cancer (PCa) progression to a therapy-resistant, lethal phenotype. We found that blockade of fibroblast growth factor receptors (FGFRs) with the receptor tyrosine kinase inhibitor dovitinib has clinical activity in a subset of men with castration-resistant PCa and bone metastases. Our integrated analyses suggest that FGF signaling mediates a positive feedback loop between PCa cells and bone cells and that blockade of FGFR1 in osteoblasts partially mediates the antitumor activity of dovitinib by improving bone quality and by blocking PCa cell-bone cell interaction. These findings account for clinical observations such as reductions in lesion size and intensity on bone scans, lymph node size, and tumor-specific symptoms without proportional declines in serum prostate-specific antigen concentration. Our findings suggest that targeting FGFR has therapeutic activity in advanced PCa and provide direction for the development of therapies with FGFR inhibitors. Copyright © 2014, American Association for the Advancement of Science.

Molecular docking studies of banana flower flavonoids as insulin receptor tyrosine kinase activators as a cure for diabetes mellitus.

PubMed

Ganugapati, Jayasree; Baldwa, Aashish; Lalani, Sarfaraz

2012-01-01

Diabetes mellitus is a metabolic disorder caused due to insulin deficiency. Banana flower is a rich source of flavonoids that exhibit anti diabetic activity. Insulin receptor is a tetramer that belongs to a family of receptor tyrosine kinases. It contains two alpha subunits that form the extracellular domain and two beta subunits that constitute the intracellular tyrosine kinase domain. Insulin binds to the extracellular region of the receptor and causes conformational changes that lead to the activation of the tyrosine kinase. This leads to autophosphorylation, a step that is crucial in insulin signaling pathway. Hence, compounds that augment insulin receptor tyrosine kinase activity would be useful in the treatment of diabetes mellitus. The 3D structure of IR tyrosine kinase was obtained from PDB database. The list of flavonoids found in banana flower was obtained from USDA database. The structures of the flavonoids were obtained from NCBI Pubchem. Docking analysis of the flavonoids was performed using Autodock 4.0 and Autodock Vina. The results indicate that few of the flavonoids may be potential activators of IR tyrosine kinase.

A Robust Error Model for iTRAQ Quantification Reveals Divergent Signaling between Oncogenic FLT3 Mutants in Acute Myeloid Leukemia*

PubMed Central

Zhang, Yi; Askenazi, Manor; Jiang, Jingrui; Luckey, C. John; Griffin, James D.; Marto, Jarrod A.

2010-01-01

The FLT3 receptor tyrosine kinase plays an important role in normal hematopoietic development and leukemogenesis. Point mutations within the activation loop and in-frame tandem duplications of the juxtamembrane domain represent the most frequent molecular abnormalities observed in acute myeloid leukemia. Interestingly these gain-of-function mutations correlate with different clinical outcomes, suggesting that signals from constitutive FLT3 mutants activate different downstream targets. In principle, mass spectrometry offers a powerful means to quantify protein phosphorylation and identify signaling events associated with constitutively active kinases or other oncogenic events. However, regulation of individual phosphorylation sites presents a challenging case for proteomics studies whereby quantification is based on individual peptides rather than an average across different peptides derived from the same protein. Here we describe a robust experimental framework and associated error model for iTRAQ-based quantification on an Orbitrap mass spectrometer that relates variance of peptide ratios to mass spectral peak height and provides for assignment of p value, q value, and confidence interval to every peptide identification, all based on routine measurements, obviating the need for detailed characterization of individual ion peaks. Moreover, we demonstrate that our model is stable over time and can be applied in a manner directly analogous to ubiquitously used external mass calibration routines. Application of our error model to quantitative proteomics data for FLT3 signaling provides evidence that phosphorylation of tyrosine phosphatase SHP1 abrogates the transformative potential, but not overall kinase activity, of FLT3-D835Y in acute myeloid leukemia. PMID:20019052

Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.

PubMed

Shinohara, M; Kodama, A; Matozaki, T; Fukuhara, A; Tachibana, K; Nakanishi, H; Takai, Y

2001-06-01

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induced tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduced cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increased it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades.

Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma.

PubMed

Marley, K; Maier, C S; Helfand, S C

2012-09-01

Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. © 2012 Blackwell Publishing Ltd.

EphA2 is a functional receptor for the growth factor progranulin.

PubMed

Neill, Thomas; Buraschi, Simone; Goyal, Atul; Sharpe, Catherine; Natkanski, Elizabeth; Schaefer, Liliana; Morrione, Andrea; Iozzo, Renato V

2016-12-05

Although the growth factor progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for progranulin. Recombinant progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for progranulin offers a new avenue for understanding the underlying mode of action of progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases. © 2016 Neill et al.

EphA2 is a functional receptor for the growth factor progranulin

PubMed Central

Neill, Thomas; Goyal, Atul; Sharpe, Catherine

2016-01-01

Although the growth factor progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for progranulin. Recombinant progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for progranulin offers a new avenue for understanding the underlying mode of action of progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases. PMID:27903606

Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain.

PubMed

Law, S F; Zhang, Y Z; Fashena, S J; Toby, G; Estojak, J; Golemis, E A

1999-10-10

HEF1, p130(Cas), and Efs define a family of multidomain docking proteins which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. HEF1 function has been specifically implicated in signaling pathways important for cell adhesion and differentiation in lymphoid and epithelial cells. While the SH3 domains and SH2-binding site domains (substrate domains) of HEF1 family proteins are well characterized and binding partners known, to date the highly conserved carboxy-terminal domains of the three proteins have lacked functional definition. In this study, we have determined that the carboxy-terminal domain of HEF1 contains a divergent helix-loop-helix (HLH) motif. This motif mediates HEF1 homodimerization and HEF1 heterodimerization with a recognition specificity similar to that of the transcriptional regulatory HLH proteins Id2, E12, and E47. We had previously demonstrated that the HEF1 carboxy-terminus expressed as a separate domain in yeast reprograms cell division patterns, inducing constitutive pseudohyphal growth. Here we show that pseudohyphal induction by HEF1 requires an intact HLH, further supporting the idea that this motif has an effector activity for HEF1, and implying that HEF1 pseudohyphal activity derives in part from interactions with yeast helix-loop-helix proteins. These combined results provide initial insight into the mode of function of the HEF1 carboxy-terminal domain and suggest that the HEF1 protein may interact with cellular proteins which control differentiation. Copyright 1999 Academic Press.

Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stratton, K.R.; Worley, P.F.; Huganir, R.L.

The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipidmore » system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation.« less

Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

USDA-ARS?s Scientific Manuscript database

Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14-3-3 proteins on SLP-76.

PubMed

Di Bartolo, Vincenzo; Montagne, Benjamin; Salek, Mogjiborahman; Jungwirth, Britta; Carrette, Florent; Fourtane, Julien; Sol-Foulon, Nathalie; Michel, Frédérique; Schwartz, Olivier; Lehmann, Wolf D; Acuto, Oreste

2007-03-19

The SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is a pivotal element of the signaling machinery controlling T cell receptor (TCR)-mediated activation. Here, we identify 14-3-3epsilon and zeta proteins as SLP-76 binding partners. This interaction was induced by TCR ligation and required phosphorylation of SLP-76 at serine 376. Ribonucleic acid interference and in vitro phosphorylation experiments showed that serine 376 is the target of the hematopoietic progenitor kinase 1 (HPK-1). Interestingly, either S376A mutation or HPK-1 knockdown resulted in increased TCR-induced tyrosine phosphorylation of SLP-76 and phospholipase C-gamma1. Moreover, an SLP-76-S376A mutant induced higher interleukin 2 gene transcription than wild-type SLP-76. These data reveal a novel negative feedback loop involving HPK-1-dependent serine phosphorylation of SLP-76 and 14-3-3 protein recruitment, which tunes T cell activation.

Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery.

PubMed

Choy, Meng S; Li, Yang; Machado, Luciana E S F; Kunze, Micha B A; Connors, Christopher R; Wei, Xingyu; Lindorff-Larsen, Kresten; Page, Rebecca; Peti, Wolfgang

2017-02-16

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales, and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in Protein Tyrosine Phosphatase 1B (PTP1B), a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways. By combining new experimental and computational data on WT-PTP1B and ≥10 PTP1B variants in multiple states, we discovered a fundamental and evolutionarily conserved CH/π switch that is critical for positioning the catalytically important WPD loop. Furthermore, our data show that PTP1B uses conformational and dynamic allostery to regulate its activity. This shows that both conformational rigidity and dynamics are essential for controlling protein activity. This connection between rigidity and dynamics at different timescales is likely a hallmark of all enzyme function. Copyright © 2017 Elsevier Inc. All rights reserved.

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ho, Meng-Chiao; Sturm, Matthew B.; Almo, Steven C.

2010-01-12

Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple {pi}-stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the {pi}-stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common featuresmore » of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H{sub 2}O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.« less

Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture

PubMed Central

New, Laura A.; Martin, Claire E.; Scott, Rizaldy P.; Platt, Mathew J.; Keyvani Chahi, Ava; Stringer, Colin D.; Lu, Peihua; Samborska, Bozena; Eremina, Vera; Takano, Tomoko; Simpson, Jeremy A.; Quaggin, Susan E.

2016-01-01

Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin–rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphonephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt nephrin tyrosine phosphorylation and Nck1/2 binding (nephrinY3F/Y3F mice). Homozygous nephrinY3F/Y3F mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, nephrinY3F/Y3F mice displayed delayed recovery in podocyte injury models. Profiling of nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton. PMID:26802179

Inhibition of endothelin- and phorbol ester-stimulated tyrosine kinase activity by corticotrophin in the rat adrenal zona glomerulosa.

PubMed Central

Kapas, S; Hinson, J P

1996-01-01

1. The experiments described in this study were carried out to investigate the role of tyrosine kinase in the acute adrenal response to peptide hormone stimulation, and to determine whether the activity of this kinase may be subject to regulation by other intracellular signalling mechanisms in the adrenal zona glomerulosa. 2. Previous studies from this laboratory have shown that angiotensin II stimulates tyrosine kinase activity in the rat adrenal cortex. This study has shown, for the first time, that endothelin-1 also stimulates tyrosine kinase activity in this tissue. 3. Using the specific inhibitor of protein kinase C (PKC) activity, Ro 31-8220, we have shown that stimulation of tyrosine kinase activity, in response to endothelin-1, angiotensin II or the phorbol ester phorbol 12-myristate 13-acetate, is at least partly dependent on increased PKC activity. 4. The data presented also provide further evidence of cross-talk between signalling systems in the adrenal cortex. Corticotrophin and its intracellular second messenger, cyclic AMP, significantly attenuate the increment in tyrosine kinase activity seen in response to each of the effectors used. 5. The results of this study provide important new evidence for the regulation of protein kinases by other intracellular second messenger systems. PMID:8611168

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies.

PubMed

Bokori-Brown, Monika; Kokkinidou, Maria C; Savva, Christos G; Fernandes da Costa, Sérgio; Naylor, Claire E; Cole, Ambrose R; Moss, David S; Basak, Ajit K; Titball, Richard W

2013-05-01

Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity. Copyright © 2013 The Protein Society.

A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9.

PubMed

Demoulin, J B; Uyttenhove, C; Van Roost, E; DeLestré, B; Donckers, D; Van Snick, J; Renauld, J C

1996-09-01

Interleukin-9 (IL-9), a T-cell-derived cytokine, interacts with a specific receptor associated with the IL-2 receptor gamma chain. In this report, we analyze the functional domains of the human IL-9 receptor transfected into mouse lymphoid cell lines. Three different functions were examined: growth stimulation in factor-dependent pro-B Ba/F3 cells, protection against dexamethasone-induced apoptosis, and Ly-6A2 induction in BW5147 lymphoma cells. The results indicated that a single tyrosine, at position 116 in the cytoplasmic domain, was required for all three activities. In addition, we observed that human IL-9 reduced the proliferation rate of transfected BW5147 cells, an effect also dependent on the same tyrosine. This amino acid was necessary for IL-9-mediated tyrosine phosphorylation of the receptor and for STAT activation but not for IRS-2/4PS activation or for JAK1 phosphorylation, which depended on a domain closer to the plasma membrane. We also showed that JAK1 was constitutively associated with the IL-9 receptor. Activated STAT complexes induced by IL-9 were found to contain STAT1, STAT3, and STAT5 transcription factors. Moreover, sequence homologies between human IL-9 receptor tyrosine 116 and tyrosines (of other receptors activating STAT3 and STAT5 were observed. Taken together, these data indicate that a single tyrosine of the IL-9 receptor, required for activation of three different STAT proteins, is necessary for distinct activities of this cytokine, including proliferative responses.

X-Ray Crystal Structure of Bone Marrow Kinase in the X Chromosome: A Tec Family Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muckelbauer, Jodi; Sack, John S.; Ahmed, Nazia

Bone marrow kinase in the X chromosome, a member of the Tec family of tyrosine kinases, plays a role in both monocyte/macrophage trafficking as well as cytokine secretion. Although the structures of Tec family kinases Bruton's tyrosine kinase and IL-2-inducible T-cell kinase are known, the crystal structures of other Tec family kinases have remained elusive. We report the X-ray crystal structures of bone marrow kinase in the X chromosome in complex with dasatinib at 2.4 {angstrom} resolution and PP2 at 1.9 {angstrom} resolution. The bone marrow kinase in the X chromosome structures reveal a typical kinase protein fold; with well-orderedmore » protein conformation that includes an open/extended activation loop and a stabilized DFG-motif rendering the kinase in an inactive conformation. Dasatinib and PP2 bind to bone marrow kinase in the X chromosome in the ATP binding pocket and display similar binding modes to that observed in other Tec and Src protein kinases. The bone marrow kinase in the X chromosome structures identify conformational elements of the DFG-motif that could potentially be utilized to design potent and/or selective bone marrow kinase in the X chromosome inhibitors.« less

Tyrosine hydroxylase expression and activity in the rat brain: differential regulation after long-term intermittent or sustained hypoxia.

PubMed

Gozal, Evelyne; Shah, Zahoor A; Pequignot, Jean-Marc; Pequignot, Jacqueline; Sachleben, Leroy R; Czyzyk-Krzeska, Maria F; Li, Richard C; Guo, Shang-Z; Gozal, David

2005-08-01

Tyrosine hydroxylase, a hypoxia-regulated gene, may be involved in tissue adaptation to hypoxia. Intermittent hypoxia, a characteristic feature of sleep apnea, leads to significant memory deficits, as well as to cortex and hippocampal apoptosis that are absent after sustained hypoxia. To examine the hypothesis that sustained and intermittent hypoxia induce different catecholaminergic responses, changes in tyrosine hydroxylase mRNA, protein expression, and activity were compared in various brain regions of male rats exposed for 6 h, 1 day, 3 days, and 7 days to sustained hypoxia (10% O(2)), intermittent hypoxia (alternating room air and 10% O(2)), or normoxia. Tyrosine hydroxylase activity, measured at 7 days, increased in the cortex as follows: sustained > intermittent > normoxia. Furthermore, activity decreased in the brain stem and was unchanged in other brain regions of sustained hypoxia-exposed rats, as well as in all regions from animals exposed to intermittent hypoxia, suggesting stimulus-specific and heterotopic catecholamine regulation. In the cortex, tyrosine hydroxylase mRNA expression was increased, whereas protein expression remained unchanged. In addition, significant differences in the time course of cortical Ser(40) tyrosine hydroxylase phosphorylation were present in the cortex, suggesting that intermittent and sustained hypoxia-induced enzymatic activity differences are related to different phosphorylation patterns. We conclude that long-term hypoxia induces site-specific changes in tyrosine hydroxylase activity and that intermittent hypoxia elicits reduced tyrosine hydroxylase recruitment and phosphorylation compared with sustained hypoxia. Such changes may not only account for differences in enzyme activity but also suggest that, with differential regional brain susceptibility to hypoxia, recruitment of different mechanisms in response to hypoxia will elicit region-specific modulation of catecholamine response.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Chin -Yuan; Lohman, Jeremy; Cao, Hongnan

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-beta-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 A resolution, respectively. SgcE6 shares a similar β-barrel fold withmore » the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few, other known FADH 2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. In conclusion, the crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH 2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.« less

A development of chimeric VEGFR2 TK inhibitor based on two ligand conformers from PDB: 1Y6A complex--medicinal chemistry consequences of a TKs analysis.

PubMed

Lintnerová, Lucia; García-Caballero, Melissa; Gregáň, Fridrich; Melicherčík, Milan; Quesada, Ana R; Dobiaš, Juraj; Lác, Ján; Sališová, Marta; Boháč, Andrej

2014-01-24

VEGFR2 is an important mediator of angiogenesis and influences fate of some cancer stem cells. Here we analysed all 34 structures of VEGFR2 TK available from PDB database. From them a complex PDB: 1Y6A has an exceptional AAZ ligand bound to TK in form of two conformers (U- and S-shaped). This observation inspired us to develop three chimeric bispyridyl VEGFR2 inhibitors by combining structural features of both AAZ conformers and/or their relative ligand AAX (PDB: 1Y6B). Our most interesting inhibitor 22SYM has an enzymatic VEGFR2 TK activity (IC50: 15.1 nM) comparable or better to the active compounds from clinical drugs Nexavar and Sutent. 22SYM inhibits growth, migration and tube formation of endothelial cells (EC) and selectively induces EC apoptosis. 22SYM also inhibits in vivo angiogenesis in Zebrafish embryo assay. Additionally to the above results, we proved here that tyrosine kinases in an inactive form possessing Type I inhibitors can adopt both a closed or an opened conformation of kinase A-loop independently on their DFG-out arrangement. We proposed here that an activity of certain Type I inhibitors (e.g. 22SYM-like) in complex with DFG-out TK can be negatively influenced by collisions with a dynamically moving TK A-loop. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Tyrosines of Human and Mouse Transferrin Covalently Labeled by Organophosphorus Agents: A New Motif for Binding to Proteins that Have No Active Site Serine

PubMed Central

Li, Bin; Schopfer, Lawrence M.; Grigoryan, Hasmik; Thompson, Charles M.; Hinrichs, Steven H.; Masson, Patrick; Lockridge, Oksana

2009-01-01

The expectation from the literature is that organophosphorus (OP) agents bind to proteins that have an active site serine. However, transferrin, a protein with no active site serine, was covalently modified in vitro by 0.5mM 10-fluoroethoxyphosphinyl-N-biotinamido pentyldecanamide, chlorpyrifos oxon, diisopropylfluorophosphate, dichlorvos, sarin, and soman. The site of covalent attachment was identified by analyzing tryptic peptides in the mass spectrometer. Tyr 238 and Tyr 574 in human transferrin and Tyr 238, Tyr 319, Tyr 429, Tyr 491, and Tyr 518 in mouse transferrin were labeled by OP. Tyrosine in the small synthetic peptide ArgTyrThrArg made a covalent bond with diisopropylfluorophosphate, chlorpyrifos oxon, and dichlorvos at pH 8.3. These results, together with our previous demonstration that albumin and tubulin bind OP on tyrosine, lead to the conclusion that OP bind covalently to tyrosine, and that OP binding to tyrosine is a new OP-binding residue. The OP-reactive tyrosines are activated by interaction with Arg or Lys. It is suggested that many proteins in addition to those already identified may be modified by OP on tyrosine. The extent to which tyrosine modification by OP can occur in vivo and the toxicological implications of such modifications require further investigation. PMID:18930948

Initiation of human myoblast differentiation via dephosphorylation of Kir2.1 K+ channels at tyrosine 242.

PubMed

Hinard, Valérie; Belin, Dominique; Konig, Stéphane; Bader, Charles Roland; Bernheim, Laurent

2008-03-01

Myoblast differentiation is essential to skeletal muscle formation and repair. The earliest detectable event leading to human myoblast differentiation is an upregulation of Kir2.1 channel activity, which causes a negative shift (hyperpolarization) of the resting potential of myoblasts. After exploring various mechanisms, we found that this upregulation of Kir2.1 was due to dephosphorylation of the channel itself. Application of genistein, a tyrosine kinase inhibitor, increased Kir2.1 activity and triggered the differentiation process, whereas application of bpV(Phen), a tyrosine phosphatase inhibitor, had the opposite effects. We could show that increased Kir2.1 activity requires dephosphorylation of tyrosine 242; replacing this tyrosine in Kir2.1 by a phenylalanine abolished inhibition by bpV(Phen). Finally, we found that the level of tyrosine phosphorylation in endogenous Kir2.1 channels is considerably reduced during differentiation when compared with proliferation. We propose that Kir2.1 channels are already present at the membrane of proliferating, undifferentiated human myoblasts but in a silent state, and that Kir2.1 tyrosine 242 dephosphorylation triggers differentiation.

Activation Loop Dynamics Determine the Different Catalytic Efficiencies of B Cell- and T Cell-Specific Tec Kinases

PubMed Central

Joseph, Raji E.; Kleino, Iivari; Wales, Thomas E.; Xie, Qian; Fulton, D. Bruce; Engen, John R.; Berg, Leslie J.; Andreotti, Amy H.

2014-01-01

Itk and Btk are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of six of the 619 amino acid residues of Itk with those of Btk was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the observed differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties depending on the cellular context. We suggest that the weaker catalytic activities observed for T cell–specific kinases is one mechanism to regulate cellular activation and prevent aberrant immune responses. PMID:23982207

Prostaglandin F(2alpha) stimulates tyrosine phosphorylation of phospholipase C-gamma1.

PubMed

Husain, Shahid; Jafri, Farahdiba

2002-10-11

In this study, we investigated the ability of prostaglandin F(2alpha) (PGF(2alpha)) to induce tyrosine phosphorylation of phospholipase C-gamma1 (PLC-gamma1) in cat iris sphincter smooth muscle (CISM) cells. PGF(2alpha)(1 microM) stimulated PLC-gamma1 tyrosine phosphorylation in a time- and dose-dependent manner with a maximum increase of 3-fold at 0.5min. The protein tyrosine kinase inhibitors, genistein, and tyrphostin A-25, blocked the stimulatory effects of PGF(2alpha), suggesting involvement of protein tyrosine kinase activity in the physiological actions of the PGF(2alpha). Furthermore, PGF(2alpha)-induced p42/p44 MAP kinase activation was also completely blocked by protein tyrosine kinase inhibitors. In summary, these findings show that PGF(2alpha) stimulates tyrosine phosphorylation of PLC-gamma1 in CISM cells and indicate that PGF(2alpha)-stimulated tyrosine phosphorylation is responsible for an early signal transduction event.

Anaplastic lymphoma kinase: role in cancer pathogenesis and small-molecule inhibitor development for therapy

PubMed Central

Webb, Thomas R; Slavish, Jake; George, Rani E; Look, A Thomas; Xue, Liquan; Jiang, Qin; Cui, Xiaoli; Rentrop, Walter B; Morris, Stephan W

2009-01-01

Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase in the insulin receptor superfamily, was initially identified in constitutively activated oncogenic fusion forms – the most common being nucleophosmin-ALK – in anaplastic large-cell lymphomas, and subsequent studies have identified ALK fusions in diffuse large B-cell lymphomas, systemic histiocytosis, inflammatory myofibroblastic tumors, esophageal squamous cell carcinomas and non-small-cell lung carcinomas. More recently, genomic DNA amplification and protein overexpression, as well as activating point mutations, of ALK have been described in neuroblastomas. In addition to those cancers for which a causative role for aberrant ALK activity is well validated, more circumstantial links implicate the full-length, normal ALK receptor in the genesis of other malignancies – including glioblastoma and breast cancer – via a mechanism of receptor activation involving autocrine and/or paracrine growth loops with the reported ALK ligands, pleiotrophin and midkine. This review summarizes normal ALK biology, the confirmed and putative roles of ALK in the development of human cancers and efforts to target ALK using small-molecule kinase inhibitors. PMID:19275511

A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation.

PubMed

Dikic, I; Tokiwa, G; Lev, S; Courtneidge, S A; Schlessinger, J

1996-10-10

The mechanisms by which mitogenic G-protein-coupled receptors activate the MAP kinase signalling pathway are poorly understood. Candidate protein tyrosine kinases that link G-protein-coupled receptors with MAP kinase include Src family kinases, the epidermal growth factor receptor, Lyn and Syk. Here we show that lysophosphatidic acid (LPA) and bradykinin induce tyrosine phosphorylation of Pyk2 and complex formation between Pyk2 and activated Src. Moreover, tyrosine phosphorylation of Pyk2 leads to binding of the SH2 domain of Src to tyrosine 402 of Pyk2 and activation of Src. Transient overexpression of a dominant interfering mutant of Pyk2 or the protein tyrosine kinase Csk reduces LPA- or bradykinin-induced activation of MAP kinase. LPA- or bradykinin-induced MAP kinase activation was also inhibited by overexpression of dominant interfering mutants of Grb2 and Sos. We propose that Pyk2 acts with Src to link Gi- and Gq-coupled receptors with Grb2 and Sos to activate the MAP kinase signalling pathway in PC12 cells.

Tumor driven by gain-of-function HER2 H878Y mutant is highly sensitive to HER2 inhibitor

PubMed Central

Hu, Zexi; Hu, Yong; Liu, Xicheng; Xi, Rongwen; Zhang, Aiqun; Liu, Deruo; Xie, Qiang; Chen, Liang

2015-01-01

HER2, a well established oncogenic member of EGFR family, is among the most intensely investigated kinase drug targets. In contrast to hotspot mutations of EGFR, few mutations of HER2 locate in activation loop within kinase domain. We previously reported the molecular mechanism underlying hyper kinase activity of HER2H878Y, a mutation located in activation loop. However, its tumorigenicity in vivo and relevant therapeutics remain to be determined. Here, we report for the first time that HER2H878Y was tumorigenic in vivo in lung adenocarcinoma transgenic mouse model. Induced expression of HER2H878Y in lung epithelial compartments resulted in formation of poorly differentiated lung adenocarcinoma with bronchioloalveolar carcinoma (BAC) features. Strikingly, we found that these tumors depended on continuous expression of HER2H878Y for maintenance. Typical HER2 downstream signaling mediators, including PLCγ1, STAT5 and AKT, were hyperactivated in HER2H878Y driven lung tumors. More importantly, administration of HKI-272, a tyrosine kinase inhibitor (TKI), efficiently shrank HER2H878Y driven tumors in transgenic mouse model. Moreover, we found that combinational treatment with HKI272 and mTOR inhibitor, Rapamycin, showed a superior cytotoxicity to H878Y mutant transformed cells and enhanced activity to elicit apoptosis and inhibit growth in situ in tumorous area. Our work therefore showed that HER2H878Y mutant was a reasonable drug target. Hence, our work supported the assessment of HKI-272/rapamycin treatment in clinical trials. PMID:26375550

Molecular Characterization of PDGFR-α/PDGF-A and c-KIT/SCF in Gliosarcomas

PubMed Central

Reis, Rui M.; Martins, Albino; Ribeiro, Susana A.; Basto, Diana; Longatto-Filho, Adhemar; Schmitt, Fernando C.; Lopes, José M.

2005-01-01

Gliosarcomas are rare and poorly characterized malignant brain tumors that exhibit a biphasic tissue pattern with areas of gliomatous and sarcomatous differentiation. These tumors are histological variants of glioblastoma, displaying a similar genetic profile and dismal prognosis. Up-regulation of PDGFR subfamily of tyrosine kinase members, PDGFR-α and c-Kit, and their intracellular effectors RAS/RAF/MAPK has a crucial role in the cancer development. In addition, signal transduction mediated by activating mutations of c-Kit and PDGFR can be effectively blocked by specific tyrosine kinase inhibitors, such as Imatinib mesylate. The aim of this study was to characterize the molecular alterations of PDGFR signaling in gliosarcomas. Six cases were analyzed by immunohistochemistry for the expression of PDGFR-α, c-Kit and their ligands PDGF-A and SCF, respectively. The cases were further evaluated for the presence of activating mutations of PDGFR-α (exons 12 and 18) and c-kit (exons 9, 11, 13, and 17), as well as B-RAF (exons 11 and 15). Expression of PDGF-A was found in all cases and co-expression of PDGFR-α was observed in three cases. Four cases showed expression of SCF, and c-Kit was observed only in one case that also expressed SCF. Generally, immunoreaction predominates in the glial component. The mutational analysis of PDGFR-α showed the presence of an IVS17-50insT intronic insertion in two cases, one of them also with a 2472C > T silent mutation; this silent mutation was also found in another case. Glioma cell line analysis of IVS17-50insT insertion showed no influence on PDGFR-α gene splicing. No mutations were detected in c-kit and B-RAF oncogenes. Our Results indicate that activating mutations of PDGFR-α, c-kit and B-RAF are absent in gliosarcomas. Nevertheless, the presence of a PDGFR-a/PDGFA and c-Kit/SCF autocrine/paracrine stimulation loop in a proportion of cases, supports the potential role of specific tyrosine kinase inhibitors in the treatment of gliosarcomas. PMID:16373964

Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

PubMed Central

Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru

2013-01-01

Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693

Structural dynamics of a methionine γ-lyase for calicheamicin biosynthesis: Rotation of the conserved tyrosine stacking with pyridoxal phosphate

DOE PAGES

Cao, Hongnan; Tan, Kemin; Wang, Fengbin; ...

2016-04-29

CalE6 from Micromonospora echinospora is a (pyridoxal 50 phosphate) PLP-dependent methionine γ-lyase involved in the biosynthesis of calicheamicins. Here, we report the crystal structure of a CalE6 2-(N-morpholino)ethanesulfonic acid complex showing ligand-induced rotation of Tyr100, which stacks with PLP, resembling the corresponding tyrosine rotation of true catalytic intermediates of CalE6 homologs. Elastic network modeling and crystallographic ensemble refinement reveal mobility of the N-terminal loop, which involves both tetrameric assembly and PLP binding. Modeling and comparative structural analysis of PLP-dependent enzymes involved in Cys/Met metabolism shine light on the functional implications of the intrinsic dynamic properties of CalE6 in catalysis andmore » holoenzyme maturation.« less

Importance of tyrosine phosphorylation in receptor kinase complexes.

PubMed

Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

2015-05-01

Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transformation by oncogenic mutants and ligand-dependent activation of FLT3 wild-type requires the tyrosine residues 589 and 591.

PubMed

Vempati, Sridhar; Reindl, Carola; Wolf, Ulla; Kern, Ruth; Petropoulos, Konstantin; Naidu, Vegi M; Buske, Christian; Hiddemann, Wolfgang; Kohl, Tobias M; Spiekermann, Karsten

2008-07-15

Mutations in the receptor tyrosine kinase FLT3 are found in up to 30% of acute myelogenous leukemia patients and are associated with an inferior prognosis. In this study, we characterized critical tyrosine residues responsible for the transforming potential of active FLT3-receptor mutants and ligand-dependent activation of FLT3-WT. We performed a detailed structure-function analysis of putative autophosphorylation tyrosine residues in the FLT3-D835Y tyrosine kinase domain (TKD) mutant. All tyrosine residues in the juxtamembrane domain (Y566, Y572, Y589, Y591, Y597, and Y599), interkinase domain (Y726 and Y768), and COOH-terminal domain (Y955 and Y969) of the FLT3-D835Y construct were successively mutated to phenylalanine and the transforming activity of these mutants was analyzed in interleukin-3-dependent Ba/F3 cells. Tyrosine residues critical for the transforming potential of FLT3-D835Y were also analyzed in FLT3 internal tandem duplication mutants (FLT3-ITD)and the FLT3 wild-type (FLT3-WT) receptor. The substitution of the tyrosine residues by phenylalanine in the juxtamembrane, interkinase, and COOH-terminal domains resulted in a complete loss of the transforming potential of FLT3-D835Y-expressing cells which can be attributed to a significant reduction of signal tranducer and activator of transcription 5 (STAT5) phosphorylation at the molecular level. Reintroduction of single tyrosine residues revealed the critical role of Y589 and Y591 in reconstituting interleukin-3-independent growth of FLT3-TKD-expressing cells. Combined mutation of Y589 and Y591 to phenylalanine also abrogated ligand-dependent proliferation of FLT3-WT and the transforming potential of FLT3-ITD-with a subsequent abrogation of STAT5 phosphorylation. We identified two tyrosine residues, Y589 and Y591, in the juxtamembrane domain that are critical for the ligand-dependent activation of FLT3-WT and the transforming potential of oncogenic FLT3 mutants.

Regulation of Src homology 2-containing tyrosine phosphatase 1 during activation of human neutrophils. Role of protein kinase C.

PubMed

Brumell, J H; Chan, C K; Butler, J; Borregaard, N; Siminovitch, K A; Grinstein, S; Downey, G P

1997-01-10

The tyrosine phosphorylation of several proteins induced in neutrophils by soluble and particulate stimuli is thought to be crucial for initiating antimicrobial responses. Although activation of tyrosine kinases is thought to mediate this event, the role of tyrosine phosphatases in the initiation and modulation of neutrophil responses remains largely undefined. We investigated the role of Src homology 2-containing tyrosine phosphatase 1 (SHP-1; also known as protein tyrosine phosphatase 1C (PTP1C), hematopoetic cell phosphatase, PTP-N6, and SHPTP-1), a phosphatase expressed primarily in hemopoietic cells, in the activation of human neutrophils. SHP-1 mRNA and protein were detected in these cells, and the enzyme was found to be predominantly localized to the cytosol in unstimulated cells. Following stimulation with neutrophil agonists such as phorbol ester, chemotactic peptide, or opsonized zymosan, a fraction of the phosphatase redistributed to the cytoskeleton. Agonist treatment also induced significant decreases (30-60%) in SHP-1 activity, which correlated temporally with increases in the cellular phosphotyrosine content. Phosphorylation of SHP-1 on serine residues was associated with the inhibition of its enzymatic activity, suggesting a causal relationship. Accordingly, both the agonist-evoked phosphorylation of SHP-1 and the inhibition of its catalytic activity were blocked by treatment with bisindolylmaleimide I, a potent and specific inhibitor of protein kinase C (PKC) activity. Immunoprecipitated SHP-1 was found to be phosphorylated efficiently by purified PKC in vitro. Such phosphorylation also caused a decrease in the phosphatase activity of SHP-1. Together, these data suggest that inhibition of SHP-1 by PKC-mediated serine phosphorylation plays a role in facilitating the accumulation of tyrosine-phosphorylated proteins following neutrophil stimulation. These findings provide a new link between the PKC and tyrosine phosphorylation branches of the signaling cascade that triggers antimicrobial responses in human neutrophils.

Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors

NASA Astrophysics Data System (ADS)

Thompson, Robert E.; Liu, Xuyu; Ripoll-Rozada, Jorge; Alonso-García, Noelia; Parker, Benjamin L.; Pereira, Pedro José Barbosa; Payne, Richard J.

2017-09-01

Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification—tyrosine sulfation—of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

The roles of the conserved tyrosine in the β2-α2 loop of the prion protein.

PubMed

Huang, Danzhi; Caflisch, Amedeo

2015-01-01

Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP(C)) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP(Sc)) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP(C) proteins have provided evidence of a conformational equilibrium between a 3(10)-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165-175). In vitro cell-free experiments of the seeded conversion of PrP(C) indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3(10)-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3(10)-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.

Molecular mechanisms of OLIG2 transcription factor in brain cancer

PubMed Central

Lian, Nathan; Kesari, Santosh

2016-01-01

Oligodendrocyte lineage transcription factor 2 (OLIG2) plays a pivotal role in glioma development. Here we conducted a comprehensive study of the critical gene regulatory networks involving OLIG2. These include the networks responsible for OLIG2 expression, its translocation to nucleus, cell cycle, epigenetic regulation, and Rho-pathway interactions. We described positive feedback loops including OLIG2: loops of epigenetic regulation and loops involving receptor tyrosine kinases. These loops may be responsible for the prolonged oncogenic activity of OLIG2. The proposed schemes for epigenetic regulation of the gene networks involving OLIG2 are confirmed by patient survival (Kaplan–Meier) curves based on the cancer genome atlas (TCGA) datasets. Finally, we elucidate the Coherent-Gene Modules (CGMs) networks—framework of OLIG2 involvement in cancer. We showed that genes interacting with OLIG2 formed eight CGMs having a set of intermodular connections. We showed also that among the genes involved in these modules the most connected hub is EGFR, then, on lower level, HSP90 and CALM1, followed by three lower levels including epigenetic genes KDM1A and NCOR1. The genes on the six upper levels of the hierarchy are involved in interconnections of all eight CGMs and organize functionally defined gene-signaling subnetworks having specific functions. For example, CGM1 is involved in epigenetic control. CGM2 is significantly related to cell proliferation and differentiation. CGM3 includes a number of interconnected helix–loop–helix transcription factors (bHLH) including OLIG2. Many of these TFs are partially controlled by OLIG2. The CGM4 is involved in PDGF-related: angiogenesis, tumor cell proliferation and differentiation. These analyses provide testable hypotheses and approaches to inhibit OLIG2 pathway and relevant feed-forward and feedback loops to be interrogated. This broad approach can be applied to other TFs. PMID:27447975

QSAR Study of p56lck Protein Tyrosine Kinase Inhibitory Activity of Flavonoid Derivatives Using MLR and GA-PLS

PubMed Central

Fassihi, Afshin; Sabet, Razieh

2008-01-01

Quantitative relationships between molecular structure and p56lck protein tyrosine kinase inhibitory activity of 50 flavonoid derivatives are discovered by MLR and GA-PLS methods. Different QSAR models revealed that substituent electronic descriptors (SED) parameters have significant impact on protein tyrosine kinase inhibitory activity of the compounds. Between the two statistical methods employed, GA-PLS gave superior results. The resultant GA-PLS model had a high statistical quality (R2 = 0.74 and Q2 = 0.61) for predicting the activity of the inhibitors. The models proposed in the present work are more useful in describing QSAR of flavonoid derivatives as p56lck protein tyrosine kinase inhibitors than those provided previously. PMID:19325836

Regulation of apoptosis of interleukin 2-dependent mouse T-cell line by protein tyrosine phosphorylation and polyamines.

PubMed

Min, A; Hasuma, T; Yano, Y; Matsui-Yuasa, I; Otani, S

1995-12-01

We examined the effect of inhibitors of tyrosine kinase and tyrosine phosphatase on DNA fragmentation, protein tyrosine phosphorylation, and polyamine metabolism in the murine T-cell line CTLL-2. When cells were exposed to herbimycin A, a specific inhibitor of tyrosine kinase (Uehara et al., 1989, Biochem. Biophys. Res. Commun., 163:803-809), in the presence of interleukin 2 (IL-2), DNA was degraded into oligonucleosomal fragments in a dose-dependent fashion. Genistein, another inhibitor of tyrosine kinase (Akiyama et al., 1987, J. Biol. Chem., 262:5592-5596), had similar effects. Exposure of CTLL-2 cells to vanadate, a tyrosine phosphatase inhibitor, blocked with the DNA fragmentation induced by herbimycin A. Tyrosine phosphorylation of 55 Kd protein was inhibited by herbimycin A, and the inhibition was reduced by vanadate. Ornithine decarboxylase (ODC) activity decreased rapidly after herbimycin A was added to CTLL-2 cell cultures, while vanadate increased ODC activity. The exogenous addition of putrescine or spermine, but not that of spermidine, attenuated herbimycin A-induced DNA fragmentation. These findings suggest that phosphorylation of tyrosine residues of 55 Kd protein prevents DNA fragmentation and that polyamines are involved in regulation of apoptosis.

Shigella flexneri 3a Outer Membrane Protein C Epitope Is Recognized by Human Umbilical Cord Sera and Associated with Protective Activity

PubMed Central

Jarząb, Anna; Witkowska, Danuta; Ziomek, Edmund; Dąbrowska, Anna; Szewczuk, Zbigniew; Gamian, Andrzej

2013-01-01

Shigella flexneri 3a is one of the five major strains of the Shigella genus responsible for dysentery, especially among children, in regions of high poverty and poor sanitation. The outer membrane proteins (OMP) of this bacterium elicit immunological responses and are considered a prime target for vaccine development. When injected into mice they elicit a protective immunological response against a lethal dose of the pathogen. The OMPs from S. flexneri 3a were isolated and resolved by two-dimension-SDS-PAGE. Two 38-kDa spots were of particular interest since in our earlier studies OMPs of such molecular mass were found to interact with umbilical cord sera. These two spots were identified as OmpC by ESI-MS/MS spectrometry. By DNA sequencing, the ompC gene from S. flexneri 3a was identical to ompC from S. flexneri 2a [Gene Bank: 24113600]. A 3D model of OmpC was built and used to predict B-cell type (discontinuous) antigenic epitopes. Six epitopes bearing the highest score were selected and the corresponding peptides were synthesized. Only the peptides representing loop V of OmpC reacted strongly with the umbilical cord serum immunoglobulins. To determine which amino acids are essential for the antigenic activity of the epitope, the loop V was scanned with a series of dodecapeptides. The peptide RYDERY was identified as a minimal sequence for the loop V epitope. Truncation at either the C- or N-terminus rendered this peptide inactive. Apart from C-terminal tyrosine, substitution of each of the remaining five amino acids with glycine, led to a precipitous loss of immunological activity. This peptide may serve as a ligand in affinity chromatography of OmpC-specific antibodies and as a component of a vaccine designed to boost human immune defenses against enterobacterial infections. PMID:23940590

An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering.

PubMed

Keedy, Daniel A; Hill, Zachary B; Biel, Justin T; Kang, Emily; Rettenmaier, T Justin; Brandao-Neto, Jose; Pearce, Nicholas M; von Delft, Frank; Wells, James A; Fraser, James S

2018-06-07

Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function. © 2018, Keedy et al.

Src-dependent Tyrosine Phosphorylation of Non-muscle Myosin Heavy Chain-IIA Restricts Listeria monocytogenes Cellular Infection*

PubMed Central

Almeida, Maria Teresa; Mesquita, Francisco S.; Cruz, Rui; Osório, Hugo; Custódio, Rafael; Brito, Cláudia; Vingadassalom, Didier; Martins, Mariana; Leong, John M.; Holden, David W.; Cabanes, Didier; Sousa, Sandra

2015-01-01

Bacterial pathogens often interfere with host tyrosine phosphorylation cascades to control host responses and cause infection. Given the role of tyrosine phosphorylation events in different human infections and our previous results showing the activation of the tyrosine kinase Src upon incubation of cells with Listeria monocytogenes, we searched for novel host proteins undergoing tyrosine phosphorylation upon L. monocytogenes infection. We identify the heavy chain of the non-muscle myosin IIA (NMHC-IIA) as being phosphorylated in a specific tyrosine residue in response to L. monocytogenes infection. We characterize this novel post-translational modification event and show that, upon L. monocytogenes infection, Src phosphorylates NMHC-IIA in a previously uncharacterized tyrosine residue (Tyr-158) located in its motor domain near the ATP-binding site. In addition, we found that other intracellular and extracellular bacterial pathogens trigger NMHC-IIA tyrosine phosphorylation. We demonstrate that NMHC-IIA limits intracellular levels of L. monocytogenes, and this is dependent on the phosphorylation of Tyr-158. Our data suggest a novel mechanism of regulation of NMHC-IIA activity relying on the phosphorylation of Tyr-158 by Src. PMID:25635050

Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM)-mediated Inhibitory Signaling is Regulated by Sequential Phosphorylation Mediated by Distinct Nonreceptor Tyrosine Kinases: A Case Study Involving PECAM-1

PubMed Central

Tourdot, Benjamin E.; Brenner, Michelle K.; Keough, Kathleen C.; Holyst, Trudy; Newman, Peter J.; Newman, Debra K.

2013-01-01

The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing non-receptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton’s tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk, and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals. PMID:23418871

Tyrosine kinome sequencing of pediatric acute lymphoblastic leukemia: a report from the Children's Oncology Group TARGET Project | Office of Cancer Genomics

Cancer.gov

TARGET researchers sequenced the tyrosine kinome and downstream signaling genes in 45 high-risk pediatric ALL cases with activated kinase signaling, including Ph-like ALL, to establish the incidence of tyrosine kinase mutations in this cohort. The study confirmed previously identified somatic mutations in JAK and FLT3, but did not find novel alterations in any additional tyrosine kinases or downstream genes. The mechanism of kinase signaling activation in this high-risk subgroup of pediatric ALL remains largely unknown.

Deciphering of ADP-induced, phosphotyrosine-dependent signaling networks in human platelets by Src-homology 2 region (SH2)-profiling.

PubMed

Schweigel, Hardy; Geiger, Jörg; Beck, Florian; Buhs, Sophia; Gerull, Helwe; Walter, Ulrich; Sickmann, Albert; Nollau, Peter

2013-03-01

Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

PubMed

Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

2016-07-01

The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.

PECAM1 regulates flow-mediated Gab1 tyrosine phosphorylation and signaling

PubMed Central

Xu, Suowen; Ha, Chang Hoon; Wang, Weiye; Xu, Xiangbin; Yin, Meimei; Jin, Felix Q.; Mastrangelo, Michael; Koroleva, Marina; Fujiwara, Keigi; Jin, Zheng Gen

2016-01-01

Endothelial dysfunction, characterized by impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued decrease of NO production, is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Laminar blood flow-mediated specific signaling cascades modulate vascular endothelial cells (ECs) structure and functions. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation in ECs, which in part confers laminar flow atheroprotective action. However, the molecular mechanisms whereby flow regulates Gab1 tyrosine phosphorylation and its downstream signaling events remain unclear. Here we show that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in flow signaling and HGF signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K decreased flow-, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs. PMID:26706435

A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.

PubMed

Shen, S H; Bastien, L; Posner, B I; Chrétien, P

1991-08-22

The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction, neoplastic transformation and control of the mitotic cycle. These mechanisms are regulated by the activities of both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases). As in the PTKs, there are two classes of PTPases: membrane associated, receptor-like enzymes and soluble proteins. Here we report the isolation of a complementary DNA clone encoding a new form of soluble PTPase, PTP1C. The enzyme possesses a large noncatalytic region at the N terminus which unexpectedly contains two adjacent copies of the Src homology region 2 (the SH2 domain) found in various nonreceptor PTKs and other cytoplasmic signalling proteins. As with other SH2 sequences, the SH2 domains of PTP1C formed high-affinity complexes with the activated epidermal growth factor receptor and other phosphotyrosine-containing proteins. These results suggest that the SH2 regions in PTP1C may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. PTPase activity may thus directly link growth factor receptors and other signalling proteins through protein-tyrosine phosphorylation.

Cadmium inhibits mouse sperm motility through inducing tyrosine phosphorylation in a specific subset of proteins.

PubMed

Wang, Lirui; Li, Yuhua; Fu, Jieli; Zhen, Linqing; Zhao, Na; Yang, Qiangzhen; Li, Sisi; Li, Xinhong

2016-08-01

Cadmium (Cd) has been reported to impair male fertility, primarily by disrupting sperm motility, but the underlying molecular mechanism remains unclear. Here we investigated the effects of Cd on sperm motility, tyrosine phosphorylation, AMP-activated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, and ATP levels in vitro. Our results demonstrated that Cd inhibited sperm motility, GAPDH activity, AMPK activity and ATP production, and induced tyrosine phosphorylation of 55-57KDa proteins. Importantly, all the parameters affected by Cd were restored to normal levels when incubated with 10μM Cd in the presence of 30μM ethylene diamine tetraacetic acid (EDTA). Interestingly, changes of tyrosine phosphorylation levels of 55-57KDa proteins are completely contrary to that of other parameters. These results suggest that Cd-induced tyrosine phosphorylation of 55-57KDa proteins might act as an engine to block intracellular energy metabolism and thus decrease sperm motility. Copyright © 2016 Elsevier Inc. All rights reserved.

Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in Platelets*

PubMed Central

Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol; Eble, Johannes A.; Ellmeier, Wilfried; Kahn, Mark; Kunapuli, Satya P.

2015-01-01

Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor. PMID:25767114

The use of the tyrosine phosphatase antagonist orthovanadate in the study of a cell proliferation inhibitor

NASA Technical Reports Server (NTRS)

Enebo, D. J.; Hanek, G.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

1993-01-01

Incubation of murine fibroblasts with orthovanadate, a global tyrosine phosphatase inhibitor, was shown to confer a "pseudo-transformed" phenotype with regard to cell morphology and growth characteristics. This alteration was manifested by both an increasing refractile appearance of the cells, consistent with many transformed cell lines, as well as an increase in maximum cell density was attained. Despite the abrogation of cellular tyrosine phosphatase activity, orthovanadate-treated cells remained sensitive to the biological activity of a naturally occurring sialoglycopeptide (SGP) cell surface proliferation inhibitor. The results indicated that tyrosine phosphatase activity, inhibited by orthovanadate, was not involved in the signal transduction pathway of the SGP.

Tyrosine Phosphorylation of the Guanine Nucleotide Exchange Factor GIV Promotes Activation of PI3K During Cell Migration

PubMed Central

Lin, Changsheng; Ear, Jason; Pavlova, Yelena; Mittal, Yash; Kufareva, Irina; Ghassemian, Majid; Abagyan, Ruben; Garcia-Marcos, Mikel; Ghosh, Pradipta

2014-01-01

GIV (Gα-interacting vesicle-associated protein; also known as Girdin), enhances Akt activation downstream of multiple growth factor– and G-protein–coupled receptors to trigger cell migration and cancer invasion. Here we demonstrate that GIV is a tyrosine phosphoprotein that directly binds to and activates phosphoinositide 3-kinase (PI3K). Upon ligand stimulation of various receptors, GIV was phosphorylated at Tyr1764 and Tyr1798 by both receptor and non-receptor tyrosine kinases. These phosphorylation events enabled direct binding of GIV to the N- and C-terminal SH2 domains of p85α, a regulatory subunit of PI3K, stabilized receptor association with PI3K, and enhanced PI3K activity at the plasma membrane to trigger cell migration. Tyrosine phosphorylation of GIV and its association with p85α increased during metastatic progression of a breast carcinoma. These results suggest a mechanism by which multiple receptors activate PI3K through tyrosine phosphorylation of GIV, thereby making the GIVPI3K interaction a potential therapeutic target within the PI3K-Akt pathway. PMID:21954290

An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor

PubMed Central

Shah, Neel H; Wang, Qi; Yan, Qingrong; Karandur, Deepti; Kadlecek, Theresa A; Fallahee, Ian R; Russ, William P; Ranganathan, Rama; Weiss, Arthur; Kuriyan, John

2016-01-01

The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens. DOI: http://dx.doi.org/10.7554/eLife.20105.001 PMID:27700984

Activated platelet-derived growth factor autocrine pathway drives the transformed phenotype of a human glioblastoma cell line.

PubMed

Vassbotn, F S; Ostman, A; Langeland, N; Holmsen, H; Westermark, B; Heldin, C H; Nistér, M

1994-02-01

Human glioblastoma cells (A172) were found to concomitantly express PDGF-BB and PDGF beta-receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up-regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U-343MGa 31L) or on Ki-ras-transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transformed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists.

Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway.

PubMed

Mittal, Yash; Pavlova, Yelena; Garcia-Marcos, Mikel; Ghosh, Pradipta

2011-09-16

GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brandao, T.; Robinson, H; Johnson, S

Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presencemore » of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.« less

Epidermal growth factor receptor and integrins control force-dependent vinculin recruitment to E-cadherin junctions.

PubMed

Sehgal, Poonam; Kong, Xinyu; Wu, Jun; Sunyer, Raimon; Trepat, Xavier; Leckband, Deborah

2018-03-20

This study reports novel findings that link E-cadherin (also known as CDH1)-mediated force-transduction signaling to vinculin targeting to intercellular junctions via epidermal growth factor receptor (EGFR) and integrins. These results build on previous findings that demonstrated that mechanically perturbed E-cadherin receptors activate phosphoinositide 3-kinase and downstream integrins in an EGFR-dependent manner. Results of this study show that this EGFR-mediated kinase cascade controls the force-dependent recruitment of vinculin to stressed E-cadherin complexes - a key early signature of cadherin-based mechanotransduction. Vinculin targeting requires its phosphorylation at tyrosine 822 by Abl family kinases (hereafter Abl), but the origin of force-dependent Abl activation had not been identified. We now present evidence that integrin activation, which is downstream of EGFR signaling, controls Abl activation, thus linking E-cadherin to Abl through a mechanosensitive signaling network. These findings place EGFR and integrins at the center of a positive-feedback loop, through which force-activated E-cadherin signals regulate vinculin recruitment to cadherin complexes in response to increased intercellular tension.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

Activation of the EBV/C3d receptor (CR2, CD21) on human B lymphocyte surface triggers tyrosine phosphorylation of the 95-kDa nucleolin and its interaction with phosphatidylinositol 3 kinase.

PubMed

Barel, M; Le Romancer, M; Frade, R

2001-03-01

We previously demonstrated that CR2 activation on human B lymphocyte surface triggered tyrosine phosphorylation of a p95 component and its interaction with p85 subunit of phosphatidylinositol 3' (PI 3) kinase. Despite identical molecular mass of 95 kDa, this tyrosine phosphorylated p95 molecule was not CD19, the proto-oncogene Vav, or the adaptator Gab1. To identify this tyrosine phosphorylated p95 component, we first purified it by affinity chromatography on anti-phosphotyrosine mAb covalently linked to Sepharose 4B, followed by polyacrylamide gel electrophoresis. Then, the isolated 95-kDa tyrosine phosphorylated band was submitted to amino acid analysis by mass spectrometry; the two different isolated peptides were characterized by amino acid sequences 100% identical with two different domains of nucleolin, localized between aa 411--420 and 611--624. Anti-nucleolin mAb was used to confirm the antigenic properties of this p95 component. Functional studies demonstrated that CR2 activation induced, within a brief span of 2 min, tyrosine phosphorylation of nucleolin and its interaction with Src homology 2 domains of the p85 subunit of PI 3 kinase and of 3BP2 and Grb2, but not with Src homology 2 domains of Fyn and Gap. These properties of nucleolin were identical with those of the p95 previously described and induced by CR2 activation. Furthermore, tyrosine phosphorylation of nucleolin was also induced in normal B lymphocytes by CR2 activation but neither by CD19 nor BCR activation. These data support that tyrosine phosphorylation of nucleolin and its interaction with PI 3 kinase p85 subunit constitute one of the earlier steps in the specific intracellular signaling pathway of CR2.

Analysis of the murine Dtk gene identifies conservation of genomic structure within a new receptor tyrosine kinase subfamily

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis, P.M.; Crosier, K.E.; Crosier, P.S.

The receptor tyrosine kinase Dtk/Tyro 3/Sky/rse/brt/tif is a member of a new subfamily of receptors that also includes Axl/Ufo/Ark and Eyk/Mer. These receptors are characterized by the presence of two immunoglobulin-like loops and two fibronectin type III repeats in their extracellular domains. The structure of the murine Dtk gene has been determined. The gene consists of 21 exons that are distributed over 21 kb of genomic DNA. An isoform of Dtk is generated by differential splicing of exons from the 5{prime} region of the gene. The overall genomic structure of Dtk is virtually identical to that determined for the humanmore » UFO gene. This particular genomic organization is likely to have been duplicated and closely maintained throughout evolution. 38 refs., 3 figs., 1 tab.« less

An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani

PubMed Central

Das, Aditi; Mandal, Chhabinath; Dasgupta, Arindam; Sengupta, Tanushri; Majumder, Hemanta K.

2002-01-01

DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes. PMID:11809893

Misfolding of major histocompatibility complex class I molecules in activated T cells allows cis-interactions with receptors and signaling molecules and is associated with tyrosine phosphorylation.

PubMed

Santos, Susana G; Powis, Simon J; Arosa, Fernando A

2004-12-17

Knowledge of the origin and biochemical status of beta(2)-microglobulin-free or misfolded major histocompatibility complex (MHC)-I molecules is essential for understanding their pleiotropic properties. Here we show that in normal human T cells, misfolding of MHC-I molecules is turned on upon activation and cell division and is proportional to the level of proliferation. Immunoprecipitation showed that a number of proteins are associated with MHC-I heavy chains at the surface of activated T cells, including the CD8alphabeta receptor and the chaperone tandem calreticulin/ERp57, associations that rely upon the existence of a pool of HC-10-reactive molecules. Biochemical analysis showed that misfolded MHC-I molecules present at the cell surface are fully glycosylated mature molecules. Importantly, misfolded MHC-I molecules are tyrosine phosphorylated and are associated with kinase activity. In vitro kinase assays followed by reprecipitation indicated that tyrosine phosphorylation of the class I heavy chain is probably mediated by a Src tyrosine kinase because Lck was found associated with HC-10 immunocomplexes. Finally, we show that inhibition of tyrosine phosphorylation by using the Src-family tyrosine kinase inhibitor PP2 resulted in enhanced release of MHC-I heavy chains from the cell surface of activated T cells and a slight down-regulation of cell surface W6/32-reactive molecules. This study provides new insights into the biology of MHC-I molecules and suggests that tyrosine phosphorylation may be involved in the regulation of MHC-I misfolding and expression.

Hemin-Graphene Derivatives with Increased Peroxidase Activities Restrain Protein Tyrosine Nitration.

PubMed

Xu, Huan; Yang, Zhen; Li, Hailing; Gao, Zhonghong

2017-12-14

Protein tyrosine nitration is implicated in the occurrence and progression of pathological conditions involving free radical reactions. It is well recognized that hemin can catalyze protein tyrosine nitration in the presence of nitrite and hydrogen peroxide. Generally, the catalytic efficiency is positively correlated to its peroxidase activity. In this study, however, it is found that the efficiency of hemin in catalyzing protein tyrosine nitration is largely suppressed after functionalization with graphene derivatives, even though its peroxidase-like activity is more than quadrupled. Further studies show that the oxidation of tyrosine is still observed for these composites; dityrosine formation, however, is greatly inhibited. Furthermore, these composites also exhibit strong effects on the oxidation of nitrite into nitrate. Therefore, we propose a mechanism in which hemin-graphene derivatives facilitate the oxidation of tyrosine and nitrite to produce tyrosyl radicals and nitrogen dioxide radicals in the presence of hydrogen peroxide, but graphene interlayers serve as barriers that hinder radical-radical coupling reactions; consequently, protein tyrosine nitration is restrained. This property of hemin-graphene derivatives, by which they catalyze substrate oxidation but suppress radical-radical coupling reactions, shows their great potential in selective oxidation procedures for byproduct removal. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulation by CD45 of the tyrosine phosphorylation of high affinity IgE receptor beta- and gamma-chains.

PubMed

Adamczewski, M; Numerof, R P; Koretzky, G A; Kinet, J P

1995-04-01

Previous studies using tyrosine phosphatase inhibitors have implicated tyrosine phosphatases in the signal transduction pathway initiated by aggregation of Fc epsilon RI, the high affinity receptor for IgE. To define more precisely a role for the tyrosine phosphatase CD45 in Fc epsilon RI-mediated signaling, we have transfected the three subunits of Fc epsilon RI into wild-type Jurkat and a CD45-deficient Jurkat derivative. Here we demonstrate that CD45 is necessary for the initiation of calcium flux through the transfected Fc epsilon RI. In contrast to the effect of phosphatase inhibitors, the tyrosine phosphorylation levels of beta and gamma after aggregation of Fc epsilon RI are surprisingly reduced, relative to wild-type Jurkat, in the CD45-deficient cells. After reconstitution of the CD45-deficient cells with a chimeric molecule containing the cytoplasmic phosphatase domains of CD45, both the base line and activation-induced tyrosine phosphorylation levels are increased. By examining Lck autophosphorylation, we find that Fc epsilon RI aggregation induces an increase in Lck enzymatic activity only in wild-type Jurkat and the CD45-deficient Jurkat reconstituted with chimeric CD45. This regulation of src-family tyrosine kinase activity may be the means by which CD45 controls aggregation-induced receptor phosphorylation.

Mutational analysis of the SRC homology 2 domain protein-tyrosine phosphatase Corkscrew.

PubMed

Allard, J D; Herbst, R; Carroll, P M; Simon, M A

1998-05-22

The SRC homology 2 (SH2) domain protein-tyrosine phosphatase, Corkscrew (CSW) is required for signaling by receptor tyrosine kinases, including the Sevenless receptor tyrosine kinase (SEV), which directs Drosophila R7 photoreceptor cell development. To investigate the role of the different domains of CSW, we constructed domain-specific csw mutations and assayed their effects on CSW function. Our results indicate that CSW SH2 domain function is essential, but either CSW SH2 domain can fulfill this requirement. We also found that CSW and activated SEV are associated in vivo in a manner that does not require either CSW SH2 domain function or tyrosine phosphorylation of SEV. In contrast, the interaction between CSW and Daughter of Sevenless, a CSW substrate, is dependent on SH2 domain function. These results suggest that the role of the CSW SH2 domains during SEV signaling is to bind Daughter of Sevenless rather than activated SEV. We also found that although CSW protein-tyrosine phosphatase activity is required for full CSW function, a catalytically inactive CSW is capable of providing partial function. In addition, we found that deletion of either the CSW protein- tyrosine phosphatase insert or the entire CSW carboxyl terminus, which includes a conserved DRK/GRB2 SH2 domain binding sequence, does not abolish CSW function.

STriatal-Enriched protein tyrosine Phosphatase (STEP) Regulates the PTPα/Fyn Signaling Pathway

PubMed Central

Xu, Jian; Kurup, Pradeep; Foscue, Ethan; Lombroso, Paul J.

2015-01-01

The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr420) or inhibit (Tyr531) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr531 and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr420 and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr789. Dephosphorylation of Tyr789 prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction of STEP61 activity increased the phosphorylation of PTPα at Tyr789, as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by ethanol administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by ethanol leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. PMID:25951993

Protein tyrosine phosphatase 1B inhibitory activity of lavandulyl flavonoids from roots of Sophora flavescens.

PubMed

Sasaki, Tatsunori; Li, Wei; Higai, Koji; Quang, Tran Hong; Kim, Young Ho; Koike, Kazuo

2014-05-01

Protein tyrosine phosphatase 1B is a non-transmembrane protein tyrosine phosphatase and major negative regulator in insulin signaling cascades, and much attention has been paid to protein tyrosine phosphatase 1B inhibitors as potential therapies for diabetes. The screening of a natural compound library led to the discovery of five lavandulyl flavonoids, which were isolated from the roots of Sophora flavescens, as novel PTP1B inhibitors: kuraridin (1), norkurarinone (2), kurarinone (3), 2'-methoxykurarinone (4), and kushenol T (5). The three most potent compounds, 1, 2, and 4 (IC50 < 30 µM), were demonstrated to be noncompetitive inhibitors of protein tyrosine phosphatase 1B based on a kinetic analysis, and they exhibited different inhibitory selectivities against four homologous protein tyrosine phosphatases (T cell protein tyrosine phosphatase, vaccinia H1-related phosphatase, and Src homology domain 2-containing protein tyrosine phosphatases 1 and 2). Compounds 1, 2, and 4 also exhibited cellular activity in the insulin signaling pathway by increasing the insulin-stimulated Akt phosphorylation level in human hepatocellular liver carcinoma HepG2 cells, suggesting their potential for new anti-insulin-resistant drug developments. Georg Thieme Verlag KG Stuttgart · New York.

DIRECT MODULATION OF THE PROTEIN KINASE A CATALYTIC SUBUNIT α BY GROWTH FACTOR RECEPTOR TYROSINE KINASES

PubMed Central

Caldwell, George B.; Howe, Alan K.; Nickl, Christian K.; Dostmann, Wolfgang R.; Ballif, Bryan A.; Deming, Paula B.

2011-01-01

The cyclic-AMP-dependent protein kinase A (PKA) regulates processes such as cell proliferation and migration following activation of growth factor receptor tyrosine kinases (RTKs), yet the signaling mechanisms that link PKA with growth factor receptors remain largely undefined. Here we report that RTKs can directly modulate the function of the catalytic subunit of PKA (PKA-C) through post-translational modification. In vitro kinase assays revealed that both the epidermal growth factor and platelet derived growth factor receptors (EGFR and PDGFR, respectively) tyrosine phosphorylate PKA-C. Mass spectrometry identified tyrosine 330 (Y330) as a receptor-mediated phosphorylation site and mutation of Y330 to phenylalanine (Y330F) all but abolished the RTK-mediated phosphorylation of PKA-C in vitro. Y330 resides within a conserved region at the C-terminal tail of PKA-C that allosterically regulates enzymatic activity. Therefore, the effect of phosphorylation at Y330 on the activity of PKA-C was investigated. The Km for a peptide substrate was markedly decreased when PKA-C subunits were tyrosine phosphorylated by the receptors as compared to un-phosphorylated controls. Importantly, tyrosine-phosphorylated PKA-C subunits were detected in cells stimulated with EGF, PDGF and FGF2 and in fibroblasts undergoing PDGF-mediated chemotaxis. These results demonstrate a direct, functional interaction between RTKs and PKA-C and identify tyrosine phosphorylation as a novel mechansim for regulating PKA activity. PMID:21866565

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

PubMed

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Distinct pathways regulate Syk protein activation downstream of immune tyrosine activation motif (ITAM) and hemITAM receptors in platelets.

PubMed

Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol; Eble, Johannes A; Ellmeier, Wilfried; Kahn, Mark; Kunapuli, Satya P

2015-05-01

Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of Tyrosine 972 as a Novel Site of Jak2 Tyrosine Kinase Phosphorylation and its Role in Jak2 Activation

PubMed Central

McDoom, Issam; Ma, Xianyue; Kirabo, Annet; Lee, Kuang-Yung; Ostrov, David A.; Sayeski, Peter P.

2013-01-01

Jak2 is a 130 kDa tyrosine kinase that is important in a number of cellular signaling pathways. Its function is intrinsically regulated by the phosphorylation of a handful of its 49 tyrosines. Here, we report that tyrosine 972 (Y972) is a novel site of Jak2 phosphorylation, and hence auto-regulation. Specifically, we found that Y972 is phosphorylated and confirmed that this residue resides on the surface of the protein. Using expression plasmids that expressed either wild type Jak2 or a full length Jak2 cDNA containing a single Y972F substitution mutation, we investigated the consequences of losing Y972 phosphorylation on Jak2 function. We determined that the loss of Y972 phosphorylation significantly reduced both Jak2 total tyrosine phosphorylation and phosphorylation of Y1007/Y1008. Additionally, Y972 phosphorylation was shown to be important for maximal kinase function. Interestingly, in response to classical cytokine activation, the Jak2-Y972F mutant exhibited a moderately impaired level of activation when compared to wild type protein. However, when Jak2 was activated via a GPCR ligand, the ability of the Y972F mutant to activate was completely lost, therefore suggesting a differential role of Y972 in Jak2 activation. Finally, we found that phosphorylation of Y972 enhances Jak2 kinase function via a mechanism that appears to stabilize the active conformation of the protein. Collectively, our results suggest that Y972 is a novel site of Jak2 phosphorylation and plays an important differential role in ligand-dependent Jak2 activation via a mechanism that involves stabilization of the Jak2 active conformation. PMID:18636744

A new member of the 4-methylideneimidazole-5-one–containing aminomutase family from the enediyne kedarcidin biosynthetic pathway

PubMed Central

Huang, Sheng-Xiong; Lohman, Jeremy R.; Huang, Tingting; Shen, Ben

2013-01-01

4-Methylideneimidazole-5-one (MIO)-containing aminomutases catalyze the conversion of l-α-amino acids to β-amino acids with either an (R) or an (S) configuration. l-Phenylalanine and l-tyrosine are the only two natural substrates identified to date. The enediyne chromophore of the chromoprotein antitumor antibiotic kedarcidin (KED) harbors an (R)-2-aza-3-chloro-β-tyrosine moiety reminiscent of the (S)-3-chloro-5-hydroxy-β-tyrosine moiety of the C-1027 enediyne chromophore, the biosynthesis of which uncovered the first known MIO-containing aminomutase, SgcC4. Comparative analysis of the KED and C-1027 biosynthetic gene clusters inspired the proposal for (R)-2-aza-3-chloro-β-tyrosine biosynthesis starting from 2-aza-l-tyrosine, featuring KedY4 as a putative MIO-containing aminomutase. Here we report the biochemical characterization of KedY4, confirming its proposed role in KED biosynthesis. KedY4 is an MIO-containing aminomutase that stereospecifically catalyzes the conversion of 2-aza-l-tyrosine to (R)-2-aza-β-tyrosine, exhibiting no detectable activity toward 2-aza-l-phenylalanine or l-tyrosine as an alternative substrate. In contrast, SgcC4, which stereospecifically catalyzes the conversion of l-tyrosine to (S)-β-tyrosine in C-1027 biosynthesis, exhibits minimal activity with 2-aza-l-tyrosine as an alternative substrate but generating (S)-2-aza-β-tyrosine, a product with the opposite stereochemistry of KedY4. This report of KedY4 broadens the scope of known substrates for the MIO-containing aminomutase family, and comparative studies of KedY4 and SgcC4 provide an outstanding opportunity to examine how MIO-containing aminomutases control substrate specificity and product enantioselectivity. PMID:23633564

A new member of the 4-methylideneimidazole-5-one-containing aminomutase family from the enediyne kedarcidin biosynthetic pathway.

PubMed

Huang, Sheng-Xiong; Lohman, Jeremy R; Huang, Tingting; Shen, Ben

2013-05-14

4-Methylideneimidazole-5-one (MIO)-containing aminomutases catalyze the conversion of L-α-amino acids to β-amino acids with either an (R) or an (S) configuration. L-phenylalanine and L-tyrosine are the only two natural substrates identified to date. The enediyne chromophore of the chromoprotein antitumor antibiotic kedarcidin (KED) harbors an (R)-2-aza-3-chloro-β-tyrosine moiety reminiscent of the (S)-3-chloro-5-hydroxy-β-tyrosine moiety of the C-1027 enediyne chromophore, the biosynthesis of which uncovered the first known MIO-containing aminomutase, SgcC4. Comparative analysis of the KED and C-1027 biosynthetic gene clusters inspired the proposal for (R)-2-aza-3-chloro-β-tyrosine biosynthesis starting from 2-aza-L-tyrosine, featuring KedY4 as a putative MIO-containing aminomutase. Here we report the biochemical characterization of KedY4, confirming its proposed role in KED biosynthesis. KedY4 is an MIO-containing aminomutase that stereospecifically catalyzes the conversion of 2-aza-L-tyrosine to (R)-2-aza-β-tyrosine, exhibiting no detectable activity toward 2-aza-L-phenylalanine or L-tyrosine as an alternative substrate. In contrast, SgcC4, which stereospecifically catalyzes the conversion of L-tyrosine to (S)-β-tyrosine in C-1027 biosynthesis, exhibits minimal activity with 2-aza-L-tyrosine as an alternative substrate but generating (S)-2-aza-β-tyrosine, a product with the opposite stereochemistry of KedY4. This report of KedY4 broadens the scope of known substrates for the MIO-containing aminomutase family, and comparative studies of KedY4 and SgcC4 provide an outstanding opportunity to examine how MIO-containing aminomutases control substrate specificity and product enantioselectivity.

Toxicological disruption of signaling homeostasis: Tyrosine phosphatses as targets

EPA Science Inventory

The protein tyrosine phosphatases (PTP) comprised a diverse group of enzymes whose activity opposes that of the tyrosine kinases. As such, the PTP have critical roles in maintaining signaling quiescence in resting cells and in restoring homeostasis by effecting signal termination...

Naringin directly activates inwardly rectifying potassium channels at an overlapping binding site to tertiapin-Q

PubMed Central

Yow, Tin T; Pera, Elena; Absalom, Nathan; Heblinski, Marika; Johnston, Graham AR; Hanrahan, Jane R; Chebib, Mary

2011-01-01

BACKGROUND G protein-coupled inwardly rectifying potassium (KIR3) channels are important proteins that regulate numerous physiological processes including excitatory responses in the CNS and the control of heart rate. Flavonoids have been shown to have significant health benefits and are a diverse source of compounds for identifying agents with novel mechanisms of action. EXPERIMENTAL APPROACH The flavonoid glycoside, naringin, was evaluated on recombinant human KIR3.1–3.4 and KIR3.1–3.2 expressed in Xenopus oocytes using two-electrode voltage clamp methods. In addition, we evaluated the activity of naringin alone and in the presence of the KIR3 channel blocker tertiapin-Q (0.5 nM, 1 nM and 3 nM) at recombinant KIR3.1–3.4 channels. Site-directed mutagenesis was used to identify amino acids within the M1–M2 loop of the KIR3.1F137S mutant channel important for naringin's activity. KEY RESULTS Naringin (100 µM) had minimal effect on uninjected oocytes but activated KIR3.1–3.4 and KIR3.1–3.2 channels. The activation by naringin of KIR3.1–3.4 channels was inhibited by tertiapin-Q in a competitive manner. An alanine-scan performed on the KIR3.1F137S mutant channel, replacing one by one aromatic amino acids within the M1–M2 loop, identified tyrosines 148 and 150 to be significantly contributing to the affinity of naringin as these mutations reduced the activity of naringin by 20- and 40-fold respectively. CONCLUSIONS AND IMPLICATIONS These results show that naringin is a direct activator of KIR3 channels and that tertiapin-Q shares an overlapping binding site on the KIR3.1–3.4. This is the first example of a ligand that activates KIR3 channels by binding to the extracellular M1–M2 linker of the channel. PMID:21391982

Tyrosine phosphorylation of dihydrolipoamide dehydrogenase as a potential cadmium target and its inhibitory role in regulating mouse sperm motility.

PubMed

Li, Xinhong; Wang, Lirui; Li, Yuhua; Fu, Jieli; Zhen, Linqing; Yang, Qiangzhen; Li, Sisi; Zhang, Yukun

2016-05-16

Cadmium (Cd) is reported to reduce sperm motility and functions. However, the molecular mechanisms of Cd-induced toxicity remain largely unknown, presenting a major knowledge gap in research on reproductive toxicology. In the present study, we identified a candidate protein, dihydrolipoamide dehydrogenase (DLD), which is a post-pyruvate metabolic enzyme, exhibiting tyrosine phosphorylation in mouse sperm exposed to Cd both in vivo and in vitro. Immunoprecipitation assay demonstrated DLD was phosphorylated in tyrosine residues without altered expression after Cd treatment, which further confirmed our identified result. However, the tyrosine phosphorylation of DLD did not participate in mouse sperm capacitation and Bovine Serum Albumin (BSA) effectively prevented the tyrosine phosphorylation of DLD. Moreover, Cd-induced tyrosine phosphorylation of DLD lowered its dehydrogenase activity and meanwhile, Nicotinamide Adenine Dinucleotide Hydrogen (NADH) content, Adenosine Triphosphate (ATP) production and sperm motility were all inhibited by Cd. Interestingly, when the tyrosine phosphorylation of DLD was blocked by BSA, the decrease of DLD activity, NADH and ATP content as well as sperm motility was also suppressed simultaneously. These results suggested that Cd-induced tyrosine phosphorylation of DLD inhibited its activity and thus suppressed the tricarboxylic acid (TCA) cycle, which resulted in the reduction of NADH and hence the ATP production generated through oxidative phosphorylation (OPHOXS). Taken together, our results revealed that Cd induced DLD tyrosine phosphorylation, in response to regulate TCA metabolic pathway, which reduced ATP levels and these negative effects led to decreased sperm motility. This study provided new understanding of the mechanisms contributing to the harmful effects of Cd on the motility and function of spermatozoa. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Tyrosine dephosphorylation enhances the therapeutic target activity of epidermal growth factor receptor (EGFR) by disrupting its interaction with estrogen receptor (ER).

PubMed

Ma, Shao; Yin, Ning; Qi, Xiaomei; Pfister, Sandra L; Zhang, Mei-Jie; Ma, Rong; Chen, Guan

2015-05-30

Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics.

Activated Cdc42-associated kinase 1 (ACK1) binds the sterile α motif (SAM) domain of the adaptor SLP-76 and phosphorylates proximal tyrosines.

PubMed

Thaker, Youg R; Recino, Asha; Raab, Monika; Jabeen, Asma; Wallberg, Maja; Fernandez, Nelson; Rudd, Christopher E

2017-04-14

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a crucial role in T cell activation by linking antigen receptor (T cell receptor, TCR) signals to downstream pathways. At its N terminus, SLP-76 has three key tyrosines (Tyr-113, Tyr-128, and Tyr-145, "3Y") as well as a sterile α motif (SAM) domain whose function is unclear. We showed previously that the SAM domain has two binding regions that mediate dimer and oligomer formation. In this study, we have identified SAM domain-carrying non-receptor tyrosine kinase, activated Cdc42-associated tyrosine kinase 1 (ACK1; also known as Tnk2, tyrosine kinase non-receptor 2) as a novel binding partner of SLP-76. Co-precipitation, laser-scanning confocal microscopy, and in situ proximity analysis confirmed the binding of ACK1 to SLP-76. Further, the interaction was induced in response to the anti-TCR ligation and abrogated by the deletion of SLP-76 SAM domain (ΔSAM) or mutation of Tyr-113, Tyr-128, and Tyr-145 to phenylalanine (3Y3F). ACK1 induced phosphorylation of the SLP-76 N-terminal tyrosines (3Y) dependent on the SAM domain. Further, ACK1 promoted calcium flux and NFAT-AP1 promoter activity and decreased the motility of murine CD4 + primary T cells on ICAM-1-coated plates, an event reversed by a small molecule inhibitor of ACK1 (AIM-100). These findings identify ACK1 as a novel SLP-76-associated protein-tyrosine kinase that modulates early activation events in T cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Activated Cdc42-associated kinase 1 (ACK1) binds the sterile α motif (SAM) domain of the adaptor SLP-76 and phosphorylates proximal tyrosines

PubMed Central

Thaker, Youg R.; Recino, Asha; Raab, Monika; Jabeen, Asma; Wallberg, Maja; Fernandez, Nelson; Rudd, Christopher E.

2017-01-01

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a crucial role in T cell activation by linking antigen receptor (T cell receptor, TCR) signals to downstream pathways. At its N terminus, SLP-76 has three key tyrosines (Tyr-113, Tyr-128, and Tyr-145, “3Y”) as well as a sterile α motif (SAM) domain whose function is unclear. We showed previously that the SAM domain has two binding regions that mediate dimer and oligomer formation. In this study, we have identified SAM domain-carrying non-receptor tyrosine kinase, activated Cdc42-associated tyrosine kinase 1 (ACK1; also known as Tnk2, tyrosine kinase non-receptor 2) as a novel binding partner of SLP-76. Co-precipitation, laser-scanning confocal microscopy, and in situ proximity analysis confirmed the binding of ACK1 to SLP-76. Further, the interaction was induced in response to the anti-TCR ligation and abrogated by the deletion of SLP-76 SAM domain (ΔSAM) or mutation of Tyr-113, Tyr-128, and Tyr-145 to phenylalanine (3Y3F). ACK1 induced phosphorylation of the SLP-76 N-terminal tyrosines (3Y) dependent on the SAM domain. Further, ACK1 promoted calcium flux and NFAT-AP1 promoter activity and decreased the motility of murine CD4+ primary T cells on ICAM-1-coated plates, an event reversed by a small molecule inhibitor of ACK1 (AIM-100). These findings identify ACK1 as a novel SLP-76-associated protein-tyrosine kinase that modulates early activation events in T cells. PMID:28188290

Activation of phosphatidylinositol-3-kinase by platelet-derived growth factor and insulin-like growth factor-1 is inhibited by a transmembrane phosphotyrosine phosphatase.

PubMed

Way, B A; Mooney, R A

1993-12-15

Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit hormone-dependent tyrosine phosphorylation and mitogenesis (Mooney, R. A., Freund, G. G., Way, B. A., and Bordwell, K. L. (1992) J. Biol. Chem. 267, 23443-23446). Here the impact of PTPase expression on insulin-like growth factor-1 (IGF-1) and platelet-derived growth factor- (PDGF) dependent activation of PI-3-K was investigated. In PTPase+ cells, IGF-1 and PDGF-dependent PI-3-K activity in antiphosphotyrosine immunoprecipitates was decreased by 62 +/- 13 and 46 +/- 17%, respectively, compared to control cells. Similar decreases in PI-3-K activity associated with anti-PDGF receptor and anti-insulin receptor substrate-1 (IRS-1) immunoprecipitates were also observed. Association of PI-3-K with the hormone-activated PDGF receptor decreased approximately 55%, paralleling its loss of activation in PTPase+ cells. Tyrosine phosphorylation of the 85-kDa subunit of PI-3-K was also inhibited. Similarly, IGF-1 dependent tyrosine phosphorylation of IRS-1 was decreased by 45%, and its association with PI-3-K was decreased by 65% in PTPase+ cells. Finally, PDGF-dependent tyrosine phosphorylation of phospholipase C-gamma 1 and GTPase-activating protein was reduced by 60-70% in the PTPase+ cells as was tyrosine phosphorylation of the PDGF receptor associated with these proteins. In summary, expression of a transmembrane PTPase decreased hormone-dependent PI-3-K activation, tyrosine phosphorylation of receptor substrates, and their association with signaling complexes. These data support a role for transmembrane PTPases in the regulation of receptor signal transduction pathways.

Redox-Regulated Pathway of Tyrosine Phosphorylation Underlies NF-κB Induction by an Atypical Pathway Independent of the 26S Proteasome

PubMed Central

Cullen, Sarah; Ponnappan, Subramaniam; Ponnappan, Usha

2015-01-01

Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated. PMID:25671697

Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways.

PubMed

Li, Hui; Yang, Duxiao; Ning, Shanglei; Xu, Yinghui; Yang, Fan; Yin, Rusha; Feng, Taihu; Han, Shouqing; Guo, Lu; Zhang, Pengju; Qu, Wenjie; Guo, Renbo; Song, Chen; Xiao, Peng; Zhou, Chengjun; Xu, Zhigang; Sun, Jin-Peng; Yu, Xiao

2018-01-01

The protein tyrosine phosphatase nonreceptor type 12 (PTPN12) is a multifunctional protein and has elicited much research attention because its decreased protein level has been associated with poor prognosis of several types of cancers. Recently, we have solved the crystal structure of the phosphatase domain of PTPN12, which disclosed a specific PTPN12-insert-loop harboring a cyclin-dependent kinase 2 (CDK2) phosphorylation site. However, the functional significance of this phosphorylation is undefined. In the present study, we found that S19 site phosphorylation of PTPN12 by CDK2 discharged its antitumor activity by down-regulation of its inhibitory role in cell migration, but not affecting its other regulatory functions. Phosphorylation of PTPN12 at the S19 site changed its substrate interface, and by doing so, selectively decreased its activity toward the human epidermal growth factor receptor 2 (HER2)- pY 1196 site, but not other HER2 phosphorylation sites or other known PTPN12 substrates. A further in-depth mechanism study revealed that the phosphorylation of PTPN12 by CDK2 impaired recruitment of the serine/threonine-protein kinase 1 (PAK1) to HER2, resulted in the blockade of the HER2-pY 1196 -PAK1-T 423 signaling pathway, thus increased tumor cell motility. Taken together, our results identified a new phosphorylation-based substrate recognition mechanism of PTPN12 by CDK2, which orchestrated signaling crosstalk between the oncogenic CDK2 and HER2 pathways. The newly identified governing mechanism of the substrate selectivity of a particular phosphatase was previously unappreciated and exemplifies how a phospho-network is precisely controlled in different cellular contexts.-Li, H., Yang, D., Ning, S., Xu, Y., Yang, F., Yin, R., Feng, T., Han, S., Guo, L., Zhang, P., Qu, W., Guo, R., Song, C., Xiao, P., Zhou, C., Xu, Z., Sun, J.-P., Yu, X. Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways. © FASEB.

Receptor Type Protein Tyrosine Phosphatase ζ-Pleiotrophin Signaling Controls Endocytic Trafficking of DNER That Regulates Neuritogenesis▿ †

PubMed Central

Fukazawa, Nobuna; Yokoyama, Seisuke; Eiraku, Mototsugu; Kengaku, Mineko; Maeda, Nobuaki

2008-01-01

Protein tyrosine phosphatase ζ (PTPζ) is a receptor type protein tyrosine phosphatase that uses pleiotrophin as a ligand. Pleiotrophin inactivates the phosphatase activity of PTPζ, resulting in the increase of tyrosine phosphorylation levels of its substrates. We studied the functional interaction between PTPζ and DNER, a Notch-related transmembrane protein highly expressed in cerebellar Purkinje cells. PTPζ and DNER displayed patchy colocalization in the dendrites of Purkinje cells, and immunoprecipitation experiments indicated that these proteins formed complexes. Several tyrosine residues in and adjacent to the tyrosine-based and the second C-terminal sorting motifs of DNER were phosphorylated and were dephosphorylated by PTPζ, and phosphorylation of these tyrosine residues resulted in the accumulation of DNER on the plasma membrane. DNER mutants lacking sorting motifs accumulated on the plasma membrane of Purkinje cells and Neuro-2A cells and induced their process extension. While normal DNER was actively endocytosed and inhibited the retinoic-acid-induced neurite outgrowth of Neuro-2A cells, pleiotrophin stimulation increased the tyrosine phosphorylation level of DNER and suppressed the endocytosis of this protein, which led to the reversal of this inhibition, thus allowing neurite extension. These observations suggest that pleiotrophin-PTPζ signaling controls subcellular localization of DNER and thereby regulates neuritogenesis. PMID:18474614

Effects of hemorrhagic hypotension on tyrosine concentrations in rat spinal cord and plasma

NASA Technical Reports Server (NTRS)

Conlay, L. A.; Maher, T. J.; Roberts, C. H.; Wurtman, R. J.

1988-01-01

Tyrosine is the precursor for catecholamine neurotransmitters. When catecholamine-containing neurons are physiologically active (as sympathoadrenal cells are in hypotension), tyrosine administration increases catecholamine synthesis and release. Since hypotension can alter plasma amino acid composition, the effects of an acute hypotensive insult on tyrosine concentrations in plasma and spinal cord were examined. Rats were cannulated and bled until the systolic blood pressure was 50 mmHg, or were kept normotensive for 1 h. Tyrosine and other large neutral amino acids (LNAA) known to compete with tyrosine for brain uptake were assayed in plasma and spinal cord. The rate at which intra-arterial (H-3)tyrosine disappeared from the plasma was also estimated in hemorrhaged and control rats. In plasma of hemorrhaged animals, both the tyrosine concentration and the tyrosine/LNAA ratio was elevated; moreover, the disappearance of (H-3)tyrosine was slowed. Tyrosine concentrations also increased in spinal cords of hemorrhaged-hypotensive rats when compared to normotensive controls. Changes in plasma amino acid patterns may thus influence spinal cord concentrations of amino acid precursors for neurotransmitters during the stress of hemorrhagic shock.

Volume-activated trimethylamine oxide efflux in red blood cells of spiny dogfish (Squalus acanthias).

PubMed

Koomoa, D L; Musch, M W; MacLean, A V; Goldstein, L

2001-09-01

The aims of this study were to determine the pathway of swelling-activated trimethylamine oxide (TMAO) efflux and its regulation in spiny dogfish (Squalus acanthias) red blood cells and compare the characteristics of this efflux pathway with the volume-activated osmolyte (taurine) channel present in erythrocytes of fishes. The characteristics of the TMAO efflux pathway were similar to those of the taurine efflux pathway. The swelling-activated effluxes of both TMAO and taurine were significantly inhibited by known anion transport inhibitors (DIDS and niflumic acid) and by the general channel inhibitor quinine. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea activated both TMAO and taurine effluxes similarly. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea also stimulated the activity of tyrosine kinases p72syk and p56lyn, although the stimulations by the latter two treatments were less than by hypotonicity. The volume activations of both TMAO and taurine effluxes were inhibited by tyrosine kinase inhibitors, suggesting that activation of tyrosine kinases may play a role in activating the osmolyte effluxes. These results indicate that the volume-activated TMAO efflux occurs via the organic osmolyte (taurine) channel and may be regulated by the volume activation of tyrosine kinases.

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal T-cell receptor (TCR) signaling.

PubMed

Ji, Qinqin; Ding, Yiyuan; Salomon, Arthur R

2015-01-01

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor-mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr(112), Tyr(128), and Tyr(145), in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr(192) of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr(440) of Fyn, Tyr(702) of PLCγ1, Tyr(204), Tyr(397), and Tyr(69) of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

SRC Homology 2 Domain-containing Leukocyte Phosphoprotein of 76 kDa (SLP-76) N-terminal Tyrosine Residues Regulate a Dynamic Signaling Equilibrium Involving Feedback of Proximal T-cell Receptor (TCR) Signaling*

PubMed Central

Ji, Qinqin; Ding, Yiyuan; Salomon, Arthur R.

2015-01-01

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor–mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr112, Tyr128, and Tyr145, in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr192 of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr440 of Fyn, Tyr702 of PLCγ1, Tyr204, Tyr397, and Tyr69 of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues. PMID:25316710

Antioxidants reverse the changes in energy metabolism of rat brain after chronic administration of L.-tyrosine.

PubMed

Teodorak, Brena P; Scaini, Giselli; Carvalho-Silva, Milena; Gomes, Lara M; Teixeira, Letícia J; Rebelo, Joyce; De Prá, Samira D T; Zeni, Neila; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

2017-04-01

Tyrosinemia type II is a rare autosomal recessive disease caused by deficiency of hepatic tyrosine aminotransferase and is associated with neurologic and development difficulties in numerous patients. Considering that the mechanisms underlying the neurological dysfunction in hypertyrosinemic patients are poorly known and that high concentrations of tyrosine provoke mitochondrial dysfunction and oxidative stress, in the present study we investigated the in vivo influence of antioxidants (N-acetylcysteine, NAC; and deferoxamine, DFX) administration on the inhibitory effects on parameters of energy metabolism in cerebral cortex, hippocampus and striatum of rats, provoked by chronic administration of L.-tyrosine. Our results showed that chronic administration of L.-tyrosine results in a marked decrease in the activity of citrate synthase in all the analyzed structures and succinate dehydrogenase activities in hippocampus and striatum, and that antioxidants administration can prevent this inhibition in hippocampus and striatum. Moreover, chronic administration of L.-tyrosine inhibited the activity of complex I, II-III and IV in the striatum, which can be prevented by antioxidant treatment. However, the co-administration of NAC plus DFX could not prevent the inhibition of creatine kinase activity in the striatum. In conclusion, the present study demonstrates that the administration of antioxidants NAC and DFX attenuates the L.-tyrosine effects on enzymes of the Krebs cycle and the mitochondrial respiratory chain, suggesting that impairment of energy metabolism can be involved with oxidative stress. These results also indicate a possible neuroprotective role for NAC and DFX as a potential adjuvant therapy to the patients with Tyrosinemia type II.

Gab1 Acts as an Adapter Molecule Linking the Cytokine Receptor gp130 to ERK Mitogen-Activated Protein Kinase

PubMed Central

Takahashi-Tezuka, Mariko; Yoshida, Yuichi; Fukada, Toshiyuki; Ohtani, Takuya; Yamanaka, Yojiro; Nishida, Keigo; Nakajima, Koichi; Hibi, Masahiko; Hirano, Toshio

1998-01-01

Gab1 has structural similarities with Drosophila DOS (daughter of sevenless), which is a substrate of the protein tyrosine phosphatase Corkscrew. Both Gab1 and DOS have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are phosphorylated. We found that Gab1 was tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-3, alpha interferon (IFN-α), and IFN-γ. Upon the stimulation of IL-6 or IL-3, Gab1 was found to form a complex with phosphatidylinositol (PI)-3 kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, revealed that neither tyrosine residues of gp130 nor its carboxy terminus was required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhanced gp130-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogated the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2 activation. Furthermore, ERK2 activation was inhibited by a dominant negative p85 PI-3 kinase, wortmannin, or a dominant negative Ras. These observations suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp130 and that SHP-2, PI-3 kinase, and Ras are involved in Gab1-mediated ERK activation. PMID:9632795

H-Ras Modulates N-Methyl-d-aspartate Receptor Function via Inhibition of Src Tyrosine Kinase Activity*

PubMed Central

Thornton, Claire; Yaka, Rami; Dinh, Son; Ron, Dorit

2005-01-01

Tyrosine phosphorylation of the NR2A and NR2B subunits of the N-methyl-d-aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous Src activity and NR2A phosphorylation, and decreased the magnitude of hip-pocampal LTP. No change was observed for NR2B. We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction. PMID:12695509

The Structure of L-Tyrosine 2,3-Aminomutase frmo the C-1027 Enediyne Antitumor Antibiotic Biosynthetic Pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Christianson,C.; Montavon, T.; Van Lanen, S.

2007-01-01

The SgcC4 L-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-{beta}-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate L-tyrosine to generate (S)-{beta}-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Heremore » we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the L-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.« less

Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphorylation required for WAVE2 activation.

PubMed

Leng, Yan; Zhang, Jinyi; Badour, Karen; Arpaia, Enrico; Freeman, Spencer; Cheung, Pam; Siu, Michael; Siminovitch, Katherine

2005-01-25

WAVE2 is a member of the Wiskott-Aldrich syndrome protein family of cytoskeletal regulatory proteins shown to link Rac activation to actin remodeling via induction of Arp 2/3 activity. WAVE2 is thought to be regulated by its positioning in a macromolecular complex also containing the Abelson-(Abl) interactor-1 (Abi-1) adaptor, but the molecular basis and biologic relevance of WAVE2 inclusion in this complex are ill defined. Here we show that Abi-1 binding to WAVE2 is mediated by discrete motifs in the Abi-1 coiled-coil and WAVE2 WAVE-homology domains and increases markedly in conjunction with Abi-1-WAVE2 translocation and colocalization at the leading edge in B16F1 cells after fibronectin stimulation. Abi-1 also couples WAVE2 to Abl after cell stimulation, an interaction that triggers Abl membrane translocation with WAVE2, Abi-1, and activated Rac, as well as Abl-mediated tyrosine phosphorylation and WAVE2 activation. By contrast, mutation of tyrosine residue Y150, identified here as the major site of Abl-mediated WAVE2 tyrosine phosphorylation, as well as disruption of WAVE2-Abi-1 binding, impairs induction of WAVE2-driven actin polymerization and its membrane translocation in association with activated Rac. Similarly, WAVE2 tyrosine phosphorylation and induction of membrane actin rearrangement are abrogated in fibroblasts lacking the Abl family kinase. Together, these data reveal that Abi-1-mediated coupling of Abl to WAVE2 promotes Abl-evoked WAVE2 tyrosine phosphorylation required to link WAVE2 with activated Rac and with actin polymerization and remodeling at the cell periphery.

Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of the trk proto-oncogene with src homology 2 domains.

PubMed

Ohmichi, M; Decker, S J; Saltiel, A R

1992-10-01

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.

c-Abl-Mediated Tyrosine Phosphorylation of the T-bet DNA-Binding Domain Regulates CD4+ T-Cell Differentiation and Allergic Lung Inflammation ▿

PubMed Central

Chen, An; Lee, Sang-Myeong; Gao, Beixue; Shannon, Stephen; Zhu, Zhou; Fang, Deyu

2011-01-01

The tyrosine kinase c-Abl is required for full activation of T cells, while its role in T-cell differentiation has not been characterized. We report that c-Abl deficiency skews CD4+ T cells to type 2 helper T cell (Th2) differentiation, and c-Abl−/− mice are more susceptible to allergic lung inflammation. c-Abl interacts with and phosphorylates T-bet, a Th1 lineage transcription factor. c-Abl-mediated phosphorylation enhances the transcriptional activation of T-bet. Interestingly, three tyrosine residues within the T-bet DNA-binding domain are the predominant sites of phosphorylation by c-Abl. Mutation of these tyrosine residues inhibits the promoter DNA-binding activity of T-bet. c-Abl regulates Th cell differentiation in a T-bet-dependent manner because genetic deletion of T-bet in CD4+ T cells abolishes c-Abl-deficiency-mediated enhancement of Th2 differentiation. Reintroduction of T-bet-null CD4+ T cells with wild-type T-bet, but not its tyrosine mutant, rescues gamma interferon (IFN-γ) production and inhibits Th2 cytokine production. Therefore, c-Abl catalyzes tyrosine phosphorylation of the DNA-binding domain of T-bet to regulate CD4+ T cell differentiation. PMID:21690296

Phosphopeptide occupancy and photoaffinity cross-linking of the v-Src SH2 domain attenuates tyrosine kinase activity.

PubMed

Garcia, P; Shoelson, S E; Drew, J S; Miller, W T

1994-12-02

Phosphorylation of c-Src at carboxyl-terminal Tyr-527 suppresses tyrosine kinase activity and transforming potential, presumably by facilitating the intramolecular interaction of the C terminus of Src with its SH2 domain. In addition, it has been shown previously that occupancy of the c-Src SH2 domain with a phosphopeptide stimulates c-Src kinase catalytic activity. We have performed analogous studies with v-Src, the transforming protein from Rous sarcoma virus, which has extensive homology with c-Src. v-Src lacks an autoregulatory phosphorylation site, and its kinase domain is constitutively active. Phosphopeptides corresponding to the sequences surrounding c-Src Tyr-527 and a Tyr-Glu-Glu-Ile motif from the hamster polyoma virus middle T antigen inhibit tyrosine kinase activity of baculovirus-expressed v-Src 2- and 4-fold, respectively. To determine the mechanism of this regulation, the Tyr-527 phosphopeptide was substituted with the photoactive amino acid p-benzoylphenylalanine at the adjacent positions (N- and C-terminal) to phosphotyrosine. These peptides photoinactivate the v-Src tyrosine kinase 5-fold in a time- and concentration-dependent manner. Furthermore, the peptides cross-link an isolated Src SH2 domain with similar rates and specificity. These data indicate that occupancy of the v-Src SH2 domain induces a conformational change that is transmitted to the kinase domain and attenuates tyrosine kinase activity.

Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B

PubMed Central

Venkatesan, Balachandar; Ghosh-Choudhury, Nandini; Das, Falguni; Mahimainathan, Lenin; Kamat, Amrita; Kasinath, Balakuntalam S.; Abboud, Hanna E.; Choudhury, Goutam Ghosh

2008-01-01

Mesangioproliferative glomerulonephritis is associated with overactive PDGF receptor signal transduction. We show that the phytoalexin resveratrol dose dependently inhibits PDGF-induced DNA synthesis in mesangial cells with an IC50 of 10 μM without inducing apoptosis. Remarkably, the increased SIRT1 deacetylase activity induced by resveratrol was not necessary for this inhibitory effect. Resveratrol significantly blocked PDGF-stimulated c-Src and Akt kinase activation, resulting in reduced cyclin D1 expression and attenuated pRb phosphorylation and cyclin-dependent kinase-2 (CDK2) activity. Furthermore, resveratrol inhibited PDGFR phosphorylation at the PI 3 kinase and Grb-2 binding sites tyrosine-751 and tyrosine-716, respectively. This deficiency in PDGFR phosphorylation resulted in significant inhibition of PI 3 kinase and Erk1/2 MAPK activity. Interestingly, resveratrol increased the activity of protein tyrosine phosphatase PTP1B, which dephosphorylates PDGF-stimulated phosphorylation at tyrosine-751 and tyrosine-716 on PDGFR with concomitant reduction in Akt and Erk1/2 kinase activity. PTP1B significantly inhibited PDGF-induced DNA synthesis without inducing apoptosis. These results for the first time provide evidence that the stilbene resveratrol targets PTP1B to inhibit PDGFR mitogenic signaling.—Venkatesan, B., Ghosh-Choudhury, N., Das, F., Mahimainathan, L., Kamat, A., Kasinath, B. S., Abboud, H. E., Choudhury, G. G. Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B. PMID:18567737

Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine.

PubMed Central

White, R; Sjöberg, M; Kalkhoven, E; Parker, M G

1997-01-01

The oestrogen receptor is a member of the nuclear receptor family of transcription factors which, on binding the steroid hormone 17beta-oestradiol, interacts with co-activator proteins and stimulates gene expression. Replacement of a single tyrosine in the hormone-binding domain generated activated forms of the receptor which stimulated transcription in the absence of hormone. This increased activation is related to a decrease in hydrophobicity and a reduction in size of the side chain of the amino acid with which the tyrosine is replaced. Ligand-independent, in common with ligand-dependent transcriptional activation, requires an amphipathic alpha-helix at the C-terminus of the ligand-binding domain which is essential for the interaction of the receptor with a number of potential co-activator proteins. In contrast to the wild-type protein, constitutively active receptors were able to bind both the receptor-interacting protein RIP-140 and the steroid receptor co-activator SRC-1 in a ligand-independent manner, although in the case of SRC-1 this was only evident when the receptors were prebound to DNA. We propose, therefore, that this tyrosine is required to maintain the receptor in a transcriptionally inactive state in the absence of hormone. Modification of this residue may generate a conformational change in the ligand-binding domain of the receptor to form an interacting surface which allows the recruitment of co-activators independent of hormone binding. This suggests that this tyrosine may be a target for a different signalling pathway which forms an alternative mechanism of activating oestrogen receptor-mediated transcription. PMID:9135157

Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a.

PubMed

Schiering, Nikolaus; Knapp, Stefan; Marconi, Marina; Flocco, Maria M; Cui, Jean; Perego, Rita; Rusconi, Luisa; Cristiani, Cinzia

2003-10-28

The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.

Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier ProteinTethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus

DOE PAGES

Chang, Chin -Yuan; Lohman, Jeremy; Cao, Hongnan; ...

2016-08-25

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-beta-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 A resolution, respectively. SgcE6 shares a similar β-barrel fold withmore » the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few, other known FADH 2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. In conclusion, the crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH 2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.« less

Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor.

PubMed

Lumb, Simon; Fleischer, Sarah J; Wiedemann, Annika; Daridon, Capucine; Maloney, Alison; Shock, Anthony; Dörner, Thomas

2016-06-01

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr(807), a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr(292) on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.

Tyrosine phosphorylation and association of Syk with Fc gamma RII in monocytic THP-1 cells.

PubMed Central

Ghazizadeh, S; Bolen, J B; Fleit, H B

1995-01-01

Although the cytoplasmic portion of the low-affinity receptor for immunoglobulin G, Fc gamma RII, does not contain a kinase domain, rapid tyrosine phosphorylation of intracellular substrates occurs in response to aggregation of the receptor. The use of specific tyrosine kinase inhibitors has suggested that these phosphorylations are required for subsequent cellular responses. We previously demonstrated the coprecipitation of a tyrosine kinase activity with Fc gamma RII, suggesting that non-receptor tyrosine kinases might associate with the cytoplasmic domain of Fc gamma RII. Anti-receptor immune complex kinase assays revealed the coprecipitation of several phosphoproteins, most notably p56/53lyn, an Src-family protein tyrosine kinase (PTK), and a 72 kDa phosphoprotein. Here we identify the 72 kDa Fc gamma RII-associated protein as p72syk (Syk), a member of a newly described family of non-receptor PTKs. A rapid and transient tyrosine phosphorylation of Syk was observed following Fc gamma RII activation. Syk was also tyrosyl-phosphorylated following aggregation of the high-affinity Fc gamma receptor, Fc gamma RI. The Fc gamma RI activation did not result in association of Syk with Fc gamma RII, implying that distinct pools of Syk are activated upon aggregation of each receptor in a localized manner. These results demonstrate a physical association between Syk and Fc gamma RII and suggest that the molecules involved in Fc gamma RII signalling are very similar to the ones utilized by multichain immune recognition receptors such as the B-cell antigen receptor and the high-affinity IgE receptor. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7530449

Enzymological Basis for Growth Inhibition by l-Phenylalanine in the Cyanobacterium Synechocystis sp. 29108

PubMed Central

Hall, Geraldine C.; Jensen, Roy A.

1980-01-01

The pattern of allosteric control in the biosynthetic pathway for aromatic amino acids provides a basis to explain vulnerability to growth inhibition by l-phenylalanine (0.2 mM or greater) in the unicellular cyanobacterium Synechocystis sp. 29108. We attribute growth inhibition to the hypersensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase to feedback inhibition by l-phenylalanine. Hyperregulation of this initial enzyme of aromatic biosynthesis depletes the supply of precursors needed for biosynthesis of l-tyrosine and l-tryptophan. Consistent with this mechanism is the total reversal of phenylalanine inhibition by a combination of tyrosine and tryptophan. Inhibited cultures also contained decreased levels of phycocyanin pigments, a characteristic previously correlated with amino acid starvation in cyanobacteria. l-Phenylalanine is a potent noncompetitive inhibitor (with both substrates) of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase, whereas l-tyrosine is a very weak inhibitor. Prephenate dehydratase also displays allosteric sensitivity to phenylalanine (inhibition) and to tyrosine (activation). Both 2-fluoro and 4-fluoro derivatives of phenylalanine were potent analog antimetabolites, and these were used in addition to l-phenylalanine as selective agents for resistant mutants. Mutants were isolated which excreted both phenylalanine and tyrosine, the consequence of an altered 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase no longer sensitive to feedback inhibition. Simultaneous insensitivity to l-tyrosine suggests that l-tyrosine acts as a weak analog mimic of l-phenylalanine at a common binding site. Prephenate dehydratase in the regulatory mutants was unaltered. Surprisingly, in view of the lack of regulation in the tyrosine branchlet of the pathway, such mutants excrete more phenylalanine than tyrosine, indicating that l-tyrosine activation dominates l-phenylalanine inhibition of prephenate dehydratase in vivo. In mutant Phe r19 the loss in allosteric sensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase was accompanied by a threefold increase in specific activity. This could suggest that existence of a modest degree of repression control (autogenous) over 3-deoxy-d-arabinoheptulosonate synthase, although other explanations are possible. Specific activities of chorismate mutase, prephenate dehydratase, shikimate/nicotinamide adenine dinucleotide phosphate dehydrogenase, and arogenate/nicotinamide adenine dinucleotide phosphate dehydrogenase in mutant Phe r19 were identical with those of the wild type. PMID:6108316

SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.

PubMed

Koch, C A; Anderson, D; Moran, M F; Ellis, C; Pawson, T

1991-05-03

Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.

N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues.

PubMed Central

Sackett, D. L.; Ruvinov, S. B.; Thompson, J.

1999-01-01

N5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr approximately equal to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the alpha,beta family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of approximately 8.5, 11 have pKa approximately 10.75, and the remaining six titrate with pKa approximately 11.3. The two residues with pKa approximately 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the beta alphabeta fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed. PMID:10548058

The Dopamine D2 Receptor Gene in Lamprey, Its Expression in the Striatum and Cellular Effects of D2 Receptor Activation

PubMed Central

Robertson, Brita; Huerta-Ocampo, Icnelia; Ericsson, Jesper; Stephenson-Jones, Marcus; Pérez-Fernández, Juan; Bolam, J. Paul; Diaz-Heijtz, Rochellys; Grillner, Sten

2012-01-01

All basal ganglia subnuclei have recently been identified in lampreys, the phylogenetically oldest group of vertebrates. Furthermore, the interconnectivity of these nuclei is similar to mammals and tyrosine hydroxylase-positive (dopaminergic) fibers have been detected within the input layer, the striatum. Striatal processing is critically dependent on the interplay with the dopamine system, and we explore here whether D2 receptors are expressed in the lamprey striatum and their potential role. We have identified a cDNA encoding the dopamine D2 receptor from the lamprey brain and the deduced protein sequence showed close phylogenetic relationship with other vertebrate D2 receptors, and an almost 100% identity within the transmembrane domains containing the amino acids essential for dopamine binding. There was a strong and distinct expression of D2 receptor mRNA in a subpopulation of striatal neurons, and in the same region tyrosine hydroxylase-immunoreactive synaptic terminals were identified at the ultrastructural level. The synaptic incidence of tyrosine hydroxylase-immunoreactive boutons was highest in a region ventrolateral to the compact layer of striatal neurons, a region where most striatal dendrites arborise. Application of a D2 receptor agonist modulates striatal neurons by causing a reduced spike discharge and a diminished post-inhibitory rebound. We conclude that the D2 receptor gene had already evolved in the earliest group of vertebrates, cyclostomes, when they diverged from the main vertebrate line of evolution (560 mya), and that it is expressed in striatum where it exerts similar cellular effects to that in other vertebrates. These results together with our previous published data (Stephenson-Jones et al. 2011, 2012) further emphasize the high degree of conservation of the basal ganglia, also with regard to the indirect loop, and its role as a basic mechanism for action selection in all vertebrates. PMID:22563388

Angiotensin II stimulates calcineurin activity in proximal tubule epithelia through AT-1 receptor-mediated tyrosine phosphorylation of the PLC-gamma1 isoform.

PubMed

Lea, Janice P; Jin, Shao G; Roberts, Brian R; Shuler, Michael S; Marrero, Mario B; Tumlin, James A

2002-07-01

Angiotensin II (AngII) contributes to the maintenance of extracellular fluid volume by regulating sodium transport in the nephron. In nonepithelial cells, activation of phospholipase C (PLC) by AT-1 receptors stimulates the generation of 1,4,5-trisphosphate (IP(3)) and the release of intracellular calcium. Calcineurin, a serine-threonine phosphatase, is activated by calcium and calmodulin, and both PLC and calcineurin have been linked to sodium transport in the proximal tubule. An examination of whether AngII activates calcineurin in a model of proximal tubule epithelia (LLC-PK1 cells) was performed; AngII increased calcineurin activity within 30 s. An examination of whether AngII activates PLC in proximal tubule epithelia was also performed after first showing that all three families of PLC isoforms are present in LLC-PK1 cells. Application of AngII increased IP(3) generation by 60% within 15 s, which coincided with AngII-induced tyrosine phosphorylation of the PLC-gamma1 isoform also observed at 15 s. AngII-induced tyrosine phosphorylation was blocked by the AT-1 receptor antagonist, Losartan. Subsequently, an inhibitor of tyrosine phosphorylation blocked the AngII-induced activation of calcineurin, as did coincubation with an inhibitor of PLC activity and with an antagonist of the AT-1 receptor. It is therefore concluded that AngII stimulates calcineurin phosphatase activity in proximal tubule epithelial cells through a mechanism involving AT-1 receptor-mediated tyrosine phosphorylation of the PLC isoform.

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography.

PubMed

Nagatsu, T; Oka, K; Kato, T

1979-07-21

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. D-Tyrosine was used for the control. alpha-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.

Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides.

PubMed

Mitsuhashi, Jun; Nakayama, Tsutomu; Narai-Kanayama, Asako

2015-01-01

Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of L-tyrosine ethyl ester in aqueous media at 30 °C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100 mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75 mM, respectively. In the reaction initiated with 100 mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15 mM, the reaction proceeded poorly; however, from 30 mM to 120 mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, L-tyrosine ethyl ester, even at 5mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes L-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate. Copyright © 2015 Elsevier Inc. All rights reserved.

SOCS2 Binds to and Regulates EphA2 through Multiple Mechanisms.

PubMed

Pilling, Carissa; Cooper, Jonathan A

2017-09-07

Suppressors of cytokine signaling (SOCS) proteins inhibit signaling by serving as substrate receptors for the Cullin5-RING E3 ubiquitin ligase (CRL5) and through a variety of CRL5-independent mechanisms. CRL5, SOCS2 and SOCS6 are implicated in suppressing transformation of epithelial cells. We identified cell proteins that interact with SOCS2 and SOCS6 using two parallel proteomics techniques: BioID and Flag affinity purification mass spectrometry. The receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) was identified as a SOCS2-interacting protein. SOCS2-EphA2 binding requires the SOCS2 SH2 domain and EphA2 activation loop autophosphorylation, which is stimulated by Ephrin A1 (EfnA1) or by phosphotyrosine phosphatase inhibition. Surprisingly, EfnA1-stimulated EphA2-SOCS2 binding is delayed until EphA2 has been internalized into endosomes. This suggests that SOCS2 binds to EphA2 in the context of endosomal membranes. We also found that SOCS2 overexpression decreases steady state levels of EphA2, consistent with increased EphA2 degradation. This effect is indirect: SOCS2 induces EfnA1 expression, and EfnA1 induces EphA2 down-regulation. Other RTKs have been reported to bind, and be regulated by, over-expressed SOCS proteins. Our data suggest that SOCS protein over-expression may regulate receptor tyrosine kinases through indirect and direct mechanisms.

Osmostress induces autophosphorylation of Hog1 via a C-terminal regulatory region that is conserved in p38α.

PubMed

Maayan, Inbal; Beenstock, Jonah; Marbach, Irit; Tabachnick, Shira; Livnah, Oded; Engelberg, David

2012-01-01

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it's removal from p38α abolishes p38α's autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation.

Osmostress Induces Autophosphorylation of Hog1 via a C-Terminal Regulatory Region That Is Conserved in p38α

PubMed Central

Maayan, Inbal; Beenstock, Jonah; Marbach, Irit; Tabachnick, Shira; Livnah, Oded; Engelberg, David

2012-01-01

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it’s removal from p38α abolishes p38α’s autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation. PMID:22984552

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

PubMed Central

Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

2015-01-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

Syk-mediated tyrosine phosphorylation of mule promotes TNF-induced JNK activation and cell death.

PubMed

Lee, C K; Yang, Y; Chen, C; Liu, J

2016-04-14

The transcription factor Miz1 negatively regulates TNF-induced JNK activation and cell death by suppressing TRAF2 K63-polyubiquitination; upon TNF stimulation, the suppression is relieved by Mule/ARF-BP1-mediated Miz1 ubiquitination and subsequent degradation. It is not known how Mule is activated by TNF. Here we report that TNF activates Mule by inducing the dissociation of Mule from its inhibitor ARF. ARF binds to and thereby inhibits the E3 ligase activity of Mule in the steady state. TNF induces tyrosine phosphorylation of Mule, which subsequently dissociates from ARF and becomes activated. Inhibition of Mule phosphorylation by silencing of the Spleen Tyrosine Kinase (Syk) prevents its dissociation from ARF, thereby inhibiting Mule E3 ligase activity and TNF-induced JNK activation and cell death. Our data provides a missing link in TNF signaling pathway that leads to JNK activation and cell death.

The Envelope Glycoprotein of Friend Spleen Focus-Forming Virus Covalently Interacts with and Constitutively Activates a Truncated Form of the Receptor Tyrosine Kinase Stk

PubMed Central

Nishigaki, Kazuo; Thompson, Delores; Hanson, Charlotte; Yugawa, Takashi; Ruscetti, Sandra

2001-01-01

The Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein, gp55, which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). SFFV gp55 has been shown to interact with the Epo receptor complex, causing constitutive activation of various signal-transducing molecules. When injected into adult mice, SFFV induces a rapid erythroleukemia, with susceptibility being determined by the host gene Fv-2, which was recently shown to be identical to the gene encoding the receptor tyrosine kinase Stk/Ron. Susceptible, but not resistant, mice encode not only full-length Stk but also a truncated form of the kinase, sf-Stk, which may mediate the biological effects of SFFV infection. To determine whether expression of SFFV gp55 leads to the activation of sf-Stk, we expressed sf-Stk, with or without SFFV gp55, in hematopoietic cells expressing the Epo receptor. Our data indicate that sf-Stk interacts with SFFV gp55 as well as gp55P, the biologically active form of the viral glycoprotein, forming disulfide-linked complexes. This covalent interaction, as well as noncovalent interactions with SFFV gp55, results in constitutive tyrosine phosphorylation of sf-Stk and its association with multiple tyrosine-phosphorylated signal-transducing molecules. In contrast, neither Epo stimulation in the absence of SFFV gp55 expression nor expression of a mutant of SFFV that cannot interact with sf-Stk was able to induce tyrosine phosphorylation of sf-Stk or its association with any signal-transducing molecules. Covalent interaction of sf-Stk with SFFV gp55 and constitutive tyrosine phosphorylation of sf-Stk can also be detected in an erythroleukemia cell line derived from an SFFV-infected mouse. Our results suggest that SFFV gp55 may mediate its biological effects in vivo by interacting with and activating a truncated form of the receptor tyrosine kinase Stk. PMID:11483734

Unprecedented Abundance of Protein Tyrosine Phosphorylation Modulates Shigella flexneri Virulence.

PubMed

Standish, Alistair James; Teh, Min Yan; Tran, Elizabeth Ngoc Hoa; Doyle, Matthew Thomas; Baker, Paul J; Morona, Renato

2016-10-09

Evidence is accumulating that protein tyrosine phosphorylation plays a crucial role in the ability of important human bacterial pathogens to cause disease. While most works have concentrated on its role in the regulation of a major bacterial virulence factor, the polysaccharide capsule, recent studies have suggested a much broader role for this post-translational modification. This prompted us to investigate protein tyrosine phosphorylation in the human pathogen Shigella flexneri. We first completed a tyrosine phosphoproteome, identifying 905 unique tyrosine phosphorylation sites on at least 573 proteins (approximately 15% of all proteins). This is the most tyrosine-phosphorylated sites and proteins in a single bacterium identified to date, substantially more than the level seen in eukaryotic cells. Most had not previously been identified and included proteins encoded by the virulence plasmid, which is essential for S. flexneri to invade cells and cause disease. In order to investigate the function of these phosphorylation sites in important virulence factors, phosphomimetic and ablative mutations were constructed in the type 3 secretion system ATPase Spa47 and the master virulence regulator VirB. This revealed that tyrosine residues phosphorylated in our study are critical for Spa47 and VirB activity, and tyrosine phosphorylation likely regulates their functional activity and subsequently the virulence of this major human pathogen. This study suggests that tyrosine phosphorylation plays a critical role in regulating a wide variety of virulence factors in the human pathogen S. flexneri and serves as a base for future studies defining its complete role. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation.

PubMed

Baker, Mark A; Hetherington, Louise; Ecroyd, Heath; Roman, Shaun D; Aitken, R John

2004-01-15

The capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type. Controversy surrounds the impact of extracellular calcium on this process, with positive and negative effects being recorded in independent publications. We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa. Under these conditions, a rise in intracellular pH was recorded, however, this event was not responsible for the observed changes in phosphotyrosine expression. Rather, the impact of calcium on tyrosine phosphorylation in these cells was associated with an unexpected change in the intracellular availability of ATP. Thus, the ATP content of both human and mouse spermatozoa fell significantly when these cells were incubated in the presence of external calcium. Furthermore, the removal of glucose, or addition of 2-deoxyglucose, decreased ATP levels within human spermatozoon populations and induced a corresponding decline in phosphotyrosine expression. In contrast, the mitochondrial inhibitor rotenone had no effect on either ATP levels or tyrosine phosphorylation. Addition of the affinity-labeling probe 8-N3 ATP confirmed our prediction that spermatozoa have many calcium-dependent ATPases. Moreover, addition of the ATPase inhibitor thapsigargin, increased intracellular calcium levels, decreased ATP and suppressed tyrosine phosphorylation. Based on these findings, the present study indicates that extracellular calcium suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP, and not by activating tyrosine phosphatases or inhibiting tyrosine kinases as has been previously suggested.

Neuromedin B receptor activation causes tyrosine phosphorylation of p125FAK by a phospholipase C independent mechanism which requires p21rho and integrity of the actin cytoskeleton.

PubMed

Tsuda, T; Kusui, T; Jensen, R T

1997-12-23

Recent studies show that tyrosine phosphorylation by a number of neuropeptides may be an important intracellular pathway in mediating changes in cell function, particularly related to growth. Neuromedin B (NMB), a mammalian bombesin related peptide, functions through a distinct receptor, the neuromedin B receptor (NMB-R), of which little is known about its cellular basis of action. In the present study we explored the ability of NMB-R activation to cause tyrosine phosphorylation of focal adhesion kinase (p125(FAK)), an important substrate for tyrosine phosphorylation by other neuropeptides. NMB caused rapid increases in p125(FAK) phosphorylation which reached maximum at 2 min in both rat C6 glioblastoma cells which possess native NMB-Rs and rat neuromedin B receptor (rNMR-R) transfected BALB 3T3 cells. NMB had a half-maximal effect was at 0.4 nM and was 30-fold more potent than gastrin-releasing peptide (GRP). The stoichiometric relationships between increased p125(FAK) tyrosine phosphorylation and other cellular processes was similar in both C6 cells and rNMB-R transfected cells. TPA (1 microM) caused 45% and the calcium ionophore, A23187, 11% of maximal tyrosine phosphorylation of p125(FAK) seen with NMB. A23187 potentiated the effect of TPA. Pretreatment with the selective PKC inhibitor, GF109203X, inhibited TPA-induced p125(FAK) tyrosine phosphorylation, but it had no effect on the NMB stimulation. Pretreatment with thapsigargin completely inhibited NMB-stimulated increases in [Ca2+]i, but had no effect on NMB-stimulation of p125(FAK) phosphorylation either alone or with GF109203X. The tyrosine kinase inhibitor, tyrphostin A25, inhibited NMB-induced phosphorylation of p125(FAK) by 52%. However, tyrphostin A25 did not inhibit NMB-stimulated increases in [3H]inositol phosphates. Cytochalasin D, an agent which disrupts actin microfilaments, inhibited BN- and TPA-induced tyrosine phosphorylation of p125(FAK) completely. In contrast, colchicine, an agent which disrupts microtubules, had no effect. Pretreatment with Clostridium botulinum C3 exoenzyme which inactivates the small GTP-binding protein rho p21, also inhibited tyrosine phosphorylation of p125(FAK) by 55%. These results demonstrate that activation of NMB-R can cause rapid tyrosine phosphorylation of p125(FAK). NMB-induced tyrosine phosphorylation of p125(FAK) is independent of NMB-induced changes in [Ca2+]i or PKC. The integrity of the actin cytoskeleton but not of microtubules is necessary for NMB-stimulated phosphorylation of p125(FAK). The ras-related small GTP-binding protein rho p21 is at least partially involved in mediating NMB-induced tyrosine phosphorylation of p125(FAK). These results suggest that similar to some other neuropeptides, activation of this pathway may be an important mechanism in mediating cellular changes by this receptor such as growth.

Computational study of Gleevec and G6G reveals molecular determinants of kinase inhibitor selectivity

DOE PAGES

Lin, Yen -Lin; Meng, Yilin; Huang, Lei; ...

2014-10-22

Gleevec is a potent inhibitor of Abl tyrosine kinase but not of the highly homologous c-Src kinase. Because the ligand binds to an inactive form of the protein in which an Asp-Phe-Gly structural motif along the activation loop adopts a so-called DFG-out conformation, it was suggested that binding specificity was controlled by a “conformational selection” mechanism. In this context, the binding affinity displayed by the kinase inhibitor G6G poses an intriguing challenge. Although it possesses a chemical core very similar to that of Gleevec, G6G is a potent inhibitor of both Abl and c-Src kinases. Both inhibitors bind to themore » DFG-out conformation of the kinases, which seems to be in contradiction with the conformational selection mechanism. To address this issue and display the hidden thermodynamic contributions affecting the binding selectivity, molecular dynamics free energy simulations with explicit solvent molecules were carried out. Relative to Gleevec, G6G forms highly favorable van der Waals dispersive interactions upon binding to the kinases via its triazine functional group, which is considerably larger than the corresponding pyridine moiety in Gleevec. Upon binding of G6G to c-Src, these interactions offset the unfavorable free energy cost of the DFG-out conformation. When binding to Abl, however, G6G experiences an unfavorable free energy penalty due to steric clashes with the phosphate-binding loop, yielding an overall binding affinity that is similar to that of Gleevec. Such steric clashes are absent when G6G binds to c-Src, due to the extended conformation of the phosphate-binding loop.« less

Coincident regulation of PKCδ in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling

PubMed Central

Hall, Kellie J.; Jones, Matthew L.; Poole, Alastair W.

2007-01-01

PKC (protein kinase C)δ plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCδ. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCδ with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCδ is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCδ. Both thrombin and PMA induce recruitment of PKCδ to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCδ, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCδ is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity. PMID:17570831

Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis.

PubMed

Mahabeleshwar, Ganapati H; Feng, Weiyi; Reddy, Kumar; Plow, Edward F; Byzova, Tatiana V

2007-09-14

The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

Striatal-enriched Protein-tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity*

PubMed Central

Xu, Jian; Kurup, Pradeep; Bartos, Jason A.; Patriarchi, Tommaso; Hell, Johannes W.; Lombroso, Paul J.

2012-01-01

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr1472. Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr402. In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr402. STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr402 and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP. PMID:22544749

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

PubMed

Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

Insights into the Folding and Unfolding Processes of Wild-Type and Mutated SH3 Domain by Molecular Dynamics and Replica Exchange Molecular Dynamics Simulations

PubMed Central

Chu, Wen-Ting; Zhang, Ji-Long; Zheng, Qing-Chuan; Chen, Lin; Zhang, Hong-Xing

2013-01-01

Src-homology regions 3 (SH3) domain is essential for the down-regulation of tyrosine kinase activity. Mutation A39V/N53P/V55L of SH3 is found to be relative to the urgent misfolding diseases. To gain insight, the human and gallus SH3 domains (PDB ID: 1NYG and 2LP5), including 58 amino acids in each protein, were selected for MD simulations (Amber11, ff99SB force field) and cluster analysis to investigate the influence of mutations on the spatial structure of the SH3 domain. It is found that the large conformational change of mutations mainly exists in three areas in the vicinity of protein core: RT loop, N-src loop, distal β-hairpin to 310 helix. The C-terminus of the mutated gallus SH3 is disordered after simulation, which represents the intermediate state of aggregation. The disappeared strong Hbond net in the mutated human and gallus systems will make these mutated proteins looser than the wild-type proteins. Additionally, by performing the REMD simulations on the gallus SH3 domain, the mutated domain is found to have an obvious effect on the unfolding process. These studies will be helpful for further aggregation mechanisms investigations on SH3 family. PMID:23734224

Insights into the folding and unfolding processes of wild-type and mutated SH3 domain by molecular dynamics and replica exchange molecular dynamics simulations.

PubMed

Chu, Wen-Ting; Zhang, Ji-Long; Zheng, Qing-Chuan; Chen, Lin; Zhang, Hong-Xing

2013-01-01

Src-homology regions 3 (SH3) domain is essential for the down-regulation of tyrosine kinase activity. Mutation A39V/N53P/V55L of SH3 is found to be relative to the urgent misfolding diseases. To gain insight, the human and gallus SH3 domains (PDB ID: 1NYG and 2LP5), including 58 amino acids in each protein, were selected for MD simulations (Amber11, ff99SB force field) and cluster analysis to investigate the influence of mutations on the spatial structure of the SH3 domain. It is found that the large conformational change of mutations mainly exists in three areas in the vicinity of protein core: RT loop, N-src loop, distal β-hairpin to 310 helix. The C-terminus of the mutated gallus SH3 is disordered after simulation, which represents the intermediate state of aggregation. The disappeared strong Hbond net in the mutated human and gallus systems will make these mutated proteins looser than the wild-type proteins. Additionally, by performing the REMD simulations on the gallus SH3 domain, the mutated domain is found to have an obvious effect on the unfolding process. These studies will be helpful for further aggregation mechanisms investigations on SH3 family.

mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR.

PubMed

Yin, Yancun; Hua, Hui; Li, Minjing; Liu, Shu; Kong, Qingbin; Shao, Ting; Wang, Jiao; Luo, Yuanming; Wang, Qian; Luo, Ting; Jiang, Yangfu

2016-01-01

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor(+/+) MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor(-/-) MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.

mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR

PubMed Central

Yin, Yancun; Hua, Hui; Li, Minjing; Liu, Shu; Kong, Qingbin; Shao, Ting; Wang, Jiao; Luo, Yuanming; Wang, Qian; Luo, Ting; Jiang, Yangfu

2016-01-01

Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor+/+ MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor−/− MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation. PMID:26584640

Exploring the mechanistic insights of Cas scaffolding protein family member 4 with protein tyrosine kinase 2 in Alzheimer's disease by evaluating protein interactions through molecular docking and dynamic simulations.

PubMed

Hassan, Mubashir; Shahzadi, Saba; Alashwal, Hany; Zaki, Nazar; Seo, Sung-Yum; Moustafa, Ahmed A

2018-05-22

Cas scaffolding protein family member 4 and protein tyrosine kinase 2 are signaling proteins, which are involved in neuritic plaques burden, neurofibrillary tangles, and disruption of synaptic connections in Alzheimer's disease. In the current study, a computational approach was employed to explore the active binding sites of Cas scaffolding protein family member 4 and protein tyrosine kinase 2 proteins and their significant role in the activation of downstream signaling pathways. Sequential and structural analyses were performed on Cas scaffolding protein family member 4 and protein tyrosine kinase 2 to identify their core active binding sites. Molecular docking servers were used to predict the common interacting residues in both Cas scaffolding protein family member 4 and protein tyrosine kinase 2 and their involvement in Alzheimer's disease-mediated pathways. Furthermore, the results from molecular dynamic simulation experiment show the stability of targeted proteins. In addition, the generated root mean square deviations and fluctuations, solvent-accessible surface area, and gyration graphs also depict their backbone stability and compactness, respectively. A better understanding of CAS and their interconnected protein signaling cascade may help provide a treatment for Alzheimer's disease. Further, Cas scaffolding protein family member 4 could be used as a novel target for the treatment of Alzheimer's disease by inhibiting the protein tyrosine kinase 2 pathway.

INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

EPA Science Inventory

Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

MECHANISM OF PROTEIN TYROSINE PHOSPHATASE INHIBITION IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

EPA Science Inventory

A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to Zn2+ inhibits protein tyrosine phosphatase (PTP) activity and leads to activation of epidermal growth factor receptor (EGFR) signaling in ...

Crystal structure of SP-PTP, a low molecular weight protein tyrosine phosphatase from Streptococcus pyogenes.

PubMed

Ku, Bonsu; Keum, Chae Won; Lee, Hye Seon; Yun, Hye-Yeoung; Shin, Ho-Chul; Kim, Bo Yeon; Kim, Seung Jun

2016-09-23

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a pathogenic bacterium that causes a variety of infectious diseases. The GAS genome encodes one protein tyrosine phosphatase, SP-PTP, which plays an essential role in the replication and virulence maintenance of GAS. Herein, we present the crystal structure of SP-PTP at 1.9 Å resolution. Although SP-PTP has been reported to have dual phosphatase specificity for both phosphorylated tyrosine and serine/threonine, three-dimensional structural analysis showed that SP-PTP shares high similarity with typical low molecular weight protein tyrosine phosphatases (LMWPTPs), which are specific for phosphotyrosine, but not with dual-specificity phosphatases, in overall folding and active site composition. In the dephosphorylation activity test, SP-PTP consistently acted on phosphotyrosine substrates, but not or only minimally on phosphoserine/phosphothreonine substrates. Collectively, our structural and biochemical analyses verified SP-PTP as a canonical tyrosine-specific LMWPTP. Copyright © 2016 Elsevier Inc. All rights reserved.

Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

PubMed

Toubiana, Julie; Rossi, Anne-Lise; Belaidouni, Nadia; Grimaldi, David; Pene, Frederic; Chafey, Philippe; Comba, Béatrice; Camoin, Luc; Bismuth, Georges; Claessens, Yann-Erick; Mira, Jean-Paul; Chiche, Jean-Daniel

2015-10-01

TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells. © The Author(s) 2015.

Disruption of striatal-enriched protein tyrosine phosphatase (STEP) function in neuropsychiatric disorders

PubMed Central

Karasawa, Takatoshi; Lombroso, Paul J.

2014-01-01

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific tyrosine phosphatase that plays a major role in the development of synaptic plasticity. Recent findings have implicated STEP in several psychiatric and neurological disorders, including Alzheimer’s disease, schizophrenia, fragile X syndrome, Huntington’s disease, stroke/ischemia, and stress-related psychiatric disorders. In these disorders, STEP protein expression levels and activity are dysregulated, contributing to the cognitive deficits that are present. In this review, we focus on the most recent findings on STEP, discuss how STEP expression and activity are maintained during normal cognitive function, and how disruptions in STEP activity contribute to a number of illnesses. PMID:25218562

Sorafenib: targeting multiple tyrosine kinases in cancer.

PubMed

Hasskarl, Jens

2014-01-01

Sorafenib (BAY 43-9006, Nexavar®) is an oral multiple tyrosine kinase inhibitor. Main targets are receptor tyrosine kinase pathways frequently deregulated in cancer such as the Raf-Ras pathway, vascular endothelial growth factor (VEGF) pathway, and FMS-like tyrosine kinase 3 (FLT3). Sorafenib was approved by the FDA in fast track for advanced renal cell cancer and hepatocellular cancer and shows good clinical activity in thyroid cancer. Multiple clinical trials are undertaken to further investigate the role of sorafenib alone or in combination for the treatment of various tumor entities.

Tyrosine kinase inhibitors suppress prostaglandin F2alpha-induced phosphoinositide hydrolysis, Ca2+ elevation and contraction in iris sphincter smooth muscle.

PubMed

Yousufzai, S Y; Abdel-Latif, A A

1998-11-06

We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F2alpha- and carbachol-induced inositol-1,4,5-trisphosphate (IP3) production, [Ca2+]i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F2alpha and carbachol induced contraction in a concentration-dependent manner with EC50 values of 0.92 x 10(-9) and 1.75 x 10(-8) M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F2alpha, but not those evoked by carbachol, on IP3 accumulation, [Ca2+]i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F2alpha analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 microM) markedly reduced (by 67%) prostaglandin F2alpha-stimulated increase in [Ca2+]i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC50 of 82 microM and increased IP3 generation in a concentration-dependent manner with an EC50 of 90 microM. The effects of vanadate were abolished by genistein (10 microM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F2alpha- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca2+]i evoked by prostaglandin F2alpha. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F2alpha receptor activation and increases in intracellular Ca2+ mobilization and identifying the tyrosine-phosphorylated substrates will provide important information about the role of protein tyrosine kinase in the mechanism of smooth muscle contraction, as well as about the mechanism of the intraocular pressure lowering effect of the prostaglandin in glaucoma patients.

Tyrosine decarboxylase activity of enterococci grown in media with different nutritional potential: tyramine and 2-phenylethylamine accumulation and tyrDC gene expression.

PubMed

Bargossi, Eleonora; Tabanelli, Giulia; Montanari, Chiara; Lanciotti, Rosalba; Gatto, Veronica; Gardini, Fausto; Torriani, Sandra

2015-01-01

The ability to accumulate tyramine and 2-phenylethylamine by two strains of Enterococcus faecalis and two strains Enterococcus faecium was evaluated in two cultural media added or not with tyrosine. All the enterococcal strains possessed a tyrosine decarboxylase (tyrDC) which determined tyramine accumulation in all the conditions tested, independently on the addition of high concentration of free tyrosine. Enterococci differed in rate and level of biogenic amines accumulation. E. faecalis EF37 and E. faecium FC12 produced tyramine in high amount since the exponential growth phase, while 2-phenylethylamine was accumulated when tyrosine was depleted. E. faecium FC12 and E. faecalis ATCC 29212 showed a slower tyraminogenic activity which took place mainly in the stationary phase up to 72 h of incubation. Moreover, E. faecalis ATCC 29212 produced 2-phenylethylamine only in the media without tyrosine added. In BHI added or not with tyrosine the tyrDC gene expression level differed considerably depending on the strains and the growth phase. In particular, the tyrDC gene expression was high during the exponential phase in rich medium for all the strains and subsequently decreased except for E. faecium FC12. Even if tyrDC presence is common among enterococci, this study underlines the extremely variable decarboxylating potential of strains belonging to the same species, suggesting strain-dependent implications in food safety.

Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

PubMed

Swetha, Medchalmi; Ramaiah, Kolluru V A

2015-11-01

Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Microvillus-Specific Protein Tyrosine Phosphatase SAP-1 Plays a Role in Regulating the Intestinal Paracellular Transport of Macromolecules.

PubMed

Mori, Shingo; Kamei, Noriyasu; Murata, Yoji; Takayama, Kozo; Matozaki, Takashi; Takeda-Morishita, Mariko

2017-09-01

The stomach cancer-associated protein tyrosine phosphatase 1 (SAP-1) is a receptor-type protein tyrosine phosphatase that is specifically expressed on the apical membrane of the intestinal epithelium. SAP-1 is known to maintain the balance of phosphorylation of proteins together with protein kinases; however, its biological function and impact on pharmacokinetics in the intestine remain unclear. The present study, therefore, aimed at clarifying the relationship between SAP-1 and the intestinal absorption behaviors of typical transporter substrates and macromolecules. The endogenous levels of glucose and total cholesterol in the blood were similar between wild-type and SAP-1-deficient mice (Sap1 -/- ), suggesting no contribution of SAP-1 to biogenic influx. Moreover, in vitro transport study with everted ileal sacs demonstrated that there was no difference in the absorption of breast cancer resistance protein, P-glycoprotein, and peptide transporter substrates between both mice. However, absorptive clearance of macromolecular model dextrans (FD-4 and FD-10) in Sap1 -/- mice was significantly higher than that in wild-type mice, and this was confirmed by the trend of increased FD-4 absorption from colonic loops of Sap1 -/- mice. Therefore, the results of this study suggest the partial contribution of SAP-1 to the regulated transport of hydrophilic macromolecules through paracellular tight junctions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

EPA Science Inventory

Prolactin-Induced Tyrosine Phosphorylation, Activation and Receptor
Association of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells.
Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental Protection
Agency, MD-72, Research Triangle Park, NC 27711, and

A critical tyrosine residue determines the uncoupling protein-like activity of the yeast mitochondrial oxaloacetate carrier.

PubMed

Luévano-Martínez, Luis A; Barba-Ostria, Carlos; Araiza-Olivera, Daniela; Chiquete-Félix, Natalia; Guerrero-Castillo, Sergio; Rial, Eduardo; Georgellis, Dimitris; Uribe-Carvajal, Salvador

2012-04-01

The mitochondrial Oac (oxaloacetate carrier) found in some fungi and plants catalyses the uptake of oxaloacetate, malonate and sulfate. Despite their sequence similarity, transport specificity varies considerably between Oacs. Indeed, whereas ScOac (Saccharomyces cerevisiae Oac) is a specific anion-proton symporter, the YlOac (Yarrowia lipolytica Oac) has the added ability to transport protons, behaving as a UCP (uncoupling protein). Significantly, we identified two amino acid changes at the matrix gate of YlOac and ScOac, tyrosine to phenylalanine and methionine to leucine. We studied the role of these amino acids by expressing both wild-type and specifically mutated Oacs in an Oac-null S. cerevisiae strain. No phenotype could be associated with the methionine to leucine substitution, whereas UCP-like activity was dependent on the presence of the tyrosine residue normally expressed in the YlOac, i.e. Tyr-ScOac mediated proton transport, whereas Phe-YlOac lost its protonophoric activity. These findings indicate that the UCP-like activity of YlOac is determined by the tyrosine residue at position 146.

Tyrosine kinase activity of EphA2 promotes its S897 phosphorylation and glioblastoma cell proliferation.

PubMed

Hamaoka, Yuho; Negishi, Manabu; Katoh, Hironori

2018-05-23

EphA2, a member of the Eph family of receptor tyrosine kinases, has been reported to promote tumor malignancy through phosphorylation of serine 897 (S897). Here, we found that overexpression of wild-type EphA2 induced S897 phosphorylation through ERK activation without growth factors or cytokines and promoted glioblastoma cell proliferation. However, overexpression of a kinase-inactive mutant of EphA2 failed to induce ERK activation, S897 phosphorylation, and promotion of glioblastoma cell proliferation. These data suggest that when overexpressed, EphA2 induces ERK activation through its tyrosine kinase activity, leading to S897 phosphorylation and promotion of glioblastoma cell proliferation. Our findings provide a new insight into how EphA2 mediates glioblastoma progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling.

PubMed

Araki, Tsuyoshi; Kawata, Takefumi; Williams, Jeffrey G

2012-07-10

SH2 domains are integral to many animal signaling pathways. By interacting with specific phosphotyrosine residues, they provide regulatable protein-protein interaction domains. Dictyostelium is the only nonmetazoan with functionally characterized SH2 domains, but the cognate tyrosine kinases are unknown. There are no orthologs of the animal tyrosine kinases, but there are very many tyrosine kinase-like kinases (TKLs), a group of kinases which, despite their family name, are classified mainly as serine-threonine kinases. STATs are transcription factors that dimerize via phosphotyrosine-SH2 domain interactions. STATc is activated by phosphorylation on Tyr922 when cells are exposed to the prestalk inducer differentiation inducing factor (DIF-1), a chlorinated hexaphenone. We show that in a null mutant for Pyk2, a tyrosine-specific TKL, exposure to DIF-1 does not activate STATc. Conversely, overexpression of Pyk2 causes constitutive STATc activation. Pyk2 phosphorylates STATc on Tyr922 in vitro and complexes with STATc both in vitro and in vivo. This demonstration that a TKL directly activates a STAT has significant implications for understanding the evolutionary origins of SH2 domain-phosphotyrosine signaling. It also has mechanistic implications. Our previous work suggested that a predicted constitutive STATc tyrosine kinase activity is counterbalanced in vivo by the DIF-1-regulated activity of PTP3, a Tyr922 phosphatase. Here we show that the STATc-Pyk2 complex is formed constitutively by an interaction between the STATc SH2 domain and phosphotyrosine residues on Pyk2 that are generated by autophosphorylation. Also, as predicted, Pyk2 is constitutively active as a STATc kinase. This observation provides further evidence for this highly atypical, possibly ancestral, STAT regulation mechanism.

Tyrosine kinase Btk regulates E-selectin-mediated integrin activation and neutrophil recruitment by controlling phospholipase C (PLC) gamma2 and PI3Kgamma pathways.

PubMed

Mueller, Helena; Stadtmann, Anika; Van Aken, Hugo; Hirsch, Emilio; Wang, Demin; Ley, Klaus; Zarbock, Alexander

2010-04-15

Selectins mediate leukocyte rolling, trigger beta(2)-integrin activation, and promote leukocyte recruitment into inflamed tissue. E-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) leads to activation of an immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway, which in turn activates the spleen tyrosine kinase (Syk). However, the signaling pathway linking Syk to integrin activation after E-selectin engagement is unknown. To identify the pathway, we used different gene-deficient mice in autoperfused flow chamber, intravital microscopy, peritonitis, and biochemical studies. We report here that the signaling pathway downstream of Syk divides into a phospholipase C (PLC) gamma2- and phosphoinositide 3-kinase (PI3K) gamma-dependent pathway. The Tec family kinase Bruton tyrosine kinase (Btk) is required for activating both pathways, generating inositol-3,4,5-trisphosphate (IP(3)), and inducing E-selectin-mediated slow rolling. Inhibition of this signal-transduction pathway diminished Galpha(i)-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster. Galpha(i)-independent neutrophil recruitment into the inflamed peritoneal cavity was reduced in Btk(-/-) and Plcg2(-/-) mice. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by E-selectin engagement.

SH2 domains: modulators of nonreceptor tyrosine kinase activity.

PubMed

Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

2009-12-01

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed that the presence of the SH2 domain is frequently required for catalytic activity, suggesting a crucial function stabilizing the active state of many nonreceptor tyrosine kinases. Recently, the structure of the SH2-kinase domain of Fes revealed that the SH2 domain stabilizes the active kinase conformation by direct interactions with the regulatory helix alphaC. Stabilizing interactions between the SH2 and the kinase domains have also been observed in the structures of active Csk and Abl. Interestingly, mutations in the SH2 domain found in human disease can be explained by SH2 domain destabilization or incorrect positioning of the SH2. Here we summarize our understanding of mechanisms that lead to tyrosine kinase activation by direct interactions mediated by the SH2 domain and discuss how mutations in the SH2 domain trigger kinase inactivation.

Exploring protein structure and dynamics through a project-oriented biochemistry laboratory module.

PubMed

Lipchock, James M; Ginther, Patrick S; Douglas, Bonnie B; Bird, Kelly E; Patrick Loria, J

2017-09-01

Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant human enzyme, protein tyrosine phosphatase 1B (PTP1B). Over the course of the semester students guide their own mutant of PTP1B from conception to characterization in a cost-effective manner and gain exposure to fundamental techniques in biochemistry, including site-directed DNA mutagenesis, bacterial recombinant protein expression, affinity column purification, protein quantitation, SDS-PAGE, and enzyme kinetics. This project-based approach allows an instructor to simulate a research setting and prepare students for productive research beyond the classroom. Potential modifications to expand or contract this module are also provided. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):403-410, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Functional and Structural Impact of Target Uridine Substitutions on the H/ACA Ribonucleoprotein Particle Pseudouridine Synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Jing; Liang, Bo; Li, Hong

2010-09-17

Box H/ACA ribonucleoprotein protein particles catalyze the majority of pseudouridylation in functional RNA. Different from stand alone pseudouridine synthases, the RNP pseudouridine synthase comprises multiple protein subunits and an RNA subunit. Previous studies showed that each subunit, regardless its location, is sensitive to the step of subunit placement at the catalytic center and potentially to the reaction status of the substrate. Here we describe the impact of chemical substitutions of target uridine on enzyme activity and structure. We found that 3-methyluridine in place of uridine inhibited its isomerization while 2{prime}-deoxyuridine or 4-thiouridine did not. Significantly, crystal structures of an archaealmore » box H/ACA RNP bound with the nonreactive and the two postreactive substrate analogues showed only subtle structural changes throughout the assembly except for a conserved tyrosine and a substrate anchoring loop of Cbf5. Our results suggest a potential role of these elements and the subunit that contacts them in substrate binding and product release.« less

Complementary Phosphorylation Sites in the Adaptor Protein SLP-76 Promote Synergistic Activation of Natural Killer Cells

PubMed Central

Kim, Hun Sik; Long, Eric O.

2013-01-01

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require the induction of synergistic signals from co-activation receptors, such as CD314 (NKG2D) and CD244 (2B4), which bind to ligands expressed on target cells. Synergy is required to overcome inhibition of the guanine nucleotide exchange factor (GEF) Vav1, a central regulator of NK cell activation, by the E3 ubiquitin ligase c-Cbl. However, the molecular basis for this synergy is unknown. Here, we showed that the adaptor protein Src homology 2 (SH2) domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) was required for this synergy, and that distinct tyrosine residues in SLP-76 were phosphorylated by each receptor of a synergistic pair. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76, each of which enables binding of SLP-76 to Vav1, was unique to receptors that stimulate ligand-dependent target cell killing, because antibody-dependent stimulation by Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that complementation of separate phospho-tyrosine targets in SLP-76 forms the basis of synergistic NK cell activation. PMID:22786724

Complementary phosphorylation sites in the adaptor protein SLP-76 promote synergistic activation of natural killer cells.

PubMed

Kim, Hun Sik; Long, Eric O

2012-07-10

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require synergistic signals from specific pairs of co-activation receptors, such as CD314 (also known as NKG2D) and CD244 (2B4), which bind to distinct ligands present on target cells. These signals are required to overcome inhibition mediated by the E3 ubiquitin ligase c-Cbl of the guanine nucleotide exchange factor Vav1, which promotes activation of NK cells. Here, we showed that the adaptor protein SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons) was required for this synergy and that distinct tyrosine residues in SLP-76 were phosphorylated by each member of a pair of synergistic receptors. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76 enabled binding of SLP-76 to Vav1. Selective phosphorylation of SLP-76 at these residues was restricted to receptors that stimulated ligand-dependent target cell killing; antibody-dependent stimulation of the Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 mutant proteins showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that combined phosphorylation of separate tyrosine residues in SLP-76 forms the basis of synergistic NK cell activation.

PTP-PEST targets a novel tyrosine site in p120 catenin to control epithelial cell motility and Rho GTPase activity.

PubMed

Espejo, Rosario; Jeng, Yowjiun; Paulucci-Holthauzen, Adriana; Rengifo-Cam, William; Honkus, Krysta; Anastasiadis, Panos Z; Sastry, Sarita K

2014-02-01

Tyrosine phosphorylation is implicated in regulating the adherens junction protein, p120 catenin (p120), however, the mechanisms are not well defined. Here, we show, using substrate trapping, that p120 is a direct target of the protein tyrosine phosphatase, PTP-PEST, in epithelial cells. Stable shRNA knockdown of PTP-PEST in colon carcinoma cells results in an increased cytosolic pool of p120 concomitant with its enhanced tyrosine phosphorylation and decreased association with E-cadherin. Consistent with this, PTP-PEST knockdown cells exhibit increased motility, enhanced Rac1 and decreased RhoA activity on a collagen substrate. Furthermore, p120 localization is enhanced at actin-rich protrusions and lamellipodia and has an increased association with the guanine nucleotide exchange factor, VAV2, and cortactin. Exchange factor activity of VAV2 is enhanced by PTP-PEST knockdown whereas overexpression of a VAV2 C-terminal domain or DH domain mutant blocks cell motility. Analysis of point mutations identified tyrosine 335 in the N-terminal domain of p120 as the site of PTP-PEST dephosphorylation. A Y335F mutant of p120 failed to induce the 'p120 phenotype', interact with VAV2, stimulate cell motility or activate Rac1. Together, these data suggest that PTP-PEST affects epithelial cell motility by controlling the distribution and phosphorylation of p120 and its availability to control Rho GTPase activity.

PTP-PEST targets a novel tyrosine site in p120 catenin to control epithelial cell motility and Rho GTPase activity

PubMed Central

Espejo, Rosario; Jeng, Yowjiun; Paulucci-Holthauzen, Adriana; Rengifo-Cam, William; Honkus, Krysta; Anastasiadis, Panos Z.; Sastry, Sarita K.

2014-01-01

ABSTRACT Tyrosine phosphorylation is implicated in regulating the adherens junction protein, p120 catenin (p120), however, the mechanisms are not well defined. Here, we show, using substrate trapping, that p120 is a direct target of the protein tyrosine phosphatase, PTP-PEST, in epithelial cells. Stable shRNA knockdown of PTP-PEST in colon carcinoma cells results in an increased cytosolic pool of p120 concomitant with its enhanced tyrosine phosphorylation and decreased association with E-cadherin. Consistent with this, PTP-PEST knockdown cells exhibit increased motility, enhanced Rac1 and decreased RhoA activity on a collagen substrate. Furthermore, p120 localization is enhanced at actin-rich protrusions and lamellipodia and has an increased association with the guanine nucleotide exchange factor, VAV2, and cortactin. Exchange factor activity of VAV2 is enhanced by PTP-PEST knockdown whereas overexpression of a VAV2 C-terminal domain or DH domain mutant blocks cell motility. Analysis of point mutations identified tyrosine 335 in the N-terminal domain of p120 as the site of PTP-PEST dephosphorylation. A Y335F mutant of p120 failed to induce the ‘p120 phenotype’, interact with VAV2, stimulate cell motility or activate Rac1. Together, these data suggest that PTP-PEST affects epithelial cell motility by controlling the distribution and phosphorylation of p120 and its availability to control Rho GTPase activity. PMID:24284071

Excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in experimental approaches of Parkinsonism.

PubMed

Fernández-Espejo, Emilio; Bis-Humbert, Cristian

2018-06-06

3-iodo-l-tyrosine might play a role in Parkinson's disease since this molecule is able, at high concentration, to inhibit tyrosine-hydroxylase activity, the rate-limiting enzyme in dopamine biosynthesis. The possible Parkinson-like effects of 3-iodo-l-tyrosine were tested on three experimental approaches in mice: cultured substantia nigra neurons, the enteric nervous system of the jejunum after intra-peritoneal infusions, and the nigrostriatal system following unilateral intrabrain injections. 3-iodo-l-tyrosine, a physiological molecule, was used at concentrations higher than its serum levels in humans. Parkinson-like signs were evaluated through abnormal aggregation of α-synuclein and tyrosine-hydroxylase, loss of tyrosine-hydroxylase-expressing and striatum-projecting neurons and fibers, reduced tyrosine-hydroxylase density, and Parkinson-like motor and non-motor deficits. The retrograde tracer FluoroGold was used in the brain model. The findings revealed that excess amounts of 3-iodo-l-tyrosine induce Parkinson-like effects in the three experimental approaches. Thus, culture neurons of substantia nigra show, after 3-iodo-l-tyrosine exposure, intracytoplasmic inclusions that express α-synuclein and tyrosine-hydroxylase. Intra-peritoneal infusions of 3-iodo-l-tyrosine cause, in the long-term, α-synuclein aggregation, thicker α-synuclein-positive fibers, and loss of tyrosine-hydroxylase-positive cells and fibers in intramural plexuses and ganglia of the jejunum. Infusion of 3-iodo-l-tyrosine into the left dorsal striata of mice damages the nigrostriatal system, as revealed through lower striatal tyrosine-hydroxylase density, reduced number of tyrosine-hydroxylase-expressing and striatum-projecting neurons in the left substantia nigra, as well as the emergence of Parkinson-like behavioral deficits such as akinesia, bradykinesia, motor disbalance, and locomotion directional bias. In conclusion, excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in cellular, enteric and brain approaches of Parkinsonism in mice. Copyright © 2018 Elsevier B.V. All rights reserved.

Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells.

PubMed

Hermans, Mirjam H A; van de Geijn, Gert-Jan; Antonissen, Claudia; Gits, Judith; van Leeuwen, Daphne; Ward, Alister C; Touw, Ivo P

2003-04-01

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Tyr704, Tyr729, Tyr744, Tyr764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation, and cell survival. However, it is unclear whether these tyrosines are equally important under more physiologic conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated G-CSF-R-deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (m0), single tyrosine "add-back" mutants, or wild-type (WT) receptors. G-CSF-induced responses were determined in primary colony assays, serial replatings, and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Tyr764 strongly enhanced proliferative responses, which was reverted by inhibition of ERK activity. Tyr729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing m0 gradually dropped compared with WT. The presence of Tyr729, but also Tyr704 and Tyr744, both involved in activation of signal transducer and activator of transcription 3 (STAT3), further reduced replating efficiencies. Conversely, Tyr764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a more than 10(4)-fold increase of colony-forming cells over m0 after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.

Photo-oxidation of tyrosine in a bio-engineered bacterioferritin 'reaction centre'-a protein model for artificial photosynthesis.

PubMed

Hingorani, Kastoori; Pace, Ron; Whitney, Spencer; Murray, James W; Smith, Paul; Cheah, Mun Hon; Wydrzynski, Tom; Hillier, Warwick

2014-10-01

The photosynthetic reaction centre (RC) is central to the conversion of solar energy into chemical energy and is a model for bio-mimetic engineering approaches to this end. We describe bio-engineering of a Photosystem II (PSII) RC inspired peptide model, building on our earlier studies. A non-photosynthetic haem containing bacterioferritin (BFR) from Escherichia coli that expresses as a homodimer was used as a protein scaffold, incorporating redox-active cofactors mimicking those of PSII. Desirable properties include: a di-nuclear metal binding site which provides ligands for bivalent metals, a hydrophobic pocket at the dimer interface which can bind a photosensitive porphyrin and presence of tyrosine residues proximal to the bound cofactors, which can be utilised as efficient electron-tunnelling intermediates. Light-induced electron transfer from proximal tyrosine residues to the photo-oxidised ZnCe6(•+), in the modified BFR reconstituted with both ZnCe6 and Mn(II), is presented. Three site-specific tyrosine variants (Y25F, Y58F and Y45F) were made to localise the redox-active tyrosine in the engineered system. The results indicate that: presence of bound Mn(II) is necessary to observe tyrosine oxidation in all BFR variants; Y45 the most important tyrosine as an immediate electron donor to the oxidised ZnCe6(•+) and that Y25 and Y58 are both redox-active in this system, but appear to function interchangebaly. High-resolution (2.1Å) crystal structures of the tyrosine variants show that there are no mutation-induced effects on the overall 3-D structure of the protein. Small effects are observed in the Y45F variant. Here, the BFR-RC represents a protein model for artificial photosynthesis. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

EGFR-SGLT1 interaction does not respond to EGFR modulators, but inhibition of SGLT1 sensitizes prostate cancer cells to EGFR tyrosine kinase inhibitors.

PubMed

Ren, Jiangong; Bollu, Lakshmi R; Su, Fei; Gao, Guang; Xu, Lei; Huang, Wei-Chien; Hung, Mien-Chie; Weihua, Zhang

2013-09-01

Overexpression of epidermal growth factor receptor (EGFR) is associated with poor prognosis in malignant tumors. Sodium/glucose co-transporter 1 (SGLT1) is an active glucose transporter that is overexpressed in many cancers including prostate cancer. Previously, we found that EGFR interacts with and stabilizes SGLT1 in cancer cells. In this study, we determined the micro-domain of EGFR that is required for its interaction with SGLT1 and the effects of activation/inactivation of EGFR on EGFR-SGLT1 interaction, measured the expression of EGFR and SGLT1 in prostate cancer tissues, and tested the effect of inhibition of SGLT1 on the sensitivity of prostate cancer cells to EGFR tyrosine inhibitors. We found that the autophosphorylation region (978-1210 amino acids) of EGFR was required for its sufficient interaction with SGLT1 and that this interaction was independent of EGFR's tyrosine kinase activity. Most importantly, the EGFR-SGLT1 interaction does not respond to EGFR tyrosine kinase modulators (EGF and tyrosine kinase inhibitors). EGFR and SGLT1 co-localized in prostate cancer tissues, and inhibition of SGLT1 by a SGLT1 inhibitor (Phlorizin) sensitized prostate cancer cells to EGFR inhibitors (Gefitinib and Erlotinib). These data suggest that EGFR in cancer cells can exist as either a tyrosine kinase modulator responsive status or an irresponsive status. SGLT1 is a protein involved in EGFR's functions that are irresponsive to EGFR tyrosine kinase inhibitors and, therefore, the EGFR-SGLT1 interaction might be a novel target for prostate cancer therapy. © 2013 Wiley Periodicals, Inc. This article is a U.S. Government work and is in the public domain in the USA.

On the role of adenylate cyclase, tyrosine kinase, and tyrosine phosphatase in the response of nerve and glial cells to photodynamic impact

NASA Astrophysics Data System (ADS)

Kolosov, Mikhail S.; Bragin, D. E.; Dergacheva, Olga Y.; Vanzha, O.; Oparina, L.; Uzdensky, Anatoly B.

2004-08-01

The role of different intercellular signaling pathways involving adenylate cyclase (AC), receptor tyrosine kinase (RTK), tyrosine and serine/threonine protein phosphatases (PTP or PP, respectively) in the response of crayfish mechanoreceptor neuron (MRN) and surrounding glial cells to photodynamic effect of aluminum phthalocyanine Photosens have been studied. AC inhibition by MDL-12330A decreased neuron lifetime, whereas AC activation by forskolin increase it. Thus, increase in cAMP produced by activated AC protects SRN against photodynamic inactivation. Similarly, RTK inhibition by genistein decreased neuron lifetime, while inhibition of PTP or PP that remove phosphate groups from proteins, prolonged neuronal activity. AC inhibition reduced photoinduced damage of the plasma membrane, and, therefore, necrosis in neuronal and glial cells. RTK inhibition protected only neurons against PDT-induced membrane permeabilization while glial cells became lesser permeable under ortovanadate-mediated PTP inhibition. AC activation also prevented PDT-induced apoptosis in glial cells. PP inhibition enhanced apoptotic processes in photosensitized glial cells. Therefore, both intercellular signaling pathways involving AC and TRK are involved in the maintenance of neuronal activity, integrity of the neuronal and glial plasma membranes and in apoptotic processes in glia under photosensitization.

Ca(2+)-sensitive tyrosine kinase Pyk2/CAK beta-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy.

PubMed

Hirotani, Shinichi; Higuchi, Yoshiharu; Nishida, Kazuhiko; Nakayama, Hiroyuki; Yamaguchi, Osamu; Hikoso, Shungo; Takeda, Toshihiro; Kashiwase, Kazunori; Watanabe, Tetsuya; Asahi, Michio; Taniike, Masayuki; Tsujimoto, Ikuko; Matsumura, Yasushi; Sasaki, Terukatsu; Hori, Masatsugu; Otsu, Kinya

2004-06-01

G-protein-coupled receptor agonists including endothelin-1 (ET-1) and phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Others and we previously reported that Rac1 signaling pathway plays an important role in this agonist-induced cardiomyocyte hypertrophy. In this study reported here, we found that a Ca(2+)-sensitive non-receptor tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2)/cell adhesion kinase beta (CAKbeta), is involved in ET-1- and PE-induced cardiomyocyte hypertrophy medicated through Rac1 activation. ET-1, PE or the Ca(2+) inophore, ionomycin, stimulated a rapid increase in tyrosine phosphorylation of Pyk2. The tyrosine phosphorylation of Pyk2 was suppressed by the Ca(2+) chelator, BAPTA. ET-1- or PE-induced increases in [(3)H]-leucine incorporation and expression of atrial natriuretic factor and the enhancement of sarcomere organization. Infection of cardiomyocytes with an adenovirus expressing a mutant Pyk2 which lacked its kinase domain or its ability to bind to c-Src, eliminated ET-1- and PE-induced hypertrophic responses. Inhibition of Pyk2 activation also suppressed Rac1 activation and reactive oxygen species (ROS) production. These findings suggest that the signal transduction pathway leading to hypertrophy involves Ca(2+)-induced Pyk2 activation followed by Rac1-dependent ROS production.

Protein tyrosine phosphatase-1B regulates the tyrosine phosphorylation of the adapter Grb2-associated binder 1 (Gab1) in the retina

PubMed Central

2013-01-01

Background Gab1 (Grb2-associated binder 1) is a key coordinator that belongs to the insulin receptor substrate-1 like family of adaptor molecules and is tyrosine phosphorylated in response to various growth factors, cytokines, and numerous other molecules. Tyrosine phosphorylated Gab1 is able to recruit a number of signaling effectors including PI3K, SHP2 and PLC-γ. In this study, we characterized the localization and regulation of tyrosine phosphorylation of Gab1 in the retina. Results Our immuno localization studies suggest that Gab1 is expressed in rod photoreceptor inner segments. We found that hydrogen peroxide activates the tyrosine phosphorylation of Gab1 ex vivo and hydrogen peroxide has been shown to inhibit the protein tyrosine phosphatase PTP1B activity. We found a stable association between the D181A substrate trap mutant of PTP1B and Gab1. Our studies suggest that PTP1B interacts with Gab1 through Tyrosine 83 and this residue may be the major PTP1B target residue on Gab1. We also found that Gab1 undergoes a light-dependent tyrosine phosphorylation and PTP1B regulates the phosphorylation state of Gab1. Consistent with these observations, we found an enhanced Gab1 tyrosine phosphorylation in PTP1B deficient mice and also in retinas treated ex vivo with a PTP1B specific allosteric inhibitor. Conclusions Our laboratory has previously reported that retinas deficient of PTP1B are resistant to light damage compared to wild type mice. Since Gab1 is negatively regulated by PTP1B, a part of the retinal neuroprotective effect we have observed previously in PTP1B deficient mice could be contributed by Gab1 as well. In summary, our data suggest that PTP1B regulates the phosphorylation state of retinal Gab1 in vivo. PMID:23521888

Effects of methamphetamine exposure on anxiety-like behavior in the open field test, corticosterone, and hippocampal tyrosine hydroxylase in adolescent and adult mice.

PubMed

Struntz, Katelyn H; Siegel, Jessica A

2018-08-01

Methamphetamine (MA) is a psychomotor stimulant drug that can alter behavior, the stress response system, and the dopaminergic system. The effects of MA can be modulated by age, however relatively little research has examined the acute effects of MA in adolescents and how the effects compare to those found in adults. The hippocampal dopamine system is altered by MA exposure and can modulate anxiety-like behavior, but the effects of MA on the hippocampal dopamine system have not been well studied, especially in adolescent animals. In order to assess potential age differences in the effects of MA exposure, this research examined the effects of acute MA exposure on locomotor and anxiety-like behavior in the open field test, plasma corticosterone levels, and hippocampal total tyrosine hydroxylase and phosphorylated tyrosine hydroxylase levels in adolescent and adult male C57BL/6 J mice. Tyrosine hydroxylase is the rate limiting enzyme in the synthesis of dopamine and was used as a marker of the hippocampal dopaminergic system. Mice were exposed to saline or 4 mg/kg MA and locomotor and anxiety-like behavior were measured in the open field test. Serum and brains were collected immediately after testing and plasma corticosterone and hippocampal total tyrosine hydroxylase and phosphorylated tyrosine hydroxylase levels measured. MA-exposed mice showed increased locomotor activity and anxiety-like behavior in the open field test compared with saline controls, regardless of age. There was no effect of MA on plasma corticosterone levels or hippocampal total tyrosine hydroxylase or phosphorylated tyrosine hydroxylase levels in either adolescent or adult mice. These data suggest that acute MA exposure during adolescence and adulthood increases locomotor activity and anxiety-like behavior but does not alter plasma corticosterone levels or hippocampal total tyrosine hydroxylase or phosphorylated tyrosine hydroxylase levels, and that these effects are not modulated by age. Copyright © 2018 Elsevier B.V. All rights reserved.

Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein.

PubMed

Yan, C; Han, R

1997-01-01

Protein tyrosine kinase (PTK) appears to be involved in the activation of signaling during cell attachment to and spreading on extracellular matrix (ECM) in the metastatic cascade. To verify the assumption that PTK inhibitors might impair ECM signaling and prevent cancer metastasis, the highly metastatic B16-BL6 mouse melanoma cells were exposed to the PTK inhibitor genistein for 3 days. The ability of the cells to invade through reconstituted basement membrane (Matrigel) and to establish experimental pulmonary metastatic foci in C57BL/6 mice decreased after genistein exposure. The genistein-treated cells were also prevented from attaching to Matrigel and spread extremely poorly on the ECM substratum. Immunoblot analysis showed that tyrosine phosphorylation of a 125-kD protein in response to cell spreading on Matrigel was suppressed in the genistein-treated cells. Adhesion-induced protein tyrosine phosphorylation represents the earlier and specific event in the activation of ECM signaling, so this result implied ECM signaling was impaired in the treated cells. With immunofluorescence microscopy, the adhesion-induced tyrosine phosphorylated proteins were located at the pericytoplasms of well-spread cells, but not at the periphery of poorly spread genistein-treated cells. Therefore, this paper suggests that genistein might impair ECM signaling and subsequently prevent cancer cells from spreading well and invading or establishing metastasis through the suppression of adhesion-induced protein tyrosine phosphorylation. PTKs and adhesion-induced protein tyrosine phosphorylation might play a role in the control of invasion and metastasis.

Tyrosine-like condensed derivatives as tyrosinase inhibitors.

PubMed

Matos, Maria João; Santana, Lourdes; Uriarte, Eugenio; Serra, Silvia; Corda, Marcella; Fadda, Maria Benedetta; Era, Benedetta; Fais, Antonella

2012-05-01

We report the pharmacological evaluation of a new series of 3-aminocoumarins differently substituted with hydroxyl groups, which have been synthesized because they include in their structures the tyrosine fragment (tyrosine-like compounds), with the aim of discovering structural features necessary for tyrosinase inhibitory activity. The synthesized compounds 4 and 7-9 were evaluated in vitro as mushroom tyrosinase inhibitors. Two of the described compounds showed lower IC50 (concentration giving 50% inhibition of tyrosinase activity) than umbelliferone, used as a reference compound. Compound 7 (IC50=53µm) was the best tyrosinase inhibitor of this small series, having an IC50 value 10-fold lower than umbelliferone. Compound 7 (3-amino-7-hydroxycoumarin) had amino and hydroxyl groups precisely mimicking the same positions that both groups occupy on the tyrosine molecule. © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.

Structure-based design of nitrosoureas containing tyrosine derivatives as potential antimelanoma agents.

PubMed

Gadjeva, Vesselina

2002-04-01

Two new nitrosoureas (TNUs), containing tyrosine derivatives as carriers of nitrosourea cytotoxic group have been synthesised. The physicochemical properties such as half-life time (tau(0.5)), alkylating and carbamoylating activities were determined. The nitrosoureas showed a higher inhibiting effect on the DOPA-oxidase activity of mushroom tyrosinase than that of the antitumour drug N'-cyclohexyl-N-(2-chloroethyl)-N-nitrosourea (lomustine, CCNU). In vitro cytotoxic effects of newly synthesised tyrosine containing nitrosoureas have been studied and compared to those of CCNU. A higher cytotoxicity to B16 melanoma cells than to YAC-1 and to lymphocytes was demonstrated for the tyrosine containing nitrosoureas in comparison with CCNU. Based on the results presented, we accept that a new trend for synthesis of more selective and less toxic nitrosourea derivatives as potential antimelanomic drugs might be developed.

rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

PubMed

Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J

1994-04-08

We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

Tyrosine Phosphorylation Regulates Maturation of Receptor Tyrosine Kinases

PubMed Central

Schmidt-Arras, Dirk-E.; Böhmer, Annette; Markova, Boyka; Choudhary, Chunaram; Serve, Hubert; Böhmer, Frank-D.

2005-01-01

Constitutive activation of receptor tyrosine kinases (RTKs) is a frequent event in human cancer cells. Activating mutations in Fms-like tyrosine kinase 3 (FLT-3), notably, internal tandem duplications in the juxtamembrane domain (FLT-3 ITD), have been causally linked to acute myeloid leukemia. As we describe here, FLT-3 ITD exists predominantly in an immature, underglycosylated 130-kDa form, whereas wild-type FLT-3 is expressed predominantly as a mature, complex glycosylated 150-kDa molecule. Endogenous FLT-3 ITD, but little wild-type FLT-3, is detectable in the endoplasmic reticulum (ER) compartment. Conversely, cell surface expression of FLT-3 ITD is less efficient than that of wild-type FLT-3. Inhibition of FLT-3 ITD kinase by small molecules, inactivating point mutations, or coexpression with the protein-tyrosine phosphatases (PTPs) SHP-1, PTP1B, and PTP-PEST but not RPTPα promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants. PMID:15831474

Protein-tyrosine-phosphatase-mediated epidermal growth factor (EGF) receptor transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells.

PubMed Central

Graness, A; Hanke, S; Boehmer, F D; Presek, P; Liebmann, C

2000-01-01

Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway which can bypass EGFR. PMID:10749673

Affinity analysis and application of dipeptides derived from l-tyrosine in plasmid purification.

PubMed

Ferreira, Soraia; Carvalho, Josué; Valente, Joana F A; Corvo, Marta C; Cabrita, Eurico J; Sousa, Fani; Queiroz, João A; Cruz, Carla

2015-12-01

The developments in the use of plasmid DNA (pDNA) in gene therapy and vaccines have motivated the search and improvement of optimized purification processes. In this context, dipeptides l-tyrosine-l-tyrosine and l-tyrosine-l-arginine are synthetized to explore their application as affinity ligands for supercoiled (sc) plasmid DNA (pDNA) purification. The synthesis is based on the protection of N-Boc-l-tyrosine, followed by condensation with l-tyrosine or l-arginine methyl esters in the presence of dicyclohexylcarbodiimide (DCC), which after hydrolysis and acidification give the afforded dipeptides. The supports are then obtained by coupling l-tyrosine, l-tyrosine-l-tyrosine and l-tyrosine-l-arginine to epoxy-activated Sepharose and are characterized by high resolution magic angle spinning (HR-MAS) NMR and Fourier transform infrared spectroscopy (FTIR). Surface plasmon resonance (SPR) biosensor is used to establish the promising ligand to be used in the chromatographic experiments and ascertain experimental conditions. Sc isoform showed the highest affinity to the dipeptides, followed by linear (ln) pDNA, being the open circular (oc) the one that promoted the lowest affinity to l-tyrosine-l-arginine. Saturation transfer difference (STD)-NMR experiments show that the interaction is mainly hydrophobic with the majority of the 5'-mononucleotides, except for 5'-GMP with l-tyrosine-l-arginine Sepharose that is mainly electrostatic. The support l-tyrosine Sepharose used in chromatographic experiments promotes the separation of native pVAX1-LacZ and pcDNA3-FLAG-p53 samples (oc+sc) by decreasing the salt concentration. The results suggest that it is possible to purify different plasmids with the l-tyrosine Sepharose, with slight adjustments in the gradient conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Tyrosine content, influx and accumulation rate, and catecholamine biosynthesis measured in vivo, in the central nervous system and in peripheral organs of the young rat. Influence of neonatal hypo- and hyperthyroidism.

PubMed

Diarra, A; Lefauconnier, J M; Valens, M; Georges, P; Gripois, D

1989-10-01

The influence of neonatal hypo- and hyperthyroidism on different aspects of tyrosine metabolism in the hypothalamus, striatum, brainstem, adrenal glands, heart and brown adipose tissue (BAT) were studied in 14-day old rats. The synthesis rate of catecholamines (CA) was also determined in vivo after the injection of labelled tyrosine. Hypothyroidism increases tyrosinaemia and endogenous tyrosine concentration in the hypothalamus and BAT. Hyperthyroidism decreases tyrosinaemia and endogenous tyrosine levels in the striatum, adrenals and heart. The accumulation rate of tyrosine determined 30 min after an intravenous injection of the labelled amino acid has been determined in the organs, together with the influx of the amino acid, determined within 20s. Hypothyroidism increases tyrosine accumulation rate in all the organs studied, and tyrosine clearance is decreased in the striatum and brainstem; together with an increased tyrosinaemia, this leads to a normal influx. The influx of tyrosine is increased in the hypothalamus. Hyperthyroidism decreases tyrosine accumulation rate in all the organs except the adrenals. These results indicate that the thyroid status of the young rat can influence tyrosine uptake mechanisms, without modifying an organ's tyrosine content. The fact that hypothyroidism increases tyrosine influx in the hypothalamus without modifying it in the brainstem and striatum reflects an heterogeneous reactivity to the lack of thyroid hormones in different brain structures. Neonatal hypothyroidism decreases the CA synthesis rate in the striatum, the heart and the interscapular brown adipose tissue, while synthesis was enhanced in the brainstem and the adrenals. It is likely that these variations in CA synthesis are due to thyroid hormone modulation of tyrosine hydroxylase activity, the enzyme which catalyses the rate limiting step in CA biosynthesis.

Platelet-derived growth factor-dependent association of the GTPase-activating protein of Ras and Src.

PubMed Central

Schlesinger, T K; Demali, K A; Johnson, G L; Kazlauskas, A

1999-01-01

Here we report that the platelet-derived growth factor beta receptor (betaPDGFR) is not the only tyrosine kinase able to associate with the GTPase-activating protein of Ras (RasGAP). The interaction of non-betaPDGFR kinase(s) with RasGAP was dependent on stimulation with platelet-derived growth factor (PDGF) and seemed to require tyrosine phosphorylation of RasGAP. Because the tyrosine phosphorylation site of RasGAP is in a sequence context that is favoured by the Src homology 2 ('SH2') domain of Src family members, we tested the possibility that Src was the kinase that associated with RasGAP. Indeed, Src interacted with phosphorylated RasGAP fusion proteins; immunodepletion of Src markedly decreased the recovery of the RasGAP-associated kinase activity. Thus PDGF-dependent tyrosine phosphorylation of RasGAP results in the formation of a complex between RasGAP and Src. To begin to address the relevance of these observations, we focused on the consequences of the interaction of Src and RasGAP. We found that a receptor mutant that did not activate Src was unable to efficiently mediate the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Taken together, these observations support the following hypothesis. When RasGAP is recruited to the betaPDGFR, it is phosphorylated and associates with Src. Once bound to RasGAP, Src is no longer able to promote the phosphorylation of PLCgamma. This hypothesis offers a mechanistic explanation for our previously published findings that the recruitment of RasGAP to the betaPDGFR attenuates the tyrosine phosphorylation of PLCgamma. Finally, these findings suggest a novel way in which RasGAP negatively regulates signal relay by the betaPDGFR. PMID:10567236

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

PubMed

Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

2008-09-05

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Growth Factor Receptor–Bound Protein 2 Contributes to (Hem)Immunoreceptor Tyrosine-Based Activation Motif–Mediated Signaling in Platelets

PubMed Central

Morowski, Martina; Schiessl, Sarah; Schäfer, Carmen M.; Watson, Stephanie K.; Hughes, Craig E.; Ackermann, Jochen A.; Radtke, Daniel; Hermanns, Heike M.; Watson, Steve P.; Nitschke, Lars; Nieswandt, Bernhard

2015-01-01

Rationale Platelets are anuclear cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity but may also cause pathological vessel occlusion. One major pathway of platelet activation is triggered by 2 receptors that signal through an (hem)immunoreceptor tyrosine-based activation motif (ITAM), the activating collagen receptor glycoprotein (GP) VI and the C-type lectin-like receptor 2 (CLEC-2). Growth factor receptor–bound protein 2 (Grb2) is a ubiquitously expressed adapter molecule involved in signaling processes of numerous receptors in different cell types, but its function in platelets and MKs is unknown. Objective We tested the hypothesis that Grb2 is a crucial adapter protein in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets. Methods and Results Here, we show that genetic ablation of Grb2 in MKs and platelets did not interfere with MK differentiation or platelet production. However, Grb2-deficiency severely impaired glycoprotein VI–mediated platelet activation because of defective stabilization of the linker of activated T-cell (LAT) signalosome and activation of downstream signaling proteins that resulted in reduced adhesion, aggregation, and coagulant activity on collagen in vitro. Similarly, CLEC-2–mediated signaling was impaired in Grb2-deficient platelets, whereas the cells responded normally to stimulation of G protein–coupled receptors. In vivo, this selective (hem) immunoreceptor tyrosine-based activation motif signaling defect resulted in prolonged bleeding times but affected arterial thrombus formation only after concomitant treatment with acetylsalicylic acid, indicating that defective glycoprotein VI signaling in the absence of Grb2 can be compensated through thromboxane A2–induced G protein–coupled receptor signaling pathways. Conclusions These results reveal an important contribution of Grb2 in (hem)immunoreceptor tyrosine-based activation motif signaling in platelets in hemostasis and thrombosis by stabilizing the LAT signalosome. PMID:24265393

Activated PLC-γ1 is Catalytically Induced at LAT but Activated PLC-γ1 is Localized at both LAT- and TCR-Containing Complexes

PubMed Central

Cruz-Orcutt, Noemi; Vacaflores, Aldo; Connolly, Sean F.; Bunnell, Stephen C.; Houtman, Jon C.D.

2014-01-01

Phospholipase C-γ1 (PLC-γ1) is a key regulator of T cell receptor (TCR)-induced signaling. Activation of the TCR enhances PLC-γ1 enzymatic function, resulting in calcium influx and the activation of PKC family members and RasGRP. The current model is that phosphorylation of LAT tyrosine 132 facilitates the recruitment of PLC-γ1, leading to its activation and function at the LAT complex. In this study, we examined the phosphorylation kinetics of LAT and PLC-γ1 and the cellular localization of activated PLC-γ1. We observed that commencement of the phosphorylation of LAT tyrosine 132 and PLC-γ1 tyrosine 783 occurred simultaneously, supporting the current model. However, once begun, PLC-γ1 activation occurred more rapidly than LAT tyrosine 132. The association of LAT and PLC-γ1 was more transient than the interaction of LAT and Grb2 and a pool of activated PLC-γ1 translocated away from LAT to cellular structures containing the TCR. These studies demonstrate that LAT and PLC-γ1 form transient interactions that catalyze the activation of PLC-γ1, but that activated PLC-γ1 resides in both LAT and TCR clusters. Together, this work highlights that our current model is incomplete and the activation and function of PLC-γ1 in T cells is highly complex. PMID:24412752

Tyrosine sulfation in a Gram-negative bacterium

PubMed Central

Han, Sang-Wook; Lee, Sang-Won; Bahar, Ofir; Schwessinger, Benjamin; Robinson, Michelle R.; Shaw, Jared B.; Madsen, James A.; Brodbelt, Jennifer S.; Ronald, Pamela C.

2015-01-01

Tyrosine sulfation, a well-characterized post-translation modification in eukaryotes, has not previously been reported in prokaryotes. Here we demonstrate that the RaxST protein from the Gram-negative bacterium, Xanthomonas oryzae pv. oryzae, is a tyrosine sulfotransferase. We used a newly developed sulfotransferase assay and ultraviolet photodissociation mass spectrometry (UVPD) to demonstrate that RaxST catalyzes sulfation of tyrosine 22 of the Xoo Ax21 (activator of XA21-mediated immunity). These results demonstrate a previously undescribed post-translational modification in a prokaryotic species with implications extending to host immune response and bacterial cell-cell communication system. PMID:23093190

Expression of receptor protein tyrosine kinase tif is regulated during leukemia cell differentiation.

PubMed

Dai, W; Pan, H Q; Ouyang, B; Greenberg, J M; Means, R T; Li, B; Cardie, J

1996-06-01

tif is a recently cloned and characterized cDNA predicting a transmembrane protein with a putative tyrosine kinase structure in its cytoplasmic domain. By analysis of the purified tif cytoplasmic domain expressed in Escherichia coli, we have demonstrated that tif is an active protein tyrosine kinase capable of autophosphorylation on tyrosine residues and this phosphorylation is inhibited by a tyrosine-specific inhibitor genistein. Northern blot analyses of various leukemia cell lines have revealed that tif mRNA expression is primarily confined to those bearing erythroid and megakaryocytic phenotypes. Megakaryocytic differentiation of K562 and HEL cells induced by phorbol 12-myristate 13-acetate is accompanied by down-regulation of tif mRNA expression. In addition, treatment of K562 and HEL with hexamethylene bis-acetamide, but not with hemin, decreases the steady-state level of tif mRNA. These combined results suggest that the receptor tyrosine kinase tif is involved in hematopoietic development.

NK cell activation: distinct stimulatory pathways counterbalancing inhibitory signals.

PubMed

Bakker, A B; Wu, J; Phillips, J H; Lanier, L L

2000-01-01

A delicate balance between positive and negative signals regulates NK cell effector function. Activation of NK cells may be initiated by the triggering of multiple adhesion or costimulatory molecules, and can be counterbalanced by inhibitory signals induced by receptors for MHC class I. A common pathway of inhibitory signaling is provided by immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic domains of these receptors which mediate the recruitment of SH2 domain-bearing tyrosine phosphate-1 (SHP-1). In contrast to the extensive progress that has been made regarding the negative regulation of NK cell function, our knowledge of the signals that activate NK cells is still poor. Recent studies of the activating receptor complexes have shed new light on the induction of NK cell effector function. Several NK receptors using novel adaptors with immunoreceptor tyrosine-based activation motifs (ITAMs) and with PI 3-kinase recruiting motifs have been implicated in NK cell stimulation.

JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes.

PubMed

Yin, T; Tsang, M L; Yang, Y C

1994-10-28

Interleukin (IL)-4 and IL-9 regulate the proliferation of T lymphocytes through interactions with their receptors. Previous studies have shown that unknown tyrosine kinases are involved in the proliferative signaling triggered by IL-4 and IL-9. Here we show that IL-4 and IL-9 induce overlapping (170, 130, and 125 kilodalton (kDa)) and distinct (45 and 88/90 kDa, respectively) protein tyrosine phosphorylation in T lymphocytes. We further identify the 170-kDa tyrosine-phosphorylated protein as 4PS/insulin receptor substrate-1-like (IRS-1L) protein and 130-kDa protein as JAK1 kinase. Furthermore, we demonstrate for the first time that JAK1 forms complexes with the IL-4 receptor and 4PS/IRS-1L protein following ligand-receptor interaction. In addition, we demonstrate that IL-9, but not IL-4, induced tyrosine phosphorylation of Stat 91 transcriptional factor. The overlapping and distinct protein tyrosine phosphorylation and activation of the same JAK1 kinase in T lymphocytes strongly suggests that IL-4 and IL-9 share the common signal transduction pathways and that the specificity for each cytokine could be achieved through the unique tyrosine-phosphorylated proteins triggered by individual cytokines.

Sesterterpenes as tubulin tyrosine ligase inhibitors. First insight of structure-activity relationships and discovery of new lead.

PubMed

Dal Piaz, Fabrizio; Vassallo, Antonio; Lepore, Laura; Tosco, Alessandra; Bader, Ammar; De Tommasi, Nunziatina

2009-06-25

Twenty-four new sesterterpenes, compounds 1-24, were isolated from the aerial parts of Salvia dominica. Their structures were elucidated by 1D and 2D NMR experiments as well as ESIMS analysis and chemical methods. The evaluation of the biological activity of Salvia dominica sesterterpenes by means of a panel of chemical and biological approaches, including chemical proteomics, surface plasmon resonance (SPR) measurements, and biochemical assays were realized. Obtained results showed that 18 out of the 24 sesterterpene lactones isolated from Salvia dominica interact with tubulin-tyrosine ligase (TTL) an enzyme involved in the tyrosination cycle of the C-terminal of tubulin, and inhibit TTL activity in cancer cells. Besides, results of our studies provided an activity/structure relationship that can be used to design effective TTL inhibitors.

Hck is a key regulator of gene expression in alternatively activated human monocytes.

PubMed

Bhattacharjee, Ashish; Pal, Srabani; Feldman, Gerald M; Cathcart, Martha K

2011-10-21

IL-13 is a Th2 cytokine that promotes alternative activation (M2 polarization) in primary human monocytes. Our studies have characterized the functional IL-13 receptor complex and the downstream signaling events in response to IL-13 stimulation in alternatively activated monocytes/macrophages. In this report, we present evidence that IL-13 induces the activation of a Src family tyrosine kinase, which is required for IL-13 induction of M2 gene expression, including 15-lipoxygenase (15-LO). Our data show that Src kinase activity regulates IL-13-induced p38 MAPK tyrosine phosphorylation via the upstream kinases MKK3 or MKK6. Our findings also reveal that the IL-13 receptor-associated tyrosine kinase Jak2 is required for the activation of both Src kinase as well as p38 MAPK. Further, we found that Src tyrosine kinase-mediated activation of p38 MAPK is required for Stat1 and Stat3 serine 727 phosphorylation in alternatively activated monocytes/macrophages. Additional studies identify Hck as the specific Src family member, stimulated by IL-13 and involved in regulating both p38 MAPK activation and p38 MAPK-mediated 15-LO expression. Finally we show that the Hck regulates the expression of other alternative state (M2)-specific genes (Mannose receptor, MAO-A, and CD36) and therefore conclude that Hck acts as a key regulator controlling gene expression in alternatively activated monocytes/macrophages.

Tyrosine kinases activate store-mediated Ca2+ entry in human platelets through the reorganization of the actin cytoskeleton.

PubMed Central

Rosado, J A; Graves, D; Sage, S O

2000-01-01

We have recently reported that store-mediated Ca(2+) entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed 'secretion-like coupling'. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca(2+) entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin- or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca(2+) entry were found to be entirely dependent on the actin cytoskeleton. PP1, an inhibitor of the Src family of proteins, partially inhibited store-mediated Ca(2+) entry. In addition, depletion of intracellular Ca(2+) stores stimulated cytoskeletal association of the cytoplasmic tyrosine kinase pp60(src), a process that was sensitive to treatment with cytochalasin D and PP1, but not to inhibition of Ras proteins using prenylcysteine analogues. Finally, combined inhibition of both Ras proteins and tyrosine kinases resulted in complete inhibition of Ca(2+) entry, suggesting that these two families of proteins have independent effects in the activation of store-mediated Ca(2+) entry in human platelets. PMID:11023829

Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation.

PubMed

Law, Nathan C; Hunzicker-Dunn, Mary E

2016-02-26

The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser(789). Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Lead acetate induces EGFR activation upstream of SFK and PKC{alpha} linkage to the Ras/Raf-1/ERK signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, C.-Y.; Wang, Y.-T.; Tzeng, D.-W.

2009-03-01

Lead acetate (Pb), a probable human carcinogen, can activate protein kinase C (PKC) upstream of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Yet, it remains unclear whether Pb activation of PKC {yields} ERK1/2 involves receptor/non-receptor tyrosine kinases and the Ras signaling transducer. Here we demonstrate a novel mechanism elicited by Pb for transmitting ERK1/2 signaling in CL3 human non-small-cell lung adenocarcinoma cells. Pb induction of higher steady-state levels of Ras-GTP was essential for increasing phospho-Raf-1{sup S338} and phospho-ERK1/2. Pre-treatment of the cells with a conventional PKC inhibitor Goe6976 or depleting PKC{alpha} using specific small interfering RNA blocked Pb induction ofmore » Ras-GTP. Pb also activated cellular tyrosine kinases. Specific pharmacological inhibitors, PD153035 for epidermal growth factor receptor (EGFR) and SU6656 for Src family tyrosine kinases (SFK), but not AG1296 for platelet-derived growth factor receptor, could suppress the Pb-induced tyrosine kinases, PKC{alpha}, Ras-GTP, phospho-Raf-1{sup S338} and phospho-ERK1/2. Furthermore, phosphorylation of tyrosines on the EGFR multiple autophosphorylation sites and the conserved SFK autophosphorylation site occurred during exposure of cells to Pb for 1-5 min and 5-30 min, respectively. Intriguingly, Pb activation of EGFR required the intrinsic kinase activity but not dimerization of the receptor. Inhibition of SFK or PKC{alpha} activities did not affect EGFR phosphorylation, while knockdown of EGFR blocked SFK phosphorylation and PKC{alpha} activation following Pb. Together, these results indicate that immediate activation of EGFR in response to Pb is obligatory for activation of SFK and PKC{alpha} and subsequent the Ras-Raf-1-MKK1/2-ERK1/2 signaling cascade.« less

Tyrosine Residues Regulate Multiple Nuclear Functions of P54nrb.

PubMed

Lee, Ahn R; Hung, Wayne; Xie, Ning; Liu, Liangliang; He, Leye; Dong, Xuesen

2017-04-01

The non-POU-domain-containing octamer binding protein (NONO; also known as p54nrb) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although tyrosine phosphorylation has been proposed to account for the multi-functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. To investigate the tyrosine phosphorylation status of p54nrb, we performed site-directed mutagenesis on the five tyrosine residues of p54nrb, replacing the tyrosine residues with phenylalanine or alanine, and immunoblotted for tyrosine phosphorylation. We then preceded with luciferase reporter assays, RNA splicing minigene assays, co-immunoprecipitation, and confocal microscopy to study the function of p54nrb tyrosine residues on transcription, RNA splicing, protein-protein interaction, and cellular localization. We found that p54nrb was not phosphorylated at tyrosine residues. Rather, it has non-specific binding affinity to anti-phosphotyrosine antibodies. However, replacement of tyrosine with phenylalanine altered p54nrb activities in transcription co-repression and RNA splicing in gene context-dependent fashions by means of differential regulation of p54nrb protein association with its interacting partners and co-regulators of transcription and splicing. These results demonstrate that tyrosine residues, regardless of phosphorylation status, are important for p54nrb function. J. Cell. Physiol. 232: 852-861, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

dDYRK2 and Minibrain interact with the chromatin remodelling factors SNR1 and TRX.

PubMed

Kinstrie, Ross; Lochhead, Pamela A; Sibbet, Gary; Morrice, Nick; Cleghon, Vaughn

2006-08-15

The DYRKs (dual specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that autophosphorylate a tyrosine residue in their activation loop by an intra-molecular mechanism and phosphorylate exogenous substrates on serine/threonine residues. Little is known about the identity of true substrates for DYRK family members and their binding partners. To address this question, we used full-length dDYRK2 (Drosophila DYRK2) as bait in a yeast two-hybrid screen of a Drosophila embryo cDNA library. Of 14 independent dDYRK2 interacting clones identified, three were derived from the chromatin remodelling factor, SNR1 (Snf5-related 1), and three from the essential chromatin component, TRX (trithorax). The association of dDYRK2 with SNR1 and TRX was confirmed by co-immunoprecipitation studies. Deletion analysis showed that the C-terminus of dDYRK2 modulated the interaction with SNR1 and TRX. DYRK family member MNB (Minibrain) was also found to co-precipitate with SNR1 and TRX, associations that did not require the C-terminus of the molecule. dDYRK2 and MNB were also found to phosphorylate SNR1 at Thr102 in vitro and in vivo. This phosphorylation required the highly conserved DH-box (DYRK homology box) of dDYRK2, whereas the DH-box was not essential for phosphorylation by MNB. This is the first instance of phosphorylation of SNR1 or any of its homologues and implicates the DYRK family of kinases with a role in chromatin remodelling.

A change in structural integrity of c-Kit mutant D816V causes constitutive signaling.

PubMed

Raghav, Pawan Kumar; Singh, Ajay Kumar; Gangenahalli, Gurudutta

2018-03-01

Several signaling pathways, ligands, and genes that regulate proliferative and self-renewal properties of the Hematopoietic Stem Cells (HSCs) have been studied meticulously. One of the signaling pathways that play a crucial role in the process of hematopoiesis is the Stem Cell Factor (SCF) mediated c-Kit pathway. The c-Kit is a Receptor Tyrosine Kinase (RTK), which is expressed in the cells including HSCs. It undergoes dimerization upon binding with its cognate ligand SCF. As a result, phosphorylation of the Juxtamembrane (JM) domain of c-Kit takes place at Tyr568 and Tyr570 residues. These phosphorylated residues become the docking sites for protein tyrosine phosphatases (PTPs) namely SHP-1 and SHP-2, which in turn cause dephosphorylation and negative regulation of the downstream signaling responsible for the cell proliferation. Interestingly, it has been reported that the mutation of c-Kit at D816V makes it independent of SCF stimulation and SHP-1/SHP-2 inhibition, thereby, causing its constitutive activation. The present study was commenced to elucidate the structural behavior of this mutation in the JM and A-loop region of c-Kit using Molecular Dynamics (MD) simulations of the wild-type and mutant c-Kit in unphosphorylated and phosphorylated states. The energy difference computed between the wild type and mutant (D816V) c-Kit, and protein-protein docking and complex analysis revealed the impact of this single residue mutation on the integrity dynamics of c-Kit that makes it independent of SHP-1/SHP-2 negative regulation. Copyright © 2018 Elsevier B.V. All rights reserved.

Neuropeptide y gates a stress-induced, long-lasting plasticity in the sympathetic nervous system.

PubMed

Wang, Qian; Wang, Manqi; Whim, Matthew D

2013-07-31

Acute stress evokes the fight-or-flight reflex, which via release of the catecholamine hormones affects the function of every major organ. Although the reflex is transient, it has lasting consequences that produce an exaggerated response when stress is reexperienced. How this change is encoded is not known. We investigated whether the reflex affects the adrenal component of the sympathetic nervous system, a major branch of the stress response. Mice were briefly exposed to the cold-water forced swim test (FST) which evoked an increase in circulating catecholamines. Although this hormonal response was transient, the FST led to a long-lasting increase in the catecholamine secretory capacity measured amperometrically from chromaffin cells and in the expression of tyrosine hydroxylase. A variety of approaches indicate that these changes are regulated postsynaptically by neuropeptide Y (NPY), an adrenal cotransmitter. Using immunohistochemistry, RT-PCR, and NPY(GFP) BAC mice, we find that NPY is synthesized by all chromaffin cells. Stress failed to increase secretory capacity in NPY knock-out mice. Genetic or pharmacological interference with NPY and Y1 (but not Y2 or Y5) receptor signaling attenuated the stress-induced change in tyrosine hydroxylase expression. These results indicate that, under basal conditions, adrenal signaling is tonically inhibited by NPY, but stress overrides this autocrine negative feedback loop. Because acute stress leads to a lasting increase in secretory capacity in vivo but does not alter sympathetic tone, these postsynaptic changes appear to be an adaptive response. We conclude that the sympathetic limb of the stress response exhibits an activity-dependent form of long-lasting plasticity.

Tyrosine assisted size controlled synthesis of silver nanoparticles and their catalytic, in-vitro cytotoxicity evaluation.

PubMed

Maddinedi, Sireesh Babu; Mandal, Badal Kumar; Anna, Kiran Kumar

2017-04-01

A simple, green approach for the size controllable preparation of silver nanoparticles (SNPs) using tyrosine as reducing and capping agent is shown here. The size of SNPs is controlled by varying the pH of tyrosine solution. The as synthesized SNPs are characterized by using XRD, UV-Visible, DLS, TEM and SAED. Zeta potential measurements revealed the stability of tyrosine capped silver nanocolloids. Furthermore, catalytic activity studies concluded that the smaller SNPs acts as good catalyst and the catalytic activity depends on size of the nanoparticles. Further, the in-vitro cytotoxicity experiments concluded that the cytotoxicity of the prepared SNPs towards mouse fibroblast (3T3) cell lines is size and dose dependent. Additionally, the present approach is substitute to the traditional methods that are being used now-a-days for size controlled synthesis of SNPs. Copyright © 2017 Elsevier B.V. All rights reserved.

N-terminal tyrosine phosphorylation of caveolin-2 negates anti-proliferative effect of transforming growth factor beta in endothelial cells

PubMed Central

Abel, Britain; Willoughby, Cara; Jang, Sungchan; Cooper, Laura; Xie, Leike; Vo-Ransdell, Chi; Sowa, Grzegorz

2012-01-01

Here we show that tyrosine phosphorylation of caveolin-2 (Cav-2) negatively regulates the anti-proliferative function of transforming growth factor beta (TGF-beta) in endothelial cells. In contrast to wild-type-Cav-2, retroviral re-expression of Y19/27F-Cav-2 in Cav-2 knockout endothelial cells did not affect anti-proliferative effect of TGF-beta compared to empty vector. Conversely, although less effective than wild-type, re-expression of S23/36A-Cav-2 reduced the effect of TGF-beta compared to empty vector. This differential effect of tyrosine and serine phosphorylation mutants of Cav-2 correlated with TGF-beta-induced Smad3 phosphorylation and transcriptional activation of plasminogen activator inhibitor-1. Thus tyrosine-phosphorylated Cav-2 counteracts anti-proliferative effect of TGF-beta in endothelial cells. PMID:22819829

Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal.

PubMed

Maeda, A; Kurosaki, M; Ono, M; Takai, T; Kurosaki, T

1998-04-20

Paired immunoglobulin-like receptor B (PIR-B) (p91) molecule has been proposed to function as an inhibitory receptor in B cells and myeloid lineage cells. We demonstrate here that the cytoplasmic region of PIR-B is capable of inhibiting B cell activation. Mutational analysis of five cytoplasmic tyrosines indicate that tyrosine 771 in the motif VxYxxL plays the most crucial role in mediating the inhibitory signal. PIR-B-mediated inhibition was markedly reduced in the SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 double-deficient DT40 B cells, whereas this inhibition was unaffected in the inositol polyphosphate 5'-phosphatase SHIP-deficient cells. These data demonstrate that PIR-B can negatively regulate B cell receptor activation and that this PIR-B-mediated inhibition requires redundant functions of SHP-1 and SHP-2.

Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation.

PubMed

Wong, Lilly; Lieser, Scot A; Miyashita, Osamu; Miller, Meghan; Tasken, Kjetil; Onuchic, Josè N; Adams, Joseph A; Woods, Virgil L; Jennings, Patricia A

2005-08-05

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kattke, Michele D.; Chan, Albert H.; Duong, Andrew

Here, many species of Gram-positive bacteria use sortase transpeptidases to covalently affix proteins to their cell wall or to assemble pili. Sortase-displayed proteins perform critical and diverse functions for cell survival, including cell adhesion, nutrient acquisition, and morphological development, among others. Based on their amino acid sequences, there are at least six types of sortases (class A to F enzymes); however, class E enzymes have not been extensively studied. Class E sortases are used by soil and freshwater-dwelling Actinobacteria to display proteins that contain a non-canonical LAXTG sorting signal, which differs from 90% of known sorting signals by substitution ofmore » alanine for proline. Here we report the first crystal structure of a class E sortase, the 1.93 Å resolution structure of the SrtE1 enzyme from Streptomyces coelicolor. The active site is bound to a tripeptide, providing insight into the mechanism of substrate binding. SrtE1 possesses β3/β4 and β6/β7 active site loops that contact the LAXTG substrate and are structurally distinct from other classes. We propose that SrtE1 and other class E sortases employ a conserved tyrosine residue within their β3/β4 loop to recognize the amide nitrogen of alanine at position P3 of the sorting signal through a hydrogen bond, as seen here. Incapability of hydrogen-bonding with canonical proline-containing sorting signals likely contributes to class E substrate specificity. Furthermore, we demonstrate that surface anchoring of proteins involved in aerial hyphae formation requires an N-terminal segment in SrtE1 that is presumably positioned within the cytoplasm. Combined, our results reveal unique features within class E enzymes that enable them to recognize distinct sorting signals, and could facilitate the development of substrate-based inhibitors of this important enzyme family.« less

Tyrosine Phosphorylation of the Pioneer Transcription Factor FoxA1 Promotes Activation of Estrogen Signaling.

PubMed

Yamaguchi, Noritaka; Shibazaki, Misato; Yamada, Chiaki; Anzai, Erina; Morii, Mariko; Nakayama, Yuji; Kuga, Takahisa; Hashimoto, Yuuki; Tomonaga, Takeshi; Yamaguchi, Naoto

2017-06-01

The pioneer transcription factor FoxA1 plays an important role in estrogen signaling by opening closed chromatin and promoting recruitment of the estrogen receptor to its target regions in DNA. In this study, we analyzed tyrosine phosphorylation of FoxA1 by the non-receptor-type tyrosine kinase c-Abl. c-Abl was shown to phosphorylate FoxA1 at multiple sites, especially in the N- and C-terminal regions. Tyr429 and Tyr464 were identified as the major phosphorylation sites in the FoxA1 C-terminal region. The phosphomimetic and nonphosphorylatable FoxA1 mutants were generated by glutamic acid and phenylalanine substitutions at these tyrosine residues, respectively. The phosphomimetic FoxA1 promoted the activation of estrogen signaling, whereas the nonphosphorylatable FoxA1 suppressed its activation. Stimulation with the epidermal growth factor, which activates c-Abl, enhanced the activation of estrogen signaling. In contrast, the c-Abl inhibitor imatinib reduced its activation. The phosphomimetic FoxA1 mutant showed a higher affinity toward histone H3 than the wild-type. These results suggest that c-Abl-mediated phosphorylation of FoxA1 promotes the activation of estrogen signaling by inducing its binding to histones. J. Cell. Biochem. 118: 1453-1461, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Tyrosine hydroxylase in the ventral tegmental area of rams with high or low libido-A role for dopamine.

PubMed

Kramer, A C; Mirto, A J; Austin, K J; Roselli, C E; Alexander, B M

2017-12-01

Dopamine synthesis in the ventral tegmental area (VTA) is necessary for the reinforcement of sexual behavior. The objective of this study determined if sexual stimuli initiates reward, and whether reward is attenuated in sexually inactive rams. Sexually active rams were exposed to urine from estrous (n=4) or ovariectomized (n=3) ewes with inactive rams (n=3) exposed to urine from estrous ewes. Following exposure, rams were exsanguinated and brains perfused. Alternating sections of the VTA were stained for Fos related antigens (FRA), tyrosine hydroxylase, and dopamine beta-hydroxylase activity. Forebrain tissue, mid-sagittal ventral to the anterior corpus callosum, was stained for dopamine D 2 receptors. Concentrations of cortisol was determined prior to and following exposure. Exposure to ovariectomized-ewe urine in sexually active rams did not influence (P=0.6) FRA expression, but fewer (P<0.05) neurons were positive for tyrosine hydroxylase in the VTA. Sexually inactive rams had fewer (P<0.05) FRA and tyrosine hydroxylase positive neurons in the VTA than sexually active rams following exposure to estrous ewe urine. VTA neurons staining positive for dopamine beta-hydroxylase did not differ by sexual activity (P=0.44) or urine exposure (P=0.07). Exposure to stimulus did not influence (P=0.46) numbers of forebrain neurons staining positive for dopamine D2 receptors in sexually active rams, but fewer (P=0.04) neurons stain positive in inactive rams. Serum concentrations of cortisol did not differ (P≥0.52) among rams prior to or following stimulus. In conclusion sexual inactivity is unlikely due to stress, but may be partially a result of decreased tyrosine hydroxylase and/or the response to dopamine. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and Targeting of Tyrosine Kinase Activity in Prostate Cancer Initiation, Progression, and Metastasis

DTIC Science & Technology

2012-10-01

accomplishments. Aim 1: Identify the specific tyrosine kinases activated during initiation and progression of genetically altered prostate cancer... Genetics Departmental Retreat (October 2011). (see appendices) • Presented research findings at the AACR Advances in Prostate Cancer Research Conference... genetic backgrounds. However, preliminary data suggests that phosphopeptides from metastatic tumors do indeed segregate from primary prostate tumors and

SRC-DEPENDENT PHOSPHORYLATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR ON TYROSINE 845 IS REQUIRED FOR ZINC-INDUCED RAS ACTIVATION

EPA Science Inventory

Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor on Tyrosine 845 Is Required for Zinc-induced Ras Activation
Weidong Wu 1 , Lee M. Graves 2 , Gordon N. Gill 3 , Sarah J. Parsons 4 , and James M. Samet 5
1 Center for Environmental Medicine and Lung Biolo...

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells.

PubMed

Kuwada, S K; Lund, K A; Li, X F; Cliften, P; Amsler, K; Opresko, L K; Wiley, H S

1998-12-01

Overexpression of the epidermal growth factor receptors (EGFR) in polarized kidney epithelial cells caused them to appear in high numbers at both the basolateral and apical cell surfaces. We utilized these cells to look for differences in the regulation and signaling of apical vs. basolateral EGFR. Apical and basolateral EGFR were biologically active and mediated EGF-induced cell proliferation to similar degrees. Receptor downregulation and endocytosis were less efficient at the apical surface, resulting in prolonged EGF-induced tyrosine kinase activity at the apical cell membrane. Tyrosine phosphorylation of EGFR substrates known to mediate cell proliferation, Src-homologous and collagen protein (SHC), extracellularly regulated kinase 1 (ERK1), and ERK2 could be induced similarly by activation of apical or basolateral EGFR. Focal adhesion kinase was tyrosine phosphorylated more by basolateral than by apical EGFR; however, beta-catenin was tyrosine phosphorylated to a much greater degree following the activation of mislocalized apical EGFR. Thus EGFR regulation and EGFR-mediated phosphorylation of certain substrates differ at the apical and basolateral cell membrane domains. This suggests that EGFR mislocalization could result in abnormal signal transduction and aberrant cell behavior.

Dephosphorylation of receptor tyrosine kinases as target of regulation by radiation, oxidants or alkylating agents.

PubMed Central

Knebel, A; Rahmsdorf, H J; Ullrich, A; Herrlich, P

1996-01-01

Several non-physiologic agents such as radiation, oxidants and alkylating agents induce ligand-independent activation of numerous receptor tyrosine kinases (RTKs) and of protein tyrosine kinases at the inner side of the plasma membrane (e.g. Dévary et al., 1992; Sachsenmaier et al., 1994; Schieven et al., 1994; Coffer et al., 1995). Here we show additional evidence for the activation of epidermal growth factor receptor (EGFR), and we show activation of v-ErbB, ErbB2 and platelet-derived growth factor receptor. As a common principle of action the inducing agents such as UVC, UVB, UVA, hydrogen peroxide and iodoacetamide inhibit receptor tyrosine dephosphorylation in a thiol-sensitive and, with the exception of the SH-alkylating agent, reversible manner. EGFR dephosphorylation can also be modulated by these non-physiologic agents in isolated plasma membranes in the presence of Triton X-100. Further, substrate (EGFR) and phosphatase have been separated: a membrane preparation of cells that have been treated with epidermal growth factor (EGF) and whose dephosphorylating enzymes have been permanently destroyed by iodoacetamide can be mixed with a membrane preparation from untreated cells which re-establishes EGFR dephosphorylation. This dephosphorylation can be modulated in vitro by UV and thiol agents. We conclude that RTKs exhibit significant spontaneous protein kinase activity; several adverse agents target (an) essential SH-group(s) carried by (a) membrane-bound protein tyrosine phosphatase(s). Images PMID:8895576

Mechanistic events underlying odorant binding protein chemoreception.

PubMed

Golebiowski, Jérôme; Antonczak, Serge; Fiorucci, Sébastien; Cabrol-Bass, Daniel

2007-05-01

Odorant binding proteins (OBP's) are small hydrophilic proteins, belonging to the lipocalin family dedicated to bind and transport small hydrophobic ligands. Despite many works, the mechanism of ligand binding, together with the functional role of these proteins remains a topic of debate and little is known at the atomic level. The present work reports a computational study of odorants capture and release by an OBP, using both constrained and unconstrained simulations, giving a glimpse on the molecular mechanism of chemoreception. The residues at the origin of the regulation of the protein door opening are identified and a tyrosine amino-acid together with other nearby residues appear to play a crucial role in allowing this event to occur. The simulations reveal that this tyrosine and the protein's L5 loop are implicated in the ligand contact with the protein and act as an anchoring point for the ligand. The protein structural features required for the ligand entry are highly conserved among many transport proteins, suggesting that this mechanism could somewhat be extended to some members of the larger family of lipocalin. (c) 2007 Wiley-Liss, Inc.

Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and specificity.

PubMed

Jin, Lily L; Wybenga-Groot, Leanne E; Tong, Jiefei; Taylor, Paul; Minden, Mark D; Trudel, Suzanne; McGlade, C Jane; Moran, Michael F

2015-03-01

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Modulation of Bacillus thuringiensis Phosphatidylinositol-Specific Phospholipase C Activity by Mutations in the Putative Dimerization Interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, X.; Shao, C; Zhang, X

2009-01-01

Cleavage of phosphatidylinositol (PI) to inositol 1,2-(cyclic)-phosphate (cIP) and cIP hydrolysis to inositol 1-phosphate by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C are activated by the enzyme binding to phosphatidylcholine (PC) surfaces. Part of this reflects improved binding of the protein to interfaces. However, crystallographic analysis of an interfacially impaired phosphatidylinositol-specific phospholipase (W47A/W242A) suggested protein dimerization might occur on the membrane. In the W47A/W242A dimer, four tyrosine residues from one monomer interact with the same tyrosine cluster of the other, forming a tight dimer interface close to the membrane binding regions. We have constructed mutant proteins in which two or more ofmore » these tyrosine residues have been replaced with serine. Phospholipid binding and enzymatic activity of these mutants have been examined to assess the importance of these residues to enzyme function. Replacing two tyrosines had small effects on enzyme activity. However, removal of three or four tyrosine residues weakened PC binding and reduced PI cleavage by the enzyme as well as PC activation of cIP hydrolysis. Crystal structures of Y247S/Y251S in the absence and presence of myo-inositol as well as Y246S/Y247S/Y248S/Y251S indicate that both mutant proteins crystallized as monomers, were very similar to one another, and had no change in the active site region. Kinetic assays, lipid binding, and structural results indicate that either (i) a specific PC binding site, critical for vesicle activities and cIP activation, has been impaired, or (ii) the reduced dimerization potential for Y246S/Y247S/Y248S and Y246S/Y247S/Y248S/Y251S is responsible for their reduced catalytic activity in all assay systems.« less

Nck-Interacting Ste20 Kinase Couples Eph Receptors to c-Jun N-Terminal Kinase and Integrin Activation

PubMed Central

Becker, Elena; Huynh-Do, Uyen; Holland, Sacha; Pawson, Tony; Daniel, Tom O.; Skolnik, Edward Y.

2000-01-01

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62dok, RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62dok most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs. PMID:10669731

Insulin Receptor Substrate (IRS)-2 negatively regulates alternative macrophage activation and allergic lung inflammation

PubMed Central

Dasgupta, Preeta; Dorsey, Nicolas J.; Li, Jiaqi; Qi, Xiulan; Smith, Elizabeth P.; Yamaji-Kegan, Kazuyo; Keegan, Achsah D.

2017-01-01

Insulin Receptor Substrate (IRS)-2 is an adaptor protein that becomes tyrosine phosphorylated in response to IL-4 and IL-13 resulting in activation of the PI-3′ kinase/Akt pathway. While the contribution of IL-4 and IL-13 to allergic lung inflammation has been studied extensively, the functional significance of the IRS2 pathway is unclear. To examine the role of IRS2 in allergic disease, we evaluated responses in IRS2-deficient mice. Deficiency of IRS2 resulted in a substantial increase in expression of a subset of genes associated with alternatively activated macrophages (AAM) in response to IL-4 or IL-13 in vitro. Moreover, IRS2+/− and IRS2−/− mice developed enhanced pulmonary inflammation, accumulation of eosinophils and AAM, and airway and vascular remodeling upon allergen stimulation in comparison to IRS2+/+ mice; this enhanced response was in part macrophage intrinsic. Loss of IRS2 led to greater phosphorylation of Akt and ribosomal S6 protein in the basal state and upon IL-4 stimulation. Thus, we identify a critical negative regulatory loop downstream of IRS2, demonstrating a previously unrecognized role for IRS2 in suppressing allergic lung inflammation and remodeling. PMID:27330190

Downregulation of PTP1B and TC-PTP phosphatases potentiate dendritic cell-based immunotherapy through IL-12/IFNγ signaling.

PubMed

Penafuerte, Claudia; Feldhammer, Matthew; Mills, John R; Vinette, Valerie; Pike, Kelly A; Hall, Anita; Migon, Eva; Karsenty, Gerard; Pelletier, Jerry; Zogopoulos, George; Tremblay, Michel L

2017-01-01

PTP1B and TC-PTP are highly related protein-tyrosine phosphatases (PTPs) that regulate the JAK/STAT signaling cascade essential for cytokine-receptor activation in immune cells. Here, we describe a novel immunotherapy approach whereby monocyte-derived dendritic cell (moDC) function is enhanced by modulating the enzymatic activities of PTP1B and TC-PTP. To downregulate or delete the activity/expression of these PTPs, we generated mice with PTP-specific deletions in the dendritic cell compartment or used PTP1B and TC-PTP specific inhibitor. While total ablation of PTP1B or TC-PTP expression leads to tolerogenic DCs via STAT3 hyperactivation, downregulation of either phosphatase remarkably shifts the balance toward an immunogenic DC phenotype due to hyperactivation of STAT4, STAT1 and Src kinase. The resulting increase in IL-12 and IFNγ production subsequently amplifies the IL-12/STAT4/IFNγ/STAT1/IL-12 positive autocrine loop and enhances the therapeutic potential of mature moDCs in tumor-bearing mice. Furthermore, pharmacological inhibition of both PTPs improves the maturation of defective moDCs derived from pancreatic cancer (PaC) patients. Our study provides a new advance in the use of DC-based cancer immunotherapy that is complementary to current cancer therapeutics.

Structure-Based Network Analysis of Activation Mechanisms in the ErbB Family of Receptor Tyrosine Kinases: The Regulatory Spine Residues Are Global Mediators of Structural Stability and Allosteric Interactions

PubMed Central

James, Kevin A.; Verkhivker, Gennady M.

2014-01-01

The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced “superacceptor” activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD) motif in the catalytic loop and the Asp-Phe-Gly (DFG) motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not limited to the ATP site, and may enhance allosteric cooperativity with the substrate binding region by increasing communication capabilities of mediating residues. PMID:25427151

Replacing the Axial Ligand Tyrosine 75 or Its Hydrogen Bond Partner Histidine 83 Minimally Affects Hemin Acquisition by the Hemophore HasAp from Pseudomonas aeruginosa

PubMed Central

2015-01-01

Hemophores from Pseudomonas aeruginosa (HasAp), Serratia marcescens (HasAsm), and Yersinia pestis (HasAyp) bind hemin between two loops. One of the loops harbors conserved axial ligand Tyr75 (Y75 loop) in all three structures, whereas the second loop (H32 loop) contains axial ligand His32 in HasAp and HasAsm, but a noncoordinating Gln32 in HasAyp. Binding of hemin to the Y75 loop of HasAp or HasAsm causes a large rearrangement of the H32 loop that allows His32 coordination. The Q32 loop in apo-HasAyp is already in the closed conformation, such that binding of hemin to the conserved Y75 loop occurs with minimal structural rearrangement and without coordinative interaction with the Q32 loop. In this study, structural and spectroscopic investigations of the hemophore HasAp were conducted to probe (i) the role of the conserved Tyr75 loop in hemin binding and (ii) the proposed requirement of the His83–Tyr75 hydrogen bond to allow the coordination of hemin by Tyr75. High-resolution crystal structures of H83A holo-HasAp obtained at pH 6.5 (0.89 Å) and pH 5.4 (1.25 Å) show that Tyr75 remains coordinated to the heme iron, and that a water molecule can substitute for Nδ of His83 to interact with the Oη atom of Tyr75, likely stabilizing the Tyr75–Fe interaction. Nuclear magnetic resonance spectroscopy revealed that in apo-Y75A and apo-H83A HasAp, the Y75 loop is disordered, and that disorder propagates to nearby elements of secondary structure, suggesting that His83 Nδ–Tyr75 Oη interaction is important to the organization of the Y75 loop in apo-HasA. Kinetic analysis of hemin loading conducted via stopped-flow UV–vis and rapid-freeze-quench resonance Raman shows that both mutants load hemin with biphasic kinetic parameters that are not significantly dissimilar from those previously observed for wild-type HasAp. When the structural and kinetic data are taken together, a tentative model emerges, which suggests that HasA hemophores utilize hydrophobic, π–π stacking, and van der Waals interactions to load hemin efficiently, while axial ligation likely functions to slow hemin release, thus allowing the hemophore to meet the challenge of capturing hemin under inhospitable conditions and delivering it selectively to its cognate receptor. PMID:24625274

Driving Protein Conformational Changes with Light: Photoinduced Structural Rearrangement in a Heterobimetallic Oxidase.

PubMed

Maugeri, Pearson T; Griese, Julia J; Branca, Rui M; Miller, Effie K; Smith, Zachary R; Eirich, Jürgen; Högbom, Martin; Shafaat, Hannah S

2018-01-31

The heterobimetallic R2lox protein binds both manganese and iron ions in a site-selective fashion and activates oxygen, ultimately performing C-H bond oxidation to generate a tyrosine-valine cross-link near the active site. In this work, we demonstrate that, following assembly, R2lox undergoes photoinduced changes to the active site geometry and metal coordination motif. Through spectroscopic, structural, and mass spectrometric characterization, the photoconverted species is found to consist of a tyrosinate-bound iron center following light-induced decarboxylation of a coordinating glutamate residue and cleavage of the tyrosine-valine cross-link. This process occurs with high quantum efficiencies (Φ = 3%) using violet and near-ultraviolet light, suggesting that the photodecarboxylation is initiated via ligand-to-metal charge transfer excitation. Site-directed mutagenesis and structural analysis suggest that the cross-linked tyrosine-162 is the coordinating residue. One primary product is observed following irradiation, indicating potential use of this class of proteins, which contains a putative substrate channel, for controlled photoinduced decarboxylation processes, with relevance for in vivo functionality of R2lox as well as application in environmental remediation.

Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets.

PubMed Central

Wonerow, Peter; Obergfell, Achim; Wilde, Jonathan I; Bobe, Régis; Asazuma, Naoki; Brdicka, Tomás; Leo, Albrecht; Schraven, Burkhart; Horejsí, Václav; Shattil, Sanford J; Watson, Steve P

2002-01-01

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis. PMID:12049640

Atomistic insights into the lung cancer-associated L755P mutation in HER2 resistance to lapatinib: a molecular dynamics study.

PubMed

Yang, Bei; Zhang, Haiping; Wang, Hao

2015-02-01

HER2, a member of the human ErbB protein family belonging to receptor tyrosine kinases, plays important roles in regulating crucial cellular processes, including cell migration, proliferation, and differentiation. A missense mutation, L755P, in the HER2 kinase domain has been involved in lung cancer in humans and exhibits reduced response to lapatinib therapy. However, the detailed mechanism of how the HER2 L755P mutation causes drug resistance to lapatinib remains elusive. Here, molecular docking, molecular dynamics (MD) simulations, binding free energy calculations [molecular mechanics and generalized Born/surface area (MM-GBSA)] were performed to reveal the mechanism of drug resistance due to the HER2 L755P mutation. MD simulations revealed that the L755P mutation caused structural changes in the regions of helix αC, the glycine-rich loop, and the activation loop, thereby leading to the loss of interactions between the solubilizing group of lapatinib and HER2. Moreover, MM-GBSA calculations suggested that hydrophobic interactions between lapatinib and HER2 contribute most to the binding affinity, and that the L755P mutation could result in a less energetically favorable HER2/lapatinib complex. This may weaken the binding of lapatinib to the mutated HER2, thereby leading to the emergence of drug resistance. This study offers a structural explanation for the effect of the L755P mutation on the HER2/lapatinib complex.

Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate

PubMed Central

Wang, Qi; Vogan, Erik M; Nocka, Laura M; Rosen, Connor E; Zorn, Julie A; Harrison, Stephen C; Kuriyan, John

2015-01-01

Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk. DOI: http://dx.doi.org/10.7554/eLife.06074.001 PMID:25699547

Synaptopodin Is a Coincidence Detector of Tyrosine versus Serine/Threonine Phosphorylation for the Modulation of Rho Protein Crosstalk in Podocytes

PubMed Central

Buvall, Lisa; Wallentin, Hanna; Sieber, Jonas; Andreeva, Svetlana; Choi, Hoon Young; Mundel, Peter

2017-01-01

Tyrosine and serine/threonine signal-transduction pathways influence many aspects of cell behavior, including the spatial and temporal regulation of the actin cytoskeleton. However, little is known about how input from diverse tyrosine and serine/threonine kinases is integrated to control Rho protein crosstalk and actin remodeling, which are critically important in podocyte health and disease. Here we unveil the proteolytically-regulated, actin organizing protein synaptopodin as a coincidence detector of tyrosine versus serine/threonine phosphorylation. We show that serine/threonine and tyrosine kinases duel for synaptopodin stability versus degradation. EGFR/Src-mediated tyrosine phosphorylation of synaptopodin in podocytes promotes binding to the serine/threonine phosphatase calcineurin. This leads to the loss of 14–3-3 binding, resulting in synaptopodin degradation, Vav2 activation, enhanced Rac1 signaling, and ultimate loss of stress fibers. Our studies reveal how synaptopodin, a single proteolytically-controlled protein, integrates antagonistic tyrosine versus serine/threonine phosphorylation events for the dynamic control of the actin cytoskeleton in podocytes. PMID:27628902

Synaptopodin Is a Coincidence Detector of Tyrosine versus Serine/Threonine Phosphorylation for the Modulation of Rho Protein Crosstalk in Podocytes.

PubMed

Buvall, Lisa; Wallentin, Hanna; Sieber, Jonas; Andreeva, Svetlana; Choi, Hoon Young; Mundel, Peter; Greka, Anna

2017-03-01

Tyrosine and serine/threonine signal-transduction pathways influence many aspects of cell behavior, including the spatial and temporal regulation of the actin cytoskeleton. However, little is known about how input from diverse tyrosine and serine/threonine kinases is integrated to control Rho protein crosstalk and actin remodeling, which are critically important in podocyte health and disease. Here we unveil the proteolytically-regulated, actin organizing protein synaptopodin as a coincidence detector of tyrosine versus serine/threonine phosphorylation. We show that serine/threonine and tyrosine kinases duel for synaptopodin stability versus degradation. EGFR/Src-mediated tyrosine phosphorylation of synaptopodin in podocytes promotes binding to the serine/threonine phosphatase calcineurin. This leads to the loss of 14-3-3 binding, resulting in synaptopodin degradation, Vav2 activation, enhanced Rac1 signaling, and ultimate loss of stress fibers. Our studies reveal how synaptopodin, a single proteolytically-controlled protein, integrates antagonistic tyrosine versus serine/threonine phosphorylation events for the dynamic control of the actin cytoskeleton in podocytes. Copyright © 2017 by the American Society of Nephrology.

Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.

PubMed Central

Furitsu, T; Tsujimura, T; Tono, T; Ikeda, H; Kitayama, H; Koshimizu, U; Sugahara, H; Butterfield, J H; Ashman, L K; Kanayama, Y

1993-01-01

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells. Images PMID:7691885

Long-Term Behavioral Recovery in Parkinsonian Rats by an HSV Vector Expressing Tyrosine Hydroxylase

PubMed Central

Naegele, Janice R.; O’Malley, Karen L.; Geller, Alfred I.

2006-01-01

One therapeutic approach to treating Parkinson’s disease is to convert endogenous striatal cells into levo-3,4-dihydroxyphenylalanine (l-dopa)–producing cells. A defective herpes simplex virus type 1 vector expressing human tyrosine hydroxylase was delivered into the partially denervated striatum of 6-hydroxydopamine–lesioned rats, used as a model of Parkinson’s disease. Efficient behavioral and biochemical recovery was maintained for 1 year after gene transfer. Biochemical recovery included increases in both striatal tyrosine hydroxylase enzyme activity and in extracellular dopamine concentrations. Persistence of human tyrosine hydroxylase was revealed by expression of RNA and immunoreactivity. PMID:7669103

Phosphorylation of KRAB-associated Protein 1 (KAP1) at Tyr-449, Tyr-458, and Tyr-517 by Nuclear Tyrosine Kinases Inhibits the Association of KAP1 and Heterochromatin Protein 1α (HP1α) with Heterochromatin*

PubMed Central

Kubota, Sho; Fukumoto, Yasunori; Aoyama, Kazumasa; Ishibashi, Kenichi; Yuki, Ryuzaburo; Morinaga, Takao; Honda, Takuya; Yamaguchi, Noritaka; Kuga, Takahisa; Tomonaga, Takeshi; Yamaguchi, Naoto

2013-01-01

Protein tyrosine phosphorylation regulates a wide range of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine phosphorylation by Src family kinases (SFKs) induces chromatin structural changes. In this study, we identify KRAB-associated protein 1 (KAP1/TIF1β/TRIM28), a component of heterochromatin, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of KAP1 is induced by several tyrosine kinases, such as Src, Lyn, Abl, and Brk. Among SFKs, Src strongly induces tyrosine phosphorylation of KAP1. Nucleus-targeted Lyn potentiates tyrosine phosphorylation of KAP1 compared with intact Lyn, but neither intact Fyn nor nucleus-targeted Fyn phosphorylates KAP1. Substitution of the three tyrosine residues Tyr-449/Tyr-458/Tyr-517, located close to the HP1 binding-motif, into phenylalanine ablates tyrosine phosphorylation of KAP1. Immunostaining and chromatin fractionation show that Src and Lyn decrease the association of KAP1 with heterochromatin in a kinase activity-dependent manner. KAP1 knockdown impairs the association of HP1α with heterochromatin, because HP1α associates with KAP1 in heterochromatin. Intriguingly, tyrosine phosphorylation of KAP1 decreases the association of HP1α with heterochromatin, which is inhibited by replacement of endogenous KAP1 with its phenylalanine mutant (KAP1-Y449F/Y458F/Y517F, KAP1–3YF). In DNA damage, KAP1–3YF repressed transcription of p21. These results suggest that nucleus-localized tyrosine kinases, including SFKs, phosphorylate KAP1 at Tyr-449/Tyr-458/Tyr-517 and inhibit the association of KAP1 and HP1α with heterochromatin. PMID:23645696

Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix.

PubMed

Kubota, Sho; Morii, Mariko; Yuki, Ryuzaburo; Yamaguchi, Noritaka; Yamaguchi, Hiromi; Aoyama, Kazumasa; Kuga, Takahisa; Tomonaga, Takeshi; Yamaguchi, Naoto

2015-04-24

Protein-tyrosine phosphorylation regulates a wide variety of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine kinases induce global nuclear structure changes, which we called chromatin structural changes. However, the mechanisms are not fully understood. In this study we identify protein kinase A anchoring protein 8 (AKAP8/AKAP95), which associates with chromatin and the nuclear matrix, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of AKAP8 is induced by several tyrosine kinases, such as Src, Fyn, and c-Abl but not Syk. Nucleus-targeted Lyn and c-Src strongly dissociate AKAP8 from chromatin and the nuclear matrix in a kinase activity-dependent manner. The levels of tyrosine phosphorylation of AKAP8 are decreased by substitution of multiple tyrosine residues on AKAP8 into phenylalanine. Importantly, the phenylalanine mutations of AKAP8 inhibit its dissociation from nuclear structures, suggesting that the association/dissociation of AKAP8 with/from nuclear structures is regulated by its tyrosine phosphorylation. Furthermore, the phenylalanine mutations of AKAP8 suppress the levels of nuclear tyrosine kinase-induced chromatin structural changes. In contrast, AKAP8 knockdown increases the levels of chromatin structural changes. Intriguingly, stimulation with hydrogen peroxide induces chromatin structural changes accompanied by the dissociation of AKAP8 from nuclear structures. These results suggest that AKAP8 is involved in the regulation of chromatin structural changes through nuclear tyrosine phosphorylation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Second generation tyrosine kinase inhibitors prevent disease progression in high-risk (high CIP2A) chronic myeloid leukaemia patients.

PubMed

Lucas, C M; Harris, R J; Holcroft, A K; Scott, L J; Carmell, N; McDonald, E; Polydoros, F; Clark, R E

2015-07-01

High cancerous inhibitor of PP2A (CIP2A) protein levels at diagnosis of chronic myeloid leukaemia (CML) are predictive of disease progression in imatinib-treated patients. It is not known whether this is true in patients treated with second generation tyrosine kinase inhibitors (2G TKI) from diagnosis, and whether 2G TKIs modulate the CIP2A pathway. Here, we show that patients with high diagnostic CIP2A levels who receive a 2G TKI do not progress, unlike those treated with imatinib (P=<0.0001). 2G TKIs induce more potent suppression of CIP2A and c-Myc than imatinib. The transcription factor E2F1 is elevated in high CIP2A patients and following 1 month of in vivo treatment 2G TKIs suppress E2F1 and reduce CIP2A; these effects are not seen with imatinib. Silencing of CIP2A, c-Myc or E2F1 in K562 cells or CML CD34+ cells reactivates PP2A leading to BCR-ABL suppression. CIP2A increases proliferation and this is only reduced by 2G TKIs. Patients with high CIP2A levels should be offered 2G TKI treatment in preference to imatinib. 2G TKIs disrupt the CIP2A/c-Myc/E2F1 positive feedback loop, leading to lower disease progression risk. The data supports the view that CIP2A inhibits PP2Ac, stabilising E2F1, creating a CIP2A/c-Myc/E2F1 positive feedback loop, which imatinib cannot overcome.

Protein tyrosine phosphatases: Ligand interaction analysis and optimisation of virtual screening.

PubMed

Ghattas, Mohammad A; Atatreh, Noor; Bichenkova, Elena V; Bryce, Richard A

2014-07-01

Docking-based virtual screening is an established component of structure-based drug discovery. Nevertheless, scoring and ranking of computationally docked ligand libraries still suffer from many false positives. Identifying optimal docking parameters for a target protein prior to virtual screening can improve experimental hit rates. Here, we examine protocols for virtual screening against the important but challenging class of drug target, protein tyrosine phosphatases. In this study, common interaction features were identified from analysis of protein-ligand binding geometries of more than 50 complexed phosphatase crystal structures. It was found that two interactions were consistently formed across all phosphatase inhibitors: (1) a polar contact with the conserved arginine residue, and (2) at least one interaction with the P-loop backbone amide. In order to investigate the significance of these features on phosphatase-ligand binding, a series of seeded virtual screening experiments were conducted on three phosphatase enzymes, PTP1B, Cdc25b and IF2. It was observed that when the conserved arginine and P-loop amide interactions were used as pharmacophoric constraints during docking, enrichment of the virtual screen significantly increased in the three studied phosphatases, by up to a factor of two in some cases. Additionally, the use of such pharmacophoric constraints considerably improved the ability of docking to predict the inhibitor's bound pose, decreasing RMSD to the crystallographic geometry by 43% on average. Constrained docking improved enrichment of screens against both open and closed conformations of PTP1B. Incorporation of an ordered water molecule in PTP1B screening was also found to generally improve enrichment. The knowledge-based computational strategies explored here can potentially inform structure-based design of new phosphatase inhibitors using docking-based virtual screening. Copyright © 2014 Elsevier Inc. All rights reserved.

Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades

PubMed Central

Jiang, Cheng-shi; Liang, Lin-fu; Guo, Yue-wei

2012-01-01

This article provides an overview of approximately 300 secondary metabolites with inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), which were isolated from various natural sources or derived from synthetic process in the last decades. The structure-activity relationship and the selectivity of some compounds against other protein phosphatases were also discussed. Potential pharmaceutical applications of several PTP1B inhibitors were presented. PMID:22941286

Effect of angiotensin II type 2 receptor on tyrosine kinase Pyk2 and c-Jun NH2-terminal kinase via SHP-1 tyrosine phosphatase activity: evidence from vascular-targeted transgenic mice of AT2 receptor.

PubMed

Matsubara, H; Shibasaki, Y; Okigaki, M; Mori, Y; Masaki, H; Kosaki, A; Tsutsumi, Y; Uchiyama, Y; Fujiyama, S; Nose, A; Iba, O; Tateishi, E; Hasegawa, T; Horiuchi, M; Nahmias, C; Iwasaka, T

2001-04-20

Angiotensin II (Ang II) has two major receptor isoforms, AT1 and AT2. AT1 transphosphorylates Ca(2+)-sensitive tyrosine kinase Pyk2 to activate c-Jun NH2-terminal kinase (JNK). Although AT2 inactivates extracellular signal-regulated kinase (ERK) via tyrosine phosphatases (PTP), the action of AT2 on Pyk2 and JNK remains undefined. Using AT2-overexpressing vascular smooth muscle cells (AT2-VSMC) from AT2-transgenic mice, we studied these undefined actions of AT2. AT1-mediated JNK activity was increased 2.2-fold by AT2 inhibition, which was abolished by orthovanadate. AT2 did not affect AT1-mediated Pyk2 phosphorylation, but attenuated c-Jun mRNA accumulation by 32%. The activity of src-homology 2 domain-containing PTP (SHP-1) was significantly upregulated 1 min after AT2 stimulation. Stable overexpression of SHP-1 dominant negative mutant in AT2-VSMC completely abolished AT2-mediated inhibition of JNK activation and c-Jun expression. These findings suggest that AT2 inhibits JNK activity by affecting the downstream signal of Pyk2 in a SHP-1-dependent manner, leading to a decrease in c-Jun expression. Copyright 2001 Academic Press.

Structural Basis for Activation of ZAP-70 by Phosphorylation of the SH2-Kinase Linker

PubMed Central

Yan, Qingrong; Barros, Tiago; Visperas, Patrick R.; Deindl, Sebastian; Kadlecek, Theresa A.; Weiss, Arthur

2013-01-01

Serial activation of the tyrosine kinases Lck and ZAP-70 initiates signaling downstream of the T cell receptor. We previously reported the structure of an autoinhibited ZAP-70 variant in which two regulatory tyrosine residues (315 and 319) in the SH2-kinase linker were replaced by phenylalanine. We now present a crystal structure of ZAP-70 in which Tyr 315 and Tyr 319 are not mutated, leading to the recognition of a five-residue sequence register error in the SH2-kinase linker of the original crystallographic model. The revised model identifies distinct roles for these two tyrosines. As seen in a recently reported structure of the related tyrosine kinase Syk, Tyr 315 of ZAP-70 is part of a hydrophobic interface between the regulatory apparatus and the kinase domain, and the integrity of this interface would be lost upon engagement of doubly phosphorylated peptides by the SH2 domains. Tyr 319 is not necessarily dislodged by SH2 engagement, which activates ZAP-70 only ∼5-fold in vitro. In contrast, phosphorylation by Lck activates ZAP-70 ∼100-fold. This difference is due to the ability of Tyr 319 to suppress ZAP-70 activity even when the SH2 domains are dislodged from the kinase domain, providing stringent control of ZAP-70 activity downstream of Lck. PMID:23530057

Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker.

PubMed

Yan, Qingrong; Barros, Tiago; Visperas, Patrick R; Deindl, Sebastian; Kadlecek, Theresa A; Weiss, Arthur; Kuriyan, John

2013-06-01

Serial activation of the tyrosine kinases Lck and ZAP-70 initiates signaling downstream of the T cell receptor. We previously reported the structure of an autoinhibited ZAP-70 variant in which two regulatory tyrosine residues (315 and 319) in the SH2-kinase linker were replaced by phenylalanine. We now present a crystal structure of ZAP-70 in which Tyr 315 and Tyr 319 are not mutated, leading to the recognition of a five-residue sequence register error in the SH2-kinase linker of the original crystallographic model. The revised model identifies distinct roles for these two tyrosines. As seen in a recently reported structure of the related tyrosine kinase Syk, Tyr 315 of ZAP-70 is part of a hydrophobic interface between the regulatory apparatus and the kinase domain, and the integrity of this interface would be lost upon engagement of doubly phosphorylated peptides by the SH2 domains. Tyr 319 is not necessarily dislodged by SH2 engagement, which activates ZAP-70 only ∼5-fold in vitro. In contrast, phosphorylation by Lck activates ZAP-70 ∼100-fold. This difference is due to the ability of Tyr 319 to suppress ZAP-70 activity even when the SH2 domains are dislodged from the kinase domain, providing stringent control of ZAP-70 activity downstream of Lck.

Tyrosine Kinase Signaling in Clear Cell and Papillary Renal Cell Carcinoma Revealed by Mass Spectrometry-Based Phosphotyrosine Proteomics

PubMed Central

Haake, Scott M.; Li, Jiannong; Bai, Yun; Kinose, Fumi; Fang, Bin; Welsh, Eric; Zent, Roy; Dhillon, Jasreman; Pow-Sang, Julio; Chen, Yian Ann; Koomen, John; Rathmell, W. Kimryn; Fishman, Mayer; Haura, Eric B.

2016-01-01

Purpose Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes. Experimental Design Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. In order to describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry-based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. Results Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, while DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. Conclusions Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology amongst kidney cancer subtypes. PMID:27220961

SH2 domain-containing protein tyrosine phosphatase 2 and focal adhesion kinase protein interactions regulate pulmonary endothelium barrier function.

PubMed

Chichger, Havovi; Braza, Julie; Duong, Huetran; Harrington, Elizabeth O

2015-06-01

Enhanced protein tyrosine phosphorylation is associated with changes in vascular permeability through formation and dissolution of adherens junctions and regulation of stress fiber formation. Inhibition of the protein tyrosine phosphorylase SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) increases tyrosine phosphorylation of vascular endothelial cadherin and β-catenin, resulting in disruption of the endothelial monolayer and edema formation in the pulmonary endothelium. Vascular permeability is a hallmark of acute lung injury (ALI); thus, enhanced SHP2 activity offers potential therapeutic value for the pulmonary vasculature in diseases such as ALI, but this has not been characterized. To assess whether SHP2 activity mediates protection against edema in the endothelium, we assessed the effect of molecular activation of SHP2 on lung endothelial barrier function in response to the edemagenic agents LPS and thrombin. Both LPS and thrombin reduced SHP2 activity, correlated with decreased focal adhesion kinase (FAK) phosphorylation (Y(397) and Y(925)) and diminished SHP2 protein-protein associations with FAK. Overexpression of constitutively active SHP2 (SHP2(D61A)) enhanced baseline endothelial monolayer resistance and completely blocked LPS- and thrombin-induced permeability in vitro and significantly blunted pulmonary edema formation induced by either endotoxin (LPS) or Pseudomonas aeruginosa exposure in vivo. Chemical inhibition of FAK decreased SHP2 protein-protein interactions with FAK concomitant with increased permeability; however, overexpression of SHP2(D61A) rescued the endothelium and maintained FAK activity and FAK-SHP2 protein interactions. Our data suggest that SHP2 activation offers the pulmonary endothelium protection against barrier permeability mediators downstream of the FAK signaling pathway. We postulate that further studies into the promotion of SHP2 activation in the pulmonary endothelium may offer a therapeutic approach for patients suffering from ALI.

The IRS-1 signaling system.

PubMed

Myers, M G; Sun, X J; White, M F

1994-07-01

Insulin-receptor substrate 1 (IRS-1) is a principal substrate of the receptor tyrosine kinase for insulin and insulin-like growth factor 1, and a substrate for a tyrosine kinase activated by interleukin 4. IRS-1 undergoes multisite tyrosine phosphorylation and mediates downstream signals by 'docking' various proteins that contain Src homology 2 domains. IRS-1 appears to be a unique molecule; however, 4PS, a protein found mainly in hemopoietic cells, may represent another member of this family.

Angiotensin II mediated signal transduction. Important role of tyrosine kinases.

PubMed

Haendeler, J; Berk, B C

2000-11-24

It has been 100 years since the discovery of renin by Bergman and Tigerstedt. Since then, numerous studies have advanced our understanding of the renin-angiotensin system. A remarkable aspect was the discovery that angiotensin II (AngII) is the central product of the renin-angiotensin system and that this octapeptide induces multiple physiological responses in different cell types. In addition to its well known vasoconstrictive effects, growing evidence supports the notion that AngII may play a central role not only in hypertension, but also in cardiovascular and renal diseases. Binding of AngII to the seven-transmembrane angiotensin II type 1 receptor is responsible for nearly all of the physiological actions of AngII. Recent studies underscore the new concept that activation of intracellular second messengers by AngII requires tyrosine phosphorylation. An increasing number of tyrosine kinases have been shown to be activated by AngII, including the Src kinase family, the focal adhesion kinase family, the Janus kinases and receptor tyrosine kinases. These actions of AngII contribute to the pathophysiology of cardiac hypertrophy and remodeling, vascular thickening, heart failure and atherosclerosis. In this review, we discuss the important role of tyrosine kinases in AngII-mediated signal transduction. Understanding the importance of tyrosine phosphorylation in AngII-stimulated signaling events may contribute to new therapies for cardiovascular and renal diseases.

Characterization of a yeast sporulation-specific P450 family protein, Dit2, using an in vitro assay to crosslink formyl tyrosine.

PubMed

Bemena, Leo D; Mukama, Omar; Wang, Ning; Gao, Xiao-Dong; Nakanishi, Hideki

2018-02-01

The outermost layer of the yeast Saccharomyces cerevisiae spore, termed the dityrosine layer, is primarily composed of bisformyl dityrosine. Bisformyl dityrosine is produced in the spore cytosol by crosslinking of two formyl tyrosine molecules, after which it is transported to the nascent spore wall and assembled into the dityrosine layer by an unknown mechanism. A P450 family protein, Dit2, is believed to mediate the crosslinking of bisformyl dityrosine molecules. To characterize Dit2 and gain insight into the biological process of dityrosine layer formation, we performed an in vitro assay to crosslink formyl tyrosine with using permeabilized cells. For an unknown reason, the production of bisformyl dityrosine could not be confirmed under our experimental conditions, but dityrosine was detected in acid hydrolysates of the reaction mixtures in a Dit2 dependent manner. Thus, Dit2 mediated the crosslinking of formyl tyrosine in vitro. Dityrosine was detected when formyl tyrosine, but not tyrosine, was used as a substrate and the reaction required NADPH as a cofactor. Intriguingly, apart from Dit2, we found that the spore wall, but not the vegetative cell wall, contains bisformyl dityrosine crosslinking activity. This activity may be involved in the assembly of the dityrosine layer. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif

PubMed Central

Lucey, Marie J.; Chen, Dongsheng; Lopez-Garcia, Jorge; Hart, Stephen M.; Phoenix, Fladia; Al-Jehani, Rajai; Alao, John P.; White, Roger; Kindle, Karin B.; Losson, Régine; Chambon, Pierre; Parker, Malcolm G.; Schär, Primo; Heery, David M.; Buluwela, Lakjaya; Ali, Simak

2005-01-01

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-α. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein–protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexes. PMID:16282588

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

PubMed

Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

2010-02-01

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

PubMed Central

Meiler, Eugenia; Nieto-Pelegrín, Elvira; Martinez-Quiles, Narcisa

2012-01-01

Background Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes. Methodology/Principal Findings In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation. Conclusions/Significance Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading. PMID:22479425

Tyrosine phosphorylation of LRP6 by Src and Fer inhibits Wnt/β-catenin signalling

PubMed Central

Chen, Qing; Su, Yi; Wesslowski, Janine; Hagemann, Anja I; Ramialison, Mirana; Wittbrodt, Joachim; Scholpp, Steffen; Davidson, Gary

2014-01-01

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of β-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6. Subject Categories Membrane & Intracellular Transport; Post-translational Modifications, Proteolysis & Proteomics PMID:25391905

Metal-catalyzed protein tyrosine nitration in biological systems.

PubMed

Campolo, Nicolás; Bartesaghi, Silvina; Radi, Rafael

2014-11-01

Protein tyrosine nitration is an oxidative postranslational modification that can affect protein structure and function. It is mediated in vivo by the production of nitric oxide-derived reactive nitrogen species (RNS), including peroxynitrite (ONOO(-)) and nitrogen dioxide ((•)NO₂). Redox-active transition metals such as iron (Fe), copper (Cu), and manganese (Mn) can actively participate in the processes of tyrosine nitration in biological systems, as they catalyze the production of both reactive oxygen species and RNS, enhance nitration yields and provide site-specificity to this process. Early after the discovery that protein tyrosine nitration can occur under biologically relevant conditions, it was shown that some low molecular weight transition-metal centers and metalloproteins could promote peroxynitrite-dependent nitration. Later studies showed that nitration could be achieved by peroxynitrite-independent routes as well, depending on the transition metal-catalyzed oxidation of nitrite (NO₂(-)) to (•)NO₂ in the presence of hydrogen peroxide. Processes like these can be achieved either by hemeperoxidase-dependent reactions or by ferrous and cuprous ions through Fenton-type chemistry. Besides the in vitro evidence, there are now several in vivo studies that support the close relationship between transition metal levels and protein tyrosine nitration. So, the contribution of transition metals to the levels of tyrosine nitrated proteins observed under basal conditions and, specially, in disease states related with high levels of these metal ions, seems to be quite clear. Altogether, current evidence unambiguously supports a central role of transition metals in determining the extent and selectivity of protein tyrosine nitration mediated both by peroxynitrite-dependent and independent mechanisms.

Genistein inhibits voltage-gated sodium currents in SCG neurons through protein tyrosine kinase-dependent and kinase-independent mechanisms.

PubMed

Jia, Zhanfeng; Jia, Yueqin; Liu, Boyi; Zhao, Zhiying; Jia, Qingzhong; Liang, Huiling; Zhang, Hailin

2008-08-01

Voltage-gated sodium channels play a crucial role in the initiation and propagation of neuronal action potentials. Genistein, an isoflavone phytoestrogen, has long been used as a broad-spectrum inhibitor of protein tyrosine kinases (PTK). In addition, genistein-induced modulation of ion channels has been described previously in the literature. In this study, we investigated the effect of genistein on voltage-gated sodium channels in rat superior cervical ganglia (SCG) neurons. The results show that genistein inhibits Na(+) currents in a concentration-dependent manner, with a concentration of half-maximal effect (IC(50)) at 9.1 +/- 0.9 microM. Genistein positively shifted the voltage dependence of activation but did not affect inactivation of the Na(+) current. The inactive genistein analog daidzein also inhibited Na(+) currents, but was less effective than genistein. The IC(50) for daidzein-induced inhibition was 20.7 +/- 0.1 microM. Vanadate, an inhibitor of protein tyrosine phosphatases, partially but significantly reversed genistein-induced inhibition of Na(+) currents. Other protein tyrosine kinase antagonists such as tyrphostin 23, an erbstatin analog, and PP2 all had small but significant inhibitory effects on Na(+) currents. Among all active and inactive tyrosine kinase inhibitors tested, genistein was the most potent inhibitor of Na(+) currents. These results suggest that genistein inhibits Na(+) currents in rat SCG neurons through two distinct mechanisms: protein tyrosine kinase-independent, and protein tyrosine kinase-dependent mechanisms. Furthermore, the Src kinase family may be involved in the basal phosphorylation of the Na(+) channel.

The discovery of a reciprocal relationship between tyrosine-kinase signaling and cullin neddylation.

PubMed

Friend, Samantha F; Peterson, Lisa K; Treacy, Eric; Stefanski, Adrianne L; Sosinowski, Tomasz; Pennock, Nathan D; Berger, Allison J; Winn, Virginia D; Dragone, Leonard L

2013-01-01

While neddylation is known to activate cullin (CUL)-RING ubiquitin ligases (CRLs), its role in regulating T cell signaling is poorly understood. Using the investigational NEDD8 activating enzyme (NAE) inhibitor, MLN4924, we found that neddylation negatively regulates T cell receptor (TCR) signaling, as its inhibition increases IL-2 production, T cell proliferation and Treg development in vitro. We also discovered that loss of CUL neddylation occurs upon TCR signaling, and CRLs negatively regulate IL-2 production. Additionally, we found that tyrosine kinase signaling leads to CUL deneddylation in multiple cell types. These studies indicate that CUL neddylation is a global regulatory mechanism for tyrosine kinase signaling.

Tyrosine kinase inhibition: A therapeutic target for the management of chronic-phase chronic myeloid leukemia

PubMed Central

Jabbour, Elias J; Cortes, Jorge E; Kantarjian, Hagop M

2014-01-01

Chronic myeloid leukemia (CML) is a hematologic neoplasm with a progressive, ultimately terminal, disease course. In most cases, CML arises owing to the aberrant formation of a chimeric gene for a constitutively active tyrosine kinase. Inhibition of the signaling activity of this kinase has proved to be a highly successful treatment target transforming the prognosis of patients with CML. New tyrosine kinase inhibitors (TKIs) continue to improve the management of CML, offering alternative options for those resistant to or intolerant of standard TKIs. Here we review the pathobiology of CML and explore emerging strategies to optimize the management of chronic-phase CML, particularly first-line treatment. PMID:24236822

A tyrosine-phosphorylated 55-kilodalton motility-associated bovine sperm protein is regulated by cyclic adenosine 3',5'-monophosphates and calcium.

PubMed

Vijayaraghavan, S; Trautman, K D; Goueli, S A; Carr, D W

1997-06-01

Sperm motility is regulated by protein phosphorylation. We have recently shown that a serine/threonine phosphatase system is involved in motility regulation. Two of the components of the phosphatase system, GSK-3 and PP1gamma2, are regulated by tyrosine phosphorylation. During our investigation of sperm tyrosine-phosphorylated proteins we discovered a 55-kDa protein whose tyrosine phosphorylation correlates closely to the motility state of sperm. This protein is tyrosine phosphorylated to a much higher degree in motile caudal than in immotile caput epididymal sperm. Motility inhibition of caudal epididymal sperm by protein kinase A (PKA) anchoring inhibition or by ionomycin-induced calcium overload led to the virtual disappearance of tyrosine phosphorylation of the 55-kDa protein. Conversely, treatment of sperm with motility activators, isobutylmethylxanthine or 8-bromo-cAMP, resulted in increased tyrosine phosphorylation of the protein. The protein was present in the soluble 100 000 x g supernatants of sperm extracts and was heat labile. Chromatography through diethylaminoethyl-cellulose and Western blot analysis showed that this 55-kDa protein is not a regulatory subunit of PKA or alpha-tubulin. Our results represent the identification of a soluble protein whose tyrosine phosphorylation varies directly with motility and suggest that motility regulation may involve cross talk between PKA, calcium, and tyrosine kinase pathways.

Protein tyrosine nitration in pea roots during development and senescence

PubMed Central

Corpas, Francisco J.

2013-01-01

Protein tyrosine nitration is a post-translational modification mediated by reactive nitrogen species (RNS) that is associated with nitro-oxidative damage. No information about this process is available in relation to higher plants during development and senescence. Using pea plants at different developmental stages (ranging from 8 to 71 days), tyrosine nitration in the main organs (roots, stems, leaves, flowers, and fruits) was analysed using immunological and proteomic approaches. In the roots of 71-day-old senescent plants, nitroproteome analysis enabled the identification a total of 16 nitrotyrosine-immunopositive proteins. Among the proteins identified, NADP-isocitrate dehydrogenase (ICDH), an enzyme involved in the carbon and nitrogen metabolism, redox regulation, and responses to oxidative stress, was selected to evaluate the effect of nitration. NADP-ICDH activity fell by 75% during senescence. Analysis showed that peroxynitrite inhibits recombinant cytosolic NADP-ICDH activity through a process of nitration. Of the 12 tyrosines present in this enzyme, mass spectrometric analysis of nitrated recombinant cytosolic NADP-ICDH enabled this study to identify the Tyr392 as exclusively nitrated by peroxynitrite. The data as a whole reveal that protein tyrosine nitration is a nitric oxide-derived PTM prevalent throughout root development and intensifies during senescence. PMID:23362300

l-Tyrosine-loaded nanoparticles increase the antitumoral activity of direct electric current in a metastatic melanoma cell model

PubMed Central

de Campos, Vânia Emerich Bucco; Teixeira, Cesar Augusto Antunes; da Veiga, Venicio Feo; Júnior, Eduardo Ricci; Holandino, Carla

2010-01-01

Inhibition of tumor growth induced by treatment with direct electric current (DC) has been reported in several models. One of the mechanisms responsible for the antitumoral activity of DC is the generation of oxidative species, known as chloramines. With the aim of increasing chloramine production in the electrolytic medium and optimizing the antitumoral effects of DC, poly(ɛ-caprolactone) (PCL) nanoparticles (NPs) loaded with the amino acid tyrosine were obtained. The physical–chemical characterization showed that the NPs presented size in nanometric range and monomodal distribution. A slightly negative electrokinetic potential was also found in both blank NPs and l-tyrosine-loaded PCL NPs. The yield of the loading process was approximately 50%. Within 3 h of dissolution assay, a burst release of about 80% l-tyrosine was obtained. The in vitro cytotoxicity of DC was significantly increased when associated with l-tyrosine-loaded NPs, using a murine multidrug-resistant melanoma cell line model. This study showed that the use of the combination of nanotechnology and DC has a promising antineoplastic potential and opens a new perspective in cancer therapy. PMID:21187948

Molecular dynamics simulation reveals how phosphorylation of tyrosine 26 of phosphoglycerate mutase 1 upregulates glycolysis and promotes tumor growth.

PubMed

Wang, Yan; Cai, Wen-Sheng; Chen, Luonan; Wang, Guanyu

2017-02-14

Phosphoglycerate mutase 1 (PGAM1) catalyzes the eighth step of glycolysis and is often found upregulated in cancer cells. To test the hypothesis that the phosphorylation of tyrosine 26 residue of PGAM1 greatly enhances its activity, we performed both conventional and steered molecular dynamics simulations on the binding and unbinding of PGAM1 to its substrates, with tyrosine 26 either phosphorylated or not. We analyzed the simulated data in terms of structural stability, hydrogen bond formation, binding free energy, etc. We found that tyrosine 26 phosphorylation enhances the binding of PGAM1 to its substrates through generating electrostatic environment and structural features that are advantageous to the binding. Our results may provide valuable insights into computer-aided design of drugs that specifically target cancer cells with PGAM1 tyrosine 26 phosphorylated.

Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation

PubMed Central

Hoyt, Laura R.; Ather, Jennifer L.; Randall, Matthew J.; DePuccio, Daniel P.; Landry, Christopher C.; Wewers, Mark D.; Gavrilin, Mikhail A.; Poynter, Matthew E.

2016-01-01

Immunosuppression is a major complication of alcoholism that contributes to increased rates of opportunistic infections and sepsis in alcoholics. The NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the pro-inflammatory cytokines IL-1β and IL-18, can be inhibited by ethanol and we sought to better understand the mechanism through which this occurs and whether chemically similar molecules exert comparable effects. We show that ethanol can specifically inhibit activation of the NLRP3 inflammasome, resulting in attenuated IL-1β and caspase-1 cleavage and secretion, as well as diminished ASC speck formation, without affecting potassium efflux, in a mouse macrophage cell line (J774), mouse bone marrow derived dendritic cells, mouse neutrophils, and human PBMCs. The inhibitory effects on the Nlrp3 inflammasome were independent of GABAA receptor activation or NMDA receptor inhibition, but was associated with decreased oxidant production. Ethanol treatment markedly decreased cellular tyrosine phosphorylation, while administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored tyrosine phosphorylation and IL-1β secretion subsequent to ATP stimulation. Furthermore, sodium orthovanadate-induced phosphorylation of ASC Y144, necessary and sufficient for Nlrp3 inflammasome activation, and secretion of phosphorylated ASC, were inhibited by ethanol. Finally, multiple alcohol-containing organic compounds exerted inhibitory effects on the Nlrp3 inflammasome, whereas 2-methylbutane (isopentane), the analogous alkane of the potent inhibitor isoamyl alcohol (isopentanol), did not. Our results demonstrate that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation through the stimulation of protein tyrosine phosphatases, an effect shared by other short-chain alcohols. PMID:27421477

Dectin-1-mediated signaling leads to characteristic gene expressions and cytokine secretion via spleen tyrosine kinase (Syk) in rat mast cells.

PubMed

Kimura, Yukihiro; Chihara, Kazuyasu; Honjoh, Chisato; Takeuchi, Kenji; Yamauchi, Shota; Yoshiki, Hatsumi; Fujieda, Shigeharu; Sada, Kiyonao

2014-11-07

Dectin-1 recognizes β-glucan and plays important roles for the antifungal immunity through the activation of spleen tyrosine kinase (Syk) in dendritic cells or macrophages. Recently, expression of Dectin-1 was also identified in human and mouse mast cells, although its physiological roles were largely unknown. In this report, rat mast cell line RBL-2H3 was analyzed to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells. Treatment of cells with Dectin-1-specific agonist curdlan induced tyrosine phosphorylation of cellular proteins and the interaction of Dectin-1 with the Src homology 2 domain of Syk. These responses depended on tyrosine phosphorylation of the hemi-immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of Dectin-1, whereas they were independent of the γ-subunit of high-affinity IgE receptor. DNA microarray and real-time PCR analyses showed that Dectin-1-mediated signaling stimulated gene expression of transcription factor Nfkbiz and inflammatory cytokines, such as monocyte chemoattractant protein-1, IL-3, IL-4, IL-13, and tumor necrosis factor (TNF)-α. The response was abrogated by pretreatment with Syk inhibitor R406. These results suggest that Syk is critical for Dectin-1-mediated activation of mast cells, although the signaling differs from that triggered by FcϵRI activation. In addition, these gene expressions induced by curdlan stimulation were specifically observed in mast cells, suggesting that Dectin-1-mediated signaling of mast cells offers new insight into the antifungal immunity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation.

PubMed

Hoyt, Laura R; Ather, Jennifer L; Randall, Matthew J; DePuccio, Daniel P; Landry, Christopher C; Wewers, Mark D; Gavrilin, Mikhail A; Poynter, Matthew E

2016-08-15

Immunosuppression is a major complication of alcoholism that contributes to increased rates of opportunistic infections and sepsis in alcoholics. The NLRP3 inflammasome, a multiprotein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the proinflammatory cytokines IL-1β and IL-18, can be inhibited by ethanol, and we sought to better understand the mechanism through which this occurs and whether chemically similar molecules exert comparable effects. We show that ethanol can specifically inhibit activation of the NLRP3 inflammasome, resulting in attenuated IL-1β and caspase-1 cleavage and secretion, as well as diminished apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, without affecting potassium efflux, in a mouse macrophage cell line (J774), mouse bone marrow-derived dendritic cells, mouse neutrophils, and human PBMCs. The inhibitory effects on the Nlrp3 inflammasome were independent of γ-aminobutyric acid A receptor activation or N-methyl-d-asparate receptor inhibition but were associated with decreased oxidant production. Ethanol treatment markedly decreased cellular tyrosine phosphorylation, whereas administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored tyrosine phosphorylation and IL-1β secretion subsequent to ATP stimulation. Furthermore, sodium orthovanadate-induced phosphorylation of ASC Y144, necessary and sufficient for Nlrp3 inflammasome activation, and secretion of phosphorylated ASC were inhibited by ethanol. Finally, multiple alcohol-containing organic compounds exerted inhibitory effects on the Nlrp3 inflammasome, whereas 2-methylbutane (isopentane), the analogous alkane of the potent inhibitor isoamyl alcohol (isopentanol), did not. Our results demonstrate that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation through the stimulation of protein tyrosine phosphatases, an effect shared by other short-chain alcohols. Copyright © 2016 by The American Association of Immunologists, Inc.

SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

PubMed

Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

1997-10-31

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

Intermediate activity of midge antifreeze protein is due to a tyrosine-rich ice-binding site and atypical ice plane affinity.

PubMed

Basu, Koli; Wasserman, Samantha S; Jeronimo, Paul S; Graham, Laurie A; Davies, Peter L

2016-04-01

An antifreeze protein (AFP) from a midge (Chironomidae) was recently discovered and modelled as a tightly wound disulfide-braced solenoid with a surface-exposed rank of stacked tyrosines. New isoforms of the midge AFP have been identified from RT-PCR and are fully consistent with the model. Although they differ in the number of 10-residue coils, the row of tyrosines that form the putative ice-binding site is conserved. Recombinant midge AFP has been produced, and the properly folded form purified by ice affinity. This monomeric AFP has a distinct circular dichroism spectrum, a melting temperature between 35 and 50 °C and is fully renaturable on cooling. Mutagenesis of the middle tyrosine in the rank of seven eliminates antifreeze activity, whereas mutation of a tyrosine off this predicted ice-binding face had no such effect. This AFP has unusual properties compared to other known AFPs. First, its freezing-point depression activity is intermediate between that of the hyperactive and moderately active AFPs. As with hyperactive AFPs, when midge AFP-bound ice crystals exceed their freezing-point depression, ice grows explosively perpendicular to the c-axis. However, midge AFP does not bind to the basal plane of ice as do hyperactive AFPs, but rather to a pyramidal plane that is at a shallower angle relative to the basal plane than binding planes of moderate AFPs. These properties distinguish midge AFP from all other ice-binding proteins and the intermediate activity level fits well to the modest challenge of protecting newly emerged adult insects from late spring frosts. Nucleotide sequences of new midge AFP isoforms are available in the GenBank database under accession numbers KU094814-8. Sequences will be released after publication. © 2016 Federation of European Biochemical Societies.

Phosphatase of Regenerating Liver 3 (PRL3) Provokes a Tyrosine Phosphoproteome to Drive Prometastatic Signal Transduction*

PubMed Central

Walls, Chad D.; Iliuk, Anton; Bai, Yunpeng; Wang, Mu; Tao, W. Andy; Zhang, Zhong-Yin

2013-01-01

Phosphatase of regenerating liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when in excess. To date, the molecular basis for PRL3 function remains an enigma, making efforts at distilling a concerted mechanism for PRL3-mediated metastatic dissemination very difficult. We previously discovered that PRL3 expressing cells exhibit a pronounced increase in protein tyrosine phosphorylation. Here we take an unbiased mass spectrometry-based approach toward identifying the phosphoproteins exhibiting enhanced levels of tyrosine phosphorylation with a goal to define the “PRL3-mediated signaling network.” Phosphoproteomic data support intracellular activation of an extensive signaling network normally governed by extracellular ligand-activated transmembrane growth factor, cytokine, and integrin receptors in the PRL3 cells. Additionally, data implicate the Src tyrosine kinase as the major intracellular kinase responsible for “hijacking” this network and provide strong evidence that aberrant Src activation is a major consequence of PRL3 overexpression. Importantly, the data support a PDGF(α/β)-, Eph (A2/B3/B4)-, and Integrin (β1/β5)-receptor array as being the predominant network coordinator in the PRL3 cells, corroborating a PRL3-induced mesenchymal-state. Within this network, we find that tyrosine phosphorylation is increased on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, STAT, and ERK activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives prometastatic molecular events through Src activation. PMID:24030100

Tyrosine kinase inhibitors and immunosuppressants perturb the myo-inositol but not the betaine cotransporter in isotonic and hypertonic MDCK cells

PubMed Central

Atta, Mohamed G.; Dahl, Stephen C.; Kwon, H. Moo; Handler, Joseph S.

2008-01-01

Background The sodium/myo-inositol cotransporter (SMIT) and the betaine cotransporter (BGT1) are essential for the accumulation of myo-inositol and betaine, and hence cell survival in a hypertonic environment. The underlying molecular mechanism involves an increase in transcription of the SMIT and BGT1 genes through binding of a trans-acting factor to enhancer elements in the 5′ flanking region of both genes, resulting in increased mRNA abundance and increased activity of the cotransporters. Current evidence regarding transcriptional and post-transcriptional regulation indicates that both cotransporters are regulated in parallel. Methods To investigate the signal transduction of hypertonic stress, we examined the effect of tyrosine kinase inhibitors and immunosuppressants on the hypertonicity-induced activity of the two cotransporters in Madin-Darby canine kidney (MDCK) cells. Results None of the agents studied affected BGT1 activity in isotonic or hypertonic conditions. Treatment of MDCK cells with genistein, a tyrosine kinase inhibitor, increased SMIT activity in hypertonic but not isotonic conditions. The stimulation of SMIT by genistein was accompanied by a parallel increase in mRNA abundance. In contrast, treating cells with tyrphostin A23, another tyrosine kinase inhibitor, or cyclosporine A, an immunosuppressant, inhibited SMIT activity in hypertonic cells. FK506, another immunosuppressant, increased SMIT activity, but only in isotonic conditions. Conclusions These results provide the first evidence of divergent regulatory pathways modulating SMIT and BGT activity. PMID:10027932

[Changes of protein tyrosine phosphorylation in erythrocyte band 3 glucose-6-phosphate dehydrogenase deficiency].

PubMed

Yu, Guoyu; Li, Jialin; Tian, Xingya; Lin, Hong; Wang, Xiaoying

2002-11-01

To explore the hemolytic mechanism of glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes in the view of phosphorylation of membrane protein. The alternation of membrane protein phosphorylation and the effect of dithiothreitol (DTT) on protein phosphorylation were analysed by Western blot technique. The activity of phosphotyrosine phosphatase (PTPs) was determined by using p-nitrophenyl phosphate as substrate. Tyrosine phosphorylation of band 3 protein was obviously enhanced in G6PD-deficient erythrocytes. The activity of PTPs was low compared to the normal erythrocytes. The level of phosphotyrosine in G6PD-deficient erythrocytes incubated with DTT was almost the same as in those without DTT. The results were consistent with the activity of PTPs. PTPs activity reduction and tyrosine phosphorylation enhancement induced by oxidation in G6PD deficiency play an important role in erythrocytes hemolysis. However, the alternation of thiol group is not the only factor affecting the activity of PTPs in G6PD-deficient erythrocytes.

ROLE OF TYROSINE-SULFATED PROTEINS IN RETINAL STRUCTURE AND FUNCTION

PubMed Central

Kanan, Y.; Al-Ubaidi, M.R.

2014-01-01

The extracellular matrix (ECM) plays a significant role in cellular and retinal health. The study of retinal tyrosine-sulfated proteins is an important first step toward understanding the role of ECM in retinal health and diseases. These secreted proteins are members of the retinal ECM. Tyrosine sulfation was shown to be necessary for the development of proper retinal structure and function. The importance of tyrosine sulfation is further demonstrated by the evolutionary presence of tyrosylprotein sulfotransferases, enzymes that catalyze proteins’ tyrosine sulfation, and the compensatory abilities of these enzymes. Research has identified four tyrosine-sulfated retinal proteins: fibulin 2, vitronectin, complement factor H (CFH), and opticin. Vitronectin and CFH regulate the activation of the complement system and are involved in the etiology of some cases of age-related macular degeneration. Analysis of the role of tyrosine sulfation in fibulin function showed that sulfation influences the protein's ability to regulate growth and migration. Although opticin was recently shown to exhibit anti-angiogenic properties, it is not yet determined what role sulfation plays in that function. Future studies focusing on identifying all of the tyrosine-sulfated retinal proteins would be instrumental in determining the impact of sulfation on retinal protein function in retinal homeostasis and diseases. PMID:25819460

Small Molecule Reversible Inhibitors of Bruton’s Tyrosine Kinase (BTK): Structure–Activity Relationships Leading to the Identification of 7-(2-Hydroxypropan-2-yl)-4-[2-methyl-3-(4-oxo-3,4-dihydroquinazolin-3-yl)phenyl]-9 H -carbazole-1-carboxamide (BMS-935177)

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Lucca, George V.; Shi, Qing; Liu, Qingjie

Bruton’s tyrosine kinase (BTK) belongs to the TEC family of nonreceptor tyrosine kinases and plays a critical role in multiple cell types responsible for numerous autoimmune diseases. This article will detail the structure–activity relationships (SARs) leading to a novel second generation series of potent and selective reversible carbazole inhibitors of BTK. With an excellent pharmacokinetic profile as well as demonstrated in vivo activity and an acceptable safety profile, 7-(2-hydroxypropan-2-yl)-4-[2-methyl-3-(4-oxo-3,4-dihydroquinazolin-3-yl)phenyl]-9H-carbazole-1-carboxamide 6 (BMS-935177) was selected to advance into clinical development.

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

PubMed Central

Dubreuil, Patrice; Letard, Sébastien; Ciufolini, Marco; Gros, Laurent; Humbert, Martine; Castéran, Nathalie; Borge, Laurence; Hajem, Bérengère; Lermet, Anne; Sippl, Wolfgang; Voisset, Edwige; Arock, Michel; Auclair, Christian; Leventhal, Phillip S.; Mansfield, Colin D.; Moussy, Alain; Hermine, Olivier

2009-01-01

Background The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. Methodology/Principal Findings In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. Conclusions Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity. PMID:19789626

YAP Tyrosine Phosphorylation and Nuclear Localization in Cholangiocarcinoma Cells is Regulated by LCK and Independent of LATS Activity.

PubMed

Sugihara, Takaaki; Werneburg, Nathan W; Hernandez, Matthew C; Yang, Lin; Kabashima, Ayano; Hirsova, Petra; Yohanathan, Lavanya; Sosa, Carlos; Truty, Mark Joseph; Vasmatzis, George; Gores, Gregory J; Smoot, Rory L

2018-06-14

The hippo pathway effector, Yes-associated protein (YAP) is a transcriptional co-activator implicated in cholangiocarcinoma (CCA) pathogenesis. YAP is known to be regulated by a serine/threonine kinase relay module (MST1/2 - LATS1/2) culminating in phosphorylation of YAP at Serine 127 (S127) and cytoplasmic sequestration. However, YAP also undergoes tyrosine phosphorylation, and the role of tyrosine phosphorylation in YAP regulation remains unclear. Herein, YAP regulation by tyrosine phosphorylation was examined in human and mouse CCA cells, as well as patient-derived xenograft (PDX) models. YAP was phosphorylated on tyrosine 357 (Y357) in CCA cell lines and PDX models. SRC family kinase (SFK) inhibition with dasatinib resulted in loss of YAPY357 phosphorylation, promoted its translocation from the nucleus to the cytoplasm, and reduced YAP target gene expression; including cell lines expressing a LATS1/2-resistant YAP mutant in which all serine residues were mutated to alanine. Consistent with these observations, precluding YAPY357 phosphorylation by site-directed mutagenesis (YAPY357F) excluded YAP from the nucleus. Targeted siRNA experiments identified LCK as the SFK that most potently mediated YAPY357 phosphorylation. Likewise, inducible CRISPR/Cas9-targeted LCK deletion decreased YAPY357 phosphorylation and its nuclear localization. The importance of LCK in CCA biology was demonstrated by clinical observations suggesting LCK expression levels were associated with early tumor recurrence following resection of CCA. Finally, dasatinib displayed therapeutic efficacy in PDX models. Demonstration of targetable, LCK-mediated YAP tyrosine phosphorylation in CCA regulating YAP's nuclear retention and oncogenic activity. Copyright ©2018, American Association for Cancer Research.

Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling

PubMed Central

Holopainen, Tanja; Räsänen, Markus; Anisimov, Andrey; Tuomainen, Tomi; Zheng, Wei; Tvorogov, Denis; Hulmi, Juha J.; Andersson, Leif C.; Cenni, Bruno; Tavi, Pasi; Mervaala, Eero; Kivelä, Riikka; Alitalo, Kari

2015-01-01

Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy. PMID:26430242

EGF-dependent re-routing of vesicular recycling switches spontaneous phosphorylation suppression to EGFR signaling

PubMed Central

Baumdick, Martin; Brüggemann, Yannick; Schmick, Malte; Xouri, Georgia; Sabet, Ola; Davis, Lloyd; Chin, Jason W; Bastiaens, Philippe IH

2015-01-01

Autocatalytic activation of epidermal growth factor receptor (EGFR) coupled to dephosphorylating activity of protein tyrosine phosphatases (PTPs) ensures robust yet diverse responses to extracellular stimuli. The inevitable tradeoff of this plasticity is spontaneous receptor activation and spurious signaling. We show that a ligand-mediated switch in EGFR trafficking enables suppression of spontaneous activation while maintaining EGFR’s capacity to transduce extracellular signals. Autocatalytic phosphorylation of tyrosine 845 on unliganded EGFR monomers is suppressed by vesicular recycling through perinuclear areas with high PTP1B activity. Ligand-binding results in phosphorylation of the c-Cbl docking tyrosine and ubiquitination of the receptor. This secondary signal relies on EGF-induced EGFR self-association and switches suppressive recycling to directional trafficking. The re-routing regulates EGFR signaling response by the transit-time to late endosomes where it is switched-off by high PTP1B activity. This ubiquitin-mediated switch in EGFR trafficking is a uniquely suited solution to suppress spontaneous activation while maintaining responsiveness to EGF. DOI: http://dx.doi.org/10.7554/eLife.12223.001 PMID:26609808

Presence of ecto-protein tyrosine phosphatase activity is vital for survival of Setaria cervi, a bovine filarial parasite.

PubMed

Singh, Neetu; Heneberg, Petr; Rathaur, Sushma

2014-10-01

The ecto protein tyrosine phosphatases (PTP) are known to play a crucial role in the pathogenesis and survival of the intracellular parasites. However, their presence and role in filarial parasites is still unknown. We found a significant amount of tyrosine phosphatase activity in the surface antigen fraction extracted from Setaria cervi (S. cervi), a bovine filarial parasite. An antibody designed against the conserved catalytic core of human protein tyrosine phosphatases, PTP1B cross reacted with a 63 kDa band in the surface antigen. We detected a significant amount of PTP activity in the intact S. cervi adult parasites as well as microfilariae in this study for the first time. This PTP may be localized on the surface of the parasite with an exposed active site available for the external substrates. The PTP activity was also inhibited by sodium orthovanadate and phenyl arsine oxide, specific inhibitors of PTP in both the life stages. The Km and Vmax for PTP in the adult parasites and microfilariae were determined to be 2.574 ± 0.14 mM; 206.3 ± 2.75 μM Pi/h/two parasites and 5.510 ± 0.59 mM; 62.27 ± 2.27 μM Pi/h/10(6) parasites respectively using O-P-L-Tyrosine as substrate. Interestingly, a positive correlation was observed between the inhibition in PTP activity and reduction in the motility/ viability of the parasites when they were subjected to the specific PTP inhibitors (Orthovanadate and Phenyl arsine oxide) for 4 h in the KRB maintenance medium. The activity was also significantly inhibited in the parasites exposed to antifilarial drug/compounds for e.g. Diethylcarbamazine, Acetylsalicylic Acid and SK7, a methyl chalcone. Therefore suggesting a possible role played by PTP in the survival of the parasite, its interaction with the host as well as in the screening of newly synthesized antifilarials/drugs.

Pseudomonas aeruginosa 4-Amino-4-Deoxychorismate Lyase: Spatial Conservation of an Active Site Tyrosine and Classification of Two Types of Enzyme

PubMed Central

O'Rourke, Patrick E. F.; Eadsforth, Thomas C.; Fyfe, Paul K.; Shepherd, Sharon M.; Hunter, William N.

2011-01-01

4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp 2 to sp 3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism. PMID:21935381

3D MHD Models of Active Region Loops

NASA Technical Reports Server (NTRS)

Ofman, Leon

2004-01-01

Present imaging and spectroscopic observations of active region loops allow to determine many physical parameters of the coronal loops, such as the density, temperature, velocity of flows in loops, and the magnetic field. However, due to projection effects many of these parameters remain ambiguous. Three dimensional imaging in EUV by the STEREO spacecraft will help to resolve the projection ambiguities, and the observations could be used to setup 3D MHD models of active region loops to study the dynamics and stability of active regions. Here the results of 3D MHD models of active region loops are presented, and the progress towards more realistic 3D MHD models of active regions. In particular the effects of impulsive events on the excitation of active region loop oscillations, and the generation, propagations and reflection of EIT waves are shown. It is shown how 3D MHD models together with 3D EUV observations can be used as a diagnostic tool for active region loop physical parameters, and to advance the science of the sources of solar coronal activity.

Early intervention of tyrosine nitration prevents vaso-obliteration and neovascularization in ischemic retinopathy.

PubMed

Abdelsaid, Mohammed A; Pillai, Bindu A; Matragoon, Suraporn; Prakash, Roshini; Al-Shabrawey, Mohamed; El-Remessy, Azza B

2010-01-01

Diabetic retinopathy and retinopathy of prematurity are blinding disorders that follow a pathological pattern of ischemic retinopathy and affect premature infants and working-age adults. Yet, the treatment options are limited to laser photocoagulation. The goal of this study is to elucidate the molecular mechanism and examine the therapeutic effects of inhibiting tyrosine nitration on protecting early retinal vascular cell death and late neovascularization in the ischemic retinopathy model. Ischemic retinopathy was developed by exposing neonatal mice to 75% oxygen [postnatal day (p) 7-p12] followed by normoxia (21% oxygen) (p12-p17). Peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride (FeTPPS) (1 mg/kg), the nitration inhibitor epicatechin (10 mg/kg) or the thiol donor N-acetylcysteine (NAC, 150 mg/kg) were administered (p7-p12) or (p7-p17). Vascular endothelial cells were incubated at hyperoxia (40% oxygen) or normoxia (21% oxygen) for 48 h. Vascular density was determined in retinal flat mounts labeled with isolectin B4. Expression of vascular endothelial growth factor, caspase-3, and poly(ADP ribose) polymerase (PARP), activation of Akt and p38 mitogen-activated protein kinase (MAPK), and tyrosine nitration of the phosphatidylinositol (PI) 3-kinase p85 subunit were analyzed by Western blot. Hyperoxia-induced peroxynitrite caused endothelial cell apoptosis as indicated by expression of cleaved caspase-3 and PARP leading to vaso-obliteration. These effects were associated with significant tyrosine nitration of the p85 subunit of PI 3-kinase, decreased Akt activation, and enhanced p38 MAPK activation. Blocking tyrosine nitration of PI 3-kinase with epicatechin or NAC restored Akt phosphorylation, and inhibited vaso-obliteration at p12 and neovascularization at p17 comparable with FeTPPS. Early inhibition of tyrosine nitration with use of epicatechin or NAC can represent safe and effective vascular-protective agents in ischemic retinopathy.

A new precursor for the preparation of 6-[18F]-fluoro-L-m-tyrosine (FMT): Efficient synthesis and comparison of radiolabeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

VanBrocklin, Henry F.; Blagoev, Milan; Hoepping, Alexander

For the electrophilic preparation of 6-[18F]-Fluoro-L-m-tyrosine (FMT), a PET tracer for measuring changes in dopaminergic function in movement disorders, a novel precursor, N-(tert-butoxycarbonyl)-3-(tert-butoxycarbonyloxy)-6-trimethylstannnyl-L-phenylalanine ethyl ester, was synthesized in four steps and 26 percent yield starting from L-m-tyrosine. FMT produced by two methods at two institutions was comparable in decay corrected yield, 25-26 percent, and quality (chemical, enantiomeric, and radiochemical purity and specific activity) as that obtained with the original N-trifluoroacetyl-3-acetyl-6-trimethylstannyl-L-m-tyrosine ethyl ester FMT precursor.

The activity and stability of the intrinsically disordered Cip/Kip protein family are regulated by non-receptor tyrosine kinases

PubMed Central

Otieno, Steve; Lelli, Moreno; Kriwacki, Richard W.

2014-01-01

The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21Cip1, p27Kip1 and p57Kip2. Their kinase inhibitory activities are mediated by a homologous N-terminal kinase-inhibitory domain (KID). The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the KID and a threonine residue within the flexible C-terminus. We show that these residues are conserved in p21 and p57, suggesting that a similar phosphorylation cascade regulates these Cdk inhibitors. However, the presence of a cyclin binding motif within its C-terminus alters the regulatory interplay between p21 and Cdk2/cyclin A, and its responses to tyrosine phosphorylation and altered p21:Cdk2/cyclin A stoichiometry. We also show that the Cip/Kip proteins can be phosphorylated in vitro by representatives of many non-receptor tyrosine kinase (NRTK) sub-families, suggesting that NRTKs may generally regulate the activity and stability of these Cdk inhibitors. Our results further suggest that the Cip/Kip proteins integrate signals from various NRTK pathways and cell cycle regulation. PMID:25463440

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells

PubMed Central

Sela, Meirav; Bogin, Yaron; Beach, Dvora; Oellerich, Thomas; Lehne, Johanna; Smith-Garvin, Jennifer E; Okumura, Mariko; Starosvetsky, Elina; Kosoff, Rachelle; Libman, Evgeny; Koretzky, Gary; Kambayashi, Taku; Urlaub, Henning; Wienands, Jürgen; Chernoff, Jonathan; Yablonski, Deborah

2011-01-01

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1. PMID:21725281

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells.

PubMed

Sela, Meirav; Bogin, Yaron; Beach, Dvora; Oellerich, Thomas; Lehne, Johanna; Smith-Garvin, Jennifer E; Okumura, Mariko; Starosvetsky, Elina; Kosoff, Rachelle; Libman, Evgeny; Koretzky, Gary; Kambayashi, Taku; Urlaub, Henning; Wienands, Jürgen; Chernoff, Jonathan; Yablonski, Deborah

2011-07-01

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1.

Characterization of two mosquito STATs, AaSTAT and CtSTAT. Differential regulation of tyrosine phosphorylation and DNA binding activity by lipopolysaccharide treatment and by Japanese encephalitis virus infection.

PubMed

Lin, Chang-Chi; Chou, Chih-Ming; Hsu, Ya-Li; Lien, Jih-Ching; Wang, Yu-Ming; Chen, Shui-Tsung; Tsai, Shu-Chuan; Hsiao, Pei-Wen; Huang, Chang-Jen

2004-01-30

Two mosquito STATs, AaSTAT and CtSTAT, have been cloned from Aedes albopictus and Culex tritaeniorhynchus mosquitoes, respectively. These two STATs are more similar to those of Drosophila, Anopheles, and mammalian STAT5 in the DNA binding and Src homology 2 domains. The mRNA transcripts are expressed at all developmental stages, and the proteins are present predominantly at the pupal and adult stages in both mosquitoes. Stimulation with lipopolysaccharide resulted in an increase of tyrosine phosphorylation and DNA binding activity of AaSTAT and CtSTAT as well as an increase of luciferase activity of a reporter gene containing Drosophila STAT binding motif in mosquito C6/36 cells. After being infected with Japanese encephalitis virus, nuclear extracts of C6/36 cells revealed a decrease of tyrosine phosphorylation and DNA binding activity of AaSTAT which could be restored by sodium orthovanadate treatment. Taking all of the data together, this is the first report to clone and characterize two mosquito STATs with 81% identity and to demonstrate a different response of tyrosine phosphorylation and DNA binding of these two STATs by lipopolysaccharide treatment and by Japanese encephalitis virus infection.

The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

PubMed

Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

1993-05-06

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

The activity and stability of the intrinsically disordered Cip/Kip protein family are regulated by non-receptor tyrosine kinases.

PubMed

Huang, Yongqi; Yoon, Mi-Kyung; Otieno, Steve; Lelli, Moreno; Kriwacki, Richard W

2015-01-30

The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21(Cip1), p27(Kip1) and p57(Kip2). Their kinase inhibitory activities are mediated by a homologous N-terminal kinase inhibitory domain. The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the kinase inhibitory domain and a threonine residue within the flexible C-terminus. We show that these residues are conserved in p21 and p57, suggesting that a similar phosphorylation cascade regulates these Cdk inhibitors. However, the presence of a cyclin binding motif within its C-terminus alters the regulatory interplay between p21 and Cdk2/cyclin A, as well as its responses to tyrosine phosphorylation and altered p21:Cdk2/cyclin A stoichiometry. We also show that the Cip/Kip proteins can be phosphorylated in vitro by representatives of many non-receptor tyrosine kinase (NRTK) sub-families, suggesting that NRTKs may generally regulate the activity and stability of these Cdk inhibitors. Our results further suggest that the Cip/Kip proteins integrate signals from various NRTK pathways and cell cycle regulation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Maintenance of murine platelet homeostasis by the kinase Csk and phosphatase CD148

PubMed Central

Di Nunzio, Giada; Smith, Christopher W.; Al Ghaithi, Rashid; van Geffen, Johanna P.; Heising, Silke; Tullemans, Bibian M. E.; Tee, Louise; Heemskerk, Johan W. M.; Tarakhovsky, Alexander

2018-01-01

Src family kinases (SFKs) coordinate the initiating and propagating activation signals in platelets, but it remains unclear how they are regulated. Here, we show that ablation of C-terminal Src kinase (Csk) and receptor-like protein tyrosine-phosphatase CD148 in mice results in a dramatic increase in platelet SFK activity, demonstrating that these proteins are essential regulators of platelet reactivity. Paradoxically, Csk/CD148-deficient mice exhibit reduced in vivo and ex vivo thrombus formation and increased bleeding following injury rather than a prothrombotic phenotype. This is a consequence of multiple negative feedback mechanisms, including downregulation of the immunoreceptor tyrosine-based activation motif (ITAM)– and hemi-ITAM–containing receptors glycoprotein VI (GPVI)-Fc receptor (FcR) γ-chain and CLEC-2, respectively and upregulation of the immunoreceptor tyrosine-based inhibition motif (ITIM)–containing receptor G6b-B and its interaction with the tyrosine phosphatases Shp1 and Shp2. Results from an analog-sensitive Csk mouse model demonstrate the unconventional role of SFKs in activating ITIM signaling. This study establishes Csk and CD148 as critical molecular switches controlling the thrombotic and hemostatic capacity of platelets and reveals cell-intrinsic mechanisms that prevent pathological thrombosis from occurring. PMID:29301754

Activation of the protein-tyrosine kinase associated with the bombesin receptor complex in small cell lung carcinomas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaudino, G.; Cirillo, D.; Naldini, L.

1988-04-01

It has been hypothesized that bombesin-like peptides produced by small cell lung carcinomas may sustain deregulated proliferation through an autocrine mechanism. The authors have shown that the neuropeptide bombesin leads to the activation of a protein-tyrosine kinase that phosphorylates a 115-kDa protein (p115) associated with the bombesin receptor complex in mouse Swiss 3T3 fibroblasts. They now report that phosphotyrosine antibodies recognize a 115-kDa protein, phosphorylated on tyrosine, in four human small cell lung carcinoma cell lines producing bombesin but not in a nonproducer variant line. p115 from detergent-treated small cell lung carcinoma cells binds to bombesin-Sepharose and can be phosphorylatedmore » on tyrosine in the presence of radiolabeled ATP and Mn{sup 2+}. As for the p115 immunoprecipitated from mouse fibroblast, the small cell lung carcinoma p115 can be phosphorylated in an immunocomplex kinase assay. However, the latter does not require the presence of exogenous bombesin for activity. Binding data, obtained by using radiolabeled ligand, suggest receptor occupancy in the cell lines producing bombesin. These observations are consistent with the hypothesis that proliferation in some human small cell lung carcinoma lines is under autocrine control, regulated through activation of bombesin receptors.« less

BTK blocks the inhibitory effects of MDM2 on p53 activity

PubMed Central

Rada, Miran; Althubiti, Mohammad; Ekpenyong-Akiba, Akang E.; Lee, Koon-Guan; Lam, Kong Peng; Fedorova, Olga; Barlev, Nickolai A.; Macip, Salvador

2017-01-01

p53 is a tumour suppressor that is activated in response to various types of stress. It is regulated by a complex pattern of over 50 different post-translational modifications, including ubiquitination by the E3 ligase MDM2, which leads to its proteasomal degradation. We have previously reported that expression of Bruton’s Tyrosine Kinase (BTK) induces phosphorylation of p53 at the N-terminus, including Serine 15, and increases its protein levels and activity. The mechanisms involved in this process are not completely understood. Here, we show that BTK also increases MDM2 and is necessary for MDM2 upregulation after DNA damage, consistent with what we have shown for other p53 target genes. Moreover, we found that BTK binds to MDM2 on its PH domain and induces its phosphorylation. This suggested a negative regulation of MDM2 functions by BTK, supported by the fact BTK expression rescued the inhibitory effects of MDM2 on p53 transcriptional activity. Indeed, we observed that BTK mediated the loss of the ubiquitination activity of MDM2, a process that was dependent on the phosphorylation functions of BTK. Our data together shows that the kinase activity of BTK plays an important role in disrupting the MDM2-p53 negative feedback loop by acting at different levels, including binding to and inactivation of MDM2. This study provides a potential mechanism to explain how BTK modulates p53 functions. PMID:29290977

Ion channel TRPV1-dependent activation of PTP1B suppresses EGFR-associated intestinal tumorigenesis

PubMed Central

de Jong, Petrus R.; Takahashi, Naoki; Harris, Alexandra R.; Lee, Jihyung; Bertin, Samuel; Jeffries, James; Jung, Michael; Duong, Jen; Triano, Amy I.; Lee, Jongdae; Niv, Yaron; Herdman, David S.; Taniguchi, Koji; Kim, Chang-Whan; Dong, Hui; Eckmann, Lars; Stanford, Stephanie M.; Bottini, Nunzio; Corr, Maripat; Raz, Eyal

2014-01-01

The intestinal epithelium has a high rate of turnover, and dysregulation of pathways that regulate regeneration can lead to tumor development; however, the negative regulators of oncogenic events in the intestinal epithelium are not fully understood. Here we identified a feedback loop between the epidermal growth factor receptor (EGFR), a known mediator of proliferation, and the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), in intestinal epithelial cells (IECs). We found that TRPV1 was expressed by IECs and was intrinsically activated upon EGFR stimulation. Subsequently, TRPV1 activation inhibited EGFR-induced epithelial cell proliferation via activation of Ca2+/calpain and resulting activation of protein tyrosine phosphatase 1B (PTP1B). In a murine model of multiple intestinal neoplasia (ApcMin/+ mice), TRPV1 deficiency increased adenoma formation, and treatment of these animals with an EGFR kinase inhibitor reversed protumorigenic phenotypes, supporting a functional association between TRPV1 and EGFR signaling in IECs. Administration of a TRPV1 agonist suppressed intestinal tumorigenesis in ApcMin/+ mice, similar to — as well as in conjunction with — a cyclooxygenase-2 (COX-2) inhibitor, which suggests that targeting both TRPV1 and COX-2 has potential as a therapeutic approach for tumor prevention. Our findings implicate TRPV1 as a regulator of growth factor signaling in the intestinal epithelium through activation of PTP1B and subsequent suppression of intestinal tumorigenesis. PMID:25083990

[Tyrosine hydroxylase in telencephalon and diencephalon of Rhodeus sericeus (Cyprinidae)].

PubMed

Pushchina, E V

2009-01-01

Immunohistochemical labeling of tyrosine hydroxylase was used to demonstrate catecholaminergic neuronal populations in the telencephalon and diencephalonof adult cypryniform fish Rhodeus sericeus. Various immunoreactive cell populations have been found in the telencephalon (ventral, central and lateral nuclei of ventral telencephalic area). Immunoreactive cells and fibers were discovered in dorsal nucleus of ventral telencephalic area and supracomissural nucleus in the caudal part of the telencephalon. In the diencephalon, periventricular nuclei (preoptic, periventricular nucleus of posterior tuberculum and periventricular organ) contained considerable TH-ergic cells. High activity of tyrosine hydroxylase was revealed in the pretectal, ventro-medial, ventro-lateral and suprachiasmatic nuclei. Periventricular hypothalamic nuclei also displayed high activity of tyrosine hydroxylase. Pseudounipolar neurons prevailed in all TH-immunereactive structures of the telencephalon and diencephalon: numerous bipolar liquor-contacting cells were discovered in the periventricular nuclei. Large pear-shaped cells and bipolar TH-ergic cells were found in posterior tuberculum. These cells may be functionally related to the dopamine-acquiring system.

Ret function in muscle stem cells points to tyrosine kinase inhibitor therapy for facioscapulohumeral muscular dystrophy.

PubMed

Moyle, Louise A; Blanc, Eric; Jaka, Oihane; Prueller, Johanna; Banerji, Christopher Rs; Tedesco, Francesco Saverio; Harridge, Stephen Dr; Knight, Robert D; Zammit, Peter S

2016-11-14

Facioscapulohumeral muscular dystrophy (FSHD) involves sporadic expression of DUX4, which inhibits myogenesis and is pro-apoptotic. To identify target genes, we over-expressed DUX4 in myoblasts and found that the receptor tyrosine kinase Ret was significantly up-regulated, suggesting a role in FSHD. RET is dynamically expressed during myogenic progression in mouse and human myoblasts. Constitutive expression of either RET9 or RET51 increased myoblast proliferation, whereas siRNA-mediated knockdown of Ret induced myogenic differentiation. Suppressing RET activity using Sunitinib, a clinically-approved tyrosine kinase inhibitor, rescued differentiation in both DUX4-expressing murine myoblasts and in FSHD patient-derived myoblasts. Importantly, Sunitinib also increased engraftment and differentiation of FSHD myoblasts in regenerating mouse muscle. Thus, DUX4-mediated activation of Ret prevents myogenic differentiation and could contribute to FSHD pathology by preventing satellite cell-mediated repair. Rescue of DUX4-induced pathology by Sunitinib highlights the therapeutic potential of tyrosine kinase inhibitors for treatment of FSHD.

A role for the tyrosine kinase ACK1 in neurotrophin signaling and neuronal extension and branching

PubMed Central

La Torre, A; del Mar Masdeu, M; Cotrufo, T; Moubarak, R S; del Río, J A; Comella, J X; Soriano, E; Ureña, J M

2013-01-01

Neurotrophins are involved in many crucial cellular functions, including neurite outgrowth, synapse formation, and plasticity. Although these events have long been known, the molecular determinants underlying neuritogenesis have not been fully characterized. Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in the brain. Here, we demonstrate that Ack1 is a molecular constituent of neurotrophin signaling cascades in neurons and PC12 cells. We report that Ack1 interacts with Trk receptors and becomes tyrosine phosphorylated and its kinase activity is increased in response to neurotrophins. Moreover, our data indicate that Ack1 acts upstream of the Akt and MAPK pathways. We show that Ack1 overexpression induces neuritic outgrowth and promotes branching in neurotrophin-treated neuronal cells, whereas the expression of Ack1 dominant negatives or short-hairpin RNAs counteract neurotrophin-stimulated differentiation. Our results identify Ack1 as a novel regulator of neurotrophin-mediated events in primary neurons and in PC12 cells. PMID:23598414

Light regulation of the insulin receptor in the retina.

PubMed

Rajala, Raju V S; Anderson, Robert E

2003-10-01

The peptide hormone insulin binds its cognate cell-surface receptors to activate a coordinated biochemical-signaling network and to induce intracellular events. The retina is an integral part of the central nervous system and is known to contain insulin receptors, although their function is unknown. This article, describes recent studies that link the photobleaching of rhodopsin to tyrosine phosphorylation of the insulin receptor and subsequent activation of phosphoinositide 3- kinase (PI3K). We recently found a light-dependent increase in tyrosine phosphorylation of the insulin receptor-beta-subunit (IR beta) and an increase in PI3K enzyme activity in isolated rod outer segments (ROS) and in anti-phosphotyrosine (PY) and anti-IR beta immunoprecipitates of retinal homogenates. The light effect, which was localized to photoreceptor neurons, is independent of insulin secretion. Our results suggest that light induces tyrosine phosphorylation of IR beta in outer-segment membranes, which leads to the binding of p85 through its N-terminal SH2 domain and the generation of PI-3,4,5-P3. We suggest that the physiological role of this process may be to provide neuroprotection of the retina against light damage by activating proteins that protect against stress-induced apoptosis. The studies linking PI3K activation through tyrosine phosphorylation of IR beta now provide physiological relevance for the presence of these receptors in the retina.

Functional hierarchy of the N-terminal tyrosines of SLP-76.

PubMed

Jordan, Martha S; Sadler, Jeffrey; Austin, Jessica E; Finkelstein, Lisa D; Singer, Andrew L; Schwartzberg, Pamela L; Koretzky, Gary A

2006-02-15

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a central role in T cell activation and T cell development. SLP-76 has three functional modules: an acidic domain with three key tyrosines, a central proline-rich domain, and a C-terminal Src homology 2 domain. Of these, mutation of the three N-terminal tyrosines (Y112, Y128, and Y145) results in the most profound effects on T cell development and function. Y112 and Y128 associate with Vav and Nck, two proteins shown to be important for TCR-induced phosphorylation of proximal signaling substrates, Ca(2+) flux, and actin reorganization. Y145 has been shown to be important for optimal association of SLP-76 with inducible tyrosine kinase, a key regulator of T cell function. To investigate further the role of the phosphorylatable tyrosines of SLP-76 in TCR signaling, cell lines and primary T cells expressing SLP-76 with mutations in individual or paired tyrosine residues were analyzed. These studies show that Tyr(145) of SLP-76 is the most critical tyrosine for both T cell function in vitro and T cell development in vivo.

Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme*

PubMed Central

Fan, Aili; Zocher, Georg; Stec, Edyta; Stehle, Thilo; Li, Shu-Ming

2015-01-01

The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C4- and C7-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C3-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis. PMID:25477507

Tyrosine Kinase Signaling in Clear Cell and Papillary Renal Cell Carcinoma Revealed by Mass Spectrometry-Based Phosphotyrosine Proteomics.

PubMed

Haake, Scott M; Li, Jiannong; Bai, Yun; Kinose, Fumi; Fang, Bin; Welsh, Eric A; Zent, Roy; Dhillon, Jasreman; Pow-Sang, Julio M; Chen, Y Ann; Koomen, John M; Rathmell, W Kimryn; Fishman, Mayer; Haura, Eric B

2016-11-15

Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes. Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. To describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry-based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, whereas DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology among kidney cancer subtypes. Clin Cancer Res; 22(22); 5605-16. ©2016 AACR. ©2016 American Association for Cancer Research.

A dual positive and negative regulation of monocyte activation by leukocyte Ig-like receptor B4 depends on the position of the tyrosine residues in its ITIMs.

PubMed

Park, Mijeong; Liu, Robert W; An, Hongyan; Geczy, Carolyn L; Thomas, Paul S; Tedla, Nicodemus

2017-05-01

The leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory cell surface receptor, primarily expressed on mono-myeloid cells. It contains 2 C-type Ig-like extracellular domains and a long cytoplasmic domain that contains three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Data suggest that LILRB4 suppresses Fc receptor-dependent monocyte functions via its ITIMs, but relative contributions of the three ITIMs are not characterised. To address this, tyrosine (Tyr) residues at positions 337, 389 and 419 were single, double or triple mutated to phenylalanine and stably transfected into a human monocytic cell line, THP-1. Intact Tyr 389 was sufficient to maximally inhibit FcγRI-mediated TNF-α production in THP-1 cells, but, paradoxically, Tyr 337 significantly enhanced TNF-α production. In contrast, bactericidal activity was significantly enhanced in mutants containing Tyr 419 , while Tyr 337 markedly inhibited bacteria killing. Taken together, these results indicate that LILRB4 might have dual inhibitory and activating functions, depending on the position of the functional tyrosine residues in its ITIMs and/or the nature of the stimuli.

Protein Tyrosine Phosphatase 1B Regulates Pyruvate Kinase M2 Tyrosine Phosphorylation*

PubMed Central

Bettaieb, Ahmed; Bakke, Jesse; Nagata, Naoto; Matsuo, Kosuke; Xi, Yannan; Liu, Siming; AbouBechara, Daniel; Melhem, Ramzi; Stanhope, Kimber; Cummings, Bethany; Graham, James; Bremer, Andrew; Zhang, Sheng; Lyssiotis, Costas A.; Zhang, Zhong-Yin; Cantley, Lewis C.; Havel, Peter J.; Haj, Fawaz G.

2013-01-01

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and adiposity and is a drug target for the treatment of obesity and diabetes. Here we identify pyruvate kinase M2 (PKM2) as a novel PTP1B substrate in adipocytes. PTP1B deficiency leads to increased PKM2 total tyrosine and Tyr105 phosphorylation in cultured adipocytes and in vivo. Substrate trapping and mutagenesis studies identify PKM2 Tyr-105 and Tyr-148 as key sites that mediate PTP1B-PKM2 interaction. In addition, in vitro analyses illustrate a direct effect of Tyr-105 phosphorylation on PKM2 activity in adipocytes. Importantly, PTP1B pharmacological inhibition increased PKM2 Tyr-105 phosphorylation and decreased PKM2 activity. Moreover, PKM2 Tyr-105 phosphorylation is regulated nutritionally, decreasing in adipose tissue depots after high-fat feeding. Further, decreased PKM2 Tyr-105 phosphorylation correlates with the development of glucose intolerance and insulin resistance in rodents, non-human primates, and humans. Together, these findings identify PKM2 as a novel substrate of PTP1B and provide new insights into the regulation of adipose PKM2 activity. PMID:23640882

Protein tyrosine phosphatase 1B (PTP1B) is dispensable for IgE-mediated cutaneous reaction in vivo.

PubMed

Yang, Ting; Xie, Zhongping; Li, Hua; Yue, Lei; Pang, Zheng; MacNeil, Adam J; Tremblay, Michel L; Tang, Jin-Tian; Lin, Tong-Jun

2016-01-01

Mast cells play a critical role in allergic reactions. The cross-linking of FcεRI-bound IgE with multivalent antigen initiates a cascade of signaling events leading to mast cell activation. It has been well-recognized that cross linking of FcεRI mediates tyrosine phosphorylation. However, the mechanism involved in tyrosine dephosphorylation in mast cells is less clear. Here we demonstrated that protein tyrosine phosphatase 1B (PTP1B)-deficient mast cells showed increased IgE-mediated phosphorylation of the signal transducer and activator of transcription 5 (STAT5) and enhanced production of CCL9 (MIP-1γ) and IL-6 in IgE-mediated mast cells activation in vitro. However, IgE-mediated calcium mobilization, β-hexaosaminidase release (degranulation), and phosphorylation of IκB and MAP kinases were not affected by PTP1B deficiency. Furthermore, PTP1B deficient mice showed normal IgE-dependent passive cutaneous anaphylaxis and late phase cutaneous reactions in vivo. Thus, PTP1B specifically regulates IgE-mediated STAT5 pathway, but is redundant in influencing mast cell function in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Skin problems and EGFR-tyrosine kinase inhibitor

PubMed Central

Kozuki, Toshiyuki

2016-01-01

Epidermal growth factor receptor inhibition is a good target for the treatment of lung, colon, pancreatic and head and neck cancers. Epidermal growth factor receptor-tyrosine kinase inhibitor was first approved for the treatment of advanced lung cancer in 2002. Epidermal growth factor receptor-tyrosine kinase inhibitor plays an essential role in the treatment of cancer, especially for patients harbouring epidermal growth factor receptor activating mutation. Hence, skin toxicity is the most concerning issue for the epidermal growth factor receptor-tyrosine kinase inhibitor treatment. Skin toxicity is bothersome and sometimes affects the quality of life and treatment compliance. Thus, it is important for physicians to understand the background and how to manage epidermal growth factor receptor-tyrosine kinase inhibitor-associated skin toxicity. Here, the author reviewed the mechanism and upfront preventive and reactive treatments for epidermal growth factor receptor inhibitor-associated skin toxicities. PMID:26826719

The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

PubMed

Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

2001-05-01

cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development. Copyright 2001 Academic Press.

Tyrosine phosphorylation of histone H2A by CK2 regulates transcriptional elongation

PubMed Central

Basnet, Harihar; Bessie Su, Xue; Tan, Yuliang; Meisenhelder, Jill; Merkurjev, Daria; Ohgi, Kenneth A.; Hunter, Tony; Pillus, Lorraine; Rosenfeld, Michael G.

2014-01-01

Post-translational histone modifications play critical roles in regulating transcription, the cell cycle, DNA replication and DNA damage repair1. The identification of new histone modifications critical for transcriptional regulation at initiation, elongation, or termination is of particular interest. Here, we report a new layer of regulation in transcriptional elongation that is conserved from yeast to mammals, based on a phosphorylation of a highly-conserved tyrosine residue, Y57, in histone H2A that is mediated by an unsuspected tyrosine kinase activity of casein kinase 2 (CK2). Mutation of H2A-Y57 in yeast or inhibition of CK2 activity impairs transcriptional elongation in yeast as well as in mammalian cells. Genome-wide binding analysis reveals that CK2α, the catalytic subunit of CK2, binds across RNA polymerase II-transcribed coding genes and active enhancers. Mutation of Y57 causes a loss of H2B mono-ubiquitylation as well as H3K4me3 and H3K79me3, histone marks associated with active transcription. Mechanistically, both CK2 inhibition and H2A-Y57F mutation enhance the H2B deubiquitylation activity of the SAGA complex, suggesting a critical role of this phosphorylation in coordinating the activity of the SAGA during transcription. Together, these results identify a new component of regulation in transcriptional elongation based on CK2-dependent tyrosine phosphorylation of the globular domain of H2A. PMID:25252977

Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor.

PubMed

Jouvin, M H; Adamczewski, M; Numerof, R; Letourneur, O; Vallé, A; Kinet, J P

1994-02-25

Nonreceptor tyrosine kinases such as the newly described 70-kDa (ZAP-70/Syk) and Src-related tyrosine kinases are coupled to a variety of receptors, including the antigen receptors on B- and T-cells and the Fc receptors for IgE (Fc epsilon RI) and IgG (Fc gamma RI, Fc gamma RIII/CD16). Various subunits of these receptors contain homologous activation motifs which appear capable of autonomously triggering cell activation. Two forms of this motif are present in the Fc epsilon RI multimeric complex: one in the beta chain and one in the gamma chain. Here we show that each of the two tyrosine kinases known to be involved in Fc epsilon RI signaling is controlled by a distinct motif-containing chain. Lyn associates with the nonactivated beta chain, whereas gamma promotes the activation of Syk. We also show that neither the beta nor the gamma motif alone can account for the full signaling capacity of the entire receptor. We propose that, upon triggering of the tetrameric receptor, Lyn already bound to beta becomes activated and phosphorylates beta and gamma; the phosphorylation of gamma induces the association of Syk with gamma and also the activation of Syk, resulting in the phosphorylation and activation of phospholipase C gamma 1. Cooperative recruitment of specific kinases by the various signaling chains found in this family of antigen receptors could represent a way to achieve the full signaling capacity of the multimeric complexes.

Alkyl isothiocyanates suppress epidermal growth factor receptor kinase activity but augment tyrosine kinase activity.

PubMed

Nomura, Takahiro; Uehara, Yoshimasa; Kawajiri, Hiroo; Ryoyama, Kazuo; Yamori, Takao; Fuke, Yoko

2009-10-01

We have reported the in vitro and in vivo anticancer activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) derived from a Japanese spice, wasabi. In order to obtain some clues about the mechanism of the anticancer activity, we have studied the effect of alkyl isothiocyanates (MITCs) on protein kinase activities. The anti-autophosphorylation activity of MITCs with respect to the epidermal growth factor (EGF)-stimulated receptor kinase of A431 epidermoid carcinoma cells was examined by incorporation of radioactive ATP into an acid-insoluble fraction. Their anti-phosphorylation activity with respect to the non-receptor protein kinase was analyzed by a standard SDS-PAGE method. All the tested MITCs interfered with the EGF-stimulated receptor kinase activity in a dose-dependent manner, although their effects were less than 1/10 of that of erbstatin in microg/ml. On the other hand, the MITCs did not interfere with non-receptor kinases (kinase A, kinase C, tyrosine kinase and calmodulin dependent kinase III), but enhanced non-receptor tyrosine kinase. A possible anticancer mechanism of MITCs may involve the suppression of EGF receptor kinase activity and augmentation of non-receptor PTK.

Characterization of free and alginate-polylysine-alginate microencapsulated Erwinia herbicola for the conversion of ammonia, pyruvate, and phenol into L-tyrosine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lloyd-George, I.; Chang, T.M.S.

1995-12-20

The whole cell tyrosine phenol-lyase activity of Erwinia herbicola was microencapsulated. The authors studied the use of this for the conversion of ammonia and pyruvate along with phenol or catechol, respectively, into L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa). The reactions are relevant to the development of new methods for the production of L-tyrosine and L-dopa. The growth of E. herbicola at temperatures from 22 C to 32 C is stable, since at these temperatures the cells grow up to the stationary phase and remain there for at least 10 h. At 37 C the cells grow rapidly, but they also enter themore » death phase rapidly. There is only limited growth of E. herbicola at 42 C. Whole cells of E. herbicola were encapsulated within alginate-polylysine-alginate microcapsules (916 {+-} 100 {micro}m, mean {+-} std. dev.). The TPL activity of the cells catalyzed the production of L-tyrosine or dihydroxyphenol-L-alanine (L-dopa) from ammonia, pyruvate, and phenol or catechol, respectively. In the production of tyrosine, an integrated equation based on an ordered ter-uni rapid equilibrium mechanism can be used to find the kinetic parameters of TPL. In an adequately stirred system, the apparent values of the kinetic parameters of whole cell TPL are equal whether the cells are free or encapsulated. The apparent K{sub M} of tyrosine varies with the amount of whole cells in the system, ranging from 0.2 to 0.3 mM. The apparent K{sub M} for phenol is 0.5 mM. The apparent K{sub M} values for pyruvate and ammonia are an order of magnitude greater for whole cells than they are for the cell free enzyme.« less

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

PubMed

Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

1995-03-01

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.

Evolution: Weevils Get Tough on Symbiotic Tyrosine.

PubMed

Dale, Colin

2017-12-04

Weevils, which represent one of the most diverse groups of terrestrial insects in nature, obtain a tough exoskeleton through the activity of an ancient bacterial symbiont with a tiny genome that serves as a factory for the production of tyrosine. Copyright © 2017 Elsevier Ltd. All rights reserved.

New compounds from acid hydrolyzed products of the fruits of Momordica charantia L. and their inhibitory activity against protein tyrosine phosphatas 1B.

PubMed

Zeng, Ke; He, Yan-Ni; Yang, Di; Cao, Jia-Qing; Xia, Xi-Chun; Zhang, Shi-Jun; Bi, Xiu-Li; Zhao, Yu-Qing

2014-06-23

Four new cucurbitane-type triterpene sapogenins, compounds 1-4, together with other eight known compounds were isolated from the acid-hydrolyzed fruits extract of Momordica charantia L. Their chemical structures were established by NMR, mass spectrometry and X-ray crystallography. Compounds 1-7 and 9-12 were evaluated for their inhibitory activities toward protein tyrosine phosphatase 1B (PTP1B), a tyrosine phosphatase that has been implicated as a key target for therapy against type II diabetes. Compounds 1, 2, 4, 7 and 9 were shown inhibitory activities of 77%, 62%, 62% 60% and 68% against PTP1B, respectively. All of these tested compounds were exhibited higher PTP1B inhibition activities than that of the Na3VO4, a known PTP1B inhibitor used as positive control in present study. Structure activity relationship (SAR) analysis indicated that the inhibition activity of PTP1B was associated with the presence and number of -OH groups. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

PTEN is a protein phosphatase that targets active PTK6 and inhibits PTK6 oncogenic signaling in prostate cancer.

PubMed

Wozniak, Darren J; Kajdacsy-Balla, Andre; Macias, Virgilia; Ball-Kell, Susan; Zenner, Morgan L; Bie, Wenjun; Tyner, Angela L

2017-11-15

PTEN activity is often lost in prostate cancer. We show that the tyrosine kinase PTK6 (BRK) is a PTEN substrate. Phosphorylation of PTK6 tyrosine 342 (PY342) promotes activation, while phosphorylation of tyrosine 447 (PY447) regulates auto-inhibition. Introduction of PTEN into a PTEN null prostate cancer cell line leads to dephosphorylation of PY342 but not PY447 and PTK6 inhibition. Conversely, PTEN knockdown promotes PTK6 activation in PTEN positive cells. Using a variety of PTEN mutant constructs, we show that protein phosphatase activity of PTEN targets PTK6, with efficiency similar to PTP1B, a phosphatase that directly dephosphorylates PTK6 Y342. Conditional disruption of Pten in the mouse prostate leads to tumorigenesis and increased phosphorylation of PTK6 Y342, and disruption of Ptk6 impairs tumorigenesis. In human prostate tumor tissue microarrays, loss of PTEN correlates with increased PTK6 PY342 and poor outcome. These data suggest PTK6 activation promotes invasive prostate cancer induced by PTEN loss.

Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site.

PubMed

Herbst, R; Munemitsu, S; Ullrich, A

1995-01-19

The transforming gene of the Hardy-Zuckerman-4 strain of feline sarcoma virus, v-kit, arose by transduction of the cellular c-kit gene, which encodes the receptor tyrosine kinase (RTK) p145c-kit. To gain insight into the molecular basis of the v-kit transforming potential, we characterized the feline c-kit by cDNA cloning. Comparison of the feline v-kit and c-kit sequences revealed, in addition to deletions of the extracellular and transmembrane domains, three additional mutations in the v-kit oncogene product: deletion of tyrosine-569 and valine-570, the exchange of aspartate at position 761 to glycine, and replacement of the C-terminal 50 amino acids by five unrelated residues. Examinations of individual v-kit mutations in the context of chimeric receptors yielded inhibitory effects for some mutants on both autophosphorylation and substrate phosphorylation functions. In contrast, deletion of tyrosine-569 and valine-570 significantly enhanced transforming and mitogenic activities of p145c-kit, while the other mutations had no significant effects. Conservation in subclass III RTKs and the identification of the corresponding residue in beta PDGF-R, Y579, as a binding site for src family tyrosine kinases suggests an important role for Y568 in kit signal regulation and the definition of its oncogenic potential. Repositioning of Y571 by an inframe two codon deletion may be the crucial alteration resulting in enhancement of v-kit oncogenic activity.

New tyrosinase inhibitory decapeptide: Molecular insights into the role of tyrosine residues.

PubMed

Ochiai, Akihito; Tanaka, Seiya; Imai, Yuta; Yoshida, Hisashi; Kanaoka, Takumi; Tanaka, Takaaki; Taniguchi, Masayuki

2016-06-01

Tyrosinase, a rate-limiting enzyme in melanin biosynthesis, catalyzes the hydroxylation of l-tyrosine to 3,4-dihydroxy-l-phenylalanine (l-dopa) (monophenolase reaction) and the subsequent oxidation of l-dopa to l-dopaquinone (diphenolase reaction). Thus, tyrosinase inhibitors have been proposed as skin-lightening agents; however, many of the existing inhibitors cannot be widely used in the cosmetic industry due to their high cytotoxicity and instability. On the other hand, some tyrosinase inhibitory peptides have been reported as safe. In this study, we found that the peptide TH10, which has a similar sequence to the characterized inhibitory peptide P4, strongly inhibits the monophenolase reaction with a half-maximal inhibitory concentration of 102 μM. Seven of the ten amino acid residues in TH10 were identical to P4; however, TH10 possesses one N-terminal tyrosine, whereas P4 contains three tyrosine residues located at its N-terminus, center, and C-terminus. Subsequent analysis using sequence-shuffled variants indicated that the tyrosine residues located at the N-terminus and center of P4 have little to no contribution to its inhibitory activity. Furthermore, docking simulation analysis of these peptides with mushroom tyrosinase demonstrated that the active tyrosine residue was positioned close to copper ions, suggesting that TH10 and P4 bind to tyrosinase as a substrate analogue. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-dependent prolonged GAB1 binding and sustained ERK2/MAPK1 activation.

PubMed

Fragale, Alessandra; Tartaglia, Marco; Wu, Jie; Gelb, Bruce D

2004-03-01

Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2 or SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. We compared the functional effects of three Noonan syndrome-causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal-regulated kinase 2 activation, which was ligand-dependent. Binding of SHP2 mutants to Grb2-associated binder-1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2-associated binder-1-FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor-mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal-regulated kinase 2 activation. Taken together, these results document that Noonan syndrome-associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N-terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen-activated protein kinase (ERK2/MAPK1) pathway in a ligand-dependent manner that required docking through Grb2-associated binder-1 (GAB1), leading to increased cell proliferation. Copyright 2004 Wiley-Liss, Inc.

Characterization of breakpoint cluster region kinase and SH2-binding activities.

PubMed

Afar, D E; Witte, O N

1995-01-01

BCR is an interesting signaling protein, whose cellular function is currently unknown. Its biochemical properties include serine kinase activity, SH2-binding activity, and a GTPase-activating activity. The SH2-binding activity is particularly interesting because it may link BCR to signaling pathways involving SH2-containing molecules. Since tyrosine phosphorylation of BCR has been detected in CML-derived cell lines and since tyrosine-phosphorylated BCR shows increased affinity toward certain SH2 domains, it seems particularly important to further characterize this activity. This chapter described a simple purification scheme for partial purification of BCR, which can be used to assess in vitro kinase and SH2-binding activities.

Novel Bruton’s tyrosine kinase inhibitors currently in development

PubMed Central

D’Cruz, Osmond J; Uckun, Fatih M

2013-01-01

Bruton’s tyrosine kinase (Btk) is intimately involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is overexpressed and constitutively active in several B-lineage lymphoid malignancies. Btk has emerged as a new antiapoptotic molecular target for treatment of B-lineage leukemias and lymphomas. Preclinical and early clinical results indicate that Btk inhibitors may be useful in the treatment of leukemias and lymphomas. PMID:23493945

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

PubMed Central

Moreno-Beltrán, Blas; Guerra-Castellano, Alejandra; Del Conte, Rebecca; García-Mauriño, Sofía M.; Díaz-Moreno, Sofía; González-Arzola, Katiuska; Santos-Ocaña, Carlos; Velázquez-Campoy, Adrián; De la Rosa, Miguel A.; Turano, Paola; Díaz-Moreno, Irene

2017-01-01

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-l-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects. PMID:28348229

A dynamically coupled allosteric network underlies binding cooperativity in Src kinase

PubMed Central

Foda, Zachariah H.; Shan, Yibing; Kim, Eric T.; Shaw, David E.; Seeliger, Markus A.

2015-01-01

Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity. PMID:25600932

Tyr phosphatase-mediated P-ERK inhibition suppresses senescence in EIA + v-raf transformed cells, which, paradoxically, are apoptosis-protected in a MEK-dependent manner.

PubMed

De Vitis, Stefania; Sonia Treglia, Antonella; Ulianich, Luca; Turco, Stefano; Terrazzano, Giuseppe; Lombardi, Angela; Miele, Claudia; Garbi, Corrado; Beguinot, Francesco; Di Jeso, Bruno

2011-02-01

Activation of the Ras-Raf-extracellular signal-regulated kinase (ERK) pathway causes not only proliferation and suppression of apoptosis but also the antioncogenic response of senescence. How these contrasting effects are reconciled to achieve cell transformation and cancer formation is poorly understood. In a system of two-step carcinogenesis (dedifferentiated PC EIA, transformed PC EIA-polyoma-middle T [PC EIA + Py] and PC EIA-v-raf [PC EIA + raf] cells], v-raf cooperated with EIA by virtue of a strong prosurvival effect, not elicited by Py-middle T, evident toward serum-deprivation-and H(2)O(2)-induced apoptosis. Apoptosis was detected by DNA fragmentation and annexin V staining. The prosurvival function of v-raf was, in part, mitogen-activated protein kinase/ERK kinase (MEK)-dependent, as shown by pharmacological MEK inhibition. The MEK-dependent antiapoptotic effect of v-raf was exerted despite a lower level of P-ERK1/2 in EIA + raf cells with respect to EIA + Py/EIA cells, which was dependent on a high tyrosine phosphatase activity, as shown by orthovanadate blockade. An ERK1/2 tyrosine phosphatase was likely involved. The high tyrosine phosphatase activity was instrumental to the complete suppression of senescence, detected by β-galactosidase activity, because tyrosine phosphatase blockade induced senescence in EIA + raf but not in EIA + Py cells. High tyrosine phosphatase activity and evasion from senescence were confirmed in an anaplastic thyroid cancer cell line. Therefore, besides EIA, EIA + raf cells suppress senescence through a new mechanism, namely, phosphatase-mediated P-ERK1/2 inhibition, but, paradoxically, retain the oncogenic effects of the Raf-ERK pathway. We propose that the survival effect of Raf is not a function of absolute P-ERK1/2 levels at a given time but is rather dynamically dependent on greater variations after an apoptotic stimulus.

Co-clustering of Fcgamma and B cell receptors induces dephosphorylation of the Grb2-associated binder 1 docking protein.

PubMed

Koncz, G; Tóth, G K; Bökönyi, G; Kéri, G; Pecht, I; Medgyesi, D; Gergely, J; Sármay, G

2001-07-01

The immunoreceptor tyrosine-based inhibitory motif (ITIM) of human type IIb Fcgamma receptor (FcgammaRIIb) is phosphorylated on its tyrosine upon co-clustering with the B cell receptor (BCR). The phosphorylated ITIM (p-ITIM) binds to the SH2 domains of polyphosphoinositol 5-phosphatase (SHIP) and the tyrosine phosphatase, SHP-2. We investigated the involvement of the molecular complex composed of the phosphorylated SHIP and FcgammaRIIb in the activation of SHP-2. As a model compound, we synthesized a bisphosphopeptide, combining the sequences of p-ITIM and the N-terminal tyrosine phosphorylated motif of SHIP with a flexible spacer. This compound bound to the recombinant SH2 domains of SHP-2 with high affinity and activated the phosphatase in an in vitro assay. These data suggest that the phosphorylated FcgammaRII-SHIP complexes formed in the intact cells may also activate SHP-2. Grb2-associated binder 1 (Gab1) is a multisite docking protein, which becomes tyrosine-phosphorylated in response to various types of signaling, including BCR. In turn it binds to the SH2 domains of SHP-2, SHIP and the p85 subunit of phosphatidyl inositol 3-kinase (PtdIns3-K) and may regulate their activity. Gab1 is a potential substrate of SHP-2, thus its binding to FcgammaRIIb may modify the Gab1-bound signaling complex. We show here that Gab1 is part of the multiprotein complex assembled by FcgammaRIIb upon its co-clustering with BCR. Gab1 may recruit SH2 domain-containing molecules to the phosphorylated FcgammaRIIb. SHP-2, activated upon the binding to FcgammaRIIb-SHIP complex, partially dephosphorylates Gab1, resulting in the release of PtdIns3-K and ultimately in the inhibition of downstream activation pathways in BCR/FcgammaRIIb co-aggregated cells.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Nancy L., E-mail: nlcho@partners.org; Lin, Chi-Iou; Du, Jinyan

Highlights: Black-Right-Pointing-Pointer Kinome profiling is a novel technique for identifying activated kinases in human cancers. Black-Right-Pointing-Pointer Src activity is increased in invasive thyroid cancers. Black-Right-Pointing-Pointer Inhibition of Src activity decreased proliferation and invasion in vitro. Black-Right-Pointing-Pointer Further investigation of Src targeted therapies in thyroid cancer is warranted. -- Abstract: Background: Novel therapies are needed for the treatment of invasive thyroid cancers. Aberrant activation of tyrosine kinases plays an important role in thyroid oncogenesis. Because current targeted therapies are biased toward a small subset of tyrosine kinases, we conducted a study to reveal novel therapeutic targets for thyroid cancer using amore » bead-based, high-throughput system. Methods: Thyroid tumors and matched normal tissues were harvested from twenty-six patients in the operating room. Protein lysates were analyzed using the Luminex immunosandwich, a bead-based kinase phosphorylation assay. Data was analyzed using GenePattern 3.0 software and clustered according to histology, demographic factors, and tumor status regarding capsular invasion, size, lymphovascular invasion, and extrathyroidal extension. Survival and invasion assays were performed to determine the effect of Src inhibition in papillary thyroid cancer (PTC) cells. Results: Tyrosine kinome profiling demonstrated upregulation of nine tyrosine kinases in tumors relative to matched normal thyroid tissue: EGFR, PTK6, BTK, HCK, ABL1, TNK1, GRB2, ERK, and SRC. Supervised clustering of well-differentiated tumors by histology, gender, age, or size did not reveal significant differences in tyrosine kinase activity. However, supervised clustering by the presence of invasive disease showed increased Src activity in invasive tumors relative to non-invasive tumors (60% v. 0%, p < 0.05). In vitro, we found that Src inhibition in PTC cells decreased cell invasion and proliferation. Conclusion: Global kinome analysis enables the discovery of novel targets for thyroid cancer therapy. Further investigation of Src targeted therapy for advanced thyroid cancer is warranted.« less

SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2

NASA Astrophysics Data System (ADS)

Naudin, Cécile; Sirvent, Audrey; Leroy, Cédric; Larive, Romain; Simon, Valérie; Pannequin, Julie; Bourgaux, Jean-François; Pierre, Josiane; Robert, Bruno; Hollande, Frédéric; Roche, Serge

2014-01-01

The adaptor SLAP is a negative regulator of receptor signalling in immune cells but its role in human cancer is ill defined. Here we report that SLAP is abundantly expressed in healthy epithelial intestine but strongly downregulated in 50% of colorectal cancer. SLAP overexpression suppresses cell tumorigenicity and invasiveness while SLAP silencing enhances these transforming properties. Mechanistically, SLAP controls SRC/EPHA2/AKT signalling via destabilization of the SRC substrate and receptor tyrosine kinase EPHA2. This activity is independent from CBL but requires SLAP SH3 interaction with the ubiquitination factor UBE4A and SLAP SH2 interaction with pTyr594-EPHA2. SRC phosphorylates EPHA2 on Tyr594, thus creating a feedback loop that promotes EPHA2 destruction and thereby self-regulates its transforming potential. SLAP silencing enhances SRC oncogenicity and sensitizes colorectal tumour cells to SRC inhibitors. Collectively, these data establish a tumour-suppressive role for SLAP in colorectal cancer and a mechanism of SRC oncogenic induction through stabilization of its cognate substrates.

Decrease in the cytosolic NADP+-dependent isocitrate dehydrogenase activity through porcine sperm capacitation.

PubMed

Katoh, Yuki; Tamba, Michiko; Matsuda, Manabu; Kikuchi, Kazuhiro; Okamura, Naomichi

2018-02-26

In order to understand the molecular mechanisms involved in the sperm capacitation, we have identified the proteins tyrosine-phosphorylated during the capacitation especially in conjunction with the regulation of the levels of reactive oxygen species (ROS) in sperm. In the present study, the effects of the tyrosine phosphorylation of cytosolic NADP + -dependent isocitrate dehydrogenase (IDPc) on its catalytic activity and on the levels of ROS in sperm have been studied. The tyrosine phosphorylated IDPc showed a significantly lowered enzymatic activity. The immunocytochemical analyses using the highly specific antisera against IDPc revealed that IDPc was mainly localized to the principal piece of the porcine sperm flagellum. As IDPc is one of the major NADPH regenerating enzymes in porcine sperm, it is strongly suggested that the decrease in IDPc activity is involved in the increased levels of ROS, which results in the induction of hyperactivated flagellar movement and capacitation. Copyright © 2018 Elsevier Inc. All rights reserved.

Disabled is a putative adaptor protein that functions during signaling by the sevenless receptor tyrosine kinase.

PubMed

Le, N; Simon, M A

1998-08-01

DRK, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by the sevenless receptor tyrosine kinase (SEV). One role of DRK is to provide a link between activated SEV and the Ras1 activator SOS. We have investigated the possibility that DRK performs other functions by identifying additional DRK-binding proteins. We show that the phosphotyrosine-binding (PTB) domain-containing protein Disabled (DAB) binds to the DRK SH3 domains. DAB is expressed in the ommatidial clusters, and loss of DAB function disrupts ommatidial development. Moreover, reduction of DAB function attenuates signaling by a constitutively activated SEV. Our biochemical analysis suggests that DAB binds SEV directly via its PTB domain, becomes tyrosine phosphorylated upon SEV activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that DAB is a novel component of the SEV signaling pathway.

Hypothalamic digoxin, hemispheric chemical dominance, and spirituality.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-03-01

The isoprenoid pathway was assessed in atheistic and spiritually inclined individuals. The pathway was also assessed in individuals with differing hemispheric dominance to assess whether hemispheric dominance has a correlation with spiritual and atheistic tendency. HMG CoA reductase activity, serum digoxin, RBC membrane Na(+)-K+ ATPase activity, serum magnesium, and tyrosine/tryptophan catabolic patterns were assessed in spiritual/atheistic individuals and in those differing hemispheric dominance. In spiritually-inclined individuals, there was increased digoxin synthesis, decreased membrane Na(+)-K+ ATPase activity, increased tryptophan catabolites (serotonin, quinolinic acid, and nicotine), and decreased tyrosine catabolites (dopamine, noradrenaline, and morphine). The pattern in spiritually-inclined individuals correlated with right hemispheric chemical dominance. In atheistic individuals there was decreased digoxin synthesis, increased membrane Na(+)-K+ ATPase activity, decreased tryptophan catabolities (serotonin, quinolinic acid, and nicotine), and increased tyrosine catabolites (dopamine, noradrenaline, and morphine). This pattern in atheistic individuals correlated with that obtained in left hemispheric chemical dominance. Hemispheric chemical dominance and hypothalamic digoxin could regulate the predisposition to spirituality or atheism.

Plant cell division is specifically affected by nitrotyrosine

PubMed Central

Jovanović, Aleksandra M.; Durst, Steffen; Nick, Peter

2010-01-01

Virtually all eukaryotic α-tubulins harbour a C-terminal tyrosine that can be reversibly removed and religated, catalysed by a specific tubulin–tyrosine carboxypeptidase (TTC) and a specific tubulin–tyrosine ligase (TTL), respectively. The biological function of this post-translational modification has remained enigmatic. 3-nitro-L-tyrosine (nitrotyrosine, NO2Tyr), can be incorporated into detyrosinated α-tubulin instead of tyrosine, producing irreversibly nitrotyrosinated α-tubulin. To gain insight into the possible function of detyrosination, the effect of NO2Tyr has been assessed in two plant model organisms (rice and tobacco). NO2Tyr causes a specific, sensitive, and dose-dependent inhibition of cell division that becomes detectable from 1 h after treatment and which is not observed with non-nitrosylated tyrosine. These effects are most pronounced in cycling tobacco BY-2 cells, where the inhibition of cell division is accompanied by a stimulation of cell length, and a misorientation of cross walls. NO2Tyr reduces the abundance of the detyrosinated form of α-tubulin whereas the tyrosinated α-tubulin is not affected. These findings are discussed with respect to a model where NO2Tyr is accepted as substrate by TTL and subsequently blocks TTC activity. The irreversibly tyrosinated α-tubulin impairs microtubular functions that are relevant to cell division in general, and cell wall deposition in particular. PMID:20018903

Huntingtin-Interacting Protein 1 Phosphorylation by Receptor Tyrosine Kinases

PubMed Central

Ames, Heather M.; Wang, Anmin A.; Coughran, Alanna; Evaul, Kristen; Huang, Sha; Graves, Chiron W.; Soyombo, Abigail A.

2013-01-01

Huntingtin-interacting protein 1 (HIP1) binds inositol lipids, clathrin, actin, and receptor tyrosine kinases (RTKs). HIP1 is elevated in many tumors, and its expression is prognostic in prostate cancer. HIP1 overexpression increases levels of the RTK epidermal growth factor receptor (EGFR) and transforms fibroblasts. Here we report that HIP1 is tyrosine phosphorylated in the presence of EGFR and platelet-derived growth factor β receptor (PDGFβR) as well as the oncogenic derivatives EGFRvIII, HIP1/PDGFβR (H/P), and TEL/PDGFβR (T/P). We identified a four-tyrosine “HIP1 phosphorylation motif” (HPM) in the N-terminal region of HIP1 that is required for phosphorylation mediated by both EGFR and PDGFβR but not by the oncoproteins H/P and T/P. We also identified a tyrosine residue (Y152) within the HPM motif of HIP1 that inhibits HIP1 tyrosine phosphorylation. The HPM tyrosines are conserved in HIP1's only known mammalian relative, HIP1-related protein (HIP1r), and are also required for HIP1r phosphorylation. Tyrosine-to-phenylalanine point mutations in the HPM of HIP1 result in proapoptotic activity, indicating that an intact HPM may be necessary for HIP1's role in cellular survival. These data suggest that phosphorylation of HIP1 by RTKs in an N-terminal region contributes to the promotion of cellular survival. PMID:23836884

Huntingtin-interacting protein 1 phosphorylation by receptor tyrosine kinases.

PubMed

Ames, Heather M; Wang, Anmin A; Coughran, Alanna; Evaul, Kristen; Huang, Sha; Graves, Chiron W; Soyombo, Abigail A; Ross, Theodora S

2013-09-01

Huntingtin-interacting protein 1 (HIP1) binds inositol lipids, clathrin, actin, and receptor tyrosine kinases (RTKs). HIP1 is elevated in many tumors, and its expression is prognostic in prostate cancer. HIP1 overexpression increases levels of the RTK epidermal growth factor receptor (EGFR) and transforms fibroblasts. Here we report that HIP1 is tyrosine phosphorylated in the presence of EGFR and platelet-derived growth factor β receptor (PDGFβR) as well as the oncogenic derivatives EGFRvIII, HIP1/PDGFβR (H/P), and TEL/PDGFβR (T/P). We identified a four-tyrosine "HIP1 phosphorylation motif" (HPM) in the N-terminal region of HIP1 that is required for phosphorylation mediated by both EGFR and PDGFβR but not by the oncoproteins H/P and T/P. We also identified a tyrosine residue (Y152) within the HPM motif of HIP1 that inhibits HIP1 tyrosine phosphorylation. The HPM tyrosines are conserved in HIP1's only known mammalian relative, HIP1-related protein (HIP1r), and are also required for HIP1r phosphorylation. Tyrosine-to-phenylalanine point mutations in the HPM of HIP1 result in proapoptotic activity, indicating that an intact HPM may be necessary for HIP1's role in cellular survival. These data suggest that phosphorylation of HIP1 by RTKs in an N-terminal region contributes to the promotion of cellular survival.

The immune-endocrine loop during aging: role of growth hormone and insulin-like growth factor-I.

PubMed

Burgess, W; Liu, Q; Zhou, J; Tang, Q; Ozawa, A; VanHoy, R; Arkins, S; Dantzer, R; Kelley, K W

1999-01-01

Why a primary lymphoid organ such as the thymus involutes during aging remains a fundamental question in immunology. Aging is associated with a decrease in plasma growth hormone (somatotropin) and IGF-I, and this somatopause of aging suggests a connection between the neuroendocrine and immune systems. Several investigators have demonstrated that treatment with either growth hormone or IGF-I restores architecture of the involuted thymus gland by reversing the loss of immature cortical thymocytes and preventing the decline in thymulin synthesis that occurs in old or GH-deficient animals and humans. The proliferation, differentiation and functions of other components of the immune system, including T and B cells, macrophages and neutrophils, also demonstrate age-associated decrements that can be restored by IGF-I. Knowledge of the mechanism by which cytokines and hormones influence hematopoietic cells is critical to improving the health of aged individuals. Our laboratory has recently demonstrated that IGF-I prevents apoptosis in promyeloid cells, which subsequently permits these cells to differentiate into neutrophils. We also demonstrated that IL-4 acts much like IGF-I to promote survival of promyeloid cells and to activate the enzyme phosphatidylinositol 3'-kinase (PI 3-kinase). However, the receptors for IGF-I and IL-4 are completely different, with the intracellular beta chains of the IGF receptor possessing intrinsic tyrosine kinase activity and the alpha and gammac subunit of the heterodimeric IL-4 receptor utilizing the Janus kinase family of nonreceptor protein kinases to tyrosine phosphorylate downstream targets. Both receptors share many of the components of the PI 3-kinase signal transduction pathway, converging at the level of insulin receptor substrate-1 or insulin receptor subtrate-2 (formally known as 4PS, or IL-4 Phosphorylated Substrate). Our investigations with IGF-I and IL-4 suggest that PI 3-kinase inhibits apoptosis by maintaining high levels of the anti-apoptotic protein Bcl-2. The sharing of common activation molecules, despite vastly different protein structures of their receptors, forms a molecular explanation for the possibility of cross talk between IL-4 and IGF-I in regulating many of the events associated with hematopoietic differentiation, proliferation and survival.

KinView: A visual comparative sequence analysis tool for integrated kinome research

PubMed Central

McSkimming, Daniel Ian; Dastgheib, Shima; Baffi, Timothy R.; Byrne, Dominic P.; Ferries, Samantha; Scott, Steven Thomas; Newton, Alexandra C.; Eyers, Claire E.; Kochut, Krzysztof J.; Eyers, Patrick A.

2017-01-01

Multiple sequence alignments (MSAs) are a fundamental analysis tool used throughout biology to investigate relationships between protein sequence, structure, function, evolutionary history, and patterns of disease-associated variants. However, their widespread application in systems biology research is currently hindered by the lack of user-friendly tools to simultaneously visualize, manipulate and query the information conceptualized in large sequence alignments, and the challenges in integrating MSAs with multiple orthogonal data such as cancer variants and post-translational modifications, which are often stored in heterogeneous data sources and formats. Here, we present the Multiple Sequence Alignment Ontology (MSAOnt), which represents a profile or consensus alignment in an ontological format. Subsets of the alignment are easily selected through the SPARQL Protocol and RDF Query Language for downstream statistical analysis or visualization. We have also created the Kinome Viewer (KinView), an interactive integrative visualization that places eukaryotic protein kinase cancer variants in the context of natural sequence variation and experimentally determined post-translational modifications, which play central roles in the regulation of cellular signaling pathways. Using KinView, we identified differential phosphorylation patterns between tyrosine and serine/threonine kinases in the activation segment, a major kinase regulatory region that is often mutated in proliferative diseases. We discuss cancer variants that disrupt phosphorylation sites in the activation segment, and show how KinView can be used as a comparative tool to identify differences and similarities in natural variation, cancer variants and post-translational modifications between kinase groups, families and subfamilies. Based on KinView comparisons, we identify and experimentally characterize a regulatory tyrosine (Y177PLK4) in the PLK4 C-terminal activation segment region termed the P+1 loop. To further demonstrate the application of KinView in hypothesis generation and testing, we formulate and validate a hypothesis explaining a novel predicted loss-of-function variant (D523NPKCβ) in the regulatory spine of PKCβ, a recently identified tumor suppressor kinase. KinView provides a novel, extensible interface for performing comparative analyses between subsets of kinases and for integrating multiple types of residue specific annotations in user friendly formats. PMID:27731453

Structural Basis of Substrate Recognition by Hematopoietic Tyrosine Phosphatase (HePTP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Critton, D.; Tortajada, A; Stetson, G

2008-01-01

Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPsmore » as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.« less

Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme.

PubMed

Torrens-Spence, Michael P; Gillaspy, Glenda; Zhao, Bingyu; Harich, Kim; White, Robert H; Li, Jianyong

2012-02-10

Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine. Copyright © 2012 Elsevier Inc. All rights reserved.

Enhanced NMDA receptor tyrosine phosphorylation and increased brain injury following neonatal hypoxia–ischemia in mice with neuronal Fyn overexpression

PubMed Central

Knox, Renatta; Zhao, Chong; Miguel-Perez, Dario; Wang, Steven; Yuan, Jinwei; Ferriero, Donna; Jiang, Xiangning

2013-01-01

The Src family kinases (SFKs) Src and Fyn are implicated in hypoxic–ischemic (HI) injury in the developing brain. However, it is unclear how these particular SFKs contribute to brain injury. Using neuron-specific Fyn overexpressing (OE) mice, we investigated the role of neuronal Fyn in neonatal brain HI. Wild type (WT) and Fyn OE mice were subjected to HI using the Vannucci model at postnatal day 7. Brains were scored five days later for evaluation of damage using cresyl violet and iron staining. Western blotting with postsynaptic density (PSD)-associated synaptic membrane proteins and co-immunoprecipitation with cortical lysates were performed at various time points after HI to determine NMDA receptor tyrosine phosphorylation and Fyn kinase activity. Fyn OE mice had significantly higher mortality and brain injury compared to their WT littermates. Neuronal Fyn overexpression led to sustained NR2A and NR2B tyrosine phosphorylation and enhanced NR2B phosphorylation at tyrosine (Y) 1472 and Y1252 in synaptic membranes. These early changes correlated with higher calpain activity 24 h after HI in Fyn OE mice relative to WT animals. Our findings suggest a role for Fyn kinase in neuronal death after neonatal HI, possibly via up-regulation of NMDA receptor tyrosine phosphorylation. PMID:23127881

Spleen tyrosine kinase mediates high glucose-induced transforming growth factor-{beta}1 up-regulation in proximal tubular epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Won Seok; Chang, Jai Won; Han, Nam Jeong

The role of spleen tyrosine kinase (Syk) in high glucose-induced intracellular signal transduction has yet to be elucidated. We investigated whether Syk is implicated in high glucose-induced transforming growth factor-{beta}1 (TGF-{beta}1) up-regulation in cultured human proximal tubular epithelial cells (HK-2 cell). High glucose increased TGF-{beta}1 gene expression through Syk, extracellular signal-regulated kinase (ERK), AP-1 and NF-{kappa}B. High glucose-induced AP-1 DNA binding activity was decreased by Syk inhibitors and U0126 (an ERK inhibitor). Syk inhibitors suppressed high glucose-induced ERK activation, whereas U0126 had no effect on Syk activation. High glucose-induced NF-{kappa}B DNA binding activity was also decreased by Syk inhibitors. Highmore » glucose increased nuclear translocation of p65 without serine phosphorylation of I{kappa}B{alpha} and without degradation of I{kappa}B{alpha}, but with an increase in tyrosine phosphorylation of I{kappa}B{alpha} that may account for the activation of NF-{kappa}B. Both Syk inhibitors and Syk-siRNA attenuated high glucose-induced I{kappa}B{alpha} tyrosine phosphorylation and p65 nuclear translocation. Depletion of p21-activated kinase 2 (Pak2) by transfection of Pak2-siRNA abolished high glucose-induced Syk activation. In summary, high glucose-induced TGF-{beta}1 gene transcription occurred through Pak2, Syk and subsequent ERK/AP-1 and NF-{kappa}B pathways. This suggests that Syk might be implicated in the diabetic kidney disease.« less

TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Feifei; Jiang, Yinan; Zheng, Qiping

Research highlights: {yields} TEC is rapidly tyrosine-phosphorylated and activated by HGF-stimulation in vivo or after partial hepatectomy in mice. {yields} TEC enhances the activity of Elk and serum response element (SRE) in HGF signaling pathway in hepatocyte. {yields} TEC promotes hepatocyte proliferation through the Erk-MAPK pathway. -- Abstract: Background/aims: TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involvedmore » in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. Methods: We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC{sup KM}) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by {sup 3}H-TdR incorporation. Results: TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. Conclusions: TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.« less

Modulation of the Fcepsilon receptor I signaling by tyrosine kinase inhibitors: search for therapeutic targets of inflammatory and allergy diseases.

PubMed

Lusková, Petra; Dráber, Petr

2004-01-01

Mast cells and basophils are major effector cells in the immunoglobulin E (IgE)-dependent allergic reactions as well as in the innate immunity. They are distributed throughout the body and, upon allergen exposure, are stimulated via the high affinity IgE receptor (FcepsilonRI) to release several pro-inflammatory mediators such as leukotrienes, immunoregulatory cytokines and histamine. FcepsilonRI-mediated signaling is initiated by tyrosine phosphorylation of FcepsilonRI subunits by Src family kinase Lyn, which is followed by an activation of Syk/Zap family kinase Syk. The activated kinases then in turn phosphorylate and activate other enzymes [phospholipase Cgamma (PLCgamma) isoforms, phosphatidylinositol-3 kinase (PI3K) isoforms, protein kinase C (PKC) isoforms, Bruton's tyrosine kinase (Btk) and others], adaptors [linker for activation of T cells (LAT), Cbl, Grb2 and others] and GTP exchange factors/GTPases (Vav, Ras, Rho, and others), and subsequently induce the mobilization of stored and extracellular Ca(2+). These and other biochemical events lead within seconds and minutes to the secretory response and later to the production of chemokines. This review is focused on the use of tyrosine kinase inhibitors specific for Src family kinases (PP1/PP2, SU6656 and CT5269), Syk kinase (piceatannol, ER-27319 and BAY 61-3606) and Btk (terreic acid and LFM-A13) for a modulation of FcepsilonRI-mediated signaling in mast cells. Potential use of the inhibitors in the treatment of inflammatory and allergy diseases as well as future directions in the development of highly specific tyrosine kinases inhibitors of new generations and their use in an intended modulation of mast cell signaling are discussed.

Role of Non Receptor Tyrosine Kinases in Hematological Malignances and its Targeting by Natural Products.

PubMed

Siveen, Kodappully S; Prabhu, Kirti S; Achkar, Iman W; Kuttikrishnan, Shilpa; Shyam, Sunitha; Khan, Abdul Q; Merhi, Maysaloun; Dermime, Said; Uddin, Shahab

2018-02-19

Tyrosine kinases belong to a family of enzymes that mediate the movement of the phosphate group to tyrosine residues of target protein, thus transmitting signals from the cell surface to cytoplasmic proteins and the nucleus to regulate physiological processes. Non-receptor tyrosine kinases (NRTK) are a sub-group of tyrosine kinases, which can relay intracellular signals originating from extracellular receptor. NRTKs can regulate a huge array of cellular functions such as cell survival, division/propagation and adhesion, gene expression, immune response, etc. NRTKs exhibit considerable variability in their structural make up, having a shared kinase domain and commonly possessing many other domains such as SH2, SH3 which are protein-protein interacting domains. Recent studies show that NRTKs are mutated in several hematological malignancies, including lymphomas, leukemias and myelomas, leading to aberrant activation. It can be due to point mutations which are intragenic changes or by fusion of genes leading to chromosome translocation. Mutations that lead to constitutive kinase activity result in the formation of oncogenes, such as Abl, Fes, Src, etc. Therefore, specific kinase inhibitors have been sought after to target mutated kinases. A number of compounds have since been discovered, which have shown to inhibit the activity of NRTKs, which are remarkably well tolerated. This review covers the role of various NRTKs in the development of hematological cancers, including their deregulation, genetic alterations, aberrant activation and associated mutations. In addition, it also looks at the recent advances in the development of novel natural compounds that can target NRTKs and perhaps in combination with other forms of therapy can show great promise for the treatment of hematological malignancies.

First cytoplasmic loop of glucagon-like peptide-1 receptor can function at the third cytoplasmic loop position of rhodopsin.

PubMed

Yamashita, Takahiro; Tose, Koji; Shichida, Yoshinori

2008-01-01

G protein-coupled receptors (GPCRs) are classified into several families based on their amino acid sequences. In family 1, GPCRs such as rhodopsin and adrenergic receptor, the structure-function relationship has been extensively investigated to demonstrate that exposure of the third cytoplasmic loop is essential for selective G protein activation. In contrast, much less is known about other families. Here we prepared chimeric mutants between Gt-coupled rhodopsin and Gi/Go- and Gs-coupled glucagon-like peptide-1 (GLP-1) receptor of family 2 and tried to identify the loop region that functions at the third cytoplasmic loop position of rhodopsin. We succeeded in expressing a mutant having the first cytoplasmic loop of GLP-1 receptor and found that this mutant activated Gi and Go efficiently but did not activate Gt. Moreover, the rhodopsin mutant having the first loop of Gs-coupled secretin receptor of family 2 decreased the Gi and Go activation efficiencies. Therefore, the first loop of GLP-1 receptor would share a similar role to the third loop of rhodopsin in G protein activation. This result strongly suggested that different families of GPCRs have maintained molecular architectures of their ancestral types to generate a common mechanism, namely exposure of the cytoplasmic loop, to activate peripheral G protein.

Mechanisms underlying the inhibitory effects of arsenic compounds on protein tyrosine phosphatase (PTP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rehman, Kanwal; Chen, Zhe; Wang, Wen Wen

2012-09-15

Arsenic binding to biomolecules is considered one of the major toxic mechanisms, which may also be related to the carcinogenic risks of arsenic in humans. At the same time, arsenic is also known to activate the phosphorylation-dependent signaling pathways including the epidermal growth factor receptor, the mitogen-activated protein kinase and insulin/insulin-like growth factor-1 pathways. These signaling pathways originate at the level of receptor tyrosine kinases whose phosphorylation status is regulated by opposing protein tyrosine phosphatase (PTP) activity. Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases and phosphatases, regulates important signaling pathways that are involvedmore » in the control of cell proliferation, adhesion and migration. In the present study, we have focused on the interaction of cellular PTPs with toxic trivalent arsenite (iAs{sup III}) and its intermediate metabolites such as monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) in vitro, and then determined the arsenic binding site in PTP by the use of recombinant PTPs (e.g., PTP1B and CD45). Interestingly, the activities of PTP1B (cytoplasm-form) or CD45 (receptor-linked form) were observed to be strongly inhibited by both methylated metabolites (i.e., MMA{sup III} and DMA{sup III}) but not by iAs{sup III}. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMA{sup III} directly bound to the active site cysteine residue of PTP1B (e.g., Cys215), resulting in inhibition of enzyme activity. These results suggest that arsenic exposure may disturb the cellular signaling pathways through PTP inactivation. Highlights: ► This study focused on the interaction of PTPs with trivalent arsenicals in vitro. ► We for the first time confirmed that DMA{sup III} strongly inhibited activity of PTP1B. ► DMA{sup III} directly bound to PTP1B, resulting in inhibition of enzyme activity.« less

Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca2+i signal in B cells

PubMed Central

Chen, Jie; Wang, Hong; Xu, Wei-Ping; Wei, Si-Si; Li, Hui Joyce; Mei, Yun-Qing; Li, Yi-Gang; Wang, Yue-Peng

2016-01-01

CD22 is a surface immunoglobulin implicated in negative regulation of B cell receptor (BCR) signaling; particularly inhibiting intracellular Ca2+ (Ca2+i)signals. Its cytoplasmic tail contains six tyrosine residues (Y773/Y783/Y817/Y828/Y843/Y863, designated Y1~Y6 respectively), including three (Y2/5/6) lying within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that serve to recruit the protein tyrosine phosphatase SHP-1 after BCR activation-induced phosphorylation. The mechanism of inhibiting Ca2+i by CD22 has been poorly understood. Previous study demonstrated that CD22 associated with plasma membrane calcium-ATPase (PMCA) and enhanced its activity (Chen, J. et al. Nat Immunol 2004;5:651-7). The association is dependent on BCR activation-induced cytoplasmic tyrosine phosphorylation, because CD22 with either all six tyrosines mutated to phenylalanines or cytoplasmic tail truncated loses its ability to associate with PMCA. However, which individual or a group of tyrosine residues determine the association and how CD22 and PMCA interacts, are still unclear. In this study, by using a series of CD22 tyrosine mutants, we found that ITIM Y2/5/6 accounts for 34.3~37.1% Ca2+i inhibition but is irrelevant for CD22/PMCA association. Non-ITIM Y4 and its YEND motif contribute to the remaining 69.4~71.7% Ca2+i inhibition and is the binding site for PMCA-associated Grb2. Grb2, independently of BCR cross-linking, is constitutively associated with and directly binds to PMCA in both chicken and human B cells. Knockout of Grb2 by CRISPR/Cas9 completely disrupted the CD22/PMCA association. Thus, our results demonstrate for the first time that in addition to previously-identified ITIM/SHP-1-dependent pathway, CD22 holds a major pathway of negative regulation of Ca2+i signal, which is ITIM/SHP-1-independent, but Y4/Grb2/PMCA-dependent. PMID:27276708

Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca2+i signal in B cells.

PubMed

Chen, Jie; Wang, Hong; Xu, Wei-Ping; Wei, Si-Si; Li, Hui Joyce; Mei, Yun-Qing; Li, Yi-Gang; Wang, Yue-Peng

2016-08-30

CD22 is a surface immunoglobulin implicated in negative regulation of B cell receptor (BCR) signaling; particularly inhibiting intracellular Ca2+ (Ca2+i)signals. Its cytoplasmic tail contains six tyrosine residues (Y773/Y783/Y817/Y828/Y843/Y863, designated Y1~Y6 respectively), including three (Y2/5/6) lying within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that serve to recruit the protein tyrosine phosphatase SHP-1 after BCR activation-induced phosphorylation. The mechanism of inhibiting Ca2+i by CD22 has been poorly understood. Previous study demonstrated that CD22 associated with plasma membrane calcium-ATPase (PMCA) and enhanced its activity (Chen, J. et al. Nat Immunol 2004;5:651-7). The association is dependent on BCR activation-induced cytoplasmic tyrosine phosphorylation, because CD22 with either all six tyrosines mutated to phenylalanines or cytoplasmic tail truncated loses its ability to associate with PMCA. However, which individual or a group of tyrosine residues determine the association and how CD22 and PMCA interacts, are still unclear. In this study, by using a series of CD22 tyrosine mutants, we found that ITIM Y2/5/6 accounts for 34.3~37.1% Ca2+i inhibition but is irrelevant for CD22/PMCA association. Non-ITIM Y4 and its YEND motif contribute to the remaining 69.4~71.7% Ca2+i inhibition and is the binding site for PMCA-associated Grb2. Grb2, independently of BCR cross-linking, is constitutively associated with and directly binds to PMCA in both chicken and human B cells. Knockout of Grb2 by CRISPR/Cas9 completely disrupted the CD22/PMCA association. Thus, our results demonstrate for the first time that in addition to previously-identified ITIM/SHP-1-dependent pathway, CD22 holds a major pathway of negative regulation of Ca2+i signal, which is ITIM/SHP-1-independent, but Y4/Grb2/PMCA-dependent.

Enzymatic characteristics of an ApaH-like phosphatase, PrpA, and a diadenosine tetraphosphate hydrolase, ApaH, from Myxococcus xanthus.

PubMed

Sasaki, Masashi; Takegawa, Kaoru; Kimura, Yoshio

2014-09-17

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap4A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards ApnA and ATP. In the presence of Mn(2+), PrpA hydrolyzed Ap4A into AMP and ATP, whereas in the presence of Co(2+) PrpA hydrolyzed Ap4A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards ApnA and ATP. Mn(2+) was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co(2+) was required for ApnA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an ApnA hydrolase, respectively. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Tyrosine phosphorylation–dependent activation of TRPC6 regulated by PLC-γ1 and nephrin: effect of mutations associated with focal segmental glomerulosclerosis

PubMed Central

Kanda, Shoichiro; Harita, Yutaka; Shibagaki, Yoshio; Sekine, Takashi; Igarashi, Takashi; Inoue, Takafumi; Hattori, Seisuke

2011-01-01

Transient receptor potential canonicals (TRPCs) play important roles in the regulation of intracellular calcium concentration. Mutations in the TRPC6 gene are found in patients with focal segmental glomerulosclerosis (FSGS), a proteinuric disease characterized by dysregulated function of renal glomerular epithelial cells (podocytes). There is as yet no clear picture for the activation mechanism of TRPC6 at the molecular basis, however, and the association between its channel activity and pathogenesis remains unclear. We demonstrate here that tyrosine phosphorylation of TRPC6 induces a complex formation with phospholipase C (PLC)-γ1, which is prerequisite for TRPC6 surface expression. Furthermore, nephrin, an adhesion protein between the foot processes of podocytes, binds to phosphorylated TRPC6 via its cytoplasmic domain, competitively inhibiting TRPC6–PLC-γ1 complex formation, TRPC6 surface localization, and TRPC6 activation. Importantly, FSGS-associated mutations render the mutated TRPC6s insensitive to nephrin suppression, thereby promoting their surface expression and channel activation. These results delineate the mechanism of TRPC6 activation regulated by tyrosine phosphorylation, and imply the cell type–specific regulation, which correlates the FSGS mutations with deregulated TRPC6 channel activity. PMID:21471003

N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene.

PubMed Central

Cross, F R; Garber, E A; Hanafusa, H

1985-01-01

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein. Images PMID:2426576

IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85.

PubMed Central

Myers, M G; Backer, J M; Sun, X J; Shoelson, S; Hu, P; Schlessinger, J; Yoakim, M; Schaffhausen, B; White, M F

1992-01-01

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains. Images PMID:1332046

The carboxy-terminal αN helix of the archaeal XerA tyrosine recombinase is a molecular switch to control site-specific recombination.

PubMed

Serre, Marie-Claude; El Arnaout, Toufic; Brooks, Mark A; Durand, Dominique; Lisboa, Johnny; Lazar, Noureddine; Raynal, Bertrand; van Tilbeurgh, Herman; Quevillon-Cheruel, Sophie

2013-01-01

Tyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding. Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.

NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 3 in apo state and in complex with inhibitor PD173074.

PubMed

Sanfelice, Domenico; Koss, Hans; Bunney, Tom D; Thompson, Gary S; Farrell, Brendan; Katan, Matilda; Breeze, Alexander L

2018-03-26

Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process, including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074. Analysis of chemical shift changes upon inhibitor binding highlights a characteristic pattern of allosteric network perturbations that is of relevance for future drug discovery activities aimed at development of conformationally-selective FGFR inhibitors.

Mass Spectral Detection of Diethoxyphospho-Tyrosine Adducts on Proteins from HEK293 Cells Using Monoclonal Antibody depY for Enrichment

PubMed Central

2018-01-01

Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins. Tyrosine in proteins that have no active site serine readily reacts with organophosphorus toxicants. We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence. Our goal in the current study was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon. Cell lysates treated with chlorpyrifos oxon were recognized by depY antibody in ELISA and capillary electrophoresis based Western blot. Tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. Liquid chromatography tandem mass spectrometry identified 116 diethoxyphospho-tyrosine peptides from 73 proteins in immunopurified samples, but found only 15 diethoxyphospho-tyrosine peptides from 12 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, histone H4, heat shock 70 kDa protein 1A/1B, heat shock protein HSP 90 β, and α-enolase, were represented by several diethoxyphospho-tyrosine peptides. It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture. The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species. Use of the depY antibody could lead to an understanding of chronic illness from organophosphorus pesticide exposure. PMID:29775289

The osmotic shock-induced glucose transport pathway in 3T3-L1 adipocytes is mediated by gab-1 and requires Gab-1-associated phosphatidylinositol 3-kinase activity for full activation.

PubMed

Janez, A; Worrall, D S; Imamura, T; Sharma, P M; Olefsky, J M

2000-09-01

Osmotic shock treatment of 3T3-L1 adipocytes causes an increase in glucose transport activity and translocation of GLUT4 protein similar to that elicited by insulin treatment. Insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, however, activation by osmotic shock was only partially blocked. Additionally, we have found that the newly identified insulin receptor substrate Gab-1 (Grb2-associated binder-1) is tyrosine-phosphorylated following sorbitol stimulation. Treatment of cells with the tyrosine kinase inhibitor genistein inhibited osmotic shock-stimulated Gab-1 phosphorylation as well as shock-induced glucose transport. Furthermore, pretreatment with the selective Src family kinase inhibitor PP2 completely inhibited the ability of sorbitol treatment to cause tyrosine phosphorylation of Gab-1. We have also shown that microinjection of anti-Gab-1 antibody inhibits osmotic shock-induced GLUT4 translocation. Furthermore, phosphorylated Gab-1 binds and activates phosphatidylinositol 3-kinase (PI3K) in response to osmotic shock. The PI3K activity associated with Gab-1 was 82% of that associated with anti-phosphotyrosine antibodies, indicating that Gab-1 is the major site for PI3K recruitment following osmotic shock stimulation. Although wortmannin only causes a partial block of osmotic shock-stimulated glucose uptake, wortmannin completely abolishes Gab-1 associated PI3K activity. This suggests that other tyrosine kinase-dependent pathways, in addition to the Gab-1-PI3K pathway, contribute to osmotic shock-mediated glucose transport. To date, Gab-1 is the first protein identified as a member of the osmotic shock signal transduction pathway.

Coronal loops and active region structure

NASA Technical Reports Server (NTRS)

Webb, D. F.; Zirin, H.

1981-01-01

Synoptic H-alpha Ca K, magnetograph and Skylab soft X-ray and EUV data were compared for the purpose of identifying the basic coronal magnetic structure of loops in a 'typical' active region and studying its evolution. A complex of activity in July 1973, especially McMath 12417, was emphasized. The principal results are: (1) most of the brightest loops connected the bright f plage to either the sunspot penumbra or to p satellite spots; no non-flaring X-ray loops end in umbrae; (2) short, bright loops had one or both ends in regions of emergent flux, strong field or high field gradients; (3) stable, strongly sheared loop arcades formed over filaments; (4) EFRs were always associated with compact X-ray arcades; and (5) loops connecting to other active regions had their bases in outlying plage of weak field strength in McM 417 where H-alpha fibrils marked the direction of the loops

The HER2 Signaling Network in Breast Cancer--Like a Spider in its Web.

PubMed

Dittrich, A; Gautrey, H; Browell, D; Tyson-Capper, A

2014-12-01

The human epidermal growth factor receptor 2 (HER2) is a major player in the survival and proliferation of tumour cells and is overexpressed in up to 30 % of breast cancer cases. A considerable amount of work has been undertaken to unravel the activity and function of HER2 to try and develop effective therapies that impede its action in HER2 positive breast tumours. Research has focused on exploring the HER2 activated phosphoinositide-3-kinase (PI3K)/AKT and rat sarcoma/mitogen-activated protein kinase (RAS/MAPK) pathways for therapies. Despite the advances, cases of drug resistance and recurrence of disease still remain a challenge to overcome. An important aspect for drug resistance is the complexity of the HER2 signaling network. This includes the crosstalk between HER2 and hormone receptors; its function as a transcription factor; the regulation of HER2 by protein-tyrosine phosphatases and a complex network of positive and negative feedback-loops. This review summarises the current knowledge of many different HER2 interactions to illustrate the complexity of the HER2 network from the transcription of HER2 to the effect of its downstream targets. Exploring the novel avenues of the HER2 signaling could yield a better understanding of treatment resistance and give rise to developing new and more effective therapies.

Gab2 Phosphorylation by RSK Inhibits Shp2 Recruitment and Cell Motility

PubMed Central

Zhang, Xiaocui; Lavoie, Genevieve; Fort, Loic; Huttlin, Edward L.; Tcherkezian, Joseph; Galan, Jacob A.; Gu, Haihua; Gygi, Steven P.; Carreno, Sebastien

2013-01-01

The scaffolding adapter protein Gab2 (Grb2-associated binder) participates in the signaling response evoked by various growth factors and cytokines. Gab2 is overexpressed in several human malignancies, including breast cancer, and was shown to promote mammary epithelial cell migration. The role of Gab2 in the activation of different signaling pathways is well documented, but less is known regarding the feedback mechanisms responsible for its inactivation. We now demonstrate that activation of the Ras/mitogen-activated protein kinase (MAPK) pathway promotes Gab2 phosphorylation on basic consensus motifs. More specifically, we show that RSK (p90 ribosomal S6 kinase) phosphorylates Gab2 on three conserved residues, both in vivo and in vitro. Mutation of these phosphorylation sites does not alter Gab2 binding to Grb2, but instead, we show that Gab2 phosphorylation inhibits the recruitment of the tyrosine phosphatase Shp2 in response to growth factors. Expression of an unphosphorylatable Gab2 mutant in mammary epithelial cells promotes an invasion-like phenotype and increases cell motility. Taken together, these results suggest that RSK is part of a negative-feedback loop that restricts Gab2-dependent epithelial cell motility. On the basis of the widespread role of Gab2 in receptor signaling, these findings also suggest that RSK plays a regulatory function in diverse receptor systems. PMID:23401857

Roles of the SH2 and SH3 domains in the regulation of neuronal Src kinase functions.

PubMed

Groveman, Bradley R; Xue, Sheng; Marin, Vedrana; Xu, Jindong; Ali, Mohammad K; Bienkiewicz, Ewa A; Yu, Xian-Min

2011-02-01

Previous studies demonstrated that intra-domain interactions between Src family kinases (SFKs), stabilized by binding of the phosphorylated C-terminus to the SH2 domain and/or binding of the SH2 kinase linker to the SH3 domain, lock the molecules in a closed conformation, disrupt the kinase active site, and inactivate SFKs. Here we report that the up-regulation of N-methyl-D-aspartate receptors (NMDARs) induced by expression of constitutively active neuronal Src (n-Src), in which the C-terminus tyrosine is mutated to phenylalanine (n-Src/Y535F), is significantly reduced by dysfunctions of the SH2 and/or SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and/or SH3 domains reduce auto-phosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDAR phosphorylation. The SH2 domain plays a greater regulatory role than the SH3 domain. Our data also show that n-Src binds directly to the C-terminus of the NMDAR NR2A subunit in vitro, with a K(D) of 108.2 ± 13.3 nM. This binding is not Src kinase activity-dependent, and dysfunctions of the SH2 and/or SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n-Src, which is important in the regulation of NMDAR functions. © 2010 The Authors Journal compilation © 2010 FEBS.

The EGFR family members sustain the neoplastic phenotype of ALK+ lung adenocarcinoma via EGR1

PubMed Central

Voena, C; Di Giacomo, F; Panizza, E; D'Amico, L; Boccalatte, F E; Pellegrino, E; Todaro, M; Recupero, D; Tabbò, F; Ambrogio, C; Martinengo, C; Bonello, L; Pulito, R; Hamm, J; Chiarle, R; Cheng, M; Ruggeri, B; Medico, E; Inghirami, G

2013-01-01

In non-small cell lung cancer (NSCLC), receptor tyrosine kinases (RTKs) stand out among causal dominant oncogenes, and the ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nonetheless, long-term RTK inhibition leads invariably to acquired resistance, tumor recurrence and metastatic dissemination. In ALK+ cell lines, inhibition of ALK signaling was associated with coactivation of several RTKs, whose pharmacological suppression reverted the partial resistance to ALK blockade. Remarkably, ERBB2 signaling synergized with ALK and contributed to the neoplastic phenotype. Moreover, the engagement of wild-type epidermal growth factor receptor or MET receptors could sustain cell viability through early growth response 1 (EGR1) and/or Erk1/2; Akt activation and EGR1 overexpression prevented cell death induced by combined ALK/RTK inhibition. Membrane expression of ERBB2 in a subset of primary naive ALK+ NSCLC could be relevant in the clinical arena. Our data demonstrate that the neoplastic phenotype of ALK-driven NSCLC relays ‘ab initio' on the concomitant activation of multiple RTK signals via autocrine/paracrine regulatory loops. These findings suggest that molecular and functional signatures are required in de novo lung cancer patients for the design of efficacious and multi-targeted ‘patient-specific' therapies. PMID:23567620

Profibrotic TGFβ responses require the cooperative action of PDGF and ErbB receptor tyrosine kinases.

PubMed

Andrianifahanana, Mahefatiana; Wilkes, Mark C; Gupta, Shiv K; Rahimi, Rod A; Repellin, Claire E; Edens, Maryanne; Wittenberger, Joshua; Yin, Xueqian; Maidl, Elizabeth; Becker, Jackson; Leof, Edward B

2013-11-01

Transforming growth factor β (TGFβ) has significant profibrotic activity both in vitro and in vivo. This reflects its capacity to stimulate fibrogenic mediators and induce the expression of other profibrotic cytokines such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF/ErbB) ligands. Here we address both the mechanisms by which TGFβ induced ErbB ligands and the physiological significance of inhibiting multiple TGFβ-regulated processes. The data document that ErbB ligand induction requires PDGF receptor (PDGFR) mediation and engages a positive autocrine/paracrine feedback loop via ErbB receptors. Whereas PDGFRs are essential for TGFβ-stimulated ErbB ligand up-regulation, TGFβ-specific signals are also required for ErbB receptor activation. Subsequent profibrotic responses are shown to involve the cooperative action of PDGF and ErbB signaling. Moreover, using a murine treatment model of bleomycin-induced pulmonary fibrosis we found that inhibition of TGFβ/PDGF and ErbB pathways with imatinib plus lapatinib, respectively, not only prevented myofibroblast gene expression to a greater extent than either drug alone, but also essentially stabilized gas exchange (oxygen saturation) as an overall measure of lung function. These observations provide important mechanistic insights into profibrotic TGFβ signaling and indicate that targeting multiple cytokines represents a possible strategy to ameliorate organ fibrosis dependent on TGFβ.

CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

USDA-ARS?s Scientific Manuscript database

Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain

USDA-ARS?s Scientific Manuscript database

In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct from one another, and thus while most animal receptor kinases are tyrosin...

Effects of 20 standard amino acids on the growth, total fatty acids production, and γ-linolenic acid yield in Mucor circinelloides.

PubMed

Tang, Xin; Zhang, Huaiyuan; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

2014-12-01

Twenty standard amino acids were examined as single nitrogen source on the growth, total fatty acids production, and yield of γ-linolenic acid (GLA) in Mucor circinelloides. Of the amino acids, tyrosine gave the highest biomass and lipid accumulation and thus resulted in a high GLA yield with respective values of 17.8 g/L, 23 % (w/w, dry cell weight, DCW), and 0.81 g/L, which were 36, 25, and 72 % higher than when the fungus was grown with ammonium tartrate. To find out the potential mechanism underlying the increased lipid accumulation of M. circinelloides when grown on tyrosine, the activity of lipogenic enzymes of the fungus during lipid accumulation phase was measured. The enzyme activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and ATP-citrate lyase were up-regulated, while NADP-isocitrate dehydrogenase was down-regulated by tyrosine during the lipid accumulation phase of the fungus which suggested that these enzymes may be involved in the increased lipid biosynthesis by tyrosine in this fungus.

The Gab1 protein is a docking site for multiple proteins involved in signaling by the B cell antigen receptor.

PubMed

Ingham, R J; Holgado-Madruga, M; Siu, C; Wong, A J; Gold, M R

1998-11-13

Gab1 is a member of the docking/scaffolding protein family which includes IRS-1, IRS-2, c-Cbl, p130(cas), and p62(dok). These proteins contain a variety of protein-protein interaction motifs including multiple tyrosine residues that when phosphorylated can act as binding sites for Src homology 2 (SH2) domain-containing signaling proteins. We show in the RAMOS human B cell line that Gab1 is tyrosine-phosphorylated in response to B cell antigen receptor (BCR) engagement. Moreover, tyrosine phosphorylation of Gab1 correlated with the binding of several SH2-containing signaling proteins to Gab1 including Shc, Grb2, phosphatidylinositol 3-kinase, and the SHP-2 tyrosine phosphatase. Far Western analysis showed that the SH2 domains of Shc, SHP-2, and the p85 subunit of phosphatidylinositol 3-kinase could bind directly to tyrosine-phosphorylated Gab1 isolated from activated RAMOS cells. In contrast, the Grb2 SH2 domain did not bind directly to Gab1 but instead to the Shc and SHP-2 associated with Gab1. We also show that Gab1 is present in the membrane-enriched particulate fraction of RAMOS cells and that Gab1/signaling protein complexes are found in this fraction after BCR engagement. Thus, tyrosine-phosphorylated Gab1 may recruit cytosolic signaling proteins to cellular membranes where they can act on membrane-bound targets. This may be a critical step in the activation of multiple BCR signaling pathways.

Catalytic Activity of the Anaerobic Tyrosine Lyase Required for Thiamine Biosynthesis in Escherichia coli*

PubMed Central

Challand, Martin R.; Martins, Filipa T.; Roach, Peter L.

2010-01-01

Thiazole synthase in Escherichia coli is an αβ heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the Cα–Cβ bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cyclization reaction catalyzed by ThiG. ThiH is a radical S-adenosylmethionine (AdoMet) enzyme that utilizes a [4Fe-4S]+ cluster to reductively cleave AdoMet, forming methionine and a 5′-deoxyadenosyl radical. Analysis of the time-dependent formation of the reaction products 5′-deoxyadenosine (DOA) and p-cresol has demonstrated catalytic behavior of the tyrosine lyase. The kinetics of product formation showed a pre-steady state burst phase, and the involvement of DOA in product inhibition was identified by the addition of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase to activity assays. This hydrolyzed the DOA and changed the rate-determining step but, in addition, substantially increased the uncoupled turnover of AdoMet. Addition of glyoxylate and ammonium inhibited the tyrosine cleavage reaction, but the reductive cleavage of AdoMet continued in an uncoupled manner. Tyrosine analogues were incubated with ThiGH, which showed a strong preference for phenolic substrates. 4-Hydroxyphenylpropionic acid analogues allowed uncoupled AdoMet cleavage but did not result in further reaction (Cα–Cβ bond cleavage). The results of the substrate analogue studies and the product inhibition can be explained by a mechanistic hypothesis involving two reaction pathways, a product-forming pathway and a futile cycle. PMID:19923213

Phosphorylation-mediated regulation of the Staphylococcus aureus secreted tyrosine phosphatase PtpA.

PubMed

Brelle, Solène; Baronian, Grégory; Huc-Brandt, Sylvaine; Zaki, Laila Gannoun; Cohen-Gonsaud, Martin; Bischoff, Markus; Molle, Virginie

2016-01-15

Due to the emergence of methicillin-resistant strains, Staphylococcus aureus has become as major public-health threat. Studies aimed at deciphering the molecular mechanism of virulence are thus required to identify new targets and develop efficient therapeutic agents. Protein phosphorylations are known to play key regulatory functions and their roles in pathogenesis are under intense scrutiny. Here we analyzed the protein tyrosine phosphatase PtpA of S. aureus, a member of the family of low molecular weight protein tyrosine phosphatases that are often secreted by pathogenic bacteria. We report for the first time that PtpA is phosphorylated in vitro by the S. aureus tyrosine kinase CapA1B2. A mass spectrometry approach allowed determining that Tyr122 and Tyr123 were the only two residues phosphorylated by this kinase. This result was confirmed by analysis of a double PtpA_Y122A/Y123A mutant that showed no phosphorylation by CapA1B2. Interestingly, PtpA phosphatase activity was abrogated in this mutant, suggesting a key regulatory function for these two tyrosine residues. This was further reinforced by the observation that CapA1B2-mediated phosphorylation significantly increased PtpA phosphatase activity. Moreover, we provide evidence that PtpA is secreted during growth of S. aureus. Together our results suggest that PtpA is an exported S. aureus signaling molecule controlled by tyrosine phosphorylation which may interfere with host cell signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

Dephosphorylation of GluN2B C-Terminal Tyrosine Residues Does Not Contribute to Acute Ethanol Inhibition of Recombinant NMDA Receptors

PubMed Central

Hughes, Benjamin A.; Smothers, Corigan T.; Woodward, John J.

2013-01-01

N-methyl-D-aspartate (NMDA) receptors are ion channels activated by the neurotransmitter glutamate and are highly expressed by neurons. These receptors are critical for excitatory synaptic signaling and inhibition of NMDA receptors leads to impaired cognition and learning. Ethanol inhibits NMDA currents at concentrations associated with intoxication and this action may underlie some of the behavioral effects of ethanol. Although numerous sites and mechanisms of action have been tested, the manner in which ethanol inhibits NMDA receptors remains unclear. Recent findings in the literature suggest that ethanol, via facilitation of tyrosine phosphatase activity, may dephosphorylate key tyrosine residues in the C-terminus of GluN2B subunits resulting in diminished channel function. To directly test this hypothesis, we engineered GluN2B mutants that contained phenylalanine in place of tyrosine at three different sites and transiently expressed them with the GluN1 subunit in human embryonic kidney (HEK) cells. Whole-cell patch clamp electrophysiology was used to record glutamate-activated currents in the absence and presence of ethanol (10–600 mM). All mutants were functional and did not differ from one another with respect to current amplitude, steady-state to peak ratio, or magnesium block. Analysis of ethanol dose-response curves showed no significant difference in IC50 values between wild-type receptors and Y1252F, Y1336F, Y1472F or triple Y-F mutants. These findings suggest that dephosphorylation of C-terminal tyrosine residues does not account for ethanol inhibition of GluN2B receptors. PMID:23357553

Transforming properties of the Huntingtin interacting protein 1/ platelet-derived growth factor beta receptor fusion protein.

PubMed

Ross, T S; Gilliland, D G

1999-08-06

We have previously reported that the Huntingtin interacting protein 1 (HIP1) gene is fused to the platelet-derived growth factor beta receptor (PDGFbetaR) gene in a patient with chronic myelomonocytic leukemia. We now show that HIP1/PDGFbetaR oligomerizes, is constitutively tyrosine-phosphorylated, and transforms the murine hematopoietic cell line, Ba/F3, to interleukin-3-independent growth. A kinase-inactive mutant is neither tyrosine-phosphorylated nor able to transform Ba/F3 cells. Oligomerization and kinase activation required the 55-amino acid carboxyl-terminal TALIN homology region but not the leucine zipper domain. Tyrosine phosphorylation of a 130-kDa protein and STAT5 correlates with transformation in cells expressing HIP1/PDGFbetaR and related mutants. A deletion mutant fusion protein that contains only the TALIN homology region of HIP1 fused to PDGFbetaR is incapable of transforming Ba/F3 cells and does not tyrosine-phosphorylate p130 or STAT5, although it is itself constitutively tyrosine-phosphorylated. We have also analyzed cells expressing Tyr --> Phe mutants of HIP1/PDGFbetaR in the known PDGFbetaR SH2 docking sites and report that none of these sites are necessary for STAT5 activation, p130 phosphorylation, or Ba/F3 transformation. The correlation of factor-independent growth of hematopoietic cells with p130 and STAT5 phosphorylation/activation in both the HIP1/PDGFbetaR Tyr --> Phe and deletion mutational variants suggests that both STAT5 and p130 are important for transformation mediated by HIP1/PDGFbetaR.

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

PubMed

Takayama, S; White, M F; Kahn, C R

1988-03-05

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.

Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.

PubMed

Bubeck Wardenburg, J; Fu, C; Jackman, J K; Flotow, H; Wilkinson, S E; Williams, D H; Johnson, R; Kong, G; Chan, A C; Findell, P R

1996-08-16

Two families of tyrosine kinases, the Src and Syk families, are required for T-cell receptor activation. While the Src kinases are responsible for phosphorylation of receptor-encoded signaling motifs and for up-regulation of ZAP-70 activity, the downstream substrates of ZAP-70 are unknown. Evidence is presented herein that the Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is a substrate of ZAP-70. Phosphorylation of SLP-76 is diminished in T cells that express a catalytically inactive ZAP-70. Moreover, SLP-76 is preferentially phosphorylated by ZAP-70 in vitro and in heterologous cellular systems. In T cells, overexpression of wild-type SLP-76 results in a hyperactive receptor, while expression of a SLP-76 molecule that is unable to be tyrosine-phosphorylated attenuates receptor function. In addition, the SH2 domain of SLP-76 is required for T-cell receptor function, although its role is independent of the ability of SLP-76 to undergo tyrosine phosphorylation. As SLP-76 interacts with both Grb2 and phospholipase C-gamma1, these data indicate that phosphorylation of SLP-76 by ZAP-70 provides an important functional link between the T-cell receptor and activation of ras and calcium pathways.

Horseradish-Peroxidase-Catalyzed Tyrosine Click Reaction.

PubMed

Sato, Shinichi; Nakamura, Kosuke; Nakamura, Hiroyuki

2017-03-02

The efficiency of protein chemical modification on tyrosine residues with N-methylluminol derivatives was drastically improved by using horseradish peroxidase (HRP). In the previous method, based on the use of hemin and H 2 O 2 , oxidative side reactions such as cysteine oxidation were problematic for functionalization of proteins selectively on tyrosine residues. Oxidative activation of N-methylluminol derivatives with a minimum amount of H 2 O 2 prevented the occurrence of oxidative side reactions under HRP-catalyzed conditions. As probes for HRP-catalyzed protein modification, N-methylluminol derivatives showed much higher efficiency than tyramide without inducing oligomerization of probe molecules. Tyrosine modification also proceeded in the presence of β-nicotinamide adenine dinucleotide (NADH, H 2 O 2 -free conditions). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Use of an iodide-specific electrode to study lactoperoxidase-catalyzed iodination of l-tyrosine.

PubMed

Threatte, R M; Fregly, M J; Field, F P; Jones, P K

1979-12-01

An in vitro method employing an iodide-specific electrode for monitoring lactoperoxidase-catalyzed iodination is described. The method utilized lactoperoxidase, potassium iodide, and a glucose--glucose oxidase system for the generation of hydrogen peroxide and l-tyrosine. As iodination of l-tyrosine proceeded, the free iodide concentration in solution decreased and was monitored by an iodide-specific electrode. The iodide electrode was reliable when compared to a 131I-method for measuring free iodide changes in solution. Increasing concentrations of resorcinol, a well-known inhibitor of thyroid peroxidase-catalyzed iodination, in the reaction mixture resulted in graded inhibition of the initial rate of lactoperoxidase-catalyzed l-tyrosine iodination. This in vitro system can be used to assess inhibitory activity of various antithyroid substances.

[Tyrosine and methionine metabolism in various states of melaninogenesis].

PubMed

Kurbanov, Kh; Spiridonova, N A

1990-01-01

Excretion with urine of tyrosine and methionine metabolites as well as the activities of enzymes involved in their metabolism are correlated with the state and type of melanin synthesized in the skin. The response of tyrosine aminotransferase to melaninogenesis induction was more pronounced in animals with predominant pheomelaninogenesis, especially after tyrosine load, while that to dopachrome oxidoreductase--in animals with predominant eumelaninogenesis and after methionine load. Glutathione reductase and cystathionine-beta-synthase responded more vigorously to methionine injections, which was especially well pronounced in animals with prominent pheomelaninogenesis and in albino animals. The metabolic "block" in melanine synthesis in albino animals seems to be observed after the 5-S-cysteinyl-DOPA synthesis, whereas the initial steps of melaninogenesis in these animals are identical to pheomelanine synthesis reactions.

Phosphorylation of tyrosine 285 of PAK1 facilitates βPIX/GIT1 binding and adhesion turnover

PubMed Central

Hammer, Alan; Oladimeji, Peter; De Las Casas, Luis E.; Diakonova, Maria

2015-01-01

The p21-activated serine-threonine kinase (PAK1) regulates cell motility and adhesion. We have previously shown that the prolactin (PRL)-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo and in vitro and identified tyrosines 153, 201, and 285 in PAK1 as sites of JAK2 tyrosyl phosphorylation. Here, we further investigate the role of the tyrosyl phosphorylated PAK1 (pTyr-PAK1) in regulation of cell adhesion. We use human breast cancer T47D cell lines that stably overexpress PAK1 wild type or PAK1 Y3F mutant in which these 3 JAK2 phosphorylation sites were mutated to phenylalanine. We demonstrate that PRL/JAK2-dependent phosphorylation of these tyrosines promotes a motile phenotype in the cells upon adhesion, participates in regulation of cell adhesion on collagen IV, and is required for maximal PAK1 kinase activity. Down-regulation of PAK1 abolishes the effect of PAK1 on cell adhesion. We show that the tyrosyl phosphorylation of PAK1 promotes PAK1 binding to β-PAK1-interacting guanine-nucleotide exchange factor (βPIX) and G protein-coupled receptor kinase-interacting target 1 (GIT1), phosphorylation of paxillin on Ser273, and formation and distribution of adhesion complexes. Using phosphospecific antibodies (Abs) directed to single phosphorylated tyrosines on PAK1, we identified Tyr285 as a site of PRL-dependent phosphorylation of PAK1 by JAK2. Furthermore, using PAK1 Y285F mutant, we provide evidence for a role of pTyr285 in cell adhesion, enhanced βPIX/GIT1 binding, and adhesion turnover. Our immunohistochemistry analysis demonstrates that pTyr285- PAK1 may modulate PAK1 signaling during tumor progression.—Hammer, A., Oladimeji, P., De La Casas, L. E., Diakonova, M. Phosphorylation of tyrosine 285 of PAK1 facilitates bPIX/GIT1 binding and adhesion turnover. PMID:25466889

Skin problems and EGFR-tyrosine kinase inhibitor.

PubMed

Kozuki, Toshiyuki

2016-04-01

Epidermal growth factor receptor inhibition is a good target for the treatment of lung, colon, pancreatic and head and neck cancers. Epidermal growth factor receptor-tyrosine kinase inhibitor was first approved for the treatment of advanced lung cancer in 2002. Epidermal growth factor receptor-tyrosine kinase inhibitor plays an essential role in the treatment of cancer, especially for patients harbouring epidermal growth factor receptor activating mutation. Hence, skin toxicity is the most concerning issue for the epidermal growth factor receptor-tyrosine kinase inhibitor treatment. Skin toxicity is bothersome and sometimes affects the quality of life and treatment compliance. Thus, it is important for physicians to understand the background and how to manage epidermal growth factor receptor-tyrosine kinase inhibitor-associated skin toxicity. Here, the author reviewed the mechanism and upfront preventive and reactive treatments for epidermal growth factor receptor inhibitor-associated skin toxicities. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Synchrotron Ultraviolet Microspectroscopy on Rat Cortical Bone: Involvement of Tyrosine and Tryptophan in the Osteocyte and Its Environment

PubMed Central

Pallu, Stéphane; Rochefort, Gael Y.; Jaffre, Christelle; Refregiers, Matthieu; Maurel, Delphine B.; Benaitreau, Delphine; Lespessailles, Eric; Jamme, Frédéric; Chappard, Christine; Benhamou, Claude-Laurent

2012-01-01

Alcohol induced osteoporosis is characterized by a bone mass decrease and microarchitecture alterations. Having observed an excess in osteocyte apoptosis, we aimed to assess the bone tissue biochemistry, particularly in the osteocyte and its environment. For this purpose, we used a model of alcohol induced osteoporosis in rats. Bone sections of cortical bone were investigated using synchrotron UV-microspectrofluorescence at subcellular resolution. We show that bone present three fluorescence peaks at 305, 333 and 385 nm, respectively corresponding to tyrosine, tryptophan and collagen. We have determined that tyrosine/collagen and tryptophan/collagen ratios were higher in the strong alcohol consumption group. Tryptophan is related to the serotonin metabolism involved in bone formation, while tyrosine is involved in the activity of tyrosine kinases and phosphatases in osteocytes. Our experiment represents the first combined synchrotron UV microspectroscopy analysis of bone tissue with a quantitative biochemical characterization in the osteocyte and surrounding matrix performed separately. PMID:22937127

Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp

2013-11-29

Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143more » flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/K{sub app} reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.« less

Src kinases and ERK activate distinct responses to Stitcher receptor tyrosine kinase signaling during wound healing in Drosophila.

PubMed

Tsarouhas, Vasilios; Yao, Liqun; Samakovlis, Christos

2014-04-15

Metazoans have evolved efficient mechanisms for epidermal repair and survival following injury. Several cellular responses and key signaling molecules that are involved in wound healing have been identified in Drosophila, but the coordination of cytoskeletal rearrangements and the activation of gene expression during barrier repair are poorly understood. The Ret-like receptor tyrosine kinase (RTK) Stitcher (Stit, also known as Cad96Ca) regulates both re-epithelialization and transcriptional activation by Grainy head (Grh) to induce restoration of the extracellular barrier. Here, we describe the immediate downstream effectors of Stit signaling in vivo. Drk (Downstream of receptor kinase) and Src family tyrosine kinases bind to the same docking site in the Stit intracellular domain. Drk is required for the full activation of transcriptional responses but is dispensable for re-epithelialization. By contrast, Src family kinases (SFKs) control both the assembly of a contractile actin ring at the wound periphery and Grh-dependent activation of barrier-repair genes. Our analysis identifies distinct pathways mediating injury responses and reveals an RTK-dependent activation mode for Src kinases and their central functions during epidermal wound healing in vivo.

Into the linker's DENN: A tyrosine's control of autophagy.

PubMed

Caplan, Steve

2017-04-28

The small GTP-binding protein Rab12 plays an important role in the initiation of starvation-induced macroautophagy (autophagy) and is activated by the guanine-nucleotide exchange factor DENND3. However, the molecular mechanism by which DENND3 becomes activated has remained elusive. Xu and McPherson now identify a novel mechanism of DENND3 intramolecular binding that is regulated by the phosphorylation of a single tyrosine residue. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

An extract of Perilla stem inhibits Src homology phosphatase-1 (SHP)-1 and influences insulin signaling.

PubMed

Peng, Liu; Lei, Zhang; Xiao-na, Xie; Deli, Wang; Jing, Sun; Yong-sen, Wang; Zhi, Wang; Shu, Xing; Jun-feng, Ma; Wan-nan, Li; Xue-qi, Fu

2015-03-01

Protein tyrosine phosphatases (PTPs) are enzymes that catalyze protein tyrosine dephosphorylation of which Src homology phosphatase-1 (SHP-1) is one of the best-validated, a widely distributed intracellular tyrosine phosphatase that contains two SH2 domains. Down regulation of SHP-1 tyrosine phosphatases was significantly increased sensitivity to insulin in insulin signaling pathway. Through in vitro enzymatic reaction kinetics experiment, we found that the extract of Perilla stem was a potential inhibitor to δSHP-1, the catalytic domain of SHP-1 protein tyrosine phosphatase, and its IC(50) was 4ug/ml, and was more sensitive towards SHP-1than other PTPs, which indicated that SHP-1 might be a target of the extract of Perilla stem. It can strengthened the level of tyrosine phosphorylation of insulin receptor (IR) and extracellular signal-regulated protein kinase (ERK) in HepG2 cells, and then activated the insulin signaling pathway through inhibiting the protein phosphorylation of SHP-1. These results demonstrated that the extract of Perilla stem could play an important role for diabetes treatment through inhibiting the level of SHP-1 in insulin signaling pathway.

Enteropathogenic Escherichia coli Use Redundant Tyrosine Kinases to Form Actin PedestalsD⃞

PubMed Central

Swimm, Alyson; Bommarius, Bettina; Li, Yue; Cheng, David; Reeves, Patrick; Sherman, Melanie; Veach, Darren; Bornmann, William; Kalman, Daniel

2004-01-01

Enteropathogenic Escherichia coli (EPEC) are deadly contaminants in water and food and induce protrusion of actin-rich membrane pedestals beneath themselves upon attachment to intestinal epithelia. EPEC then causes intestinal inflammation, diarrhea, and, among children, death. Here, we show that EPEC uses multiple tyrosine kinases for formation of pedestals, each of which is sufficient but not necessary. In particular, we show that Abl and Arg, members of the Abl family of tyrosine kinases, localize and are activated in pedestals. We also show that pyrido[2,3-d]pyrimidine (PD) compounds, which inhibit Abl, Arg, and related kinases, block pedestal formation. Finally, we show that Abl and Arg are sufficient for pedestal formation in the absence of other tyrosine kinase activity, but they are not necessary. Our results suggest that additional kinases that are sensitive to inhibition by PD also can suffice. Together, these results suggest that EPEC has evolved a mechanism to use any of several functionally redundant tyrosine kinases during pathogenesis, perhaps facilitating its capacity to infect different cell types. Moreover, PD compounds are being developed to treat cancers caused by dysregulated Abl. Our results raise the possibility that PD may be useful in treating EPEC infections, and because PD affects host and not bacterium, selecting resistant strains may be far less likely than with conventional antibiotics. PMID:15155808

Identification of functional VEGF receptors on human platelets.

PubMed

Selheim, Frode; Holmsen, Holm; Vassbotn, Flemming S

2002-02-13

Platelets secrete platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) upon stimulation. We have demonstrated that platelets have functionally active PDGF alpha-receptors, a transmembrane tyrosine kinase involved in negative feedback regulation. Here we demonstrate the presence of the related VEGF receptors fms-like tyrosine kinase-1 and kinase-insert domain region on human platelets. VEGF itself did not cause platelet aggregation. However, addition of exogenous VEGF to SFRLLN or thrombin-stimulated platelets potentiated platelet aggregation. Moreover, thrombin-induced phosphoinositide 3-kinase and mitogen-activated protein kinase activity were enhanced in the presence of VEGF.

Influence of neonatal and adult hyperthyroidism on behavior and biosynthetic capacity for norepinephrine, dopamine and 5-hydroxytryptamine in rat brain.

PubMed

Rastogi, R B; Singhal, R L

1976-09-01

In neonatal rats, administration of l-triiodothyronine (10 mug/100 g/day) for 30 days presented signs of hyperthyroidism which included accelerated development of a variety of physical and behavioral characteristics accompanying maturation. The spontaneous motor activity was increased by 69%. Exposure of developing rats to thyroid hormone significantly increased the endogenous concentration of striatal tyrosine and the activity of tyrosine hydroxylase as well as the levels of dopamine in several brain regions. The concentration of striatal homovanillic acid and 3,4-dihydroxyphenylacetic acid, the chief metabolites of dopamine, was also increased and the magnitude of change was greater than the rise in dopamine. Despite increases in the activity of tyrosine hydroxylase and the availability of the substrate tyrosine, the steady-state levels of norepinephrine remained unaltered in various regions of brain except in cerebellum. Futhermore, neonatal hyperthyroidism significantly increased the levels of midbrain tryptophan and tryptophan hydroxylase activity but produced no change in 5-hydroxytryptamine levels of several discrete brain regions, except hypothalamus and cerebellum where its concentration was slightly decreased. However, the 5-hydroxyindoleacetic acid levels were enhanced in hypothalamus, ponsmedulla, midbrain, striatum and hippocampus. The elevated levels of 5-hydroxyindoleacetic acid did not seem to be due to increased intraneuronal deamination of 5-hydroxytryptamine since monoamine oxidase activity was not affected in cerebral cortex and midbrain of hyperthyroid rats. The data demonstrate that hyperthyroidism significantly increased the synthesis as well as the utilization of catecholamines and 5-hydroxytryptamine in maturing brain. Since the mature brain is known to respond differently to thyroid hormone action than does the developing brain, the effect of L-triiodothyronine treatment on various putative neurohumors also was examined in adult rats. Whereas administration of l-triiodothyronine (10 mug/100 g/day) for 30 days to 120-day-old rats increased the levels of tyrosine by 23% and of tryptophan by 43%, no appreciable change was noted in tryptophan hydroxylase activity. In contrast to neonatal hyperthyroidism, excess of thyroid hormone in adult rats failed to produce any change in motor activity and tended to decrease striatal tyrosine hydroxylase activity only slightly. The concentration of dopamine remained unchanged in all regions of the brain except in midbrain where it rose by 19%. Whereas norepinephrine concentration was altered in hypothalamus, pons-medulla and midbrain, the levels of 5-hydroxytryptamine and its metabolite, 5-hydroxyindoleacetic acid, were significantly decreased in striatum and cerebellum. Since dopaminergic and noradrenergic neurons are the critical components of the motor system, the possibility exists that elevated behavioral activity in young L-triiodothyronine-treated animals might be associated with increased turnover of catecholamines in neuronal tissue.

Hey1- and p53-dependent TrkC proapoptotic activity controls neuroblastoma growth.

PubMed

Ménard, Marie; Costechareyre, Clélia; Ichim, Gabriel; Blachier, Jonathan; Neves, David; Jarrosson-Wuilleme, Loraine; Depping, Reinhard; Koster, Jan; Saintigny, Pierre; Mehlen, Patrick; Tauszig-Delamasure, Servane

2018-05-01

The neurotrophin-3 (NT-3) receptor tropomyosin receptor kinase C (TrkC/NTRK3) has been described as a dependence receptor and, as such, triggers apoptosis in the absence of its ligand NT-3. This proapoptotic activity has been proposed to confer a tumor suppressor activity to this classic tyrosine kinase receptor (RTK). By investigating interacting partners that might facilitate TrkC-induced cell death, we have identified the basic helix-loop-helix (bHLH) transcription factor Hey1 and importin-α3 (karyopherin alpha 4 [KPNA4]) as direct interactors of TrkC intracellular domain, and we show that Hey1 is required for TrkC-induced apoptosis. We propose here that the cleaved proapoptotic portion of TrkC intracellular domain (called TrkC killer-fragment [TrkC-KF]) is translocated to the nucleus by importins and interacts there with Hey1. We also demonstrate that Hey1 and TrkC-KF transcriptionally silence mouse double minute 2 homolog (MDM2), thus contributing to p53 stabilization. p53 transcriptionally regulates the expression of TrkC-KF cytoplasmic and mitochondrial interactors cofactor of breast cancer 1 (COBRA1) and B cell lymphoma 2-associated X (BAX), which will subsequently trigger the intrinsic pathway of apoptosis. Of interest, TrkC was proposed to constrain tumor progression in neuroblastoma (NB), and we demonstrate in an avian model that TrkC tumor suppressor activity requires Hey1 and p53.

SLP-76 mediates and maintains activation of the Tec family kinase ITK via the T cell antigen receptor-induced association between SLP-76 and ITK.

PubMed

Bogin, Yaron; Ainey, Carmit; Beach, Dvora; Yablonski, Deborah

2007-04-17

ITK (IL-2-inducible T cell kinase), a Tec family protein tyrosine kinase (PTK), is one of three PTKs required for T cell antigen receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1). Like Src and Abl family PTKs, ITK adopts an inactive, "closed" conformation, and its conversion to the active conformation is not well understood, nor have its direct substrates been identified. In a side-by-side comparison of ITK and ZAP-70 (zeta chain-associated protein kinase of 70 kDa), ITK efficiently phosphorylated Y(783) and Y(775) of PLC-gamma1, two phosphorylation sites that are critical for its activation, whereas ZAP-70 did not. SLP-76 (SH2-domain-containing leukocyte protein of 76 kDa), an adaptor required for TCR-induced activation of PLC-gamma1, was required for the phosphorylation of both PLC-gamma1 sites in intact cells. Furthermore, this event depended on the N-terminal tyrosines of SLP-76. Likewise, SLP-76, particularly its N-terminal tyrosines, was required for TCR-induced tyrosine phosphorylation and activation of ITK but was not required for the phosphorylation or activation of ZAP-70. Both ZAP-70 and ITK phosphorylated SLP-76 in vitro; thus, both PTKs are potential regulators of SLP-76, but only ITK is regulated by SLP-76. Upon TCR stimulation, a small fraction of ITK bound to SLP-76. This fraction, however, encompassed most of the catalytically active ITK. Catalytic activity was lost upon mild elution of ITK from the SLP-76-nucleated complex but was restored upon reconstitution of the complex. We propose that SLP-76 is required for ITK activation; furthermore, an ongoing physical interaction between SLP-76 and ITK is required to maintain ITK in an active conformation.

AXL mediates resistance to cetuximab therapy.

PubMed

Brand, Toni M; Iida, Mari; Stein, Andrew P; Corrigan, Kelsey L; Braverman, Cara M; Luthar, Neha; Toulany, Mahmoud; Gill, Parkash S; Salgia, Ravi; Kimple, Randall J; Wheeler, Deric L

2014-09-15

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical outcome. In this study, we show that overexpression of the oncogenic receptor tyrosine kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated, and tightly associated with EGFR expression in cells resistant to cetuximab (Ctx(R) cells). Using RNAi methods and novel AXL-targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation, and MAPK signaling in Ctx(R) cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in Ctx(R) cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft models, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL-targeting drugs to treat cetuximab-resistant cancers. Cancer Res; 74(18); 5152-64. ©2014 AACR. ©2014 American Association for Cancer Research.

The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

NASA Astrophysics Data System (ADS)

Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

2015-03-01

The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

Discovery of a Novel Series of Inhibitors of Lymphoid Tyrosine Phosphatase with Activity in Human T Cells†

PubMed Central

Stanford, Stephanie M.; Krishnamurthy, Divya; Falk, Matthew D.; Messina, Rossella; Debnath, Bikash; Li, Sheng; Liu, Tong; Kazemi, Roza; Dahl, Russell; He, Yantao; Yu, Xiao; Chan, Andrew C.; Zhang, Zhong-Yin; Barrios, Amy M.; Woods, Virgil L.; Neamati, Nouri; Bottini, Nunzio

2011-01-01

The lymphoid tyrosine phosphatase LYP, encoded by the PTPN22 gene, is a critical regulator of signaling in T cells and recently emerged as a candidate target for therapy of autoimmune diseases. Here, by library screening, we identified a series of noncompetitive inhibitors of LYP that showed activity in primary T cells. Kinetic analysis confirmed that binding of the compounds to the phosphatase is nonmutually exclusive with respect to a known bidentate competitive inhibitor. The mechanism of action of the lead inhibitor compound 4e was studied by a combination of hydrogen/deuterium-exchange mass spectrometry and molecular modeling. The results suggest that the inhibitor interacts critically with a hydrophobic patch located outside the active site of the phosphatase. Targeting of secondary allosteric sites is viewed as a promising yet unexplored approach to develop pharmacological inhibitors of protein tyrosine phosphatases. Our novel scaffold could be a starting point to attempt development of “nonactive site” anti-LYP pharmacological agents. PMID:21341673

Negative regulation of glial engulfment activity by Draper terminates glial responses to axon injury

PubMed Central

Logan, Mary A.; Hackett, Rachel; Doherty, Johnna; Sheehan, Amy; Speese, Sean D.; Freeman, Marc R.

2012-01-01

Neuronal injury elicits potent cellular responses from glia, but molecular pathways modulating glial activation, phagocytic function, and termination of reactive responses remain poorly defined. Here we show that positive or negative regulation of glial reponses to axon injury are molecularly encoded by unique isoforms of the Drosophila engulfment receptor Draper. Draper-I promotes engulfment of axonal debris through an immunoreceptor tyrosine-based activation motif (ITAM). In contrast, Draper-II, an alternative splice variant, potently inhibits glial engulfment function. Draper-II suppresses Draper-I signaling through a novel immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain and the tyrosine phosphatase Corkscrew (Csw). Intriguingly, loss of Draper-II/Csw signaling prolongs expression of glial engulfment genes after axotomy and reduces the ability of glia to respond to secondary axotomy. Our work highlights a novel role for Draper-II in inhibiting glial responses to neurodegeneration, and indicates a balance of opposing Draper-I/-II signaling events is essential to maintain glial sensitivity to brain injury. PMID:22426252

Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium.

PubMed

Amsler, K; Kuwada, S K

1999-01-01

Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-gamma protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.

Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

PubMed Central

Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R

1996-01-01

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261

Itk tyrosine kinase substrate docking is mediated by a nonclassical SH2 domain surface of PLCgamma1.

PubMed

Min, Lie; Joseph, Raji E; Fulton, D Bruce; Andreotti, Amy H

2009-12-15

Interleukin-2 tyrosine kinase (Itk) is a Tec family tyrosine kinase that mediates signaling processes after T cell receptor engagement. Activation of Itk requires recruitment to the membrane via its pleckstrin homology domain, phosphorylation of Itk by the Src kinase, Lck, and binding of Itk to the SLP-76/LAT adapter complex. After activation, Itk phosphorylates and activates phospholipase C-gamma1 (PLC-gamma1), leading to production of two second messengers, DAG and IP(3). We have previously shown that phosphorylation of PLC-gamma1 by Itk requires a direct, phosphotyrosine-independent interaction between the Src homology 2 (SH2) domain of PLC-gamma1 and the kinase domain of Itk. We now define this docking interface using a combination of mutagenesis and NMR spectroscopy and show that disruption of the Itk/PLCgamma1 docking interaction attenuates T cell signaling. The binding surface on PLCgamma1 that mediates recognition by Itk highlights a nonclassical binding activity of the well-studied SH2 domain providing further evidence that SH2 domains participate in important signaling interactions beyond recognition of phosphotyrosine.

Molecular mechanism of the Syk activation switch.

PubMed

Tsang, Emily; Giannetti, Anthony M; Shaw, David; Dinh, Marie; Tse, Joyce K Y; Gandhi, Shaan; Ho, Hoangdung; Wang, Sandra; Papp, Eva; Bradshaw, J Michael

2008-11-21

Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340-356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.

Structure-activity relationships of N-beta-phenylpropionyl-L-tyrosine and its derivatives on the inhibition of an identifiable giant neurone of an African giant snail (Achatina fulica Férussac).

PubMed Central

Ariyoshi, Y.; Takeuchi, H.

1982-01-01

1 Inhibitory effects of N-beta-phenylpropionyl-L-tyrosine, N-beta-phenylpropionyl-L-tryptophan and their derivatives on an identifiable giant neurone, TAN (tonically autoactive neurone) of an African giant snail (Achatina fulica Férussac) were examined in an attempt to elucidate which structural features are necessary to produce the effect. 2 Of the compounds examined, N-beta-cyclohexylpropionyl-L-tyrosine showed the strongest effect. Its critical concentration (c.c.) was 3 X 10(-8)-10(-7)M, about ten times lower than that of N-beta-phenylpropionyl-L-tyrosine (c.c., 3 X 10(-7)-10(-6)M). N-beta-cyclohexylpropionyl-L-tryptophan (c.c., 10(-6)M) had an effect almost similar to that of N-beta-phenylpropionyl-L-tryptophan (c.c., 10(-6)M). 3 N-beta-Phenylpropionyl-N-methyl-L-tyrosine had no effect at a high concentration. 4 Effects of N-beta-phenylpropionyl-L-tyrosine amide (c.c., 3 X 10(-7)-10(-6)M) and N-beta-phenylpropionyl-L-tryptophan amide (c.c., 10(-6)M) were very similar to those of N-beta-phenylpropionyl-L-tyrosine and N-beta-phenylpropionyl-L-tryptophan respectively. 5 N-beta-Phenylpropionyl-p-amino-L-phenylalanine (c.c., 3 X 10(-5)-10(-4)M) and N-beta-phenylpropionyl-p-chloro-L-phenylalanine (c.c., 10(-4)M) had only a weak effect. 6 It is proposed that the structural features producing the effect are as follows: the active compound has a phenyl or a cyclohexyl group (hydrophobic binding group), after a suitable distance a peptide bond (proton donor and proton acceptor), adjacently a carbonyl group (proton acceptor), and a phenolic hydroxyl or an indolyl imino group (proton donor) in the molecule. PMID:7150871

Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcgamma receptors and tyrosine kinases

PubMed Central

1996-01-01

In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H+ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H+ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H+ exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform- specific antibodies confirmed the presence of NHE-1 protein in neutrophils. Since phagocytosis involves Fcgamma (FcgammaR) and complement receptors such as CR3 (a beta2 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against FcgammaRs (FcgammaRII > FcgammaRIII) but not beta2 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-beta2 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE- 1 activation involved cooperation between integrins and FcgammaRs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcgammaR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-I activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca2+ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc-gammaR cross-linking. In summary, Fc-gammaRs and beta2 integrins cooperate in activation of NHE- 1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation. PMID:8601583

Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium.

PubMed

Murata, Yoji; Kotani, Takenori; Supriatna, Yana; Kitamura, Yasuaki; Imada, Shinya; Kawahara, Kohichi; Nishio, Miki; Daniwijaya, Edwin Widyanto; Sadakata, Hisanobu; Kusakari, Shinya; Mori, Munemasa; Kanazawa, Yoshitake; Saito, Yasuyuki; Okawa, Katsuya; Takeda-Morishita, Mariko; Okazawa, Hideki; Ohnishi, Hiroshi; Azuma, Takeshi; Suzuki, Akira; Matozaki, Takashi

2015-08-04

Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.

Protein tyrosine phosphatases as potential therapeutic targets

PubMed Central

He, Rong-jun; Yu, Zhi-hong; Zhang, Ruo-yu; Zhang, Zhong-yin

2014-01-01

Protein tyrosine phosphorylation is a key regulatory process in virtually all aspects of cellular functions. Dysregulation of protein tyrosine phosphorylation is a major cause of human diseases, such as cancers, diabetes, autoimmune disorders, and neurological diseases. Indeed, protein tyrosine phosphorylation-mediated signaling events offer ample therapeutic targets, and drug discovery efforts to date have brought over two dozen kinase inhibitors to the clinic. Accordingly, protein tyrosine phosphatases (PTPs) are considered next-generation drug targets. For instance, PTP1B is a well-known targets of type 2 diabetes and obesity, and recent studies indicate that it is also a promising target for breast cancer. SHP2 is a bona-fide oncoprotein, mutations of which cause juvenile myelomonocytic leukemia, acute myeloid leukemia, and solid tumors. In addition, LYP is strongly associated with type 1 diabetes and many other autoimmune diseases. This review summarizes recent findings on several highly recognized PTP family drug targets, including PTP1B, Src homology phosphotyrosyl phosphatase 2(SHP2), lymphoid-specific tyrosine phosphatase (LYP), CD45, Fas associated phosphatase-1 (FAP-1), striatal enriched tyrosine phosphatases (STEP), mitogen-activated protein kinase/dual-specificity phosphatase 1 (MKP-1), phosphatases of regenerating liver-1 (PRL), low molecular weight PTPs (LMWPTP), and CDC25. Given that there are over 100 family members, we hope this review will serve as a road map for innovative drug discovery targeting PTPs. PMID:25220640

The docking protein Gab1 is the primary mediator of EGF-stimulated activation of the PI-3K/Akt cell survival pathway

PubMed Central

Mattoon, Dawn R; Lamothe, Betty; Lax, Irit; Schlessinger, Joseph

2004-01-01

Background Gab1 is a docking protein that recruits phosphatidylinositol-3 kinase (PI-3 kinase) and other effector proteins in response to the activation of many receptor tyrosine kinases (RTKs). As the autophosphorylation sites on EGF-receptor (EGFR) do not include canonical PI-3 kinase binding sites, it is thought that EGF stimulation of PI-3 kinase and its downstream effector Akt is mediated by an indirect mechanism. Results We used fibroblasts isolated from Gab1-/- mouse embryos to explore the mechanism of EGF stimulation of the PI-3 kinase/Akt anti-apoptotic cell signaling pathway. We demonstrate that Gab1 is essential for EGF stimulation of PI-3 kinase and Akt in these cells and that these responses are mediated by complex formation between p85, the regulatory subunit of PI-3 kinase, and three canonical tyrosine phosphorylation sites on Gab1. Furthermore, complex formation between Gab1 and the protein tyrosine phosphatase Shp2 negatively regulates Gab1 mediated PI-3 kinase and Akt activation following EGF-receptor stimulation. We also demonstrate that tyrosine phosphorylation of ErbB3 may lead to recruitment and activation of PI-3 kinase and Akt in Gab1-/- MEFs. Conclusions The primary mechanism of EGF-induced stimulation of the PI-3 kinase/Akt anti-apoptotic pathway occurs via the docking protein Gab1. However, in cells expressing ErbB3, EGF and neuroregulin can stimulate PI-3 kinase and Akt activation in a Gab1-dependent or Gab1-independent manner. PMID:15550174

Mechanisms of resistance to imatinib in CML patients: a paradigm for the advantages and pitfalls of molecularly targeted therapy.

PubMed

Ritchie, E; Nichols, G

2006-12-01

One of the challenges of cancer therapeutics is to discover targets unique to the tumor cell population. Constitutively activated tyrosine kinases play a role in the malignant phenotype in a number of different cancers. While the kinases may be present in the normal cell, the cancer cell is often dependent upon the activation of the kinase for the maintenance of malignant growth. Inhibition of kinase activation may therefore selectively inhibit malignant proliferation. In the case of chronic myelogenous leukemia (CML), the activated tyrosine kinase (BCR-ABL) is due to a chromosomal translocation that defines this disease, and is necessary for malignant transformation. Imatinib mesylate (Gleevec, Novartis) is a small molecule tyrosine kinase inhibitor, developed through the chemical modification to be selected for a small number of tyrosine kinases present in human cells. This agent is also orally bioavailable and has been found to be effective in clinical trials. We have learned much through the clinical use of this agent. 1) Specific targeting of activated signal transduction pathways may be effective in inhibiting cancer cells. 2) Cancer cells may not only be inherently resistant to small molecule inhibitors, but may also develop resistance after exposure to the inhibitor. 3) Increased knowledge regarding critical signal transduction pathways, the structure of the molecules that are being targeted and the inhibitors themselves, will allow us to understand resistance as it develops and create new molecules to bypass resistance. We will discuss imatinib as an important example of the success and pitfalls of targeted therapeutics for cancer.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ying-Nan P.; LaMarche, Matthew J.; Chan, Ho Man

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1, 2, 3, 4, 5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2, 3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuatormore » (BTLA) immune checkpoint pathways6, 7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8, 9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.« less

Induction of Tyrosine Phosphorylation of UV-Activated EGFR by the Beta-Human Papillomavirus Type 8 E6 Leads to Papillomatosis.

PubMed

Taute, Stefanie; Pfister, Herbert J; Steger, Gertrud

2017-01-01

Epidemiological evidence is accumulating that beta-human papillomaviruses (HPV) synergize with UV-light in the development of precancerous actinic keratosis, and cutaneous squamous cell carcinomas (cSCC), one of the most common cancers in the Caucasian population. We previously demonstrated the tumorigenic activity of beta-HPV type 8 (HPV8) in the skin of transgenic mice and its cooperation with UV-light. Analysis of underlying mechanisms now showed that in keratinocytes expressing the HPV8E6 protein a transient increase of tyrosine phosphorylated epidermal growth factor receptor (EGFR) in response to UV-irradiation occurred, while EGFR tyrosine phosphorylation, i.e., receptor tyrosine kinase (RTK)-activity was hardly affected in empty vector control cells. FACS and immunofluorescences revealed that the EGFR was internalized into early endosomes in response to UV-exposure in both, HPV8E6 positive and in control cells, yet with a higher rate in the presence of HPV8E6. Moreover, only in HPV8E6 expressing keratinocytes the EGFR was further sorted into CD63+ intraluminal vesicles, indicative for trafficking to late endosomes. The latter requires the ubiquitination of the EGFR, and in correlation, we could show that only in HPV8E6 positive keratinocytes the EGFR was ubiquitinated upon UV-exposure. HPV8E6 and tyrosine phosphorylated EGFR directly interacted which was enhanced by UV-irradiation. The treatment of K14-HPV8E6 transgenic mice with Canertinib, an inhibitor of the RTK-activity of the EGFR, suppressed skin papilloma growth in response to UV-irradiation. This confirms the crucial role of the RTK-activity of the EGFR in HPV8E6 and UV-mediated papillomatosis in transgenic mice. Taken together, our results demonstrate that HPV8E6 alters the signaling of the UV-activated EGFR and this is a critical step in papilloma formation in response to UV-light in transgenic mice. Our results provide a molecular basis how a beta-HPV type may support early steps of skin tumor formation in cooperation with UV-light.

Induction of Tyrosine Phosphorylation of UV-Activated EGFR by the Beta-Human Papillomavirus Type 8 E6 Leads to Papillomatosis

PubMed Central

Taute, Stefanie; Pfister, Herbert J.; Steger, Gertrud

2017-01-01

Epidemiological evidence is accumulating that beta-human papillomaviruses (HPV) synergize with UV-light in the development of precancerous actinic keratosis, and cutaneous squamous cell carcinomas (cSCC), one of the most common cancers in the Caucasian population. We previously demonstrated the tumorigenic activity of beta-HPV type 8 (HPV8) in the skin of transgenic mice and its cooperation with UV-light. Analysis of underlying mechanisms now showed that in keratinocytes expressing the HPV8E6 protein a transient increase of tyrosine phosphorylated epidermal growth factor receptor (EGFR) in response to UV-irradiation occurred, while EGFR tyrosine phosphorylation, i.e., receptor tyrosine kinase (RTK)-activity was hardly affected in empty vector control cells. FACS and immunofluorescences revealed that the EGFR was internalized into early endosomes in response to UV-exposure in both, HPV8E6 positive and in control cells, yet with a higher rate in the presence of HPV8E6. Moreover, only in HPV8E6 expressing keratinocytes the EGFR was further sorted into CD63+ intraluminal vesicles, indicative for trafficking to late endosomes. The latter requires the ubiquitination of the EGFR, and in correlation, we could show that only in HPV8E6 positive keratinocytes the EGFR was ubiquitinated upon UV-exposure. HPV8E6 and tyrosine phosphorylated EGFR directly interacted which was enhanced by UV-irradiation. The treatment of K14-HPV8E6 transgenic mice with Canertinib, an inhibitor of the RTK-activity of the EGFR, suppressed skin papilloma growth in response to UV-irradiation. This confirms the crucial role of the RTK-activity of the EGFR in HPV8E6 and UV-mediated papillomatosis in transgenic mice. Taken together, our results demonstrate that HPV8E6 alters the signaling of the UV-activated EGFR and this is a critical step in papilloma formation in response to UV-light in transgenic mice. Our results provide a molecular basis how a beta-HPV type may support early steps of skin tumor formation in cooperation with UV-light. PMID:29176966

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jenei, Veronika; Andersson, Tommy; Jakus, Judit

E3B1, a human homologue of the mouse gene product Abi-1, has been implicated in growth-factor-mediated regulation of the small GTPases p21{sup Ras} and Rac. E3b1 is a regulator of Rac because it can form a complex with Sos-1 and eps8, and such a Sos-1-e3B1-eps8 complex serves as a guanine nucleotide exchange factor for Rac. In the present study, we found that overexpression of e3B1 in NIH3T3/EGFR cells sensitized EGF-induced activation of Rac1, whereas it had no impact on EGF-induced activation of p21{sup Ras}. Remarkably, we found that EGF-induced activation of the p21{sup Ras}-related GTPase Rap1 was also sensitized in NIH3T3/EGFR-e3B1more » cells. Thus, in NIH3T3/EGFR-e3B1 cells, maximal EGF-induced activation of Rap1 occurs with a dose of EGF much lower than in NIH3T3/EGFR cells. We also report that overexpression of e3B1 in NIH3T3/EGFR cells renders EGF-induced activation of Rap1 completely dependent on Src tyrosine kinases but not on c-Abl. However, EGF-induced tyrosine phosphorylation of the Rap GEF C3G occurred regardless of whether e3B1 was overexpressed or not, and this did not involve Src tyrosine kinases. Accordingly, we propose that overexpression of e3B1 in NIH3T3/EGFR cells leads to mobilization of Src tyrosine kinases that participate in EGF-induced activation of Rap1 and inhibition of cell proliferation.« less

Molecular mechanism of ERK dephosphorylation by striatal-enriched protein tyrosine phosphatase (STEP)

PubMed Central

Li, Hui; Li, Kang-shuai; Su, Jing; Chen, Lai-Zhong; Xu, Yun-Fei; Wang, Hong-Mei; Gong, Zheng; Cui, Guo-Ying; Yu, Xiao; Wang, Kai; Yao, Wei; Xin, Tao; Li, Min-Yong; Xiao, Kun-Hong; An, Xiao-fei; Huo, Yuqing; Xu, Zhi-gang; Sun, Jin-Peng; Pang, Qi

2013-01-01

Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward pNPP, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both KIM and KIS of STEP were required for ERK interaction. In addition to the N-terminal KIS region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders. PMID:24117863

BCR-ABL1-like acute lymphoblastic leukaemia: From bench to bedside.

PubMed

Boer, Judith M; den Boer, Monique L

2017-09-01

Acute lymphoblastic leukaemia (ALL) occurs in approximately 1:1500 children and is less frequently found in adults. The most common immunophenotype of ALL is the B cell lineage and within B cell precursor ALL, specific genetic aberrations define subtypes with distinct biological and clinical characteristics. With more advanced genetic analysis methods such as whole genome and transcriptome sequencing, novel genetic subtypes have recently been discovered. One novel class of genetic aberrations comprises tyrosine kinase-activating lesions, including translocations and rearrangements of tyrosine kinase and cytokine receptor genes. These newly discovered genetic aberrations are harder to detect by standard diagnostic methods such as karyotyping, fluorescent in situ hybridisation (FISH) or polymerase chain reaction (PCR) because they are diverse and often cryptic. These lesions involve one of several tyrosine kinase genes (among others, v-abl Abelson murine leukaemia viral oncogene homologue 1 (ABL1), v-abl Abelson murine leukaemia viral oncogene homologue 2 (ABL2), platelet-derived growth factor receptor beta polypeptide (PDGFRB)), each of which can be fused to up to 15 partner genes. Together, they compose 2-3% of B cell precursor ALL (BCP-ALL), which is similar in size to the well-known fusion gene BCR-ABL1 subtype. These so-called BCR-ABL1-like fusions are mutually exclusive with the sentinel translocations in BCP-ALL (BCR-ABL1, ETV6-RUNX1, TCF3-PBX1, and KMT2A (MLL) rearrangements) and have the promising prospect to be sensitive to tyrosine kinase inhibitors similar to BCR-ABL1. In this review, we discuss the types of tyrosine kinase-activating lesions discovered, and the preclinical and clinical evidence for the use of tyrosine kinase inhibitors in the treatment of this novel subtype of ALL. Copyright © 2017 Elsevier Ltd. All rights reserved.

The domain architecture of the PtkA, the first tyrosine kinase from Mycobacterium tuberculosis differs from the conventional kinase architecture.

PubMed

Niesteruk, Anna; Jonker, Hendrik R A; Richter, Christian; Linhard, Verena; Sreeramulu, Sridhar; Schwalbe, Harald

2018-06-08

The discovery that MptpA (low-molecular-weight protein tyrosine phosphatase A) from Mycobacterium tuberculosis ( Mtb ) has an essential role for Mtb virulence has motivated research of tyrosine-specific phosphorylation in Mtb and other pathogenic bacteria. The phosphatase activity of MptpA is regulated via phosphorylation on Tyr-128 and Tyr-129. Thus far, only a single tyrosine-specific kinase, protein tyrosine kinase A (PtkA), encoded by the Rv2232 gene has been identified within the Mtb genome. MptpA undergoes phosphorylation by PtkA. PtkA is an atypical bacterial tyrosine kinase, as its sequence differs from the sequence consensus within this family. The lack of structural information on PtkA hampers the detailed characterization of the MptpA-PtkA interaction. Here, using NMR spectroscopy, we provide a detailed structural characterization of the PtkA architecture and describe its intra- and intermolecular interactions with MptpA. We found that PtkA's domain architecture differs from the conventional kinase architecture and is composed of two domains, the N-terminal highly flexible IDD PtkA and the C-terminal rigid KCD PtkA The interaction studies between the two domains together with the structural model of the IDD-KCD complex proposed in this study reveals that the IDD is unstructured and highly dynamic, allowing for a "fly-casting" like mechanism of transient interactions with the rigid KCD. This interaction modulates the accessibility of the KCD active site. In general, the structural and functional knowledge of PtkA gained in this study, is crucial for understanding the MptpA-PtkA interactions, catalytic mechanism and the role of kinase-phosphatase regulatory system in Mtb virulence. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Targeting the tyrosine kinase signalling pathways for treatment of immune-mediated glomerulonephritis: from bench to bedside and beyond

PubMed Central

Ma, Terry King-Wing; McAdoo, Stephen P.

2017-01-01

Glomerulonephritis (GN) affects patients of all ages and is an important cause of morbidity and mortality. Non-selective immunosuppressive drugs have been used in immune-mediated GN but often result in systemic side effects and occasionally fatal infective complications. There is increasing evidence from both preclinical and clinical studies that abnormal activation of receptor and non-receptor tyrosine kinase signalling pathways are implicated in the pathogenesis of immune-mediated GN. Activation of spleen tyrosine kinase (SYK), Bruton's tyrosine kinase (BTK), platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR) and discoidin domain receptor 1 (DDR1) have been demonstrated in anti-GBM disease. SYK is implicated in the pathogenesis of ANCA-associated GN. SYK, BTK, PDGFR, EFGR, DDR1 and Janus kinase are implicated in the pathogenesis of lupus nephritis. A representative animal model of IgA nephropathy (IgAN) is lacking. Based on the results from in vitro and human renal biopsy study results, a phase II clinical trial is ongoing to evaluate the efficacy and safety of fostamatinib (an oral SYK inhibitor) in high-risk IgAN patient. Various tyrosine kinase inhibitors (TKIs) have been approved for cancer treatment. Clinical trials of TKIs in GN may be justified given their long-term safety data. In this review we will discuss the current unmet medical needs in GN treatment and research as well as the current stage of development of TKIs in GN treatment and propose an accelerated translational research approach to investigate whether selective inhibition of tyrosine kinase provides a safer and more efficacious option for GN treatment. PMID:28391340

Targeting the tyrosine kinase signalling pathways for treatment of immune-mediated glomerulonephritis: from bench to bedside and beyond.

PubMed

Ma, Terry King-Wing; McAdoo, Stephen P; Tam, Frederick Wai Keung

2017-01-01

Glomerulonephritis (GN) affects patients of all ages and is an important cause of morbidity and mortality. Non-selective immunosuppressive drugs have been used in immune-mediated GN but often result in systemic side effects and occasionally fatal infective complications. There is increasing evidence from both preclinical and clinical studies that abnormal activation of receptor and non-receptor tyrosine kinase signalling pathways are implicated in the pathogenesis of immune-mediated GN. Activation of spleen tyrosine kinase (SYK), Bruton's tyrosine kinase (BTK), platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR) and discoidin domain receptor 1 (DDR1) have been demonstrated in anti-GBM disease. SYK is implicated in the pathogenesis of ANCA-associated GN. SYK, BTK, PDGFR, EFGR, DDR1 and Janus kinase are implicated in the pathogenesis of lupus nephritis. A representative animal model of IgA nephropathy (IgAN) is lacking. Based on the results from in vitro and human renal biopsy study results, a phase II clinical trial is ongoing to evaluate the efficacy and safety of fostamatinib (an oral SYK inhibitor) in high-risk IgAN patient. Various tyrosine kinase inhibitors (TKIs) have been approved for cancer treatment. Clinical trials of TKIs in GN may be justified given their long-term safety data. In this review we will discuss the current unmet medical needs in GN treatment and research as well as the current stage of development of TKIs in GN treatment and propose an accelerated translational research approach to investigate whether selective inhibition of tyrosine kinase provides a safer and more efficacious option for GN treatment. © The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA.

A protein tyrosine phosphatase-like protein from baculovirus has RNA 5′-triphosphatase and diphosphatase activities

PubMed Central

Takagi, Toshimitsu; Taylor, Gregory S.; Kusakabe, Takahiro; Charbonneau, Harry; Buratowski, Stephen

1998-01-01

The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5′-phosphatase. BVP sequentially removes γ and β phosphates from the 5′ end of triphosphate-terminated RNA, leaving a 5′-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA. PMID:9707557

Crystal structure of the Streptomyces coelicolor sortase E1 transpeptidase provides insight into the binding mode of the novel class E sorting signal

DOE PAGES

Kattke, Michele D.; Chan, Albert H.; Duong, Andrew; ...

2016-12-09

Here, many species of Gram-positive bacteria use sortase transpeptidases to covalently affix proteins to their cell wall or to assemble pili. Sortase-displayed proteins perform critical and diverse functions for cell survival, including cell adhesion, nutrient acquisition, and morphological development, among others. Based on their amino acid sequences, there are at least six types of sortases (class A to F enzymes); however, class E enzymes have not been extensively studied. Class E sortases are used by soil and freshwater-dwelling Actinobacteria to display proteins that contain a non-canonical LAXTG sorting signal, which differs from 90% of known sorting signals by substitution ofmore » alanine for proline. Here we report the first crystal structure of a class E sortase, the 1.93 Å resolution structure of the SrtE1 enzyme from Streptomyces coelicolor. The active site is bound to a tripeptide, providing insight into the mechanism of substrate binding. SrtE1 possesses β3/β4 and β6/β7 active site loops that contact the LAXTG substrate and are structurally distinct from other classes. We propose that SrtE1 and other class E sortases employ a conserved tyrosine residue within their β3/β4 loop to recognize the amide nitrogen of alanine at position P3 of the sorting signal through a hydrogen bond, as seen here. Incapability of hydrogen-bonding with canonical proline-containing sorting signals likely contributes to class E substrate specificity. Furthermore, we demonstrate that surface anchoring of proteins involved in aerial hyphae formation requires an N-terminal segment in SrtE1 that is presumably positioned within the cytoplasm. Combined, our results reveal unique features within class E enzymes that enable them to recognize distinct sorting signals, and could facilitate the development of substrate-based inhibitors of this important enzyme family.« less

Tyrosine kinase receptor status in endometrial stromal sarcoma: an immunohistochemical and genetic-molecular analysis.

PubMed

Cossu-Rocca, Paolo; Contini, Marcella; Uras, Maria Gabriela; Muroni, Maria Rosaria; Pili, Francesca; Carru, Ciriaco; Bosincu, Luisanna; Massarelli, Giovannino; Nogales, Francisco F; De Miglio, Maria Rosaria

2012-11-01

Endometrial stromal sarcomas (ESS) are rare uterine malignant mesenchymal neoplasms, which are currently treated by surgery, as effective adjuvant therapies have not yet been established. Tyrosine kinase inhibitors have rarely been applied in ESS therapy, with few reports describing imatinib responsivity. The aim of this study was to analyze the status of different tyrosine kinase receptors in an ESS series, in order to evaluate their potential role as molecular targets. Immunohistochemistry was performed for EGFR, c-KIT, PDGFR-α, PDGFR-β, and ABL on 28 ESS. EGFR, PDGFR-α, and PDGFR-β gene expression was investigated by real-time polymerase chain reaction (qRT-PCR) on selected cases. "Hot-spot" mutations were screened for on EGFR, c-KIT, PDGFR-α, and PDGFR-β genes, by sequencing. All analysis was executed from formalin-fixed, paraffin-embedded specimens. Immunohistochemical overexpression of 2 or more tyrosine kinase receptors was observed in 18 of 28 tumors (64%), whereas only 5 tumors were consistently negative. Gene expression profiles were concordant with immunohistochemical overexpression in only 1 tumor, which displayed both high mRNA levels and specific immunoreactivity for PDGFR-α, and PDGFR-β. No activating mutations were found on the tumors included in the study. This study confirms that TKRs expression is frequently observed in ESS. Considering that the responsiveness to tyrosine kinase inhibitors is known to be related to the presence of specific activating mutations or gene over-expression, which are not detectable in ESS, TKRs immunohistochemical over-expression alone should not be considered as a reliable marker for targeted therapies in ESS. Specific post-translational abnormalities, responsible for activation of TKRs, should be further investigated.

Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism.

PubMed

Miki, Yuta; Pogni, Rebecca; Acebes, Sandra; Lucas, Fátima; Fernández-Fueyo, Elena; Baratto, Maria Camilla; Fernández, María I; de los Ríos, Vivian; Ruiz-Dueñas, Francisco J; Sinicropi, Adalgisa; Basosi, Riccardo; Hammel, Kenneth E; Guallar, Victor; Martínez, Angel T

2013-06-15

LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr181) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H2O2 and VA) lacked this lag, and H2O2-LiP (H2O2-treated LiP) was inactive. MS analyses revealed that VA-LiP includes one VA molecule covalently bound to the side chain of Tyr181, whereas H2O2-LiP contains a hydroxylated Tyr181. No adduct is formed in the Y171N variant. Molecular docking showed that VA binding is favoured by sandwich π stacking with Tyr181 and Phe89. EPR spectroscopy after peroxide activation of the pre-treated LiPs showed protein radicals other than the tyrosine radical found in pristine LiP, which were assigned to a tyrosine-VA adduct radical in VA-LiP and a dihydroxyphenyalanine radical in H2O2-LiP. Both radicals are able to oxidize large low-redox-potential substrates, but H2O2-LiP is unable to oxidize high-redox-potential substrates. Transient-state kinetics showed that the tyrosine-VA adduct strongly promotes (>100-fold) substrate oxidation by compound II, the rate-limiting step in catalysis. The novel activation mechanism is involved in ligninolysis, as demonstrated using lignin model substrates. The present paper is the first report on autocatalytic modification, resulting in functional alteration, among class II peroxidases.

The KIM-family protein-tyrosine phosphatases use distinct reversible oxidation intermediates: Intramolecular or intermolecular disulfide bond formation.

PubMed

Machado, Luciana E S F; Shen, Tun-Li; Page, Rebecca; Peti, Wolfgang

2017-05-26

The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H 2 O 2 , which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H 2 O 2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxidation of monohydric phenol substrates by tyrosinase. An oximetric study.

PubMed

Naish-Byfield, S; Riley, P A

1992-11-15

The purity of commercially available mushroom tyrosinase was investigated by non-denaturing PAGE. Most of the protein in the preparation migrated as a single band under these conditions. This band contained both tyrosinase and dopa oxidase activity. No other activity of either classification was found in the preparation. Oxygen consumption by tyrosinase during oxidation of the monohydric phenol substrates tyrosine and 4-hydroxyanisole (4HA) was monitored by oximetry in order to determine the stoichiometry of the reactions. For complete oxidation, the molar ratio of oxygen: 4HA was 1:1. Under identical conditions, oxidation of tyrosine required 1.5 mol of oxygen/mol of tyrosine. The additional oxygen uptake during tyrosine oxidation is due to the internal cyclization of dopaquinone to form cyclodopa, which undergoes a redox reaction with dopaquinone to form dopachrome and dopa, which is then oxidized by the enzyme, leading to an additional 0.5 mol of oxygen/mol of original substrate. Oxygen consumption for complete oxidation of 200 nmol of 4HA was constant over a range of concentrations of tyrosinase of 33-330 units/ml of substrate. The maximum rate of reaction was directly proportional to the concentration of tyrosinase, whereas the length of the lag phase decreased non-linearly with increasing tyrosinase concentration. Activation of the enzyme by exposure to citrate was not seen, nor was the lag phase abolished by exposure of the enzyme to low pH. Michaelis-Menten analysis of tyrosinase in which the lag phase is abolished by pre-exposure of the enzyme to a low concentration of dithiothreitol gave Km values for tyrosine and 4HA of 153 and 20 microM respectively.

Positive And Negative Feedback Loops Coupled By Common Transcription Activator And Repressor

NASA Astrophysics Data System (ADS)

Sielewiesiuk, Jan; Łopaciuk, Agata

2015-03-01

Dynamical systems consisting of two interlocked loops with negative and positive feedback have been studied using the linear analysis of stability and numerical solutions. Conditions for saddle-node bifurcation were formulated in a general form. Conditions for Hopf bifurcations were found in a few symmetrical cases. Auto-oscillations, when they exist, are generated by the negative feedback repressive loop. This loop determines the frequency and amplitude of oscillations. The positive feedback loop of activation slightly modifies the oscillations. Oscillations are possible when the difference between Hilll's coefficients of the repression and activation is sufficiently high. The highly cooperative activation loop with a fast turnover slows down or even makes the oscillations impossible. The system under consideration can constitute a component of epigenetic or enzymatic regulation network.

Skylab observations of X-ray loops connecting separate active regions. [solar activity

NASA Technical Reports Server (NTRS)

Chase, R. C.; Krieger, A. S.; Svestka, Z.; Vaiana, G. S.

1976-01-01

One hundred loops interconnecting 94 separate active solar regions detectable in soft X-rays were identified during the Skylab mission. While close active regions are commonly interconnected with loops, the number of such interconnections decreases steeply for longer distances; the longest interconnecting loop observed in the Skylab data connected regions separated by 37 deg. Several arguments are presented which support the point of view that this is the actual limit of the size of magnetic interconnections between active regions. No sympathetic flares could be found in the interconnected regions. These results cast doubt on the hypothesis that accelerated particles can be guided in interconnecting loops from one active region to another over distances of 100 deg or more and eventually produce sympathetic flares in them.

Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase.

PubMed Central

Zohn, I E; Yu, H; Li, X; Cox, A D; Earp, H S

1995-01-01

In GN4 rat liver epithelial cells, angiotensin II (Ang II) and other agonists which activate phospholipase C stimulate tyrosine kinase activity in a calcium-dependent, protein kinase C (PKC)-independent manner. Since Ang II also produces a proliferative response in these cells, we investigated downstream signaling elements traditionally linked to growth control by tyrosine kinases. First, Ang II, like epidermal growth factor (EGF), stimulated AP-1 binding activity in a PKC-independent manner. Because increases in AP-1 can reflect induction of c-Jun and c-Fos, we examined the activity of the mitogen-activated protein (MAP) kinase family members Erk-1 and -2 and the c-Jun N-terminal kinase (JNK), which are known to influence c-Jun and c-Fos transcription. Ang II stimulated MAP kinase (MAPK) activity but only approximately 50% as effectively as EGF; again, these effects were independent of PKC. Ang II also produced a 50- to 200-fold activation of JNK in a PKC-independent manner. Unlike its smaller effect on MAPK, Ang II was approximately four- to sixfold more potent in activating JNK than EGF was. Although others had reported a lack of calcium ionophore-stimulated JNK activity in lymphocytes and several other cell lines, we examined the role of calcium in GN4 cells. The following results suggest that JNK activation in rat liver epithelial cells is at least partially Ca(2+) dependent: (i) norepinephrine and vasopressin hormones that increase inositol 1,4,5-triphosphate stimulated JNK; (ii) both thapsigargin, a compound that produces an intracellular Ca(2+) signal, and Ca(2+) ionophores stimulated a dramatic increase in JNK activity (up to 200-fold); (iii) extracellular Ca(2+) chelation with ethylene glycol tetraacetic acid (EGTA) inhibited JNK activation by ionophore and intracellular chelation with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl-ester (BAPTA-AM) partially inhibited JNK activation by Ang II or thapsigargin; and (iv) JNK activation by Ang II was inhibited by pretreatment of cells with thapsigargin and EGTA, a procedure which depletes intracellular Ca(2+) stores. JNK activation following Ang II stimulation did not involve calmodulin; either W-7 nor calmidizolium, in concentrations sufficient to inhibit Ca(2+)/calmodulin-dependent kinase II, blocked JNK activation by Ang II. In contrast, genistein, in concentrations sufficient to inhibit Ca(2+)-dependent tyrosine phosphorylation, prevented Ang II and thapsigargin-induced JNK activation. In summary, in GN4 rat liver epithelial cells, Ang II stimulates JNK via a novel Ca(2+)-dependent pathway. The inhibition by genistein suggest that Ca(2+)-dependent tyrosine phosphorylation may modulate the JNK pathway in a cell type-specific manner, particularly in cells with a readily detectable Ca(2+)-regulated tyrosine kinase. PMID:7565768

The Prevalence and Use of Walking Loops in Neighborhood Parks: A National Study.

PubMed

Cohen, Deborah A; Han, Bing; Evenson, Kelly R; Nagel, Catherine; McKenzie, Thomas L; Marsh, Terry; Williamson, Stephanie; Harnik, Peter

2017-02-01

Previous studies indicate that the design of streets and sidewalks can influence physical activity among residents. Park features also influence park use and park-based physical activity. Although individuals can walk on streets and sidewalks, walking loops in parks offer a setting to walk in nature and to avoid interruptions from traffic. Here we describe the use of walking loops in parks and compare the number of park users and their physical activity in urban neighborhood parks with and without walking loops. We analyzed data from the National Study of Neighborhood Parks in which a representative sample of neighborhood parks (n = 174) from 25 U.S. cities with > 100,000 population were observed systematically to document facilities and park users by age group and sex. We compared the number of people and their physical activity in parks with and without walking loops, controlling for multiple factors, including park size, facilities, and population density. Overall, compared with parks without walking loops, on average during an hourly observation, parks with walking loops had 80% more users (95% CI: 42, 139%), and levels of moderate-to-vigorous physical activity were 90% higher (95% CI: 49, 145%). The additional park use and park-based physical activity occurred not only on the walking loops but throughout the park. Walking loops may be a promising means of increasing population level physical activity. Further studies are needed to confirm a causal relationship. Citation: Cohen DA, Han B, Evenson KR, Nagel C, McKenzie TL, Marsh T, Williamson S, Harnik P. 2017. The prevalence and use of walking loops in neighborhood parks: a national study. Environ Health Perspect 125:170-174; http://dx.doi.org/10.1289/EHP293.

The Prevalence and Use of Walking Loops in Neighborhood Parks: A National Study

PubMed Central

Cohen, Deborah A.; Han, Bing; Evenson, Kelly R.; Nagel, Catherine; McKenzie, Thomas L.; Marsh, Terry; Williamson, Stephanie; Harnik, Peter

2016-01-01

Background: Previous studies indicate that the design of streets and sidewalks can influence physical activity among residents. Park features also influence park use and park-based physical activity. Although individuals can walk on streets and sidewalks, walking loops in parks offer a setting to walk in nature and to avoid interruptions from traffic. Objectives: Here we describe the use of walking loops in parks and compare the number of park users and their physical activity in urban neighborhood parks with and without walking loops. Methods: We analyzed data from the National Study of Neighborhood Parks in which a representative sample of neighborhood parks (n = 174) from 25 U.S. cities with > 100,000 population were observed systematically to document facilities and park users by age group and sex. We compared the number of people and their physical activity in parks with and without walking loops, controlling for multiple factors, including park size, facilities, and population density. Results: Overall, compared with parks without walking loops, on average during an hourly observation, parks with walking loops had 80% more users (95% CI: 42, 139%), and levels of moderate-to-vigorous physical activity were 90% higher (95% CI: 49, 145%). The additional park use and park-based physical activity occurred not only on the walking loops but throughout the park. Conclusions: Walking loops may be a promising means of increasing population level physical activity. Further studies are needed to confirm a causal relationship. Citation: Cohen DA, Han B, Evenson KR, Nagel C, McKenzie TL, Marsh T, Williamson S, Harnik P. 2017. The prevalence and use of walking loops in neighborhood parks: a national study. Environ Health Perspect 125:170–174; http://dx.doi.org/10.1289/EHP293 PMID:27517530

Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Rae-Kwon; Yoon, Chang-Hwan; Hyun, Kyung-Hwan

2010-11-26

Research highlights: {yields} Activation of Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the fractionated radiation-induced expansion of glioma stem-like cells. {yields} Inhibition of LCK prevents acquisition of fractionated radiation-induced resistance to chemotherapeutic treatment. {yields} LCK activity is critical for the maintenance of self-renewal in glioma stem-like cells. -- Abstract: Brain cancers frequently recur or progress as focal masses after treatment with ionizing radiation. Radiation used to target gliomas may expand the cancer stem cell population and enhance the aggressiveness of tumors; however, the mechanisms underlying the expansion of cancer stem cell population after radiation have remained unclear. In thismore » study, we show that LCK (lymphocyte-specific protein tyrosine kinase) is involved in the fractionated radiation-induced expansion of the glioma-initiating cell population and acquisition of resistance to anticancer treatments. Fractionated radiation caused a selective increase in the activity of LCK, a Src family non-receptor tyrosine kinase. The activities of other Src family kinases Src, Fyn, and Lyn were not significantly increased. Moreover, knockdown of LCK expression with a specific small interfering RNA (siRNA) effectively blocked fractionated radiation-induced expansion of the CD133{sup +} cell population. siRNA targeting of LCK also suppressed fractionated radiation-induced expression of the glioma stem cell marker proteins CD133, Nestin, and Musashi. Expression of the known self-renewal-related proteins Notch2 and Sox2 in glioma cells treated with fractionated radiation was also downregulated by LCK inhibition. Moreover, siRNA-mediated knockdown of LCK effectively restored the sensitivity of glioma cells to cisplatin and etoposide. These results indicate that the non-receptor tyrosine kinase LCK is critically involved in fractionated radiation-induced expansion of the glioma-initiating cell population and decreased cellular sensitivity to anticancer treatments. These findings may provide pivotal insights in the context of fractionated radiation-based therapeutic interventions in brain cancer.« less

Decrease of cocaine, but not heroin, self-administration and relapse by the tyrosine kinase inhibitor masitinib in male Sprague Dawley rats.

PubMed

Belin-Rauscent, A; Lacoste, J; Hermine, O; Moussy, A; Everitt, B J; Belin, David

2018-05-01

Accumulating evidence shows that cocaine, and also heroin, influence several tyrosine kinases, expressed in neurons and in non-neuronal populations such as microglia, astrocytes and mast-cells. Drug-induced activation of mast cells both triggers inflammatory processes in the brain mediated by the glial cells they activate, and facilitates histamine release which may directly influence the dopamine system. Thus, by triggering the activation and degranulation of mast cells dependent on the tyrosine kinase c-kit and Fyn, the latter being also involved in NMDA-dependent synaptic plasticity, cocaine and heroin may indirectly influence the neural mechanisms that mediate their reinforcing properties. Masitinib, a novel tyrosine kinase inhibitor with high selectivity for c-Kit, Fyn and Lyn, may alter the aberrant consequences of the activation of these tyrosine kinases by cocaine and heroin. We investigated in rats the effect of a chronic oral treatment with masitinib (20 mg/kg) on the reinforcing and motivational properties of self-administered cocaine (250 μg/infusion) and heroin (40 μg/infusion). Three different cohorts of rats were trained instrumentally to respond for cocaine, heroin or food under continuous reinforcement. In each group, we assessed the influence of chronic daily treatment with masitinib on the maintenance of instrumental responding and intake and the motivation for the reinforcer. Thus, masitinib and vehicle-treated rats were challenged to adapt to high behavioural demand, to respond under a progressive ratio schedule of reinforcement and to reinstate instrumental responding after extinction and/or abstinence. Masitinib selectively decreased cocaine intake, the motivation for cocaine and the subsequent propensity to respond for cocaine under extinction, while having no effect on instrumental responding for heroin or food. The present findings suggest masitinib, a drug with proven efficacy in CNS disorders, could represent a novel treatment for cocaine addiction provided its influence on the reinforcing and incentive properties of the drug is confirmed.

Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism

Treesearch

Yuta Miki; Rebecca Pogni; Sandra Acebes; Fatima Lucas; Elena Fernandez-Fueyo; Maria Camilla Baratto; Maria I. Fernandez; Vivian De Los Rios; Francisco J. Ruiz-duenas; Adalgisa Sinicropi; Riccardo Basosi; Kenneth E. Hammel; Victor Guallar; Angel T. Martinez

2013-01-01

LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr181) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H2O2...