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Sample records for active tyrosine kinases

  1. Activity-based kinase profiling of approved tyrosine kinase inhibitors.

    PubMed

    Kitagawa, Daisuke; Yokota, Koichi; Gouda, Masaki; Narumi, Yugo; Ohmoto, Hiroshi; Nishiwaki, Eiji; Akita, Kensaku; Kirii, Yasuyuki

    2013-02-01

    The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K(m) for ATP. In addition, profiling at a physiological ATP concentration (1 mm) was carried out, and the IC(50) values of the inhibitors against each kinase were compared with the estimated plasma-free concentration (calculated from published pharmacokinetic parameters of plasma C(trough) and C(max) values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off-target kinases in drug side effects. Thus, large-scale kinase profiling at both K(m) and physiological ATP concentrations could be useful in characterizing the targets and off-targets of kinase inhibitors. PMID:23279183

  2. Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases.

    PubMed

    Chen, Sen; Jiang, Xinnong; Gewinner, Christina A; Asara, John M; Simon, Nicholas I; Cai, Changmeng; Cantley, Lewis C; Balk, Steven P

    2013-05-28

    The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr⁵⁷⁷ subsequent to its Src-mediated phosphorylation at Tyr⁵⁷⁶. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰, as well as Tyr¹¹⁸⁵, and downstream phosphorylation of the kinase AKT at Thr³⁰⁸ were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser⁴⁷³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases. PMID:23716717

  3. Potential sites of CFTR activation by tyrosine kinases.

    PubMed

    Billet, Arnaud; Jia, Yanlin; Jensen, Timothy J; Hou, Yue-Xian; Chang, Xiu-Bao; Riordan, John R; Hanrahan, John W

    2016-05-01

    The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation. PMID:26645934

  4. Asymmetric Tyrosine Kinase Arrangements in Activation or Autophosphorylation of Receptor Tyrosine Kinases

    SciTech Connect

    J Bae; J Schlessinger

    2011-12-31

    Receptor tyrosine kinases (RTKs) play important roles in the control of many cellular processes including cell proliferation, cell adhesion, angiogenesis, and apoptosis. Ligand-induced dimerization of RTKs leads to autophosphorylation and activation of RTKs. Structural studies have shown that while isolated ectodomains of several RTKs form symmetric dimers the isolated cytoplasmic kinase domains of epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) form asymmetric dimers during their activation. Binding of one kinase molecule of EGFR to a second kinase molecule asymmetrically leads to stimulation of kinase activity and enhanced autophosphorylation. Furthermore, the structures of the kinase domain of FGFR1 and FGFR2 reveal the formation of asymmetric interfaces in the processes of autophosphorylation at their specific phosphotyrosine (pY) sites. Disruption of asymmetric dimer interface of EGFR leads to reduction in enzymatic activity and drastic reduction of autophosphorylation of FGFRs in ligandstimulated live cells. These studies demonstrate that asymmetric dimer formation is as a common phenomenon critical for activation and autophosphorylation of RTKs.

  5. Redundant kinase activation and resistance of EGFR-tyrosine kinase inhibitors

    PubMed Central

    Luo, Min; Fu, Li-Wu

    2014-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic effects against that tumors harboring EGFR activating mutations in the EGFR intracytoplasmic tyrosine kinase domain and resulted in cell apoptosis. Unfortunately, a number of patients ultimately developed resistance by multiple mechanisms. Thus, elucidation of the mechanism of resistance to EGFR-TKIs can provide strategies for blocking or reversing the situation. Recent studies suggested that redundant kinase activation plays pivotal roles in escaping from the effects of EGFR-TKIs. Herein, we aimed to characterize several molecular events involved in the resistance to EGFR-TKIs mediated by redundant kinase activation. PMID:25520855

  6. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    SciTech Connect

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.

    2007-01-01

    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  7. Receptor tyrosine kinases: mechanisms of activation and signaling

    PubMed Central

    Hubbard, Stevan R.; Miller, W. Todd

    2008-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication. These single-pass transmembrane receptors, which bind polypeptide ligands — mainly growth factors — play key roles in processes such as cellular growth, differentiation, metabolism and motility. Recent progress has been achieved towards an understanding of the precise (and varied) mechanisms by which RTKs are activated by ligand binding and by which signals are propagated from the activated receptors to downstream targets in the cell. PMID:17306972

  8. eps15, a novel tyrosine kinase substrate, exhibits transforming activity.

    PubMed Central

    Fazioli, F; Minichiello, L; Matoskova, B; Wong, W T; Di Fiore, P P

    1993-01-01

    An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals. Images PMID:7689153

  9. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    PubMed Central

    Mezquita, Belén; Mezquita, Pau; Pau, Montserrat; Mezquita, Jovita; Mezquita, Cristóbal

    2014-01-01

    One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer. PMID:24709904

  10. Estrus cycle effect on muscle tyrosine kinase activity in bitches.

    PubMed

    Gomes Pöppl, Álan; Costa Valle, Sandra; Hilário Díaz González, Félix; de Castro Beck, Carlos Afonso; Kucharski, Luiz Carlos; Silveira Martins Da Silva, Roselis

    2012-03-01

    Estrus cycle is a well recognized cause of insulin resistance in bitches. The insulin receptor (IR) as well as the insulin-like growth factor-I receptor belong to the same subfamily of tyrosine kinase (TK) receptors. The objective of this study was to evaluate basal TK activity in muscle tissue of bitches during the estrus cycle. Twenty-four bitches were used in the study (7 in anestrus, 7 in estrus, and 10 in diestrus). Muscle samples, taken after spaying surgery to determine TK activity, were immediately frozen in liquid nitrogen and then stored at -80°C until the membranes were prepared by sequential centrifugation after being homogenized. TK activity was determined by Poly (Glu 4:Tyr 1) phosphorylation and expressed in cpm/μg of protein. TK activity was significantly lower (P < 0.001) in the animals in estrus (104.5 ± 11.9 cpm/μg of protein) and diestrus (94.5 ± 16.9 cpm/μg of protein) when compared with bitches in anestrus (183.2 ± 39.2 cpm/μg of protein). These results demonstrate, for the first time, lower basal TK activity in the muscle tissue of female dogs during estrus and diestrus, which may represent lower insulin signaling capacity, opening a new field of investigation into the molecular mechanisms of insulin resistance in dogs. PMID:22139063

  11. Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers

    PubMed Central

    Maruyama, Ichiro N.

    2014-01-01

    Receptor tyrosine kinases (RTKs) play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR) family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights. PMID:24758840

  12. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors

    PubMed Central

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak

    2014-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials. PMID:25337443

  13. Protein-tyrosine phosphorylation interaction network in Bacillus subtilis reveals new substrates, kinase activators and kinase cross-talk

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ventroux, Magali; Derouiche, Abderahmane; Bidnenko, Vladimir; Mijakovic, Ivan; Noirot-Gros, Marie-Françoise

    2014-01-01

    Signal transduction in eukaryotes is generally transmitted through phosphorylation cascades that involve a complex interplay of transmembrane receptors, protein kinases, phosphatases and their targets. Our previous work indicated that bacterial protein-tyrosine kinases and phosphatases may exhibit similar properties, since they act on many different substrates. To capture the complexity of this phosphorylation-based network, we performed a comprehensive interactome study focused on the protein-tyrosine kinases and phosphatases in the model bacterium Bacillus subtilis. The resulting network identified many potential new substrates of kinases and phosphatases, some of which were experimentally validated. Our study highlighted the role of tyrosine and serine/threonine kinases and phosphatases in DNA metabolism, transcriptional control and cell division. This interaction network reveals significant crosstalk among different classes of kinases. We found that tyrosine kinases can bind to several modulators, transmembrane or cytosolic, consistent with a branching of signaling pathways. Most particularly, we found that the division site regulator MinD can form a complex with the tyrosine kinase PtkA and modulate its activity in vitro. In vivo, it acts as a scaffold protein which anchors the kinase at the cell pole. This network highlighted a role of tyrosine phosphorylation in the spatial regulation of the Z-ring during cytokinesis. PMID:25374563

  14. Dimerization mediated through a leucine zipper activates the oncogenic potential of the met receptor tyrosine kinase.

    PubMed Central

    Rodrigues, G A; Park, M

    1993-01-01

    Oncogenic activation of the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase involves a genomic rearrangement that generates a hybrid protein containing tpr-encoded sequences at its amino terminus fused directly to the met-encoded receptor kinase domain. Deletion of Tpr sequences abolishes the transforming ability of this protein, implicating this region in oncogenic activation. We demonstrate, by site-directed mutagenesis and coimmunoprecipitation experiments, that a leucine zipper motif within Tpr mediates dimerization of the tpr-met product and is essential for the transforming activity of the met oncogene. By analogy with ligand-stimulated activation of receptor tyrosine kinases, we propose that constitutive dimerization mediated by a leucine zipper motif within Tpr is responsible for oncogenic activation of the Met kinase. The possibility that this mechanism of activation represents a paradigm for a class of receptor tyrosine kinase oncogenes activated by DNA rearrangement is discussed. Images PMID:8413267

  15. Characterization of a Mn sup 2+ -dependent membrane serine kinase that is activated by tyrosine phosphorylation

    SciTech Connect

    Singh, T.J. )

    1991-03-11

    It is hypothesized that the insulin receptor (IR) tyrosine kinase may directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases as well as their modes of activation are unclear. The authors have described a serine kinase from rat liver membranes that copurifies with the IR on wheat germ agglutinin (WGA)-sepharose. The kinase is activated after phosphorylation of the WGA-sepharose-purified fraction by casein kinase-1, casein kinase-2, or casein kinase-3. A tyrosine kinase, possibly IR tyrosine kinase, also participates in the activation process since a phosphotyrosine phosphatase inhibitor such as vanadate, p-nitrophenyl phosphate, or phosphotyrosine is required in reaction mixtures for activation to be observed. By contrast, phosphoserine and phosphothreonine do not support activation. The activated kinase can use IR {beta}-subunit, myelin basic protein (MBP), and histones as substrates. IR {beta}-subunit phosphorylation was stimulated by MBP, histones, and polylysine, and inhibited by heparin and poly(glu, tyr). The kinase prefers Mn{sup 2+} over Mg{sup 2+} as a metal cofactor.

  16. Pyridazinone derivatives displaying highly potent and selective inhibitory activities against c-Met tyrosine kinase.

    PubMed

    Liu, Yang; Jin, Shiyu; Peng, Xia; Lu, Dong; Zeng, Limin; Sun, Yiming; Ai, Jing; Geng, Meiyu; Hu, Youhong

    2016-01-27

    Over activation of c-Met tyrosine kinase is known to promote tumorigenesis and metastasis, as well as to cause therapeutic resistance. Herein we describe the design, synthesis and biological activities of novel, ATP-competitive, c-Met tyrosine kinase inhibitors that are members of the 6-aryl-2-(3-(heteroarylamino)benzyl)pyridazinone family. A structure-activity relationship (SAR) study of these substances led to identification of pyridazinone 19 as a highly selective and potent c-Met tyrosine inhibitor, which displays favorable pharmacokinetic properties in mice and significant antitumor activity against a c-Met driven EBC-1 tumor xenograft. PMID:26698536

  17. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    SciTech Connect

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F.

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  19. Multiplexed tyrosine kinase activity detection in cancer cells using hydrogel immobilized substrate

    PubMed Central

    Powers, Alicia D.; Han, Wenquing; Liu, Bi; Palecek, Sean P.

    2013-01-01

    Kinases play a key role in cellular signaling, and the overactivation or overexpression of these kinases has been linked to a variety of cancers. Tyrosine kinase inhibitors treat the mechanism of these cancers by targeting the specific kinases that are overactive. Some patients, however, do not respond to these inhibitors or develop resistance to these inhibitors during treatment. Additionally, even within cancers of the same tissue type, different kinases may be overactive in different patients. For example, some lung cancers overexpress epidermal growth factor receptor (EGFR) and respond to EGFR inhibitors, while other lung cancers do not overexpress EGFR and receive no benefit from this treatment. Even among patients exhibiting EGFR overexpression, some do not respond to EGFR kinase inhibitors because other kinases, such as Met kinase, are also overactivated. Here we describe a quantitative and specific multiplexed microfluidic assay using a hydrogel immobilized substrate for measuring the kinase activity of Met and Abl kinase from cancer cells. We immobilized kinase specific substrates into macroporous hydrogel micropillars in microchannels. These microchannels were incubated with 6 µl of a kinase reaction solution containing cancer cell lysate and measured kinase activity via fluorescence detection of a phosphotyrosine antibody. We showed that the assay can specifically measure the activity of both Met and Abl kinase within one microchannel with potential to measure the activity of as many as 5 kinases within one microchannel. The assay also detected Met kinase inhibition from lysates of cancer cells grown in the Met kinase inhibitor PHA665752. PMID:23624904

  20. DNA sequence, structure, and tyrosine kinase activity of the Drosophila melanogaster abelson proto-oncogene homolog

    SciTech Connect

    Henkemeyer, M.J.; Bennett, R.L.; Gertler, F.B.; Hoffmann, F.M.

    1988-02-01

    The authors report their molecular characterization of the Drosophila melanogaster Abelson gene (abl), a gene in which recessive loss-of-function mutations result in lethality at the pupal stage of development. This essential gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type 1b 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region was expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues was observed with an antiphosphotyrosine antibody. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development.

  1. Enhancement of cytosolic tyrosine kinase activity by propylthiouracil-induced hyperplasia in the rat thyroid.

    PubMed

    Polychronakos, C; Piscina, R; Fantus, I G

    1989-01-01

    Hyperplasia of the thyroid gland induced by propylthiouracil (PTU) is a well established model of rapid cell proliferation in vivo. Recent evidence indicates that tyrosine kinase activity is associated with growth factor receptors and oncogene protein products and may have an important regulatory action in the control of cell growth. Thus, we examined tyrosine kinase activity in rat thyroid membrane and cytosol preparations at rest and during PTU-induced hyperplasia. Although kinase activity was present in a crude microsomal membrane preparation, no change was observed during thyroid growth. In contrast, tyrosine kinase activity assayed with the artificial substrate poly(Glu,Na:Tyr) 4:1 was present in normal rat thyroid cytosol and increased 2- to 6-fold during the rapid phase of hyperplasia in the first 5-10 days of PTU treatment. It declined to control values by day 15, when the size and DNA content of the thyroid reached a plateau. Preincubation of the cytosolic preparations with several peptides known to bind to and activate growth factor receptor tyrosine kinases failed to enhance the activity, suggesting, along with the cytosolic localization, that the activity was distinct from these receptors. By gel filtration chromatography and polyacrylamide gel electrophoresis, tyrosine kinase activity was associated with a 55 kDa protein. Partial purification over a poly(Glu,Na:Tyr)4:1-Sepharose column, yielded a protein that appeared capable of autophosphorylation. It is suggested that this tyrosine kinase plays a role in mediating the growth-promoting effects of this model of thyroid cell hyperplasia. PMID:2909378

  2. Contractile effects of angiotensin peptides in rat aorta are differentially dependent on tyrosine kinase activity.

    PubMed

    Petrescu, G; Costuleanu, M; Slatineanu, S M; Costuleanu, N; Foia, L; Costuleanu, A

    2001-09-01

    It has been suggested that tyrosine kinase activity participates in the regulation of signal transduction associated with angiotensin II (Ang II)-induced pharmaco-mechanical coupling in rat aortic smooth muscle. We further tested the effects of genistein, a tyrosine-kinase inhibitor, and its inactive analogue, daidzein, on angiotensin I (Ang I), angiotensin III (Ang III) and angiotensin IV (Ang IV) contractions, as compared with those on Ang II. Genistein partially inhibited Ang II- and Ang I-induced contractions. The genistein-induced inhibition was more evident on Ang III and especially important on Ang IV contractile effects. Thus, Ang IV- and Ang III-induced contractions seem to be more dependent on tyrosine kinase activity than those evoked by Ang II or Ang I. Daidzein did not significantly affect the contractile effects of any of angiotensin peptides tested. These results clearly suggest that the inhibition of the action of angiotensin peptides actions by genistein is mediated by inhibition of endogenous tyrosine kinase activity. Furthermore, our data show that the type and/or intensity of tyrosine kinase activity is differentially associated with the contractile effects of different angiotensin peptides in rat aorta. Nifedipine, a blocker of membrane L-type Ca2+ channels, strongly inhibited Ang IV-induced contractions. At the same time, it significantly inhibited Ang III contractile effects as compared with Ang II and Ang I contractions. Meanwhile, we observed a close relationship between calcium influx and tyrosine kinase phosphorylation activity under the stimulatory effects of angiotensin peptides. Furthermore, genistein did not significantly influence the phasic contractions induced by angiotensin peptides in Ca2+-free Krebs-Henseleit solution. Thus, it appears that Ca2+ influx, rather than the release of Ca2+ from IP3-sensitive stores, may play a major role in the contractile effects of angiotensin peptides in rat aorta via tyrosine kinase activation

  3. Discovering the first tyrosine kinase

    PubMed Central

    Hunter, Tony

    2015-01-01

    In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month. PMID:26130799

  4. Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling.

    PubMed

    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

    2015-10-20

    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

  5. 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

  6. Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

    PubMed Central

    Romer, L H; McLean, N; Turner, C E; Burridge, K

    1994-01-01

    Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes. Images PMID:8049526

  7. c-Abl Tyrosine Kinase Adopts Multiple Active Conformational States in Solution

    PubMed Central

    2016-01-01

    Protein tyrosine kinases of the Abl family have diverse roles in normal cellular regulation and drive several forms of leukemia as oncogenic fusion proteins. In the crystal structure of the inactive c-Abl kinase core, the SH2 and SH3 domains dock onto the back of the kinase domain, resulting in a compact, assembled state. This inactive conformation is stabilized by the interaction of the myristoylated N-cap with a pocket in the C-lobe of the kinase domain. Mutations that perturb these intramolecular interactions result in kinase activation. Here, we present X-ray scattering solution structures of multidomain c-Abl kinase core proteins modeling diverse active states. Surprisingly, the relative positions of the regulatory N-cap, SH3, and SH2 domains in an active myristic acid binding pocket mutant (A356N) were virtually identical to those of the assembled wild-type kinase core, indicating that Abl kinase activation does not require dramatic reorganization of the downregulated core structure. In contrast, the positions of the SH2 and SH3 domains in a clinically relevant imatinib-resistant gatekeeper mutant (T315I) appear to be reconfigured relative to their positions in the wild-type protein. Our results demonstrate that c-Abl kinase activation can occur either with (T315I) or without (A356N) global allosteric changes in the core, revealing the potential for previously unrecognized signaling diversity. PMID:27166638

  8. Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity.

    PubMed Central

    Lee, S K; Su, B; Maher, S E; Bothwell, A L

    1994-01-01

    To characterize the function of the Ly-6A antigen in T cell activation, antisense Ly-6 RNA was expressed in a stably transfected antigen-specific T cell clone. Reduced Ly-6A expression results in inhibition of responses to antigen, anti-TCR (anti-T cell receptor) crosslinking and concanavalin A plus recombinant interleukin 1 and causes impairment of in vitro fyn tyrosine kinase activity. More substantial reduction of Ly-6A results in reduction of TCR expression. Analysis of mRNA species indicates that the reduction is specific for the TCR beta chain. These data demonstrate that Ly-6A may regulate TCR expression and may be involved in early events of T cell activation via regulation of fyn tyrosine kinase activity. Images PMID:8187770

  9. Activation of Protein Tyrosine Kinases by Coxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

    PubMed Central

    Meconi, Sonia; Capo, Christian; Remacle-Bonnet, Maryse; Pommier, Gilbert; Raoult, Didier; Mege, Jean-Louis

    2001-01-01

    Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) by C. burnetii in THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflects C. burnetii virulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation in C. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced by C. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, prevented C. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulent C. burnetii but had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation by C. burnetii negatively regulates bacterial uptake by interfering with cytoskeleton organization. PMID:11254615

  10. A p60v-src-related tyrosine kinase in the acetylcholine receptor-rich membranes of Narke japonica: association and dissociation of phosphatidylinositol kinase activity.

    PubMed

    Fukami, Y; Owada, M K; Sumi, M; Hayashi, F

    1986-09-14

    We have isolated a tyrosine-specific protein kinase from the acetylcholine receptor (AChR)-rich membranes of the electric ray Narke japonica. The enzyme is immunologically related to p60v-src, the product of the transforming gene of Rous sarcoma virus. A substantial phosphatidylinositol (PI) kinase activity was associated with this enzyme when it was purified through tyrosine-agarose affinity chromatography used previously for the purification of p60v-src. However, by subsequent chromatography on casein-agarose, most of the associated PI kinase activity was separated from the tyrosine kinase activity. The results suggest that the tyrosine-specific protein kinase in the AChR-rich membranes of N. japonica has no intrinsic PI kinase activity. PMID:3094516

  11. 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

  12. The Src-family tyrosine kinase inhibitor PP1 interferes with the activation of ribosomal protein S6 kinases.

    PubMed Central

    Shah, O Jameel; Kimball, Scot R; Jefferson, Leonard S

    2002-01-01

    Considerable biochemical and pharmacological evidence suggests that the activation of ribosomal protein S6 kinases (S6Ks) by activated receptor tyrosine kinases involves multiple co-ordinated input signals. However, the identities of many of these inputs remain poorly described, and their precise involvement in S6K activation has been the subject of great investigative effort. In the present study, we have shown that 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), a selective inhibitor of the Src family of non-receptor tyrosine kinases, interferes with the activation of 70 and 85 kDa S6K gene products (p70S6K1 and p85S6K1) by insulin, insulin-like growth factor 1, sodium orthovanadate and activated alleles of phosphoinositide 3-kinase and H-Ras. PP1 also impedes the activation of AKT/protein kinase B and the extracellular signal-regulated protein kinases 1 and 2 by these various stimuli. Insulin-like growth factor 1 was observed to induce a sustained increase in c-Src autophosphorylation as revealed using anti-phospho-Y416 antisera, but this effect was absent from the cells treated with PP1. To conclude, an activated allele of p70S6K1 is compared with the wild-type allele, resistant to inhibition by PP1 when co-expressed with phosphoinositide-dependent kinase 1 (PDK1), suggesting that PP1 affects p70S6K1 via a PDK1-independent pathway. Thus activation of Src may supply a necessary signal for the activation of p70S6K1 and possibly other S6Ks. PMID:12014987

  13. Identification of therapeutic targets in ovarian cancer through active tyrosine kinase profiling

    PubMed Central

    Ocaña, Alberto; Pandiella, Atanasio

    2015-01-01

    The activation status of a set of pro-oncogenic tyrosine kinases in ovarian cancer patient samples was analyzed to define potential therapeutic targets. Frequent activation of HER family receptor tyrosine kinases, especially HER2, was observed. Studies in ovarian cancer cell lines confirmed the activation of HER2. Moreover, knockdown of HER2 caused a strong inhibition of their proliferation. Analyses of the action of agents that target HER2 indicated that the antibody drug conjugate trastuzumab-emtansine (T-DM1) caused a substantial antitumoral effect in vivo and in vitro, and potentiated the action of drugs used in the therapy of ovarian cancer. T-DM1 provoked cell cycle arrest in mitosis, and caused the appearance of aberrant mitotic spindles in cells treated with the drug. Biochemical experiments confirmed accumulation of the mitotic markers phospho-Histone H3 and phospho-BUBR1 in cells treated with the drug. Prolonged treatment of ovarian cancer cells with T-DM1 provoked the appearance of multinucleated cells which later led to cell death. Together, these data indicate that HER2 represents an important oncogene in ovarian cancer, and suggest that targeting this tyrosine kinase with T-DM1 may be therapeutically effective, especially in ovarian tumors with high content of HER2. PMID:26336133

  14. Structure of Escherichia coli tyrosine Kinase Etk Reveals a Novel Activation Mechanism

    SciTech Connect

    Lee,D.; Zheng, J.; She, Y.; Jia, Z.

    2008-01-01

    While protein tyrosine (Tyr) kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. The inner-membrane Wzc/Etk protein belongs to the bacterial PTK family, which has an important function in regulating the polymerization and transport of virulence-determining capsular polysaccharide (CPS). The kinase uses a unique two-step activation process involving intra-phosphorylation of a Tyr residue, although the molecular mechanism remains unknown. Herein, we report the first crystal structure of a bacterial PTK, the C-terminal kinase domain of Escherichia coli Tyr kinase (Etk) at 2.5-Angstroms resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting mass spectrometric evidence of Etk, a unique activation mechanism is proposed that involves the phosphorylated Tyr residue, Y574, at the active site and its specific interaction with a previously unidentified key Arg residue, R614, to unblock the active site. Both in vitro kinase activity and in vivo antibiotics resistance studies using structure-guided mutants further support the novel activation mechanism.

  15. rlk/TXK Encodes Two Forms of a Novel Cysteine String Tyrosine Kinase Activated by Src Family Kinases

    PubMed Central

    Debnath, Jayantha; Chamorro, Mario; Czar, Michael J.; Schaeffer, Edward M.; Lenardo, Michael J.; Varmus, Harold E.; Schwartzberg, Pamela L.

    1999-01-01

    Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR. PMID:9891083

  16. Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice.

    PubMed

    Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

    2016-01-01

    Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

  17. Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice

    PubMed Central

    Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

    2016-01-01

    Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

  18. Structure and activation of MuSK, a receptor tyrosine kinase central to neuromuscular junction formation.

    PubMed

    Hubbard, Stevan R; Gnanasambandan, Kavitha

    2013-10-01

    MuSK (muscle-specific kinase) is a receptor tyrosine kinase that plays a central signaling role in the formation of neuromuscular junctions (NMJs). MuSK is activated in a complex spatio-temporal manner to cluster acetylcholine receptors on the postsynaptic (muscle) side of the synapse and to induce differentiation of the nerve terminal on the presynaptic side. The ligand for MuSK is LRP4 (low-density lipoprotein receptor-related protein-4), a transmembrane protein in muscle, whose binding affinity for MuSK is potentiated by agrin, a neuronally derived heparan-sulfate proteoglycan. In addition, Dok7, a cytoplasmic adaptor protein, is also required for MuSK activation in vivo. This review focuses on the physical interplay between these proteins and MuSK for activation and downstream signaling, which culminates in NMJ formation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases. PMID:23467009

  19. Structure-activity relationship study of EphB3 receptor tyrosine kinase inhibitors

    PubMed Central

    Qiao, Lixin; Choi, Sungwoon; Case, April; Gainer, Thomas G.; Seyb, Kathleen; Glicksman, Marcie A.; Lo, Donald C.; Stein, Ross L.; Cuny, Gregory D.

    2009-01-01

    A structure-activity relationship study for a 2-chloroanilide derivative of pyrazolo[1,5-a]pyridine revealed that increased EphB3 kinase inhibitory activity could be accomplished by retaining the 2-chloroanilide and introducing a phenyl or small electron donating substituents to the 5-position of the pyrazolo[1,5-a]pyridine. In addition, replacement of the pyrazolo[1,5-a]pyridine with imidazo[1,2-a]pyridine was well tolerated and resulted in enhanced mouse liver microsome stability. The structure-activity relationship for EphB3 inhibition of both heterocyclic series was similar. Kinase inhibitory activity was also demonstrated for representative analogs in cell culture. An analog (32, LDN-211904) was also profiled for inhibitory activity against a panel of two hundred and eighty eight kinases and found to be quite selective for tyrosine kinases. Overall, these studies provide useful molecular probes for examining the in vitro, cellular and potentially in vivo kinase-dependent function of EphB3 receptor. PMID:19783434

  20. Human biliverdin reductase: a member of the insulin receptor substrate family with serine/threonine/tyrosine kinase activity.

    PubMed

    Lerner-Marmarosh, Nicole; Shen, Jenny; Torno, Michael D; Kravets, Anatoliy; Hu, Zhenbo; Maines, Mahin D

    2005-05-17

    We describe here the tyrosine kinase activity of human biliverdin reductase (BVR) and its potential role in the insulin-signaling pathway. BVR is both a substrate for insulin receptor (IR) tyrosine kinase (IRK) activity and a kinase for serine phosphorylation of IR substrate 1 (IRS-1). Our previous studies have revealed serine/threonine kinase activity of BVR. Y198, in the YMKM motif found in the C-terminal domain of BVR, is shown to be a substrate for insulin-activated IRK. This motif in IRS proteins provides a docking site for proteins that contain a Src homology 2 domain. Additionally, Y228 in the YLSF sequence and Y291 are IRK substrates; the former sequence provides optimum recognition motif in the tyrosine phosphatase, SHP-1, and for SHC (Src homology 2 domain containing transfroming protein 1). BVR autophosphorylates N-terminal tyrosines Y72 and Y83. Serine residues in IRS-1 are targets for BVR phosphorylation, and point mutation of serine residues in the kinase domain of the reductase inhibits phosphotransferase activity. Because tyrosine phosphorylation of IRS-1 activates the insulin signaling pathway and serine phosphorylation of IRS-1 blocks insulin action, our findings that insulin increases BVR tyrosine phosphorylation and that there is an increase in glucose uptake in response to insulin when expression of BVR is "knocked down" by small interfering RNA suggest a potential role for BVR in the insulin signaling pathway. PMID:15870194

  1. Structure and Activation of MuSK, a Receptor Tyrosine Kinase Central to Neuromuscular Junction Formation

    PubMed Central

    Hubbard, Stevan R.; Gnanasambandan, Kavitha

    2014-01-01

    MuSK (muscle-specific kinase) is a receptor tyrosine kinase that plays a central signaling role in formation of neuromuscular junctions (NMJs). MuSK is activated in a complex spatio-temporal manner to cluster acetylcholine receptors on the postsynaptic (muscle) side of the synapse and to induce differentiation of the nerve terminal on the presynaptic side. The ligand for MuSK is LRP4 (low-density lipoprotein receptor-related protein-4), a transmembrane protein in muscle, whose binding affinity for MuSK is potentiated by agrin, a neuronally derived heparan-sulfate proteoglycan. In addition, Dok7, a cytoplasmic adaptor protein, is also required for MuSK activation in vivo. This review focuses on the physical interplay between these proteins and MuSK for activation and downstream signaling, which culminates in NMJ formation. PMID:23467009

  2. Nuclear localization of Lyn tyrosine kinase mediated by inhibition of its kinase activity

    SciTech Connect

    Ikeda, Kikuko; Nakayama, Yuji; Togashi, Yuuki; Obata, Yuuki; Kuga, Takahisa; Kasahara, Kousuke; Fukumoto, Yasunori; Yamaguchi, Naoto

    2008-11-01

    Src-family kinases, cytoplasmic enzymes that participate in various signaling events, are found at not only the plasma membrane but also subcellular compartments, such as the nucleus, the Golgi apparatus and late endosomes/lysosomes. Lyn, a member of the Src-family kinases, is known to play a role in DNA damage response and cell cycle control in the nucleus. However, it is still unclear how the localization of Lyn to the nucleus is regulated. Here, we investigated the mechanism of the distribution of Lyn between the cytoplasm and the nucleus in epitheloid HeLa cells and hematopoietic THP-1 cells. Lyn was definitely detected in purified nuclei by immunofluorescence and immunoblotting analyses. Nuclear accumulation of Lyn was enhanced upon treatment of cells with leptomycin B (LMB), an inhibitor of Crm1-mediated nuclear export. Moreover, Lyn mutants lacking the sites for lipid modification were highly accumulated in the nucleus upon LMB treatment. Intriguingly, inhibition of the kinase activity of Lyn by SU6656, Csk overexpression, or point mutation in the ATP-binding site induced an increase in nuclear Lyn levels. These results suggest that Lyn being imported into and rapidly exported from the nucleus preferentially accumulates in the nucleus by inhibition of the kinase activity and lipid modification.

  3. Cross-talk between receptors with intrinsic tyrosine kinase activity and alpha1b-adrenoceptors.

    PubMed Central

    del Carmen Medina, L; Vázquez-Prado, J; García-Sáinz, J A

    2000-01-01

    The effect of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) on the phosphorylation and function of alpha(1b)-adrenoceptors transfected into Rat-1 fibroblasts was studied. EGF and PDGF increased the phosphorylation of these adrenoceptors. The effect of EGF was blocked by tyrphostin AG1478 and that of PDGF was blocked by tyrphostin AG1296, inhibitors of the intrinsic tyrosine kinase activities of the receptors for these growth factors. Wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the alpha(1b)-adrenoceptor phosphorylation induced by EGF but not that induced by PDGF. Inhibition of protein kinase C blocked the adrenoceptor phosphorylation induced by EGF and PDGF. The ability of noradrenaline to increase [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding in membrane preparations was used as an index of the functional coupling of the alpha(1b)-adrenoceptors and G-proteins. Noradrenaline-stimulated [(35)S]GTP[S] binding was markedly decreased in membranes from cells pretreated with EGF or PDGF. Our data indicate that: (i) activation of EGF and PDGF receptors induces phosphorylation of alpha(1b)-adrenoceptors, (ii) phosphatidylinositol 3-kinase is involved in the EGF response, but does not seem to play a major role in the action of PDGF, (iii) protein kinase C mediates this action of both growth factors and (iv) the phosphorylation of alpha(1b)-adrenoceptors induced by EGF and PDGF is associated with adrenoceptor desensitization. PMID:10947955

  4. Antibacterial and EGFR-tyrosine kinase inhibitory activities of polyhydroxylated xanthones from Garcinia succifolia.

    PubMed

    Duangsrisai, Susawat; Choowongkomon, Kiattawee; Bessa, Lucinda J; Costa, Paulo M; Amat, Nurmuhammat; Kijjoa, Anake

    2014-01-01

    Chemical investigation of the methanol extract of the wood of Garcinia succifolia Kurz (Clusiaceae) led to the isolation of 1,5-dihydroxyxanthone (1), 1,7-dihydroxyxanthone (2), 1,3,7-trihydroxyxanthone (3), 1,5,6-trihydroxyxanthone (4), 1,6,7-trihydroxyxanthone (5), and 1,3,6,7-tetrahydroxyxanthone (6). All of the isolated xanthones were evaluated for their antibacterial activity against bacterial reference strains, two Gram-positive (Staphylococcus aureus ATTC 25923, Bacillus subtillis ATCC 6633) and two Gram-negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853), and environmental drug-resistant isolates (S. aureus B1, Enteroccoccus faecalis W1, and E. coli G1), as well as for their epidermal growth factor receptor (EGFR) of tyrosine kinase inhibitory activity. Only 1,5,6-trihydroxy-(4), 1,6,7-trihydroxy-(5), and 1,3,6,7-tetrahydroxyxanthones (6) exhibited antibacterial activity against Gram-positive bacteria, however none was active against vancomycin-resistant E. faecalis. Additionally, 1,7-dihydroxyxanthone (2) showed synergism with oxacillin, but not with ampicillin. On the other hand, only 1,5-dihydroxyxanthone (1) and 1,7-dihydroxyxanthone (2) were found to exhibit the EGFR-tyrosine kinase inhibitory activity, with IC50 values of 90.34 and 223 nM, respectively. PMID:25460314

  5. Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

    PubMed Central

    Migliaccio, A; Di Domenico, M; Castoria, G; de Falco, A; Bontempo, P; Nola, E; Auricchio, F

    1996-01-01

    The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor. Images PMID:8635462

  6. erbB3 Is an Active Tyrosine Kinase Capable of Homo- and Heterointeractions

    PubMed Central

    Steinkamp, Mara P.; Low-Nam, Shalini T.; Yang, Shujie; Lidke, Keith A.; Lidke, Diane S.

    2014-01-01

    Often considered to be a “dead” kinase, erbB3 is implicated in escape from erbB-targeted cancer therapies. Here, heregulin stimulation is shown to markedly upregulate kinase activity in erbB3 immunoprecipitates. Intact, activated erbB3 phosphorylates tyrosine sites in an exogenous peptide substrate, and this activity is abolished by mutagenesis of lysine 723 in the catalytic domain. Enhanced erbB3 kinase activity is linked to heterointeractions with catalytically active erbB2, since it is largely blocked in cells pretreated with lapatinib or pertuzumab. erbB2 activation of erbB3 is not dependent on equal surface levels of these receptors, since it occurs even in erbB3-transfected CHO cells with disproportionally small amounts of erbB2. We tested a model in which transient erbB3/erbB2 heterointeractions set the stage for erbB3 homodimers to be signaling competent. erbB3 homo- and heterodimerization events were captured in real time on live cells using single-particle tracking of quantum dot probes bound to ligand or hemagglutinin tags on recombinant receptors. PMID:24379439

  7. DMBA induces tyrosine phosphorylation of PLC-[gamma]1 and activates the tyrosine kinases lck and fyn in the HPB-ALL human T-cell line

    SciTech Connect

    Archuleta, M.M.; Schieven, G.L.; Ledbetter, J.A.; Burchiel, S.W. . Coll. of Pharmacy)

    1993-01-01

    Previous studies in this laboratory have demonstrated that DMBA alters biochemical events associated with lymphocyte activation including formation of the second messenger IP[sub 3] and the release of intracellular Ca[sup 2+]. The purpose of the present studies was to evaluate the mechanisms by which DMBA induces IP[sub 3] formation and Ca[sup 2+] release by examining phosphorylation of membrane associated proteins and activation of protein tyrosine kinases lck and fyn. These studies demonstrated that exposure of HPB-ALL cells to 10[mu]M DMBA resulted in a time- and dose-dependent increase in tyrosine phosphorylation of PLC-[gamma]1 that correlated with our earlier findings of IP[sub 3] formation and Ca[sup 2+] release. These results indicate that the effects of DMBA on the PI-PLC signaling pathway are in part, the result of DMBA-induced tyrosine phosphorylation of the PLC-[gamma]1 enzyme. The mechanism of DMBA- induced tyrosine phosphorylation of PLC-[gamma]1 may be due to activation of fyn or lck kinase activity, since it was found that DMBA increased the activity of these PTKs by more than 2-fold. Therefore, these studies demonstrate that DMBA may disrupt T cell activation by stimulating PTK activation with concomitant tyrosine phosphorylation of PLC-[gamma]1, release of IP[sub 3], and mobilization of intracellular Ca[sup 2+].

  8. DMBA induces tyrosine phosphorylation of PLC-{gamma}1 and activates the tyrosine kinases lck and fyn in the HPB-ALL human T-cell line

    SciTech Connect

    Archuleta, M.M.; Schieven, G.L.; Ledbetter, J.A.; Burchiel, S.W.

    1993-02-01

    Previous studies in this laboratory have demonstrated that DMBA alters biochemical events associated with lymphocyte activation including formation of the second messenger IP{sub 3} and the release of intracellular Ca{sup 2+}. The purpose of the present studies was to evaluate the mechanisms by which DMBA induces IP{sub 3} formation and Ca{sup 2+} release by examining phosphorylation of membrane associated proteins and activation of protein tyrosine kinases lck and fyn. These studies demonstrated that exposure of HPB-ALL cells to 10{mu}M DMBA resulted in a time- and dose-dependent increase in tyrosine phosphorylation of PLC-{gamma}1 that correlated with our earlier findings of IP{sub 3} formation and Ca{sup 2+} release. These results indicate that the effects of DMBA on the PI-PLC signaling pathway are in part, the result of DMBA-induced tyrosine phosphorylation of the PLC-{gamma}1 enzyme. The mechanism of DMBA- induced tyrosine phosphorylation of PLC-{gamma}1 may be due to activation of fyn or lck kinase activity, since it was found that DMBA increased the activity of these PTKs by more than 2-fold. Therefore, these studies demonstrate that DMBA may disrupt T cell activation by stimulating PTK activation with concomitant tyrosine phosphorylation of PLC-{gamma}1, release of IP{sub 3}, and mobilization of intracellular Ca{sup 2+}.

  9. Hypotonicity stimulates renal epithelial sodium transport by activating JNK via receptor tyrosine kinases.

    PubMed

    Taruno, Akiyuki; Niisato, Naomi; Marunaka, Yoshinori

    2007-07-01

    We previously reported that hypotonic stress stimulated transepithelial Na(+) transport via a pathway dependent on protein tyrosine kinase (PTK; Niisato N, Van Driessche W, Liu M, Marunaka Y. J Membr Biol 175: 63-77, 2000). However, it is still unknown what type of PTK mediates this stimulation. In the present study, we investigated the role of receptor tyrosine kinase (RTK) in the hypotonic stimulation of Na(+) transport. In renal epithelial A6 cells, we observed inhibitory effects of AG1478 [an inhibitor of the EGF receptor (EGFR)] and AG1296 [an inhibitor of the PDGF receptor (PDGFR)] on both the hypotonic stress-induced stimulation of Na(+) transport and the hypotonic stress-induced ligand-independent activation of EGFR. We further studied whether hypotonic stress activates members of the MAP kinase family, ERK1/2, p38 MAPK, and JNK/SAPK, via an RTK-dependent pathway. The present study indicates that hypotonic stress induced phosphorylation of ERK1/2 and JNK/SAPK, but not p38 MAPK, that the hypotonic stress-induced phosphorylation of ERK1/2 and JNK/SAPK was diminished by coapplication of AG1478 and AG1296, and that only JNK/SAPK was involved in the hypotonic stimulation of Na(+) transport. A further study using cyclohexamide (a protein synthesis inhibitor) suggests that both RTK and JNK/SAPK contributed to the protein synthesis-independent early phase in hypotonic stress-induced Na(+) transport, but not to the protein synthesis-dependent late phase. The present study also suggests involvement of phosphatidylinositol 3-kinase (PI3-kinase) in RTK-JNK/SAPK cascade-mediated Na(+) transport. These observations indicate that 1) hypotonic stress activates JNK/SAPK via RTKs in a ligand-independent pathway, 2) the RTK-JNK/SAPK cascade acts as a mediator of hypotonic stress for stimulation of Na(+) transport, and 3) PI3-kinase is involved in the RTK-JNK/SAPK cascade for the hypotonic stress-induced stimulation of Na(+) transport. PMID:17344192

  10. 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

  11. Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

    PubMed Central

    Böcker, Dietmar

    2001-01-01

    MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [ P 32 ]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 ( IC 50 =51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [ H 3 ]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but

  12. BCR first exon sequences specifically activate the BCR/ABL tyrosine kinase oncogene of Philadelphia chromosome-positive human leukemias

    SciTech Connect

    Muller, A.J.; Witte, O.N. ); Young, J.C.; Pendergast, A.; Pondel, M. ); Landau, N.R.; Littman, D.R. )

    1991-04-01

    The c-abl proto-oncogene encodes a cytoplasmic tyrosine kinase which is homologous to the src gene product in its kinase domain and in the upstream kinase regulatory domains SH2 (src homology region 2) and SH3 (src homology region 3). The murine v-abl oncogene product has lost the SH3 domain as a consequence of N-terminal fusion of gag sequences. Deletion of the SH3 domain is sufficient to render the murine c-abl proto-oncogene product transforming when myristylated N-terminal membrane localization sequences are also present. In contrast, the human BCR/ABL oncogene of the Philadelphia chromosome translocation has an intact SH3 domain and its product is not myristylated at the N terminus. To analyze the contribution of BCR-encoded sequences to BCR/ABL-mediated transformation, the effects of a series of deletions and substitutions were assessed in fibroblast and hematopoietic-cell transformation assays. BCR first-exon sequences specifically potentiate transformation and tyrosine kinase activation when they are fused to the second exon of otherwise intact c-ABL. This suggests that BCR-encoded sequences specifically interfere with negative regulation of the ABL-encoded tyrosine kinase, which would represent a novel mechanism for the activation of nonreceptor tyrosine kinase-encoding proto-oncogenes.

  13. Delta Opioid activation of the Mitogen-activated protein kinase cascade does not require transphosphorylation of Receptor Tyrosine Kinases

    PubMed Central

    Kramer, H Kenneth; Onoprishvili, Irma; Andria, Matthew L; Hanna, Kayane; Sheinkman, Karina; Haddad, Lisa B; Simon, Eric J

    2002-01-01

    Background In this study, we investigated the mechanism(s) by which delta opioids induce their potent activation of extracellular signal-regulated protein kinases (ERKs) in different cell lines expressing the cloned δ-opioid receptor (δ-OR). While it has been known for some time that OR stimulation leads to the phosphorylation of both ERK isoforms, the exact progression of events has remained elusive. Results Our results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed δ-ORs are stimulated by the δ-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET). Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET. These results appear to rule out any structural or catalytic role for the EGFR in the δ-opioid-mediated MAPK cascade. To confirm these results, we used C6 glioma cells, a cell line devoid of the EGFR. In δ-OR-expressing C6 glioma cells, opioids produce a robust phosphorylation of ERK 1 and 2, whereas EGF has no stimulatory effect. Furthermore, antagonists to the RTKs that are endogenously expressed in C6 glioma cells (insulin receptor (IR) and platelet-derived growth factor receptor (PDGFR)) were unable to reduce opioid-mediated ERK activation. Conclusion Taken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated δ-OR, itself, is likely to act as its own signalling scaffold. PMID:11897012

  14. Activation of tyrosine kinase c-Abl contributes to α-synuclein-induced neurodegeneration.

    PubMed

    Brahmachari, Saurav; Ge, Preston; Lee, Su Hyun; Kim, Donghoon; Karuppagounder, Senthilkumar S; Kumar, Manoj; Mao, Xiaobo; Shin, Joo Ho; Lee, Yunjong; Pletnikova, Olga; Troncoso, Juan C; Dawson, Valina L; Dawson, Ted M; Ko, Han Seok

    2016-08-01

    Aggregation of α-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and α-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote α-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates α-synuclein-induced neuropathology. In mice expressing a human α-synucleinopathy-associated mutation (hA53Tα-syn mice), deletion of the gene encoding c-Abl reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53Tα-syn mice accelerated α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 α-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 α-synuclein in brain tissues and Lewy bodies of PD patients compared with age-matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of α-synuclein at tyrosine 39 enhances α-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in α-synuclein-induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 α-synuclein is a potential disease indicator for PD and related α-synucleinopathies. PMID:27348587

  15. Overcoming mutagenicity and ion channel activity: optimization of selective spleen tyrosine kinase inhibitors.

    PubMed

    Ellis, J Michael; Altman, Michael D; Bass, Alan; Butcher, John W; Byford, Alan J; Donofrio, Anthony; Galloway, Sheila; Haidle, Andrew M; Jewell, James; Kelly, Nancy; Leccese, Erica K; Lee, Sandra; Maddess, Matthew; Miller, J Richard; Moy, Lily Y; Osimboni, Ekundayo; Otte, Ryan D; Reddy, M Vijay; Spencer, Kerrie; Sun, Binyuan; Vincent, Stella H; Ward, Gwendolyn J; Woo, Grace H C; Yang, Chiming; Houshyar, Hani; Northrup, Alan B

    2015-02-26

    Development of a series of highly kinome-selective spleen tyrosine kinase (Syk) inhibitors with favorable druglike properties is described. Early leads were discovered through X-ray crystallographic analysis, and a systematic survey of cores within a selected chemical space focused on ligand binding efficiency. Attenuation of hERG ion channel activity inherent within the initial chemotype was guided through modulation of physicochemical properties including log D, PSA, and pKa. PSA proved most effective for prospective compound design. Further profiling of an advanced compound revealed bacterial mutagenicity in the Ames test using TA97a Salmonella strain, and subsequent study demonstrated that this mutagenicity was pervasive throughout the series. Identification of intercalation as a likely mechanism for the mutagenicity-enabled modification of the core scaffold. Implementation of a DNA binding assay as a prescreen and models in DNA allowed resolution of the mutagenicity risk, affording molecules with favorable potency, selectivity, pharmacokinetic, and off-target profiles. PMID:25625541

  16. Structural features that specify tyrosine kinase activity deduced from homology modeling of the epidermal growth factor receptor.

    PubMed Central

    Knighton, D R; Cadena, D L; Zheng, J; Ten Eyck, L F; Taylor, S S; Sowadski, J M; Gill, G N

    1993-01-01

    To identify structural features that distinguish protein-tyrosine kinases from protein-serine kinases, a molecular model of the kinase domain of epidermal growth factor receptor was constructed by substituting its amino acid sequence for the amino acid sequence of the catalytic subunit of cAMP-dependent protein kinase in a 2.7-A refined crystallographic model. General folding was conserved as was the configuration of invariant residues at the active site. Two sequence motifs that distinguish the two families correspond to loops that converge at the active site of the enzyme. A conserved arginine in the catalytic loop is proposed to interact with the gamma phosphate of ATP. The second loop provides a binding surface that positions the tyrosine of the substrate. A positively charged surface provides additional sites for substrate recognition. Images Fig. 2 Fig. 3 Fig. 4 PMID:8389462

  17. Separate domains of the insulin receptor contain sites of autophosphorylation and tyrosine kinase activity

    SciTech Connect

    Goren, H.J.; White, M.F.; Khan, C.R.

    1987-04-21

    The authors have studied the structure and function of the solubilized insulin receptor before and after partial proteolytic digestion to define domains in the ..beta..-subunit that undergo autophosphorylation and contain the tyrosine kinase activity. Wheat germ agglutinin purified insulin receptor from Fao cells was digested briefly at 22/sup 0/C with low concentrations of trypsin, staphylococcal V8 protease, or elastase. Autophosphorylation of the ..beta..-subunit was carried out before and after digestion, and the (/sup 32/P)phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and analyzed by tryptic peptide mapping by use of reverse-phase high-performance liquid chromatography. The 85-kDa fragment was not immunoprecipitated by an antibody directed against the C-terminal domain of the ..beta..-subunit (..cap alpha..Pep-1), indicating that this region of the receptor was lost. The 85-kDa fragment contained about half of the (/sup 32/P)phosphate originally found in the ..beta..-subunit, and tryptic peptide mapping showed that two major tryptic phosphopeptides (previously called pY2 and pY3) were removed. Three other tryptic phosphopeptides (pY1, pY1a, and pY4) were found in the 85- and 70-kDa fragments. To determined the structural requirements for kinase activity, the insulin receptor was subjected to tryptic digestion for 30 s-30 min, such that the receptor was composed exclusively of 85- and 70-kDa fragments of the ..beta..-subunit. The 85-kDa fragment exhibited autophosphorylation at pY1, pY1a, and pY4. Both the 85- and 70-kDa fragments phosphorylated tyrosine residues in a synthetic decapeptide that has the sequence of the C-terminal domain of the ..beta..-subunit of human insulin rare in the receptor.

  18. Novel Small Molecule Activators of the Trk Family of Receptor Tyrosine Kinases

    PubMed Central

    Obianyo, Obiamaka; Ye, Keqiang

    2012-01-01

    The Tropomyosin-related kinase (Trk) receptors are a subset of the receptor tyrosine kinase family with an important functionality in the regulation of neurotrophic signaling in the peripheral and central nervous system. As the receptors are able to mediate neuronal survival by associating with their respective neurotrophin ligands, many studies have focused on the therapeutic potential of generating small-molecule mimetic compounds that elicit agonistic effects similar to those of the natural protein ligands. To this end, various structure-based studies have led to the generation of bivalent peptide-based agonists and antibodies that selectively initiate Trk receptor signaling; however, these compounds do not possess the ideal characteristics of a potential drug. Additionally, the reliance of structure-based data to generate the compound libraries, limits the potential identification of novel chemical structures with desirable activity. Therefore, subsequent investigations utilized a cell-based apoptotic screen to facilitate the analysis of large, diverse chemical libraries of small molecules and quickly identify compounds with Trk-dependent antiapoptotic activity. Herein, we describe the Trk agonists that have been identified by this screening methodology and summarize their in vitro and in vivo neurotrophic activity as well as their efficacy in various neurological disease models, implicating their future utility as therapeutic compounds. PMID:22982231

  19. 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

  20. 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

  1. Cell signaling by receptor-tyrosine kinases

    PubMed Central

    Lemmon, Mark A.; Schlessinger, Joseph

    2010-01-01

    Recent structural studies of receptor tyrosine kinases (RTKs) have revealed unexpected diversity in the mechanisms of their activation by growth factor ligands. Strategies for inducing dimerization by ligand binding are surprisingly diverse, as are mechanisms that couple this event to activation of the intracellular tyrosine kinase domains. As our understanding of these details becomes increasingly sophisticated, it provides an important context for therapeutically countering the effects of pathogenic RTK mutations in cancer and other diseases. Much remains to be learned, however, about the complex signaling networks downstream from RTKs and how alterations in these networks are translated into cellular responses. PMID:20602996

  2. Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains.

    PubMed Central

    Koyasu, S; Tse, A G; Moingeon, P; Hussey, R E; Mildonian, A; Hannisian, J; Clayton, L K; Reinherz, E L

    1994-01-01

    To define the T-cell receptor signal transduction motif, we have transfected human and murine T-cell lines with a chimeric receptor consisting of the extracellular and transmembrane domains of human CD8 alpha and the membrane-proximal portion of CD3 zeta containing at its C terminus either an 18-amino acid segment (NQLYNELNLGRREEYDVL) or alanine-scanning point mutant derivatives. Crosslinking of the extracellular domain of the chimera is sufficient to initiate Ca2+ flux, interleukin 2 production, and tyrosine phosphorylation of cellular proteins including the chimera. Subsequently, the chimera becomes associated with several tyrosine-phosphorylated proteins, among them the 70-kDa protein tyrosine kinase ZAP70. Mutational data identify the T-cell activation motif as Y(X)2L(X)7Y(X)2L and show that each of the four designated residues is necessary for the above activation events. Recombinant protein containing the two tandem SH2 domains derived from ZAP70 binds to a synthetic peptide corresponding to the above 18-amino acid motif but only when both tyrosines are phosphorylated; in contrast, little or no binding is observed to monophosphorylated or nonphosphorylated analogues. These results imply that after receptor crosslinking in T cells, and by inference also in B cells and mast cells, the motif is phosphorylated on both tyrosine residues, thereafter serving as a docking site for protein tyrosine kinases containing tandem SH2 domains. Images PMID:7517560

  3. Spatio-temporally precise activation of engineered receptor tyrosine kinases by light

    PubMed Central

    Grusch, Michael; Schelch, Karin; Riedler, Robert; Reichhart, Eva; Differ, Christopher; Berger, Walter; Inglés-Prieto, Álvaro; Janovjak, Harald

    2014-01-01

    Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that sense growth factors and hormones and regulate a variety of cell behaviours in health and disease. Contactless activation of RTKs with spatial and temporal precision is currently not feasible. Here, we generated RTKs that are insensitive to endogenous ligands but can be selectively activated by low-intensity blue light. We screened light-oxygen-voltage (LOV)-sensing domains for their ability to activate RTKs by light-activated dimerization. Incorporation of LOV domains found in aureochrome photoreceptors of stramenopiles resulted in robust activation of the fibroblast growth factor receptor 1 (FGFR1), epidermal growth factor receptor (EGFR) and rearranged during transfection (RET). In human cancer and endothelial cells, light induced cellular signalling with spatial and temporal precision. Furthermore, light faithfully mimicked complex mitogenic and morphogenic cell behaviour induced by growth factors. RTKs under optical control (Opto-RTKs) provide a powerful optogenetic approach to actuate cellular signals and manipulate cell behaviour. PMID:24986882

  4. Bruton's tyrosine kinase is essential for NLRP3 inflammasome activation and contributes to ischaemic brain injury

    PubMed Central

    Ito, Minako; Shichita, Takashi; Okada, Masahiro; Komine, Ritsuko; Noguchi, Yoshiko; Yoshimura, Akihiko; Morita, Rimpei

    2015-01-01

    Inflammasome activation has been implicated in various inflammatory diseases including post-ischaemic inflammation after stroke. Inflammasomes mediate activation of caspase-1, which subsequently induces secretion of pro-inflammatory cytokines such as IL-1β and IL-18, as well as a form of cell death called pyroptosis. In this study, we report that Bruton's tyrosine kinase (BTK) is an essential component of the NLRP3 inflammasome, in which BTK physically interacts with ASC and NLRP3. Inhibition of BTK by pharmacological or genetic means severely impairs activation of the NLRP3 inflammasome. The FDA-approved BTK inhibitor ibrutinib (PCI-32765) efficiently suppresses infarct volume growth and neurological damage in a brain ischaemia/reperfusion model in mice. Ibrutinib inhibits maturation of IL-1β by suppressing caspase-1 activation in infiltrating macrophages and neutrophils in the infarcted area of ischaemic brain. Our study indicates that BTK is essential for NLRP3 inflammasome activation and could be a potent therapeutic target in ischaemic stroke. PMID:26059659

  5. Global phosphotyrosine survey in triple-negative breast cancer reveals activation of multiple tyrosine kinase signaling pathways

    PubMed Central

    Ma, Binyun; Liu, Ren; Renuse, Santosh; Sahasrabuddhe, Nandini A.; Chen, Lily; Chaerkady, Raghothama; Kim, Min-Sik; Zhong, Jun; Jelinek, Christine; Barbhuiya, Mustafa A.; Leal-Rojas, Pamela; Yang, Yi; Kashyap, Manoj Kumar; Marimuthu, Arivusudar; Ling, Min; Fackler, Mary Jo; Merino, Vanessa; Zhang, Zhen; Zahnow, Cynthia A.; Gabrielson, Edward; Stearns, Vered; Roa, Juan Carlos; Sukumar, Saraswati; Gill, Parkash S.; Pandey, Akhilesh

    2015-01-01

    Breast cancer is the most prevalent cancer in women worldwide. About 15–20% of all breast cancers are triple negative breast cancer (TNBC) and are often highly aggressive when compared to other subtypes of breast cancers. To better characterize the biology that underlies the TNBC phenotype, we profiled the phosphotyrosine proteome of a panel of twenty-six TNBC cell lines using quantitative high resolution Fourier transform mass spectrometry. A heterogeneous pattern of tyrosine kinase activation was observed based on 1,789 tyrosine-phosphorylated peptides identified from 969 proteins. One of the tyrosine kinases, AXL, was found to be activated in a majority of aggressive TNBC cell lines and was accompanied by a higher level of AXL expression. High levels of AXL expression are correlated with a significant decrease in patient survival. Treatment of cells bearing activated AXL with a humanized AXL antibody inhibited cell proliferation and migration in vitro, and tumor growth in mice. Overall, our global phosphoproteomic analysis provided new insights into the heterogeneity in the activation status of tyrosine kinase pathways in TNBCs. Our approach presents an effective means of identifying important novel biomarkers and targets for therapy such as AXL in TNBC. PMID:26356563

  6. Net Increase of platelet membrane tyrosine specific-protein kinase activity by phorbol myristate acetate

    SciTech Connect

    Ishihara, Noriko; Sakamoto, Hikaru; Iwama, Minako; Kobayashi, Bonro )

    1990-01-01

    Tyrosine protein kinase (TPK) activity in rabbit platelets after stimulation by phorbol myristate acetate (PMA) or thrombin was directly estimated by {sup 32}P incorporation from ({gamma}-{sup 32})ATP into synthetic peptide angiotensin II. By PMA-treatment a net increase of TPK activity was obtained, while thrombin acted on the TPK quickly but stimulation was limited within the range attained by the control after lengthy incubation. The responsive TPK to these stimulators was localized mainly in membrane but much less in cytosol. The specific activity of the particulate TPK was low in the sonicate of control ice cold platelets but increased about 6-fold when the platelets were incubated at 37{degree}C. On a brief contact of platelets with PMA at 37{degrees}C the TPK was fully activated and reached a maximum value about 130% of the control. Determination of phosphotyrosine phosphatase in the stimulated platelet sonicate revealed that its participation in the above described increase of {sup 32}P-incorporation was meagre. The quick response suggested a possible role of TPK in the signal transduction through the platelet cell membrane.

  7. Tyrosine Kinase Inhibition: An Approach to Drug Development

    NASA Astrophysics Data System (ADS)

    Levitzki, Alexander; Gazit, Aviv

    1995-03-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.

  8. Tyrosines 868, 966, and 972 in the Kinase Domain of JAK2 Are Autophosphorylated and Required for Maximal JAK2 Kinase Activity

    PubMed Central

    Argetsinger, Lawrence S.; Stuckey, Jeanne A.; Robertson, Scott A.; Koleva, Rositsa I.; Cline, Joel M.; Marto, Jarrod A.; Myers, Martin G.; Carter-Su, Christin

    2010-01-01

    Janus kinase 2 (JAK2) is activated by a majority of cytokine family receptors including receptors for GH, leptin, and erythropoietin. To identify novel JAK2-regulatory and/or -binding sites, we set out to identify autophosphorylation sites in the kinase domain of JAK2. Two-dimensional phosphopeptide mapping of in vitro autophosphorylated JAK2 identified tyrosines 868, 966, and 972 as sites of autophosphorylation. Phosphorylated tyrosines 868 and 972 were also identified by mass spectrometry analysis of JAK2 activated by an erythropoietin-bound chimeric erythropoietin receptor/leptin receptor. Phosphospecific antibodies suggest that the phosphorylation of all three tyrosines increases in response to GH. Compared with wild-type JAK2, which is constitutively active when overexpressed, JAK2 lacking tyrosine 868, 966, or 972 has substantially reduced activity. Coexpression with GH receptor and protein tyrosine phosphatase1B allowed us to investigate GH-dependent activation of these mutated JAK2s in human embryonic kidney 293T cells. All three mutated JAK2s are activated by GH, although to a lesser extent than wild-type JAK2. The three mutated JAK2s also mediate GH activation of signal transducer and activator of transcription 3 (Stat3), signal transducer and activator of transcription 5b (Stat5b) and ERK1, but at reduced levels. Coexpression with Src-homology 2B1β (SH2B1β), like coexpression with GH-bound GH receptor, partially restores the activity of all three JAK2 mutants. Based on these results and the crystal structure of the JAK2 kinase domain, we hypothesize that small changes in the conformation of the regions of JAK2 surrounding tyrosines 868, 966, and 972 due to e.g. phosphorylation, binding to a ligand-bound cytokine receptor, and/or binding to Src-homology 2B1, may be essential for JAK2 to assume a maximally active conformation. PMID:20304997

  9. Downregulation of the Ras–Mitogen-Activated Protein Kinase Pathway by the EphB2 Receptor Tyrosine Kinase Is Required for Ephrin-Induced Neurite Retraction

    PubMed Central

    Elowe, Sabine; Holland, Sacha J.; Kulkarni, Sarang; Pawson, Tony

    2001-01-01

    Activation of the EphB2 receptor tyrosine kinase by clustered ephrin-B1 induces growth cone collapse and neurite retraction in differentiated NG108 neuronal cells. We have investigated the cytoplasmic signaling events associated with EphB2-induced cytoskeletal reorganization in these neuronal cells. We find that unlike other receptor tyrosine kinases, EphB2 induces a pronounced downregulation of GTP-bound Ras and consequently of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. A similar inhibition of the Ras-MAPK pathway was observed on stimulation of endogenous EphB2 in COS-1 cells. Inactivation of Ras, induced by ephrin B1 stimulation of NG108 neuronal cells, requires EphB2 tyrosine kinase activity and is blocked by a truncated form of p120-Ras GTPase-activating protein (p120-RasGAP), suggesting that EphB2 signals through the SH2 domain protein p120-RasGAP to inhibit the Ras-MAPK pathway. Suppression of Ras activity appears functionally important, since expression of a constitutively active variant of Ras impaired the ability of EphB2 to induce neurite retraction. In addition, EphB2 attenuated the elevation in ERK activation induced by attachment of NG108 cells to fibronectin, indicating that the EphB2 receptor can modulate integrin signaling to the Ras GTPase. These results suggest that a primary function of EphB2, a member of the most populous family of receptor tyrosine kinases, is to inactivate the Ras-MAPK pathway in a fashion that contributes to cytoskeletal reorganization and adhesion responses in neuronal growth cones. PMID:11585923

  10. The tyrosine kinase inhibitor bafetinib inhibits PAR2-induced activation of TRPV4 channels in vitro and pain in vivo

    PubMed Central

    Grace, M S; Lieu, T; Darby, B; Abogadie, F C; Veldhuis, N; Bunnett, N W; McIntyre, P

    2014-01-01

    BACKGROUND AND PURPOSE Protease-activated receptor 2 (PAR2) is expressed on nociceptive neurons, and can sensitize transient receptor potential (TRP) ion channels to amplify neurogenic inflammation and pain. The mechanisms by which this occurs are not fully understood. PAR2 causes receptor-operated activation of TRPV4 channels and TRPV4 null mice have attenuated PAR2-stimulated neurogenic inflammation and mechanical hyperalgesia. Here we investigate the intracellular signalling mechanisms underlying PAR2-induced TRPV4 channel activation and pain. EXPERIMENTAL APPROACH Responses of non-transfected and TRPV4-transfected HEK293 cells to agonists of PAR2 (trypsin and SLIGRL) and TRPV4 channels (GSK1016790A) were determined using calcium imaging. Inhibitors of TRPV4 channels (HC067047), sarcoendoplasmic reticulum calcium transport ATPase (thapsigargin), Gαq (UBO-QIC), tyrosine kinases (bafetinib and dasatinib) or PI3 kinases (wortmannin and LY294002) were used to investigate signalling mechanisms. In vivo effects of tyrosine kinase inhibitors on PAR2-induced mechanical hyperalgesia were assessed in mice. KEY RESULTS In non-transfected HEK293 cells, PAR2 activation transiently increased intracellular calcium ([Ca2+]i). Functional expression of TRPV4 channels caused a sustained increase of [Ca2+]i, inhibited by HC067047, bafetinib and wortmannin; but not by thapsigargin, UBO-QIC, dasatinib or LY294002. Bafetinib but not dasatinib inhibited PAR2-induced mechanical hyperalgesia in vivo. CONCLUSIONS AND IMPLICATIONS This study supports a role for tyrosine kinases in PAR2-mediated receptor-operated gating of TRPV4 channels, independent of Gαq stimulation. The ability of a tyrosine kinase inhibitor to diminish PAR2-induced activation of TRPV4 channels and consequent mechanical hyperalgesia identifies bafetinib (which is in development in oncology) as a potential novel analgesic therapy. PMID:24779362

  11. Receptor Tyrosine Kinases with Intracellular Pseudokinase Domains

    PubMed Central

    Mendrola, Jeannine M.; Shi, Fumin; Park, Jin H.; Lemmon, Mark A.

    2013-01-01

    As with other groups of protein kinases, approximately 10% of the receptor tyrosine kinases (RTKs) in the human proteome contain intracellular pseudokinases that lack one or more conserved catalytically important residues. These include ErbB3, a member of the epidermal growth factor receptor (EGFR) family, and a series of unconventional Wnt receptors. We recently showed that, despite its reputation as a pseudokinase, the ErbB3 tyrosine kinase domain (TKD) does retain significant – albeit weak – kinase activity. This led us to suggest that a subgroup of RTKs may be able to signal even with very inefficient kinases. Recent work suggests that this is not the case, however. Other pseudokinase RTKs have not revealed significant kinase activity, and mutations that impair ErbB3’s weak kinase activity have not so far been found to exhibit signaling defects. These findings therefore point to models in which the TKDs of pseudokinase RTKs participate in receptor signaling by allosterically regulating associated kinases (such as ErbB3 regulation of ErbB2) and/or function as regulated ‘scaffolds’ for other intermolecular interactions central to signal propagation. Further structural and functional studies – particularly of the pseudokinase RTKs involved in Wnt signaling – are required to shed new light on these intriguing signaling mechanisms. PMID:23863174

  12. Defect in tyrosine kinase activity of the insulin receptor from a patient with insulin resistance and acanthosis nigricans.

    PubMed

    Yamamoto, R; Shiba, T; Tobe, K; Shibasaki, Y; Koshio, O; Izumi, T; Odawara, M; Mikami, Y; Matsuura, N; Akanuma, Y

    1990-04-01

    We report here a defect in tyrosine kinase activity of the insulin receptor from an insulin-resistant patient with acanthosis nigricans using cultured Ebstein-Barr virus (EBV)-transformed B-lymphocytes. As judged by affinity labeling and immunoblotting, the alpha- and beta-subunits of insulin receptors from the patient's lymphocytes exhibited the same mol wt as those from control subjects. Lectin-purified extracts from lymphocytes of the patient and the control subjects containing the same insulin-binding capacity were assayed for autophosphorylation and the ability to phosphorylate histone H2B. The degree of insulin-dependent autophosphorylation and the tyrosine kinase activity of the insulin receptor from the patient's lymphocytes were decreased to 15% and 13%, respectively, in a cell-free system. The insulin-dependent autophosphorylation of the insulin receptor was also impaired in intact EBV lymphocytes from the patient. Consistent with these results, we found that one of this patient's alleles had a mutation in which valine is substituted for Gly996, the third glycine in the conserved Gly-X-Gly-X-X-Gly motif in the kinase domain. Thus, it seems likely that the defect in tyrosine kinase activity of the insulin receptor cause the insulin resistance in this patient. The EBV lymphocyte can be a good system to detect genetically determined abnormalities in the insulin receptor. PMID:2180980

  13. The mixed-lineage kinase DLK undergoes Src-dependent tyrosine phosphorylation and activation in cells exposed to vanadate or platelet-derived growth factor (PDGF).

    PubMed

    Daviau, Alex; Di Fruscio, Marco; Blouin, Richard

    2009-04-01

    Some data in the literature suggest that serine/threonine phosphorylation is required for activation of the mixed-lineage kinases (MLKs), a subgroup of mitogen-activated protein kinase kinase kinases (MAPKKKs). In this report, we demonstrate that the MLK family member DLK is activated and concurrently tyrosine-phosphorylated in cells exposed to the protein tyrosine phosphatase inhibitor vanadate. Tyrosine phosphorylation appears crucial for activation as incubation of vanadate-activated DLK molecules with a tyrosine phosphatase substantially reduced DLK enzymatic activity. Interestingly, the effects of vanadate on DLK are completely blocked by treatment with a Src family kinase inhibitor, PP2, or the expression of short hairpin RNA (shRNA) directed against Src. DLK also fails to undergo vanadate-stimulated tyrosine phosphorylation and activation in fibroblasts which lack expression of Src, Yes and Fyn, but reintroduction of wild-type Src or Fyn followed by vanadate treatment restores this response. In addition to vanadate, stimulation of cells with platelet-derived growth factor (PDGF) also induces tyrosine phosphorylation and activation of DLK by a Src-dependent mechanism. DLK seems important for PDGF signaling because its depletion by RNA interference substantially reduces PDGF-stimulated ERK and Akt kinase activation. Thus, our findings suggest that Src-dependent tyrosine phosphorylation of DLK may be important for regulation of its activity, and they support a role for DLK in PDGF signaling. PMID:19146952

  14. Receptor Tyrosine Kinases in Drosophila Development

    PubMed Central

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  15. Identification of the human pim-1 gene product as a 33-kilodalton cytoplasmic protein with tyrosine kinase activity

    SciTech Connect

    Telerman, A.; Amson, R.; Zakut-Houri, R.; Givol, D.

    1988-04-01

    The human pim-1 gene was recently identified as a new putative oncogene located on chromosome 6p21, a region showing karyotypic abnormalities in particular leukemias. In the present work the authors characterized the pim protein product. In vitro translation of positively selected poly(A)/sup +/ mRNA indicates that this gene encodes a 33-kilodalton protein. Anti-pim antibodies were raised against a fused TrpE-pim protein induced in a bacterial expression vector. This antibody immunoprecipitated a 33-kilodalton protein from in vivo (/sup 35/S)methionine-labeled K562 and KCl myelogenous origin cell lines. This protein was localized to the cytoplasm, and in vivo labeling as well as in vitro kinase assay suggests that it is a phosphoprotein with tyrosine kinase activity. This was further confirmed by performing autophosphorylation directly on a p33/sup pim/-containing gel band cut out after sodium dodecyl sulfate-polyacrylamide gel electrphoresis. The results imply that the tyrosine kinase activity of pim can be recovered after boiling the pim-1 protein in sample buffer: a feature not described yet for this class of protein. These results suggest that pim-1 is a new member of the subgroup of oncogenes encoding tyrosine kinases.

  16. The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

    PubMed Central

    Gu, Changkyu; Park, Soochul

    2001-01-01

    Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via α5β1- or β3 integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110γ isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110γ. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110γ mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110γ PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion. PMID:11416136

  17. Insulin binding and receptor tyrosine kinase activity in skeletal muscle of carnivorous and omnivorous fish.

    PubMed

    Párrizas, M; Planas, J; Plisetskaya, E M; Gutiérrez, J

    1994-06-01

    We characterized the insulin receptors in skeletal muscle from several fish species with different nutritional preferences: brown trout (Salmo trutta fario), gilthead sea bream (Sparus aurata), tilapia (Tilapia mossambica), and carp (Cyprinus carpio), semipurified by affinity chromatography (wheat germ agglutinin-agarose). Total specific binding and number of receptors per unit weight of piscine white skeletal muscle were lower than those values found in mammalian skeletal muscle. The same parameters in carp muscle receptor preparations were severalfold higher than in trout muscle (binding capacity 440 +/- 47 fmol/mg glycoprotein in carp and 82 +/- 23 fmol/mg glycoprotein in trout). Piscine insulin receptors phosphorylated exogenous substrate poly(Glu,Tyr) but less so than mammalian receptors. Tyrosine kinase activity of receptors, calculated as percent of 32P incorporated into substrate in the presence of insulin compared with basal incorporation, was also highest in carp (210 +/- 4%) and lowest in trout (150 +/- 2%). In both trout and carp deprived of food for 15 days, specific binding of insulin decreased. Nevertheless, differences between the two species were retained. Our results demonstrate that particular properties of insulin receptors in fish skeletal muscle may be related to nutritional preferences. This finding coincides with the phenomenon of differential glucose tolerance in fish: carnivorous fish, such as trout, are less tolerant, whereas omnivorous fish, such as carp, readily utilize a carbohydrate-rich diet. PMID:8024051

  18. Src-family tyrosine kinases in activation of ERK-1 and p85/p110-phosphatidylinositol 3-kinase by G/CCKB receptors.

    PubMed

    Daulhac, L; Kowalski-Chauvel, A; Pradayrol, L; Vaysse, N; Seva, C

    1999-07-16

    We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc.Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src. IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin. PMID:10400698

  19. Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide-induced nitric oxide synthase activity in RAW 264.7 murine macrophages.

    PubMed Central

    Paul, A; Pendreigh, R H; Plevin, R

    1995-01-01

    1. In RAW 264.7 macrophages, lipopolysaccharide (LPS) and gamma-interferon (IFN gamma) alone or in combination stimulated the induction of nitric oxide synthase (iNOS) activity and increased the expression of the 130 kDa isoform of NOS. 2. LPS-induced NOS activity was reduced by incubation with CD14 neutralising antibodies and abolished in macrophages deprived of serum. 3. LPS stimulated a small increase in protein kinase C (PKC) activity in RAW 264.7 macrophages which was dependent on the presence of serum. However, IFN gamma did not potentiate LPS-stimulated PKC activity. 4. The protein kinase C inhibitor, Ro-318220, abolished both LPS- and IFN gamma-stimulated protein kinase C activity and the induction of NOS activity. 5. LPS- and IFN gamma-induced NOS activity was reduced by the tyrosine kinase inhibitor genestein. Genestein also reduced LPS-stimulated protein kinase C activity but did not affect the response to the protein kinase C activator, tetradecanoylphorbol acetate (TPA). 6. Nicotinamide, an inhibitor of poly-ADP ribosylation, abolished LPS- and IFN gamma-induced NOS activity. 7. Brefeldin A, an inhibitor of a factor which stimulates nucleotide exchange activity on the 21 kDa ADP-ribosylation factor, ARF, reduced LPS- and IFN gamma-induced NOS activity by approximately 80%. 8. These results suggest the involvement of protein kinase C, tyrosine kinase and poly-ADP ribosylation pathways in the regulation of the induction of nitric oxide synthase in RAW 264.7 macrophages by LPS and IFN gamma. Images Figure 2 PMID:7533621

  20. Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction.

    PubMed

    Bergeron, John J M; Di Guglielmo, Gianni M; Dahan, Sophie; Dominguez, Michel; Posner, Barry I

    2016-06-01

    Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated. PMID:27023845

  1. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor.

    PubMed

    Lotz-Jenne, Carina; Lüthi, Urs; Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie

    2016-05-01

    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered "off-target" effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo. PMID:27036020

  2. A point mutation at tyrosine-809 in the human colony-stimulating factor 1 receptor impairs mitogenesis without abrogating tyrosine kinase activity, association with phosphatidylinositol 3-kinase, or induction of c-fos and junB genes

    SciTech Connect

    Roussel, M.F. ); Shurtleff, S.A.; Downing, J.R. ); Sherr, C.J. Saint Jude Children's Research Hospital, Memphis, TN )

    1990-09-01

    Substitution of phenylalanine for tyrosine-809 in the human colony-stimulating factor 1 receptor (CSF-1R) inhibited its ability to transduce ligand-dependent mitogenic signals in mouse NIH 3T3 cells. When combined with an activating mutation at codon 301 that induces constitutive CSF-1R tyrosine kinase activity, the codon 809 mutation suppressed ligand-independent cell transformation. Comparative mapping tryptic phosphopeptides from mutant and wild-type CSF-1R indicated that tyrosine-809 is a site of ligand-dependent receptor phosphorylation in vivo. The mutant receptor was active as a tyrosine kinase in vitro and in vivo, underwent CSF-1-dependent association with a phosphatidylinositol 3-kinase, and induced expression of the protooncogenes c-fos and junB, underscoring its ability to trigger some of the known cellular responses to CSF-1. The mutant receptor is likely to be impaired in its ability to interact with critical cellular effectors whose activity is required for mitogenesis.

  3. Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

    PubMed Central

    Hart, Kristen C.; Robertson, Scott C.; Donoghue, Daniel J.

    2001-01-01

    Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3. PMID:11294897

  4. Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B-lymphocyte precursors.

    PubMed Central

    Uckun, F M; Schieven, G L; Tuel-Ahlgren, L M; Dibirdik, I; Myers, D E; Ledbetter, J A; Song, C W

    1993-01-01

    Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lymphocyte precursors, irradiation with gamma-rays leads to (i) stimulation of phosphatidylinositol turnover; (ii) downstream activation by covalent modification of multiple serine-specific protein kinases, including protein kinase C; and (iii) activation of nuclear factor kappa B. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A. Thus, tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation. Images PMID:8419931

  5. Activation of the protein-tyrosine kinase associated with the bombesin receptor complex in small cell lung carcinomas

    SciTech Connect

    Gaudino, G.; Cirillo, D.; Naldini, L.; Rossino, P.; Comoglio, P.M. )

    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 phosphorylated 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.

  6. Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src

    PubMed Central

    Anguita, Estefanía; Benaim, Gustavo; Villalobo, Antonio

    2015-01-01

    Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred. PMID:26058065

  7. Slik and the receptor tyrosine kinase Breathless mediate localized activation of Moesin in terminal tracheal cells.

    PubMed

    Ukken, Fiona Paul; Aprill, Imola; JayaNandanan, N; Leptin, Maria

    2014-01-01

    A key element in the regulation of subcellular branching and tube morphogenesis of the Drosophila tracheal system is the organization of the actin cytoskeleton by the ERM protein Moesin. Activation of Moesin within specific subdomains of cells, critical for its interaction with actin, is a tightly controlled process and involves regulatory inputs from membrane proteins, kinases and phosphatases. The kinases that activate Moesin in tracheal cells are not known. Here we show that the Sterile-20 like kinase Slik, enriched at the luminal membrane, is necessary for the activation of Moesin at the luminal membrane and regulates branching and subcellular tube morphogenesis of terminal cells. Our results reveal the FGF-receptor Breathless as an additional necessary cue for the activation of Moesin in terminal cells. Breathless-mediated activation of Moesin is independent of the canonical MAP kinase pathway. PMID:25061859

  8. A role for Hsp90 in cell cycle control: Wee1 tyrosine kinase activity requires interaction with Hsp90.

    PubMed Central

    Aligue, R; Akhavan-Niak, H; Russell, P

    1994-01-01

    Wee1 protein kinase regulates the length of G2 phase by carrying out the inhibitory tyrosyl phosphorylation of Cdc2-cyclin B kinase. Mutations were isolated that suppressed the G2 cell cycle arrest caused by overproduction of Wee1. One class of swo (suppressor of wee1 overproduction) mutation, exemplified by swo1-26, also caused a temperature sensitive lethal phenotype in a wee1+ background. The swo1+ gene encodes a member of the Hsp90 family of stress proteins. Swo1 is essential for viability at all temperatures. Swo1 coimmunoprecipitates with Wee1, showing that the two proteins interact. The swo1-26 mutant undergoes premature mitosis when grown at a semi-permissive temperature. These data strongly indicate that formation of active Wee1 tyrosine kinase requires interaction with Swo1, perhaps in a manner analogous to the previously demonstrated interaction between Hsp90 and v-src tyrosine kinase. These observations demonstrate a unexpected role for Hsp90 in cell cycle control. Images PMID:7813446

  9. Adenosine-dependent activation of tyrosine hydroxylase is defective in adenosine kinase-deficient PC12 cells.

    PubMed Central

    Erny, R; Wagner, J A

    1984-01-01

    (R)-N6-Phenylisopropyladenosine (PIA) stimulates dopa production 3- to 5-fold in PC12 cells, with a half-maximal effective concentration (EC50) of 50 nM. This increase can be explained by a stable activation of tyrosine hydroxylase [TyrOHase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] when it is phosphorylated by a cAMP-dependent protein kinase. The activation of TyrOHase is mediated by the adenosine-dependent activation of adenylate cyclase (EC50 = 600 nM). PIA (10 microM) is as effective as cholera toxin or dibutyryl cAMP in activating TyrOHase in wild-type cells. Adenosine kinase-deficient mutants of PC12 were found to be resistant to PIA-dependent activation of TyrOHase (EC50 = 100-1000 nM). This phenomenon was explored in detail in one adenosine kinase-deficient mutant and was shown to occur because the mutant was resistant to the adenosine-dependent activation of adenylate cyclase. In this mutant, TyrOHase was activated 14-fold by cholera toxin, suggesting that activated TyrOHase is about 14 times as active as unactivated TyrOHase. These studies with kinase-deficient PC12 cells provide genetic evidence that adenosine-dependent activation of TyrOHase is mediated by acute increases in cAMP. When the adenosine receptor found on PC12 cells is expressed in vivo, it might function as either a presynaptic (i.e., localized on the nerve terminal) or a postsynaptic (i.e., localized on the cell body or dendrite) receptor that regulates rates of transmitter synthesis in response to cell activity. PMID:6146982

  10. Bisphenol A accelerates capacitation-associated protein tyrosine phosphorylation of rat sperm by activating protein kinase A.

    PubMed

    Wan, Xiaofeng; Ru, Yanfei; Chu, Chen; Ni, Zimei; Zhou, Yuchuan; Wang, Shoulin; Zhou, Zuomin; Zhang, Yonglian

    2016-06-01

    Bisphenol A (BPA) is a synthetic estrogen-mimic chemical. It has been shown to affect many reproductive endpoints. However, the effect of BPA on the mature sperm and the mechanism of its action are not clear yet. Here, our in vitro studies indicated that BPA could accelerate sperm capacitation-associated protein tyrosine phosphorylation in time- and dose-dependent manners. In vivo, the adult male rats exposed to a high dose of BPA could result in a significant increase in sperm activity. Further investigation demonstrated that BPA could accelerate capacitation-associated protein tyrosine phosphorylation even if sperm were incubated in medium devoid of BSA, HCO3 (-), and Ca(2+) However, this action of BPA stimulation could be blocked by H89, a highly selective blocker of protein kinase A (PKA), but not by KH7, a specific inhibitor of adenylyl cyclase. These data suggest that BPA may activate PKA to affect sperm functions and male fertility. PMID:27174873

  11. Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Non-Small Cell Lung Cancer Cells Acquire Resistance to the ALK Inhibitor Alectinib by Activating Alternative Receptor Tyrosine Kinases.

    PubMed

    Isozaki, Hideko; Ichihara, Eiki; Takigawa, Nagio; Ohashi, Kadoaki; Ochi, Nobuaki; Yasugi, Masayuki; Ninomiya, Takashi; Yamane, Hiromichi; Hotta, Katsuyuki; Sakai, Katsuya; Matsumoto, Kunio; Hosokawa, Shinobu; Bessho, Akihiro; Sendo, Toshiaki; Tanimoto, Mitsune; Kiura, Katsuyuki

    2016-03-15

    Crizotinib is the standard of care for advanced non-small cell lung cancer (NSCLC) patients harboring the anaplastic lymphoma kinase (ALK) fusion gene, but resistance invariably develops. Unlike crizotinib, alectinib is a selective ALK tyrosine kinase inhibitor (TKI) with more potent antitumor effects and a favorable toxicity profile, even in crizotinib-resistant cases. However, acquired resistance to alectinib, as for other TKIs, remains a limitation of its efficacy. Therefore, we investigated the mechanisms by which human NSCLC cells acquire resistance to alectinib. We established two alectinib-resistant cell lines that did not harbor the secondary ALK mutations frequently occurring in crizotinib-resistant cells. One cell line lost the EML4-ALK fusion gene, but exhibited increased activation of insulin-like growth factor-1 receptor (IGF1R) and human epidermal growth factor receptor 3 (HER3), and overexpressed the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF1R and HER3 signaling overcame resistance to alectinib in this cell line. The second alectinib-resistant cell line displayed stimulated HGF autocrine signaling that promoted MET activation and remained sensitive to crizotinib treatment. Taken together, our findings reveal two novel mechanisms underlying alectinib resistance that are caused by the activation of alternative tyrosine kinase receptors rather than by secondary ALK mutations. These studies may guide the development of comprehensive treatment strategies that take into consideration the various approaches ALK-positive lung tumors use to withstand therapeutic insult. PMID:26719536

  13. The Bacterial Tyrosine Kinase Activator TkmA Contributes to Biofilm Formation Largely Independently of the Cognate Kinase PtkA in Bacillus subtilis

    PubMed Central

    Gao, Tantan; Greenwich, Jennifer; Li, Yan

    2015-01-01

    ABSTRACT In Bacillus subtilis, biosynthesis of exopolysaccharide (EPS), a key biofilm matrix component, is regulated at the posttranslational level by the bacterial tyrosine kinase (BY-kinase) EpsB. EpsB, in turn, relies on the cognate kinase activator EpsA for activation. A concerted role of a second BY-kinase–kinase activator pair, PtkA and TkmA, respectively in biofilm formation was also indicated in previous studies. However, the exact functions of PtkA and TkmA in biofilm formation remain unclear. In this work, we show that the kinase activator TkmA contributes to biofilm formation largely independently of the cognate kinase, PtkA. We further show that the biofilm defect caused by a ΔtkmA mutation can be rescued by complementation by epsA, suggesting a functional overlap between TkmA and EpsA and providing a possible explanation for the role of TkmA in biofilm formation. We also show that the importance of TkmA in biofilm formation depends largely on medium conditions; the biofilm defect of ΔtkmA is very severe in the biofilm medium LBGM (lysogenic broth [LB] supplemented with 1% [vol/vol] glycerol and 100 μM MnSO4) but marginal in another commonly used biofilm medium, MSgg (5 mM potassium phosphate [pH 7.0], MOPS [100 mM morpholinepropanesulfonic acid] [pH 7.0], 2 mM MgCl2, 700 μM CaCl2, 50 μM MnCl2, 50 μM FeCl3, 1 μM ZnCl2, 2 μM thiamine, 0.5% glycerol, 0.5% glutamic acid, 50 μg/ml tryptophan, 50 μg/ml threonine, and 50 μg/ml phenylalanine). The molecular basis for the medium dependence is likely due to differential expression of tkmA and epsA in the two different media and complex regulation of these genes by both Spo0A and DegU. Our studies provide genetic evidence for possible cross talk between a BY-kinase activator (TkmA) and a noncognate kinase (EpsB) and an example of how environmental conditions may influence such cross talk in regulating biofilm formation in B. subtilis. IMPORTANCE In bacteria, biosynthesis of secreted polysaccharides

  14. Investigating Molecular Mechanisms of Activation and Mutation of the HER2 Receptor Tyrosine Kinase through Computational Modeling and Simulation

    PubMed Central

    Telesco, Shannon E.; Shih, Andrew; Liu, Yingting; Radhakrishnan, Ravi

    2011-01-01

    Human epidermal growth factor receptor 2 (HER2)/ErbB2 is a receptor tyrosine kinase belonging to the EGFR/ErbB family and is overexpressed in 20–30% of human breast cancers. Since there is a growing effort to develop pharmacological inhibitors of the HER2 kinase for the treatment of breast cancer, it is clinically valuable to rationalize how specific mutations impact the molecular mechanism of receptor activation. Although several crystal structures of the 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 (A-loop). In our studies, discussed here, we have investigated the mechanisms that are important in HER2 kinase domain regulation and compared them with the other ErbB family members, namely EGFR and ErbB4, to determine the molecular basis for HER2’s unique mode of activation. We apply computational simulation techniques at the atomic level and at the electronic structure (quantum mechanical) level to elucidate details of the mechanisms governing the kinase domains of these ErbB members. Through analysis of our simulation results, we have discovered potential regulatory mechanisms common to EGFR, HER2, and ErbB4, including a tight coupling between the A-loop and catalytic loop that may contribute to alignment of residues required for catalysis in the active kinase. We further postulate an autoinhibitory mechanism whereby the inactive kinase is stabilized through sequestration of catalytic residues. In HER2, we also predict a role for phosphorylated Y877 in bridging a network of hydrogen bonds that fasten the A-loop in its active conformation, suggesting that HER2 may be unique among the ErbB members in requiring A-loop tyrosine phosphorylation for functionality. In EGFR, HER2, and ErbB4, we discuss the possible effects of activating mutations. Delineation of the activation

  15. Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists.

    PubMed Central

    Nahas, N; Molski, T F; Fernandez, G A; Sha'afi, R I

    1996-01-01

    The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulation of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated. This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mitogen-activated protein (MAP) kinase. This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody immunoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP kinase antibody, and conversely, anti-p38 MAP kinase antibody immunoprecipitates a 38 kDa protein that can be recognized by anti-PY antibody. Moreover, this tyrosine phosphorylated protein is found associated entirely with the cytosol. It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum. This conclusion is based on three experimental findings. First, soluble fractions isolated from LPS-stimulated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and protein kinase A inhibitor peptides. This effect is similar to the effect produced by the commercially available phosphorylated and activated MAPKAP kinase-2 (MAP kinase activated protein kinase-2). Secondly, a 27 kDa protein that aligns with a protein recognized by anti-hsp27 antibody is phosphorylated upon LPS stimulation of intact human neutrophils prelabelled with radioactive phosphate. Lastly, immune complex protein kinase assays, using [gamma-32P]ATP and activating transcription factor 2 (ATF2) as substrates, showed increased p38 MAP kinase activity from LPS-stimulated human neutrophils. The phosphorylation and activation of this p38 MAP kinase can be affected by both G-protein-coupled receptors such as platelet-activating factor (PAF) and non-G-protein-coupled receptors such as the cytokine

  16. Activation of HER3 Interferes with Antitumor Effects of Axl Receptor Tyrosine Kinase Inhibitors: Suggestion of Combination Therapy1

    PubMed Central

    Torka, Robert; Pénzes, Kinga; Gusenbauer, Simone; Baumann, Christine; Szabadkai, István; Őrfi, Lászlȯ; Kéri, György; Ullrich, Axel

    2014-01-01

    The Axl receptor tyrosine kinase (RTK) has been established as a strong candidate for targeted therapy of cancer. However, the benefits of targeted therapies are limited due to acquired resistance and activation of alternative RTKs. Therefore, we asked if cancer cells are able to overcome targeted Axl therapies. Here, we demonstrate that inhibition of Axl by short interfering RNA or the tyrosine kinase inhibitor (TKI) BMS777607 induces the expression of human epidermal growth factor receptor 3 (HER3) and the neuregulin 1(NRG1)–dependent phosphorylation of HER3 in MDA-MB231 and Ovcar8 cells. Moreover, analysis of 20 Axl-expressing cancer cell lines of different tissue origin indicates a low basal phosphorylation of RAC-α serine/threonine-protein kinase (AKT) as a general requirement for HER3 activation on Axl inhibition. Consequently, phosphorylation of AKT arises as an independent biomarker for Axl treatment. Additionally, we introduce phosphorylation of HER3 as an independent pharmacodynamic biomarker for monitoring of anti-Axl therapy response. Inhibition of cell viability by BMS777607 could be rescued by NRG1-dependent activation of HER3, suggesting an escape mechanism by tumor microenvironment. The Axl-TKI MPCD84111 simultaneously blocked Axl and HER2/3 signaling and thereby prohibited HER3 feedback activation. Furthermore, dual inhibition of Axl and HER2/3 using BMS777607 and lapatinib led to a significant inhibition of cell viability in Axl-expressing MDA-MB231 and Ovcar8 cells. Therefore, we conclude that, in patient cohorts with expression of Axl and low basal activity of AKT, a combined inhibition of Axl and HER2/3 kinase would be beneficial to overcome acquired resistance to Axl-targeted therapies. PMID:24862757

  17. The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage.

    PubMed

    Reuven, Nina; Adler, Julia; Porat, Ziv; Polonio-Vallon, Tilman; Hofmann, Thomas G; Shaul, Yosef

    2015-07-01

    The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser(46) in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser(46), and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate. PMID:25944899

  18. Bruton tyrosine kinase (Btk) suppresses osteoblastic differentiation.

    PubMed

    Kaneshiro, Shoichi; Ebina, Kosuke; Shi, Kenrin; Yoshida, Kiyoshi; Otsuki, Dai; Yoshikawa, Hideki; Higuchi, Chikahisa

    2015-09-01

    The Tec family of nonreceptor tyrosine kinases has been shown to play a key role in inflammation and bone destruction. Bruton tyrosine kinase (Btk) has been the most widely studied because of its critical role in B cells. Furthermore, recent evidence has demonstrated that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. The role of Btk in osteoblastic differentiation has not been well elucidated. In this study, we demonstrated the role of Btk in osteoblastic differentiation and investigated the effects of a Btk inhibitor on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells, primary calvarial osteoblasts, and bone marrow stromal ST2 cells. Btk expression was detected in all three cell lines. Btk inhibition stimulated mRNA expression of osteoblastic markers (alkaline phosphatase, osteocalcin, and osterix) and promoted mineralization of the extracellular matrix. In addition, Btk knockdown caused increased mRNA expression of osteoblastic markers. Furthermore, Btk inhibition suppressed the phosphorylation of mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NFκB), and protein kinase Cα (PKCα). Our results indicate that Btk may regulate osteoblastic differentiation through the MAPK, NFκB, and PKCα signaling pathways. PMID:25230818

  19. pH regulation of an egg cortex tyrosine kinase.

    PubMed

    Jiang, W P; Veno, P A; Wood, R W; Peaucellier, G; Kinsey, W H

    1991-07-01

    Fertilization of the echinoderm egg is known to result in the phosphorylation, on tyrosine, of a high-molecular-weight cortical protein (HMWCP) localized in the egg cortex. Studies using various parthenogenic agents indicate that this phosphorylation event occurs in response to the alkaline shift in cytoplasmic pHi which normally occurs 1 to 2 min after fertilization. In the present study, the purified egg cell surface complex was used as in vitro system to determine whether a small alkaline shift in pH, such as occurs upon fertilization, could stimulate the activity of the egg cortex-associated tyrosine kinase toward endogenous protein substrates. The results demonstrated that the cell surface complex is highly enriched in a tyrosine kinase activity which accounts for the majority of the protein kinase activity in this preparation. The activity of this tyrosine kinase toward the HMWCP and other cortical proteins was highly dependent on pH over the range pH 6.8 to 7.3. This indicates that the fertilization-associated change in cytoplasmic pH would be sufficient to trigger increased tyrosine phosphorylation of the high-molecular-weight cortical protein in vivo. The regulation of tyrosine phosphorylation by small changes in pH represents a novel control mechanism in which a tyrosine protein kinase may act as a pH-sensitive transducer. PMID:2060713

  20. Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase.

    PubMed Central

    Peraldi, P; Zhao, Z; Filloux, C; Fischer, E H; Van Obberghen, E

    1994-01-01

    Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP. Images PMID:8197172

  1. Insulin-induced tyrosine dephosphorylation of paxillin and focal adhesion kinase requires active phosphotyrosine phosphatase 1D.

    PubMed Central

    Ouwens, D M; Mikkers, H M; van der Zon, G C; Stein-Gerlach, M; Ullrich, A; Maassen, J A

    1996-01-01

    Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor beta-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(L-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D. PMID:8809054

  2. Ocular Toxicity of Tyrosine Kinase Inhibitors

    PubMed Central

    Davis, Mary Elizabeth

    2016-01-01

    Purpose/Objectives To review common tyrosine kinase inhibitors, as well as their ocular side effects and management. Data Sources A comprehensive literature search was conducted using cINahl®, Pubmed, and cochrane databases for articles published since 2004 with the following search terms: ocular toxicities, tyrosine kinase inhibitors, ophthalmology, adverse events, eye, and vision. Data Synthesis Tyrosine kinase inhibitors can cause significant eye toxicity. Conclusions Given the prevalence of new tyrosine kinase inhibitor therapies and the complexity of possible pathogenesis of ocular pathology, oncology nurses can appreciate the occurrence of ocular toxicities and the role of nursing in the management of these problems. Implications for Nursing Knowledge of the risk factors and etiology of ocular toxicity of targeted cancer therapies can guide nursing assessment, enhance patient education, and improve care management. Including a review of eye symptoms and vision issues in nursing assessment can enhance early detection and treatment of ocular toxicity. PMID:26906134

  3. Platelet-activating factor attenuation of long-term potentiation in rat hippocampal slices via protein tyrosine kinase signaling.

    PubMed

    Reiner, Benjamin; Wang, Wenwei; Liu, Jianuo; Xiong, Huangui

    2016-02-26

    It is well established that HIV-1-infected mononuclear phagocytes release platelet activating factor (PAF) and elevated levels of PAF have been detected in blood and in the cerebrospinal fluid (CSF) of acquired immunodeficiency syndrome (AIDS) patients with HIV-associated neurocognitive disorders (HAND). It is our hypothesis that the elevated levels of PAF alter long-term potentiation (LTP) in the hippocampus, leading to neurocognitive dysfunction. To test this hypothesis, we studied the effects of PAF on LTP in the CA1 region of rat hippocampal slices. Our results showed incubation of hippocampal slices with PAF attenuated LTP. The PAF-mediated attenuation was blocked by ginkgolide B, a PAF receptor antagonist, suggesting PAF attenuation of LTP via PAF receptors. Application of lyso-PAF, an inactive PAF analog, had no apparent effect on LTP. Further investigation revealed an involvement of tyrosine kinase in PAF attenuation of LTP, which was demonstrated by lavendustin A (a specific protein tyrosine kinase inhibitor) blockage of PAF attenuation of LTP. As LTP is widely considered as the cellular and synaptic basis for learning and memory, the attenuation of LTP by PAF may contribute at least in part to the HAND pathogenesis. PMID:26808643

  4. Specific Oncogenic Activity of the Src-Family Tyrosine Kinase c-Yes in Colon Carcinoma Cells

    PubMed Central

    Paquay de Plater, Ludmilla; Edmonds, Thomas; David, Géraldine; Jan, Michel; de Montrion, Catherine; Cogé, Francis; Léonce, Stéphane; Burbridge, Michael; Bruno, Alain; Boutin, Jean A.; Lockhart, Brian; Roche, Serge; Cruzalegui, Francisco

    2011-01-01

    c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src. PMID:21390316

  5. 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. PMID:26584640

  6. Antagonistic regulation of swelling-activated Cl− current in rabbit ventricle by Src and EGFR protein tyrosine kinases

    PubMed Central

    Ren, Zuojun; Baumgarten, Clive M.

    2005-01-01

    Regulation of swelling-activated Cl− current (ICl,swell) is complex, and multiple signaling cascades are implicated. To determine whether protein tyrosine kinase (PTK) modulates ICl,swell and to identify the PTK involved, we studied the effects of a broad-spectrum PTK inhibitor (genistein), selective inhibitors of Src (PP2, a pyrazolopyrimidine) and epidermal growth factor receptor (EGFR) kinase (PD-153035), and a protein tyrosine phosphatase (PTP) inhibitor (orthovanadate). ICl,swell evoked by hyposmotic swelling was increased 181 ± 17% by 100 μM genistein, and the genistein-induced current was blocked by the selective ICl,swell blocker tamoxifen (10 μM). Block of Src with PP2 (10 μM) stimulated tamoxifen-sensitive ICl,swell by 234 ± 27%, mimicking genistein, whereas the inactive analog of PP2, PP3 (10 μM), had no effect. Moreover, block of PTP by orthovanadate (1 mM) inhibited ICl,swell and prevented its stimulation by PP2. In contrast with block of Src, block of EGFR kinase with PD-153035 (20 nM) inhibited ICl,swell. Several lines of evidence argue that the PP2-stimulated current was ICl,swell: 1) the stimulation was volume dependent, 2) the current was blocked by tamoxifen, 3) the current outwardly rectified with both symmetrical and physiological Cl− gradients, and 4) the current reversed near the Cl− equilibrium potential. To rule out contributions of other currents, Cd2+ (0.2 mM) and Ba2+ (1 mM) were added to the bath. Surprisingly, Cd2+ suppressed the decay of Cd2+ plus Ba2+ eliminated time-dependent ICl,swell, and currents between −100 and −100 mV. Nevertheless, these divalent ions did not eliminate ICl,swell or prevent its stimulation by PP2. The results indicate that tyrosine phosphorylation controls ICl,swell, and regulation of ICl,swell by the Src and EGFR kinase families of PTK is antagonistic. PMID:15681694

  7. 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. PMID:22487216

  8. Antifibrotic and anti-inflammatory activity of the tyrosine kinase inhibitor nintedanib in experimental models of lung fibrosis.

    PubMed

    Wollin, Lutz; Maillet, Isabelle; Quesniaux, Valérie; Holweg, Alexander; Ryffel, Bernhard

    2014-05-01

    The tyrosine kinase inhibitor nintedanib (BIBF 1120) is in clinical development for the treatment of idiopathic pulmonary fibrosis. To explore its mode of action, nintedanib was tested in human lung fibroblasts and mouse models of lung fibrosis. Human lung fibroblasts expressing platelet-derived growth factor (PDGF) receptor-α and -β were stimulated with platelet-derived growth factor BB (homodimer) (PDGF-BB). Receptor activation was assessed by autophosphorylation and cell proliferation by bromodeoxyuridine incorporation. Transforming growth factor β (TGFβ)-induced fibroblast to myofibroblast transformation was determined by α-smooth muscle actin (αSMA) mRNA analysis. Lung fibrosis was induced in mice by intratracheal bleomycin or silica particle administration. Nintedanib was administered every day by gavage at 30, 60, or 100 mg/kg. Preventive nintedanib treatment regimen started on the day that bleomycin was administered. Therapeutic treatment regimen started at various times after the induction of lung fibrosis. Bleomycin caused increased macrophages and lymphocytes in the bronchoalveolar lavage (BAL) and elevated interleukin-1β (IL-1β), tissue inhibitor of metalloproteinase-1 (TIMP-1), and collagen in lung tissue. Histology revealed chronic inflammation and fibrosis. Silica-induced lung pathology additionally showed elevated BAL neutrophils, keratinocyte chemoattractant (KC) levels, and granuloma formation. Nintedanib inhibited PDGF receptor activation, fibroblast proliferation, and fibroblast to myofibroblast transformation. Nintedanib significantly reduced BAL lymphocytes and neutrophils but not macrophages. Furthermore, interleukin-1β, KC, TIMP-1, and lung collagen were significantly reduced. Histologic analysis showed significantly diminished lung inflammation, granuloma formation, and fibrosis. The therapeutic effect was dependent on treatment start and duration. Nintedanib inhibited receptor tyrosine kinase activation and the proliferation and

  9. Brk/Protein tyrosine kinase 6 phosphorylates p27KIP1, regulating the activity of cyclin D-cyclin-dependent kinase 4.

    PubMed

    Patel, Priyank; Asbach, Benedikt; Shteyn, Elina; Gomez, Cindy; Coltoff, Alexander; Bhuyan, Sadia; Tyner, Angela L; Wagner, Ralf; Blain, Stacy W

    2015-05-01

    Cyclin D and cyclin-dependent kinase 4 (cdk4) are overexpressed in a variety of tumors, but their levels are not accurate indicators of oncogenic activity because an accessory factor such as p27(Kip1) is required to assemble this unstable dimer. Additionally, tyrosine (Y) phosphorylation of p27 (pY88) is required to activate cdk4, acting as an "on/off switch." We identified two SH3 recruitment domains within p27 that modulate pY88, thereby modulating cdk4 activity. Via an SH3-PXXP interaction screen, we identified Brk (breast tumor-related kinase) as a high-affinity p27 kinase. Modulation of Brk in breast cancer cells modulates pY88 and increases resistance to the cdk4 inhibitor PD 0332991. An alternatively spliced form of Brk (Alt Brk) which contains its SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor, identifying a potentially targetable regulatory region within p27. Brk is overexpressed in 60% of breast carcinomas, suggesting that this facilitates cell cycle progression by modulating cdk4 through p27 Y phosphorylation. p27 has been considered a tumor suppressor, but our data strengthen the idea that it should also be considered an oncoprotein, responsible for cyclin D-cdk4 activity. PMID:25733683

  10. The heterotrimeric G q protein-coupled angiotensin II receptor activates p21 ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes.

    PubMed Central

    Sadoshima, J; Izumo, S

    1996-01-01

    p21 ras plays as important role in cell proliferation, transformation and differentiation. Recently, the requirement of p21 ras has been suggested for cellular responses induced by stimulation of heterotrimeric G protein-coupled receptors. However, it remains to be determined how agonists for G protein-coupled receptors activate p21 ras in metazoans. We show here that stimulation of the G q protein-coupled angiotensin II (Ang II) receptor causes activation of p21 ras in cardiac myocytes. The p21 ras activation by Ang II is mediated by an increase in the guanine nucleotide exchange activity, but not by an inhibition of the GTPase-activating protein. Ang II causes rapid tyrosine phosphorylation of Shc and its association with Grb2 and mSos-1, a guanine nucleotide exchange factor of p21 ras. This leads to translocation of mSos-1 to the membrane fraction. Shc associates with the SH3 domain of Fyn whose tyrosine kinase activity is activated by Ang II with a similar time course as that of tyrosine phosphorylation of Shc. Ang II-induced increase in the guanine nucleotide exchange activity was inhibited by a peptide ligand specific to the SH3 domain of the Src family tyrosine kinases. These results suggest that an agonist for a pertussis toxin-insensitive G protein-coupled receptor may initiate the cross-talk with non-receptor-type tyrosine kinases, thereby activating p21 ras using a similar mechanism as receptor tyrosine kinase-induced p21 ras activation. Images PMID:8631299

  11. A novel nonreceptor tyrosine kinase, Srm: cloning and targeted disruption.

    PubMed Central

    Kohmura, N; Yagi, T; Tomooka, Y; Oyanagi, M; Kominami, R; Takeda, N; Chiba, J; Ikawa, Y; Aizawa, S

    1994-01-01

    We have isolated a novel nonreceptor tyrosine kinase, Srm, that maps to the distal end of chromosome 2. It has SH2, SH2', and SH3 domains and a tyrosine residue for autophosphorylation in the kinase domain but lacks an N-terminal glycine for myristylation and a C-terminal tyrosine which, when phosphorylated, suppresses kinase activity. These are structural features of the recently identified Tec family of nonreceptor tyrosine kinases. The Srm N-terminal unique domain, however, lacks the structural characteristics of the Tec family kinases, and the sequence similarity is highest to Src in the SH region. The expression of two transcripts is rather ubiquitous and changes according to tissue and developmental stage. Mutant mice were generated by gene targeting in embryonic stem cells but displayed no apparent phenotype as in mutant mice expressing Src family kinases. These results suggest that Srm constitutes a new family of nonreceptor tyrosine kinases that may be redundant in function. Images PMID:7935409

  12. Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

    PubMed Central

    Kontos, Christopher D.; Stauffer, Thomas P.; Yang, Wen-Pin; York, John D.; Huang, Liwen; Blanar, Michael A.; Meyer, Tobias; Peters, Kevin G.

    1998-01-01

    Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2’s role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival. PMID:9632797

  13. TOPK promotes lung cancer resistance to EGFR tyrosine kinase inhibitors by phosphorylating and activating c-Jun

    PubMed Central

    Wang, Tao; Wang, Ting; Niu, Mengjie; Zhang, Shengli; Jia, Lintao; Li, Shengqing

    2016-01-01

    Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) have shown promising clinical efficacy in non-squamous non-small cell lung cancer (NSCLC); however, resistance is frequently observed in malignant cells, operating through a mechanism that remains largely unknown. The present study shows that T-lymphokine-activated killer cell-originated protein kinase (TOPK) is upregulated in NSCLC and excessively activated in TKI-refractory cells. TOPK dictates the responsiveness of lung cancers to the EGFR-targeted TKI gefitinib through the transcription factor AP-1 component c-Jun. TOPK binds directly to and phosphorylates c-Jun, which consequently activates the transcription of AP-1 target genes, including CCND1 and CDC2. TOPK silencing sensitizes EGFR-TKI-resistant lung cancer cells to gefitinib and increases gefitinib efficacy in preclinical lung adenocarcinoma xenograft models. These findings represent a novel mechanism of lung cancer resistance to TKIs and suggest that TOPK may have value both as a predictive biomarker and as a therapeutic target: TOPK-targeted therapy may synergize with EGFR-targeted therapy in lung cancers. PMID:26745678

  14. Protein tyrosine kinase activity is essential for Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes.

    PubMed Central

    Zheng, L; Nibbering, P H; Zomerdijk, T P; van Furth, R

    1994-01-01

    Our previous study revealed that the intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma receptor I (Fc gamma RI) or Fc gamma RII is a phospholipase C (PLC)-dependent process. The aim of the present study was to investigate whether protein tyrosine kinase (PTK) activity plays a role in the Fc gamma R-mediated intracellular killing of bacteria and activation of PLC in these cells. The results showed that phagocytosis of bacteria by monocytes was not affected by the PTK inhibitors genistein and tyrphostin-47. The intracellular killing of S. aureus by monocytes after cross-linking Fc gamma RII or Fc gamma RII with anti-Fc gamma R monoclonal antibody and a bridging antibody or with human immunoglobulin G (IgG) was inhibited by these compounds in a dose-dependent fashion. The production of O2- by monocytes after stimulation with IgG or IgG-opsonized S. aureus was almost completely blocked by the PTK inhibitor. These results indicate that inhibition of PTK impairs the oxygen-dependent bactericidal mechanisms of monocytes. Genistein and tyrphostin-47, which do not affect the enzymatic activity of purified PLC, prevented activation of PLC after cross-linking Fc gamma RI or Fc gamma RII, measured as an increase in the intracellular inositol 1,4,5-trisphosphate concentration. Cross-linking Fc gamma RI or Fc gamma RII induced rapid tyrosine phosphorylation of several proteins in monocytes, one of which was identified as PLC-gamma 1, and the phosphorylation could be completely blocked by PTK inhibitors, leading to the conclusion that activation of PLC after cross-linking Fc gamma R in monocytes is regulated by PTK activity. Together, these results demonstrate that PTK activity is essential for the activation of PLC which is involved in the Fc gamma R-mediated intracellular killing of S. aureus by human monocytes. Images PMID:7927687

  15. Platelet-activating factor increases VE-cadherin tyrosine phosphorylation in mouse endothelial cells and its association with the PtdIns3'-kinase

    PubMed Central

    Hudry-Clergeon, Hélène; Stengel, Dominique; Ninio, Ewa; Vilgrain, Isabelle

    2005-01-01

    Platelet-activating-Factor (PAF), a potent inflammatory mediator, is involved in endothelial permeability. This study was designed to characterize PAF receptor (PAF-R) expression and its specific contribution to the modifications of adherens junctions in mouse endothelial cells. We demonstrated that PAF-R was expressed in mouse endothelial cells and was functionally active in stimulating p42/p44 MAPK and phosphatidylinositol 3-kinase (PtdIns3′-kinase)/Akt activities. Treatment of cells with PAF induced a rapid, time- and dose-dependent (10−7 to 10−10M) increase in tyrosine phosphorylation of a subset of proteins ranging from 90 kDa to 220 kDa, including the VE-cadherin, the latter effect being prevented by the tyrosine kinase inhibitors, herbimycin A and bis-tyrphostin. Furthermore, we demonstrated that PAF promoted formation of multimeric aggregates of VE-cadherin with PtdIns3′-kinase which was also inhibited by herbimycin and bis-tyrphostin. Finally, we showed by immunostaining of endothelial cells VE-cadherin, that PAF dissociated adherens junctions. The present data provide the first evidence that the treatment of endothelial cells with PAF promoted activation of tyrosine kinases and the VE-cadherin tyrosine phosphorylation and PtdIns3′-kinase association, that ultimately lead to the dissociation of adherens junctions. Physical association between PtdIns3′-kinase, serving as a docking protein, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of PAF-induced cellular activation. PMID:15791001

  16. Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926.

    PubMed Central

    McLees, A; Graham, A; Malarkey, K; Gould, G W; Plevin, R

    1995-01-01

    In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7741705

  17. Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926.

    PubMed

    McLees, A; Graham, A; Malarkey, K; Gould, G W; Plevin, R

    1995-05-01

    In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line. PMID:7741705

  18. EGFR tyrosine kinase regulates human small-conductance Ca2+-activated K+ (hSKCa1) channels expressed in HEK-293 cells.

    PubMed

    Wu, Wei; Sun, Hai-Ying; Deng, Xiu-Ling; Li, Gui-Rong

    2013-05-15

    SKCa (small-conductance Ca(2+)-activated K(+)) channels are widely distributed in different tissues, including the brain, pancreatic islets and myocardium and play an important role in controlling electrical activity and cellular functions. However, intracellular signal modulation of SKCa channels is not fully understood. The present study was designed to investigate the potential regulation of hSKCa1 (human SKCa1) channels by PTKs (protein tyrosine kinases) in HEK (human embryonic kidney)-293 cells expressing the hSKCa1 (KCNN1) gene using approaches of whole-cell patch voltage-clamp, immunoprecipitation, Western blotting and mutagenesis. We found that the hSKCa1 current was inhibited by the broad-spectrum PTK inhibitor genistein, the selective EGFR (epidermal growth factor receptor) kinase inhibitors T25 (tyrphostin 25) and AG556 (tyrphostin AG 556), but not by the Src-family kinases inhibitor PP2. The inhibitory effect of these PTK inhibitors was significantly antagonized by the PTP (protein tyrosine phosphatase) inhibitor orthovanadate. The tyrosine phosphorylation level of hSKCa1 channels was reduced by genistein, T25 or AG556. The reduced tyrosine phosphorylation was countered by orthovanadate. Interestingly, the Y109F mutant hSKCa1 channel lost the inhibitory response to T25 or AG556, and showed a dramatic reduction in tyrosine phosphorylation levels and a reduced current density. These results demonstrate the novel information that hSKCa1 channels are inhibited by genistein, T25 and AG556 via EGFR tyrosine kinase inhibition, which is related to the phosphorylation of Tyr(109) in the N-terminus. This effect may affect electrical activity and cellular functions in brain, pancreatic islets and myocardium. PMID:23496660

  19. Receptor Tyrosine Kinases, TYRO3, AXL, and MER, Demonstrate Distinct Patterns and Complex Regulation of Ligand-induced Activation*

    PubMed Central

    Tsou, Wen-I; Nguyen, Khanh-Quynh N.; Calarese, Daniel A.; Garforth, Scott J.; Antes, Anita L.; Smirnov, Sergey V.; Almo, Steve C.; Birge, Raymond B.; Kotenko, Sergei V.

    2014-01-01

    TYRO3, AXL, and MER receptors (TAMs) are three homologous type I receptor-tyrosine kinases that are activated by endogenous ligands, protein S (PROS1) and growth arrest-specific gene 6 (GAS6). These ligands can either activate TAMs as soluble factors, or, in turn, opsonize phosphatidylserine (PS) on apoptotic cells (ACs) and serve as bridging molecules between ACs and TAMs. Abnormal expression and activation of TAMs have been implicated in promoting proliferation and survival of cancer cells, as well as in suppressing anti-tumor immunity. Despite the fact that TAM receptors share significant similarity, little is known about the specificity of interaction between TAM receptors and their ligands, particularly in the context of ACs, and about the functional diversity of TAM receptors. To study ligand-mediated activation of TAMs, we generated a series of reporter cell lines expressing chimeric TAM receptors. Using this system, we found that each TAM receptor has a unique pattern of interaction with and activation by GAS6 and PROS1, which is also differentially affected by the presence of ACs, PS-containing lipid vesicles and enveloped virus. We also demonstrated that γ-carboxylation of ligands is essential for the full activation of TAMs and that soluble immunoglobulin-like TAM domains act as specific ligand antagonists. These studies demonstrate that, despite their similarity, TYRO3, AXL, and MER are likely to perform distinct functions in both immunoregulation and the recognition and removal of ACs. PMID:25074926

  20. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases

    SciTech Connect

    Kanner, S.B.; Reynolds, A.B.; Vines, R.R.; Parsons, J.T. )

    1990-05-01

    Cellular transformation by oncogenic retroviruses encoding protein tyrosine kinases coincides with the tyrosine-specific phosphorylation of multiple protein substrates. Previous studies have shown that tyrosine phosphorylation of a protein of 120 kDa, p120, correlated with src transformation in chicken embryo fibroblasts. Additionally, the authors previously identified two phosphotyrosine-containing cellular proteins, p130 and p110, that formed stable complexes with activated variants of pp60{sup src}, the src-encoded tyrosine kinase. To study transformation-relevant tyrosine kinase substrates, they have generated monoclonal antibodies to individual tyrosine phosphoproteins, including p130, p120, p110, and five additional phosphoproteins (p210, p125, p118, p85, and p185/p64). These antibodies detected several of the same tyrosine phosphoproteins in chicken embryo fibroblasts transformed by avian retroviruses Y73 and CT10, encoding the yes and crk oncogenes, respectively. Protein substrates in mouse, rat, hamster, and human cells overexpressing activated variants of chicken pp60{sup src} were also detected by several of the monoclonal antibodies.

  1. Src tyrosine kinase signaling antagonizes nuclear localization of FOXO and inhibits its transcription factor activity.

    PubMed

    Bülow, Margret H; Bülow, Torsten R; Hoch, Michael; Pankratz, Michael J; Jünger, Martin A

    2014-01-01

    Biochemical experiments in mammalian cells have linked Src family kinase activity to the insulin signaling pathway. To explore the physiological link between Src and a central insulin pathway effector, we investigated the effect of different Src signaling levels on the Drosophila transcription factor dFOXO in vivo. Ectopic activation of Src42A in the starved larval fatbody was sufficient to drive dFOXO out of the nucleus. When Src signaling levels were lowered by means of loss-of-function mutations or pharmacological inhibition, dFOXO localization was shifted to the nucleus in growing animals, and transcription of the dFOXO target genes d4E-BP and dInR was induced. dFOXO loss-of-function mutations rescued the induction of dFOXO target gene expression and the body size reduction of Src42A mutant larvae, establishing dFOXO as a critical downstream effector of Src signaling. Furthermore, we provide evidence that the regulation of FOXO transcription factors by Src is evolutionarily conserved in mammalian cells. PMID:24513978

  2. Complexity of Receptor Tyrosine Kinase Signal Processing

    PubMed Central

    Volinsky, Natalia; Kholodenko, Boris N.

    2013-01-01

    Our knowledge of molecular mechanisms of receptor tyrosine kinase (RTK) signaling advances with ever-increasing pace. Yet our understanding of how the spatiotemporal dynamics of RTK signaling control specific cellular outcomes has lagged behind. Systems-centered experimental and computational approaches can help reveal how overlapping networks of signal transducers downstream of RTKs orchestrate specific cell-fate decisions. We discuss how RTK network regulatory structures, which involve the immediate posttranslational and delayed transcriptional controls by multiple feed forward and feedback loops together with pathway cross talk, adapt cells to the combinatorial variety of external cues and conditions. This intricate network circuitry endows cells with emerging capabilities for RTK signal processing and decoding. We illustrate how mathematical modeling facilitates our understanding of RTK network behaviors by unraveling specific systems properties, including bistability, oscillations, excitable responses, and generation of intricate landscapes of signaling activities. PMID:23906711

  3. The anti-esophageal cancer cell activity by a novel tyrosine/phosphoinositide kinase inhibitor PP121.

    PubMed

    Peng, Yi; Zhou, Yajuan; Cheng, Long; Hu, Desheng; Zhou, Xiaoyi; Wang, Zhaohua; Xie, Conghua; Zhou, Fuxiang

    2015-09-11

    Here we explored the potential effect of PP121, a novel dual inhibitor of tyrosine and phosphoinositide kinases, against human esophageal cancer cells. We showed that PP121 exerted potent cytotoxic effect in primary (patient-derived) and established (Eca-109, TE-1 and TE-3 lines) esophageal cancer cells, possibly through activating caspase-3-dependnent apoptosis. PP121 was, however, non-cytotoxic to the normal human esophageal epithelial cells (EECs). At the molecular level, we showed that PP121 blocked Akt-mTOR (mammalian target of rapamycin) activation in esophageal cancer cells, which was restored by introducing a constitutively-active Akt (CA-Akt). Yet, CA-Akt only partly inhibited cytotoxicity by PP121 in Eca-109 cells. Importantly, we showed that PP121 inhibited nuclear factor kappa B (NFκB) signaling activation in esophageal cancer cells, which appeared independent of Akt-mTOR blockage. In vivo, oral administration of PP121 remarkably inhibited Eca-109 xenograft growth in nude mice, and significantly improved mice survival. Further, the immunohistochemistry (IHC) and Western blot assays analyzing xenografted tumors showed that PP121 inhibited Akt-mTOR and NFκB activations in vivo. Together, we demonstrate that PP121 potently inhibits esophageal cancer cells in vitro and in vivo, possibly through concurrently inhibiting Akt-mTOR and NFκB signalings. PMID:26235881

  4. Intrinsic resistance to EGFR tyrosine kinase inhibitors in advanced non-small-cell lung cancer with activating EGFR mutations

    PubMed Central

    Wang, Jun; Wang, Baocheng; Chu, Huili; Yao, Yunfeng

    2016-01-01

    Identifying activating EGFR mutations is a useful predictive strategy that helps select a population of advanced non-small-cell lung cancer (NSCLC) patients for treatment with EGFR tyrosine kinase inhibitors (TKIs). Patients with sensitizing EGFR mutations (predominantly an in-frame deletion in exon 19 and an L858R substitution) are highly responsive to first-generation EGFR TKIs, such as gefitinib and erlotinib, and show improved progression-free survival without serious side effects. However, all patients with activating EGFR mutations who are initially responsive to EGFR TKIs eventually develop acquired resistance after a median progression-free survival of 10–16 months, followed by disease progression. Moreover, ~20%–30% of NSCLC patients have no objective tumor regression on initial EGFR TKI treatment, although they harbor an activating EGFR mutation. These patients represent an NSCLC subgroup that is defined as having intrinsic or primary resistance to EGFR TKIs. Different mechanisms of acquired EGFR TKI resistance have been identified, and several novel compounds have been developed to reverse acquired resistance, but little is known about EGFR TKI intrinsic resistance. In this review, we summarize the latest findings involving mechanisms of intrinsic resistance to EGFR TKIs in advanced NSCLC with activating EGFR mutations and present possible therapeutic strategies to overcome this resistance. PMID:27382309

  5. Exploration of the sequence specificity of pp60c-src tyrosine kinase. Minimal peptide sequence required for maximal activity.

    PubMed

    Edison, A M; Barker, S C; Kassel, D B; Luther, M A; Knight, W B

    1995-11-10

    The minimum length required for phosphorylation of a peptide by pp60c-src tyrosine kinase (srcTK) was delineated in this work. Budde (M.D. Anderson University of Texas, personal communication) suggested that the peptide (FGE)3Y(GEF)2GD (peptide I) was a "good" srcTK substrate. Peptide I yielded a 251-fold higher kcat/Km than RRLIEDAEYAARRG, a peptide substrate based upon the autophosphorylation site of srcTK. This was due to a 38-fold lower Km and a 6.6-fold increase in kcat.N-terminal truncation of up to 8 residues in a series of peptides yielded only a 3-fold decrease in activity. Removal of the final N-terminal residue resulted in a 10-fold loss in substrate activity, primarily as a result of an increase in the Km. C-terminal truncations ending in the amide yielded no significant loss in activity until the Y + 3 residue was removed, which resulted in a 73-fold decrease in kcat/Km relative to peptide I. The latter was due primarily to an increase in Km. The results from peptides truncated on both termini suggest that subsite recognition N- and C-terminal relative to the site of phosphorylation can be examined independently. In addition, the observation that only 5 residues are required for significant substrate activity suggests that small molecule inhibitors based upon interactions with the phosphoacceptor site may be developed. PMID:7592964

  6. Multicolored, Tb³⁺-Based Antibody-Free Detection of Multiple Tyrosine Kinase Activities.

    PubMed

    Lipchik, Andrew M; Perez, Minervo; Cui, Wei; Parker, Laurie L

    2015-08-01

    Kinase signaling is a major mechanism driving many cancers. While many inhibitors have been developed and are employed in the clinic, resistance due to crosstalk and pathway reprogramming is an emerging problem. High-throughput assays to detect multiple pathway kinases simultaneously could better model these complex relationships and enable drug development to combat this type of resistance. We developed a strategy to take advantage of time-resolved luminescence of Tb(3+)-chelated phosphotyrosine-containing peptides, which facilitated efficient energy transfer to small molecule fluorophores conjugated to the peptides to produce orthogonally colored biosensors for two different kinases. This enabled multiplexed detection with high signal-to-noise in a high-throughput-compatible format. This proof-of-concept study provides a platform that could be applied to other lanthanide metal and fluorophore combinations to achieve even greater multiplexing without the need for phosphospecific antibodies. PMID:26207839

  7. Tyrosine Kinase Inhibitors and Diabetes: A Novel Treatment Paradigm?

    PubMed

    Fountas, Athanasios; Diamantopoulos, Leonidas-Nikolaos; Tsatsoulis, Agathocles

    2015-11-01

    Deregulation of protein tyrosine kinase (PTK) activity is implicated in various proliferative conditions. Multi-target tyrosine kinase inhibitors (TKIs) are increasingly used for the treatment of different malignancies. Recently, several clinical cases of the reversal of both type 1 and 2 diabetes mellitus (T1DM, T2DM) during TKI administration have been reported. Experimental in vivo and in vitro studies have elucidated some of the mechanisms behind this effect. For example, inhibition of Abelson tyrosine kinase (c-Abl) results in β cell survival and enhanced insulin secretion, while platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) inhibition leads to improvement in insulin sensitivity. In addition, inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) reduces the degree of islet cell inflammation (insulitis). Therefore, targeting several PTKs may provide a novel approach for correcting the pathophysiologic disturbances of diabetes. PMID:26492832

  8. Augmented EGF receptor tyrosine kinase activity impairs vascular function by NADPH oxidase-dependent mechanism in type 2 diabetic mouse.

    PubMed

    Kassan, Modar; Ait-Aissa, Karima; Ali, Maha; Trebak, Mohamed; Matrougui, Khalid

    2015-10-01

    We previously determined that augmented EGFR tyrosine kinase (EGFRtk) impairs vascular function in type 2 diabetic mouse (TD2). Here we determined that EGFRtk causes vascular dysfunction through NADPH oxidase activity in TD2. Mesenteric resistance arteries (MRA) from C57/BL6 and db-/db- mice were mounted in a wired myograph and pre-incubated for 1h with either EGFRtk inhibitor (AG1478) or exogenous EGF. The inhibition of EGFRtk did not affect the contractile response to phenylephrine-(PE) and thromboxane-(U46619) or endothelium-dependent relaxation (EDR) to acetylcholine in MRA from control group. However, in TD2 mice, AG1478 reduced the contractile response to U46619, improved vasodilatation and reduced p22phox-NADPH expression, but had no effect on the contractile response to PE. The incubation of MRA with exogenous EGF potentiated the contractile response to PE in MRA from control and diabetic mice. However, EGF impaired the EDR and potentiated the vasoconstriction to U46619 only in the control group. Interestingly, NADPH oxidase inhibition in the presence of EGF restored the normal contraction to PE and improved the EDR but had no effect on the potentiated contraction to U46619. Vascular function improvement was associated with the rescue of eNOS and Akt and reduction in phosphorylated Rho-kinase, NOX4 mRNA levels, and NADPH oxidase activity. MRA from p47phox-/- mice incubated with EGF potentiated the contraction to U46619 but had no effect to PE or ACh responses. The present study provides evidence that augmented EGFRtk impairs vascular function by NADPH oxidase-dependent mechanism. Therefore, EGFRtk and oxidative stress should be potential targets to treat vascular dysfunction in TD2. PMID:26036345

  9. Ovary ecdysteroidogenic hormone requires a receptor tyrosine kinase to activate egg formation in the mosquito Aedes aegypti.

    PubMed

    Vogel, Kevin J; Brown, Mark R; Strand, Michael R

    2015-04-21

    Mosquitoes are major disease vectors because most species must feed on blood from a vertebrate host to produce eggs. Blood feeding by the vector mosquito Aedes aegypti triggers the release of two neurohormones, ovary ecdysteroidogenic hormone (OEH) and insulin-like peptides (ILPs), which activate multiple processes required for egg formation. ILPs function by binding to the insulin receptor, which activates downstream components in the canonical insulin signaling pathway. OEH in contrast belongs to a neuropeptide family called neuroparsins, whose receptor is unknown. Here we demonstrate that a previously orphanized receptor tyrosine kinase (RTK) from A. aegypti encoded by the gene AAEL001915 is an OEH receptor. Phylogenetic studies indicated that the protein encoded by this gene, designated AAEL001915, belongs to a clade of RTKs related to the insulin receptor, which are distinguished by an extracellular Venus flytrap module. Knockdown of AAEL001915 by RNAi disabled OEH-mediated egg formation in A. aegypti. AAEL001915 was primarily detected in the mosquito ovary in association with follicular epithelial cells. Both monomeric and dimeric AAEL001915 were detected in mosquito ovaries and transfected Drosophila S2 cells. Functional assays further indicated that OEH bound to dimeric AAEL001915, which resulted in downstream phosphorylation of Ak strain transforming factor (Akt). We hypothesize that orthologs of AAEL001915 in other insects are neuroparsin receptors. PMID:25848040

  10. Ovary ecdysteroidogenic hormone requires a receptor tyrosine kinase to activate egg formation in the mosquito Aedes aegypti

    PubMed Central

    Vogel, Kevin J.; Brown, Mark R.; Strand, Michael R.

    2015-01-01

    Mosquitoes are major disease vectors because most species must feed on blood from a vertebrate host to produce eggs. Blood feeding by the vector mosquito Aedes aegypti triggers the release of two neurohormones, ovary ecdysteroidogenic hormone (OEH) and insulin-like peptides (ILPs), which activate multiple processes required for egg formation. ILPs function by binding to the insulin receptor, which activates downstream components in the canonical insulin signaling pathway. OEH in contrast belongs to a neuropeptide family called neuroparsins, whose receptor is unknown. Here we demonstrate that a previously orphanized receptor tyrosine kinase (RTK) from A. aegypti encoded by the gene AAEL001915 is an OEH receptor. Phylogenetic studies indicated that the protein encoded by this gene, designated AAEL001915, belongs to a clade of RTKs related to the insulin receptor, which are distinguished by an extracellular Venus flytrap module. Knockdown of AAEL001915 by RNAi disabled OEH-mediated egg formation in A. aegypti. AAEL001915 was primarily detected in the mosquito ovary in association with follicular epithelial cells. Both monomeric and dimeric AAEL001915 were detected in mosquito ovaries and transfected Drosophila S2 cells. Functional assays further indicated that OEH bound to dimeric AAEL001915, which resulted in downstream phosphorylation of Ak strain transforming factor (Akt). We hypothesize that orthologs of AAEL001915 in other insects are neuroparsin receptors. PMID:25848040

  11. The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm.

    PubMed

    Alvau, Antonio; Battistone, Maria Agustina; Gervasi, Maria Gracia; Navarrete, Felipe A; Xu, Xinran; Sánchez-Cárdenas, Claudia; De la Vega-Beltran, Jose Luis; Da Ros, Vanina G; Greer, Peter A; Darszon, Alberto; Krapf, Diego; Salicioni, Ana Maria; Cuasnicu, Patricia S; Visconti, Pablo E

    2016-07-01

    Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2(-/-) mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro. PMID:27226326

  12. Inhibition of Bcr serine kinase by tyrosine phosphorylation.

    PubMed Central

    Liu, J; Wu, Y; Ma, G Z; Lu, D; Haataja, L; Heisterkamp, N; Groffen, J; Arlinghaus, R B

    1996-01-01

    The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired. PMID:8622703

  13. The anti-esophageal cancer cell activity by a novel tyrosine/phosphoinositide kinase inhibitor PP121

    SciTech Connect

    Peng, Yi; Zhou, Yajuan; Cheng, Long; Hu, Desheng; Zhou, Xiaoyi; Wang, Zhaohua; Xie, Conghua; Zhou, Fuxiang

    2015-09-11

    Here we explored the potential effect of PP121, a novel dual inhibitor of tyrosine and phosphoinositide kinases, against human esophageal cancer cells. We showed that PP121 exerted potent cytotoxic effect in primary (patient-derived) and established (Eca-109, TE-1 and TE-3 lines) esophageal cancer cells, possibly through activating caspase-3-dependnent apoptosis. PP121 was, however, non-cytotoxic to the normal human esophageal epithelial cells (EECs). At the molecular level, we showed that PP121 blocked Akt-mTOR (mammalian target of rapamycin) activation in esophageal cancer cells, which was restored by introducing a constitutively-active Akt (CA-Akt). Yet, CA-Akt only partly inhibited cytotoxicity by PP121 in Eca-109 cells. Importantly, we showed that PP121 inhibited nuclear factor kappa B (NFκB) signaling activation in esophageal cancer cells, which appeared independent of Akt-mTOR blockage. In vivo, oral administration of PP121 remarkably inhibited Eca-109 xenograft growth in nude mice, and significantly improved mice survival. Further, the immunohistochemistry (IHC) and Western blot assays analyzing xenografted tumors showed that PP121 inhibited Akt-mTOR and NFκB activations in vivo. Together, we demonstrate that PP121 potently inhibits esophageal cancer cells in vitro and in vivo, possibly through concurrently inhibiting Akt-mTOR and NFκB signalings. - Highlights: • PP121 is cytotoxic against primary and established esophageal cancer cells. • PP121 induces caspase-3-dependnent apoptosis in esophageal cancer cells. • PP121 blocks Akt-mTOR activation in esophageal cancer cells. • PP121 inhibits NFκB activation, independent of Akt-mTOR blockage. • PP121 inhibits Eca-109 xenograft growth and Akt-mTOR/NFκB activation in vivo.

  14. Tyrosine kinase gene rearrangements in epithelial malignancies

    PubMed Central

    Shaw, Alice T.; Hsu, Peggy P.; Awad, Mark M.; Engelman, Jeffrey A.

    2014-01-01

    Chromosomal rearrangements that lead to oncogenic kinase activation are observed in many epithelial cancers. These cancers express activated fusion kinases that drive the initiation and progression of malignancy, and often have a considerable response to small-molecule kinase inhibitors, which validates these fusion kinases as ‘druggable’ targets. In this Review, we examine the aetiologic, pathogenic and clinical features that are associated with cancers harbouring oncogenic fusion kinases, including anaplastic lymphoma kinase (ALK), ROS1 and RET. We discuss the clinical outcomes with targeted therapies and explore strategies to discover additional kinases that are activated by chromosomal rearrangements in solid tumours. PMID:24132104

  15. Dimerization of the EphA1 receptor tyrosine kinase transmembrane domain: Insights into the mechanism of receptor activation.

    PubMed

    Chavent, Matthieu; Chetwynd, Alan P; Stansfeld, Phillip J; Sansom, Mark S P

    2014-10-28

    EphA1 is a receptor tyrosine kinase (RTK) that plays a key role in developmental processes, including guidance of the migration of axons and cells in the nervous system. EphA1, in common with other RTKs, contains an N-terminal extracellular domain, a single transmembrane (TM) α-helix, and a C-terminal intracellular kinase domain. The TM helix forms a dimer, as seen in recent NMR studies. We have modeled the EphA1 TM dimer using a multiscale approach combining coarse-grain (CG) and atomistic molecular dynamics (MD) simulations. The one-dimensional potential of mean force (PMF) for this system, based on interhelix separation, has been calculated using CG MD simulations. This provides a view of the free energy landscape for helix-helix interactions of the TM dimer in a lipid bilayer. The resulting PMF profiles suggest two states, consistent with a rotation-coupled activation mechanism. The more stable state corresponds to a right-handed helix dimer interacting via an N-terminal glycine zipper motif, consistent with a recent NMR structure (2K1K). A second metastable state corresponds to a structure in which the glycine zipper motif is not involved. Analysis of unrestrained CG MD simulations based on representative models from the PMF calculations or on the NMR structure reveals possible pathways of interconversion between these two states, involving helix rotations about their long axes. This suggests that the interaction of TM helices in EphA1 dimers may be intrinsically dynamic. This provides a potential mechanism for signaling whereby extracellular events drive a shift in the repopulation of the underlying TM helix dimer energy landscape. PMID:25286141

  16. PYK2: a calcium-sensitive protein tyrosine kinase activated in response to fertilization of the zebrafish oocyte.

    PubMed

    Sharma, Dipika; Kinsey, William H

    2013-01-01

    Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone. PMID:23084926

  17. The novel tyrosine kinase inhibitor AKN-028 has significant antileukemic activity in cell lines and primary cultures of acute myeloid leukemia

    PubMed Central

    Eriksson, A; Hermanson, M; Wickström, M; Lindhagen, E; Ekholm, C; Jenmalm Jensen, A; Löthgren, A; Lehmann, F; Larsson, R; Parrow, V; Höglund, M

    2012-01-01

    Aberrantly expressed tyrosine kinases have emerged as promising targets for drug development in acute myeloid leukemia (AML). We report that AKN-028, a novel tyrosine kinase inhibitor (TKI), is a potent FMS-like receptor tyrosine kinase 3 (FLT3) inhibitor (IC50=6 nℳ), causing dose-dependent inhibition of FLT3 autophosphorylation. Inhibition of KIT autophosphorylation was shown in a human megakaryoblastic leukemia cell line overexpressing KIT. In a panel of 17 cell lines, AKN-028 showed cytotoxic activity in all five AML cell lines included. AKN-028 triggered apoptosis in MV4-11 by activation of caspase 3. In primary AML samples (n=15), AKN-028 induced a clear dose-dependent cytotoxic response (mean IC50 1 μℳ). However, no correlation between antileukemic activity and FLT3 mutation status, or to the quantitative expression of FLT3, was observed. Combination studies showed synergistic activity when cytarabine or daunorubicin was added simultaneously or 24 h before AKN-028. In mice, AKN-028 demonstrated high oral bioavailability and antileukemic effect in primary AML and MV4-11 cells, with no major toxicity observed in the experiment. In conclusion, AKN-028 is a novel TKI with significant preclinical antileukemic activity in AML. Possible sequence-dependent synergy with standard AML drugs and good oral bioavailability has made it a candidate drug for clinical trials (ongoing). PMID:22864397

  18. Mechanisms of tyrosine hydroxylase regulation in striatal synaptosomes: effects of activation of cyclic AMP-dependent protein kinase

    SciTech Connect

    Colby, K.A.

    1987-01-01

    The regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis, was examined in synaptosomes prepared from rat corpus striatum. Exposure of striatal synaptosomes to dibutyryl-cyclic AMP (dbcAMP) causes an increase in the maximal velocity of TH, but does not change the K/sub m/ of the enzyme for the synthetic cofactor, 2-amino-4-hydroxy-6-methyl-tetrahydropterin. Activation of TH by synaptosomal exposure to dbcAMP also causes a decrease in the pH sensitivity and an increase in the thermolability of the enzyme. Striatal synaptosomes were used to examine the in vitro phosphorylation of TH. Under the protocol developed as part of this work, TH in synaptosomes can be labelled with /sup 32/P. This is the first report of in vitro labelling of TH in a biochemically intact CNS preparation. Under certain protocols, treatment of synaptosomes with dbcAMP causes an increase in the /sup 32/P labelling of TH. These results are consistent with the notion that dbcAMP produces changes in the physical properties of TH by activating cAMP-dependent protein kinase which subsequently phosphorylates TH. In vivo electrical stimulation of the rat medial forebrain bundle causes an activation of striatal TH as well as an decrease in the pH sensitivity of the enzyme. Since similar changes are produced upon activation of snaptosomal TH by dbcAMP, it is likely that phosphorylation of TH is involved in the increase in TH activity that is associated with neuronal depolarization.

  19. Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology.

    PubMed

    Challa, Anil Kumar; Chatti, Kiranam

    2013-09-01

    Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome. PMID:23234507

  20. Conservation and Early Expression of Zebrafish Tyrosine Kinases Support the Utility of Zebrafish as a Model for Tyrosine Kinase Biology

    PubMed Central

    Challa, Anil Kumar

    2013-01-01

    Abstract Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome. PMID:23234507

  1. Tyrosine phosphorylation-dependent activation of phosphatidylinositide 3-kinase occurs upstream of Ca2+-signalling induced by Fcgamma receptor cross-linking in human neutrophils.

    PubMed Central

    Vossebeld, P J; Homburg, C H; Schweizer, R C; Ibarrola, I; Kessler, J; Koenderman, L; Roos, D; Verhoeven, A J

    1997-01-01

    The effect of wortmannin on IgG-receptor (FcgammaR)-mediated stimulation of human neutrophils was investigated. The Ca2+ influx induced by clustering of both Fcgamma receptors was inhibited by wortmannin, as was the release of Ca2+ from intracellular stores. Wortmannin also inhibited, with the same efficacy, the accumulation of Ins(1,4,5)P3 observed after FcgammaR stimulation, but did not affect the increase in Ins(1,4,5)P3 induced by the chemotactic peptide, formyl-methionine-leucine-phenylalanine. Because wortmannin is, in the concentrations used here, an inhibitor of PtdIns 3-kinase, these results suggested a role for PtdIns 3-kinase upstream of Ca2+ signalling, induced by FcgammaR cross-linking. Support for this notion was obtained by investigating the effect of another inhibitor of PtdIns 3-kinase, LY 294002, and by studying the kinetics of PtdIns 3-kinase activation. We found translocation of PtdIns 3-kinase to the plasma membrane and increased PtdIns 3-kinase activity in the membrane as soon as 5 s after FcgammaR cross-linking, even before the onset of the Ca2+ response. Moreover, the translocation of PtdIns 3-kinase to the plasma membrane was inhibited by co-cross-linking of either FcgammaRIIa and FcgammaRIIIb with the tyrosine phosphatase, CD45, indicating a requirement for protein tyrosine phosphorylation in the recruitment of PtdIns 3-kinase to the plasma membrane. Taken together, our results suggest a role for PtdIns 3-kinase in early signal transduction events after FcgammaR cross-linking in human neutrophils. PMID:9173906

  2. Validation of in vivo pharmacodynamic activity of a novel PDGF receptor tyrosine kinase inhibitor using immunohistochemistry and quantitative image analysis.

    PubMed

    D'Andrea, Michael R; Mei, Jay M; Tuman, Robert W; Galemmo, Robert A; Johnson, Dana L

    2005-08-01

    With the advent of agents directed against specific molecular targets in drug discovery, it has become imperative to show a compound's cellular impact on the intended biomolecule in vivo. The objective of the present study was to determine if we could develop an assay to validate the in vivo effects of a compound. Hence, we investigated the in vivo pharmacodynamic activity of JNJ-10198409, a relatively selective inhibitor of platelet-derived growth factor receptor tyrosine kinase (PDGF-RTK), in tumor tissues after administering the compound orally in a nude mouse xenograft model of human LoVo colon cancer. We developed a novel assay to quantify the in vivo anti-PDGF-RTK activity of the inhibitor in tumor tissue by determining the phosphorylation status of phospholipase Cgamma1 (PLCgamma1), a key downstream cellular molecule in the PDGF-RTK signaling cascade. We used two antibodies, one specific for the total (phosphorylated and unphosphorylated forms) PLCgamma1 (pan-PLCgamma1) and the other, specific for phosphorylated form of PLCgamma1 (ph-PLCgamma1) to immunohistochemically detect their expression in tumor tissues. Computer-assisted image analysis was then used to directly compare the ratio of ph-PLCgamma1 to pan-PLCgamma1 immunolabeling intensities in serial sections (5 mum) of tumors obtained from vehicle- and JNJ-10198409-treated tumor-bearing mice. Our data showed statistically significant, dose-dependent differences in the ph-PLC/pan-PLC ratio among the four treatment groups (vehicle, 25, 50, and 100 mg/kg b.i.d.). These results confirmed this compound's ability to suppress PDGF-RTK downstream signaling in tumor tissues in vivo. In addition to this specific application of this in vivo validation approach to those targets that use PLCgamma as a downstream signaling partner, these methods may also benefit other drug discovery targets. PMID:16093435

  3. MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors

    PubMed Central

    Kubo, Takafumi; Yamamoto, Hiromasa; Lockwood, William W.; Valencia, Ilse; Soh, Junichi; Peyton, Michael; Jida, Masaru; Otani, Hiroki; Fujii, Tetsuya; Ouchida, Mamoru; Takigawa, Nagio; Kiura, Katsuyuki; Shimizu, Kenji; Date, Hiroshi; Minna, John D.; Varella-Garcia, Marileila; Lam, Wan L.; Gazdar, Adi F.; Toyooka, Shinichi

    2009-01-01

    We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real-time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in-situ hybridization. Total and phospho-MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High-level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR-TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho-MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho-MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA-mediated knockdown of EGFR abolished phospho-MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho-MET expression in 2 cell lines with amplified MET gene was not down-regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC. PMID:19117057

  4. Novel Anthraquinone-based Derivatives as Potent Inhibitors for Receptor Tyrosine Kinases

    PubMed Central

    Stasevych, M.; Zvarych, V.; Lunin, V.; Halenova, T.; Savchuk, O.; Dudchak, O.; Vovk, M.; Novikov, V.

    2015-01-01

    The influence of new derivatives of 9,10-anthraquinone with benzoylthiourea, thiazole, triazole and amino acid fragments on the activity of membrane-associated tyrosine kinases was investigated. Inhibitors of protein tyrosine kinase activity of the membrane fraction, as promising agents to search for new potential anticancer agents among the studied compounds, were discovered. PMID:26798182

  5. The Next Wave of EGFR Tyrosine Kinase Inhibitors Enter the Clinic.

    PubMed

    Politi, Katerina; Ayeni, Deborah; Lynch, Thomas

    2015-06-01

    The T790M mutation in EGFR accounts for approximately half of all lung cancer cases with acquired resistance to the current clinical EGFR tyrosine kinase inhibitors. In tyrosine kinase inhibitor-resistant lung tumors, rociletinib and AZD9291 are highly active when T790M is present and modestly active when T790M is absent. PMID:26058074

  6. Therapeutic drug monitoring and tyrosine kinase inhibitors

    PubMed Central

    Herviou, Pauline; Thivat, Emilie; Richard, Damien; Roche, Lucie; Dohou, Joyce; Pouget, Mélanie; Eschalier, Alain; Durando, Xavier; Authier, Nicolas

    2016-01-01

    The therapeutic activity of drugs can be optimized by establishing an individualized dosage, based on the measurement of the drug concentration in the serum, particularly if the drugs are characterized by an inter-individual variation in pharmacokinetics that results in an under- or overexposure to treatment. In recent years, several tyrosine kinase inhibitors (TKIs) have been developed to block intracellular signaling pathways in tumor cells. These oral drugs are candidates for therapeutic drug monitoring (TDM) due to their high inter-individual variability for therapeutic and toxic effects. Following a literature search on PubMed, studies on TKIs and their pharmacokinetic characteristics, plasma quantification and inter-individual variability was studied. TDM is commonly used in various medical fields, including cardiology and psychiatry, but is not often applied in oncology. Plasma concentration monitoring has been thoroughly studied for imatinib, in order to evaluate the usefulness of TDM. The measurement of plasma concentration can be performed by various analytical techniques, with liquid chromatography-mass spectrometry being the reference method. This method is currently used to monitor the efficacy and tolerability of imatinib treatments. Although TDM is already being used for imatinib, additional studies are required in order to improve this practice with the inclusion of other TKIs. PMID:27446421

  7. A New Family of Receptor Tyrosine Kinases with a Venus Flytrap Binding Domain in Insects and Other Invertebrates Activated by Aminoacids

    PubMed Central

    Ahier, Arnaud; Rondard, Philippe; Gouignard, Nadège; Khayath, Naji; Huang, Siluo; Trolet, Jacques; Donoghue, Daniel J.; Gauthier, Monique; Pin, Jean-Philippe; Dissous, Colette

    2009-01-01

    Background Tyrosine kinase receptors (RTKs) comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT) linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor. Methods and Findings Here we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR), were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABAB receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development. Conclusion The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases. PMID:19461966

  8. Regulation of alternative macrophage activation in the liver following acetaminophen intoxication by stem cell-derived tyrosine kinase

    SciTech Connect

    Gardner, Carol R.; Hankey, Pamela; Mishin, Vladimir; Francis, Mary; Yu, Shan; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-07-15

    Stem cell-derived tyrosine kinase (STK) is a transmembrane receptor reported to play a role in macrophage switching from a classically activated/proinflammatory phenotype to an alternatively activated/wound repair phenotype. In the present studies, STK{sup −/−} mice were used to assess the role of STK in acetaminophen-induced hepatotoxicity as evidence suggests that the pathogenic process involves both of these macrophage subpopulations. In wild type mice, centrilobular hepatic necrosis and increases in serum transaminase levels were observed within 6 h of acetaminophen administration (300 mg/kg, i.p.). Loss of STK resulted in a significant increase in sensitivity of mice to the hepatotoxic effects of acetaminophen and increased mortality, effects independent of its metabolism. This was associated with reduced levels of hepatic glutathione, rapid upregulation of inducible nitric oxide synthase, and prolonged induction of heme oxygenase-1, suggesting excessive oxidative stress in STK{sup −/−} mice. F4/80, a marker of mature macrophages, was highly expressed on subpopulations of Kupffer cells in livers of wild type, but not STK{sup −/−} mice. Whereas F4/80{sup +} macrophages rapidly declined in the livers of wild type mice following acetaminophen intoxication, they increased in STK{sup −/−} mice. In wild type mice hepatic expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-12, products of classically activated macrophages, increased after acetaminophen administration. Monocyte chemotactic protein-1 (MCP-1) and its receptor, CCR2, as well as IL-10, mediators involved in recruiting and activating anti-inflammatory/wound repair macrophages, also increased in wild type mice after acetaminophen. Loss of STK blunted the effects of acetaminophen on expression of TNFα, IL-1β, IL-12, MCP-1 and CCR2, while expression of IL-10 increased. Hepatic expression of CX3CL1, and its receptor, CX3CR1 also increased in STK{sup −/−} mice

  9. 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

  10. Comparative Analysis of Mutant Tyrosine Kinase Chemical Rescue†

    PubMed Central

    Muratore, Kathryn E.; Seeliger, Markus A.; Wang, Zhihong; Fomina, Dina; Neiswinger, Johnathan; Havranek, James J.; Baker, David; Kuriyan, John; Cole, Philip A.

    2009-01-01

    Protein tyrosine kinases are critical cell signaling enzymes. These enzymes have a highly conserved Arg residue in their catalytic loop which is present two residues or four residues downstream from an absolutely conserved Asp catalytic base. Prior studies on protein tyrosine kinases Csk and Src revealed the potential for chemical rescue of catalytically-deficient mutant kinases (Arg to Ala mutations) by small diamino compounds, particularly imidazole, however the potency and efficiency of rescue was greater for Src. This current study further examines the structural and kinetic basis of rescue for mutant Src as compared to mutant Abl tyrosine kinase. An X-ray crystal structure of R388A Src revealed the surprising finding that a histidine residue of the N-terminus of a symmetry-related kinase inserts into the active site of the adjacent Src and mimics the hydrogen bonding pattern seen in wild-type protein tyrosine kinases. Abl R367A shows potent and efficient rescue more comparable to Src, even though its catalytic loop is more like that of Csk. Various enzyme redesigns of the active sites indicate that the degree and specificity of rescue is somewhat flexible, but the overall properties of the enzymes and rescue agents play an overarching role. The newly discovered rescue agent 2-aminoimidazole is about as efficient as imidazole in rescuing R/A Src and Abl. Rate vs. pH studies with these imidazole analogs suggest that the protonated imidazolium is the preferred form for chemical rescue, consistent with structural models. The efficient rescue seen with mutant Abl points to the potential of this approach to be used effectively to analyze Abl phosphorylation pathways in cells. PMID:19260709

  11. Cross talk of tyrosine kinases with the DNA damage signaling pathways

    PubMed Central

    Mahajan, Kiran; Mahajan, Nupam P.

    2015-01-01

    Tyrosine kinases respond to extracellular and intracellular cues by activating specific cellular signaling cascades to regulate cell cycle, growth, proliferation, differentiation and survival. Likewise, DNA damage response proteins (DDR) activated by DNA lesions or chromatin alterations recruit the DNA repair and cell cycle checkpoint machinery to restore genome integrity and cellular homeostasis. Several new examples have been uncovered in recent studies which reveal novel epigenetic and non-epigenetic mechanisms by which tyrosine kinases interact with DDR proteins to dictate cell fate, i.e. survival or apoptosis, following DNA damage. These studies reveal the ability of tyrosine kinases to directly regulate the activity of DNA repair and cell cycle check point proteins by tyrosine phosphorylation. In addition, tyrosine kinases epigenetically regulate DNA damage signaling pathways by modifying the core histones as well as chromatin modifiers at critical tyrosine residues. Thus, deregulated tyrosine kinase driven epigenomic alterations have profound implications in cancer, aging and genetic disorders. Consequently, targeting oncogenic tyrosine kinase induced epigenetic alterations has gained significant traction in overcoming cancer cell resistance to various therapies. This review discusses mechanisms by which tyrosine kinases interact with DDR pathways to regulate processes critical for maintaining genome integrity as well as clinical strategies for targeted cancer therapies. PMID:26546517

  12. Cross talk of tyrosine kinases with the DNA damage signaling pathways.

    PubMed

    Mahajan, Kiran; Mahajan, Nupam P

    2015-12-15

    Tyrosine kinases respond to extracellular and intracellular cues by activating specific cellular signaling cascades to regulate cell cycle, growth, proliferation, differentiation and survival. Likewise, DNA damage response proteins (DDR) activated by DNA lesions or chromatin alterations recruit the DNA repair and cell cycle checkpoint machinery to restore genome integrity and cellular homeostasis. Several new examples have been uncovered in recent studies which reveal novel epigenetic and non-epigenetic mechanisms by which tyrosine kinases interact with DDR proteins to dictate cell fate, i.e. survival or apoptosis, following DNA damage. These studies reveal the ability of tyrosine kinases to directly regulate the activity of DNA repair and cell cycle check point proteins by tyrosine phosphorylation. In addition, tyrosine kinases epigenetically regulate DNA damage signaling pathways by modifying the core histones as well as chromatin modifiers at critical tyrosine residues. Thus, deregulated tyrosine kinase driven epigenomic alterations have profound implications in cancer, aging and genetic disorders. Consequently, targeting oncogenic tyrosine kinase induced epigenetic alterations has gained significant traction in overcoming cancer cell resistance to various therapies. This review discusses mechanisms by which tyrosine kinases interact with DDR pathways to regulate processes critical for maintaining genome integrity as well as clinical strategies for targeted cancer therapies. PMID:26546517

  13. The novel receptor tyrosine kinase Axl is constitutively active in B-cell chronic lymphocytic leukemia and acts as a docking site of nonreceptor kinases: implications for therapy

    PubMed Central

    Secreto, Charla; Boysen, Justin; Sassoon, Traci; Shanafelt, Tait D.; Mukhopadhyay, Debabrata; Kay, Neil E.

    2011-01-01

    Recently, we detected that chronic lymphocytic leukemia (CLL) B-cell–derived microvesicles in CLL plasma carry a constitutively phosphorylated novel receptor tyrosine kinase (RTK), Axl, indicating that Axl was acquired from the leukemic B cells. To examine Axl status in CLL, we determined the expression of phosphorylated-Axl (P-Axl) in freshly isolated CLL B cells by Western blot analysis. We detected differential levels of P-Axl in CLL B cells, and further analysis showed that expression of P-Axl was correlated with the other constitutively phosphorylated kinases, including Lyn, phosphoinositide-3 kinase, SyK/ζ-associated protein of 70 kDa, phospholipase C γ2 in CLL B cells. We found that these intracellular signaling molecules were complexed with P-Axl in primary CLL B cells. When Axl and Src kinases were targeted by a Src/Abl kinase inhibitor, bosutinib (SKI-606), or a specific-inhibitor of Axl (R428), robust induction of CLL B-cell apoptosis was observed in both a dose- and time-dependent manner. Therefore, we have identified a novel RTK in CLL B cells which appears to work as a docking site for multiple non-RTKs and drives leukemic cell survival signals. These findings highlight a unique target for CLL treatment. PMID:21135257

  14. The upregulation of NR2A-containing N-methyl-D-aspartate receptor function by tyrosine phosphorylation of postsynaptic density 95 via facilitating Src/proline-rich tyrosine kinase 2 activation.

    PubMed

    Zhao, Chao; Du, Cai-Ping; Peng, Yan; Xu, Zhen; Sun, Chang-Cheng; Liu, Yong; Hou, Xiao-Yu

    2015-04-01

    The activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors is required for long-term potentiation (LTP) of synaptic transmission. Postsynaptic density 95 (PSD-95) serves as a scaffold protein that tethers NMDA receptor subunits, kinases, and signal molecules. Our previous study proves that PSD-95 is a substrate of Src/Fyn and identifies Y523 on PSD-95 as a principal phosphorylation site. In this paper, we try to define an involvement and molecular consequences of PSD-95 phosphorylation by Src in NMDA receptor regulation. We found that either NMDA or chemical LTP induction leads to rapid phosphorylation of PSD-95 by Src in cultured cortical neurons. The phosphorylation of Y523 on PSD-95 potentiates NR2A-containing NMDA receptor current amplitude, implying an important role of Src-mediated PSD-95 phosphorylation in NMDA receptor activation. Comparing to wild-type PSD-95, overexpression of nonphosphorylatable mutant PSD-95Y523F attenuated the NMDA-stimulated NR2A tyrosine phosphorylation that enhances electrophysiological responses of NMDA receptor channels, while did not affect the membrane localization of NR2A subunits. PSD-95Y523D, a phosphomimetic mutant of PSD-95, induced NR2A tyrosine phosphorylation even if there was no NMDA treatment. In addition, the deficiency of Y523 phosphorylation on PSD-95 impaired the facilitatory effect of PSD-95 on the activation of Src and proline-rich tyrosine kinase 2 (Pyk2) and decreased the binding of Pyk2 with PSD-95. These results indicate that PSD-95 phosphorylation by Src facilitates the integration of Pyk2 to PSD-95 signal complex, the activation of Pyk2/Src, as well as the subsequent tyrosine phosphorylation of NR2A, which ultimately results in the upregulation of NMDA receptor function and synaptic transmission. PMID:24981431

  15. 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

  16. Activation of the lutropin/choriogonadotropin receptor (LHR) in MA-10 cells leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) by a pathway that involves Src family kinases*

    PubMed Central

    Mizutani, Tetsuya; Shiraishi, Koji; Welsh, Toni; Ascoli, Mario

    2006-01-01

    We show that activation of the endogenous or recombinant LHR in mouse Leydig tumor cells (MA-10 cells) leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) and one of its substrates (paxillin). Using specific antibodies to the five tyrosine residues of FAK that become phosphorylated we show that activation of the LHR increases the phosphorylation of Tyr576 and Tyr577 but it does not affect the phosphorylation of Tyr397, Tyr861 or Tyr925. Because FAK is a prominent substrate for the Src family of tyrosine kinases (SFKs) we tested for their involvement in the LHR-mediated phosphorylation of FAK-Tyr576. Src is not detectable in MA-10 cells, but two other prominent members of this family (Fyn and Yes) are present. The LHR-mediated phosphorylation of FAK-Tyr576 is readily inhibited by PP2 (a pharmacological inhibitor of SFKs) and by dominant-negative mutants of SKFs. Moreover, activation of the LHR in MA-10 cells results in the stimulation of the activity of Fyn and Yes and overexpression of either of these two tyrosine kinases enhances the LHR-mediate phosphorylation of FAK-Tyr576. Studies involving activation of other G protein-coupled receptors, overexpression of the different Gα subunits, and the use of second messenger analogs suggest that the LHR-induced phosphorylation of FAK-Tyr576 in MA-10 cells is mediated by SFKs, and that this family of kinases is, in turn, independently or cooperatively activated by the LHR-induced stimulation of Gs and Gq/11-mediated pathways. PMID:16293639

  17. SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

    SciTech Connect

    Buchanan, Sean G.; Hendle, Jorg; Lee, Patrick S.; Smith, Christopher R.; Bounaud, Pierre-Yves; Jessen, Katti A.; Tang, Crystal M.; Huser, Nanni H.; Felce, Jeremy D.; Froning, Karen J.; Peterman, Marshall C.; Aubol, Brandon E.; Gessert, Steve F.; Sauder, J. Michael; Schwinn, Kenneth D.; Russell, Marijane; Rooney, Isabelle A.; Adams, Jason; Leon, Barbara C.; Do, Tuan H.; Blaney, Jeff M.; Sprengeler, Paul A.; Thompson, Devon A.; Smyth, Lydia; Pelletier, Laura A.; Atwell, Shane; Holme, Kevin; Wasserman, Stephen R.; Emtage, Spencer; Burley, Stephen K.; Reich, Siegfried H.

    2010-01-12

    The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC{sub 50} of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition.

  18. The Ror receptor tyrosine kinase family.

    PubMed

    Forrester, W C

    2002-01-01

    Receptor tyrosine kinases (RTKs) participate in numerous developmental decisions. Ror RTKs are a family of orphan receptors that are related to muscle specific kinase (MuSK) and Trk neurotrophin receptors. MuSK assembles acetylcholine receptors at the neuromuscular junction, and Trk receptors function in the developing nervous system (reviewed in [3-5]). Rors have been identified in nematodes, insects and mammals. Recent studies have begun to shed light on Ror function during development. In most species, Rors are expressed in many tissue types during development. Analyses of mutants that are defective in the single nematode Ror demonstrate a role in cell migration and in orienting cell polarity. Mice lacking one of the two Ror gene products display defects in bone and heart formation. Similarly, two different human bone development disorders, dominant brachydactyly B and recessive Robinow syndrome, result from mutations in one of the human Ror genes. PMID:11846036

  19. Tyrosine kinase activity of a chimeric insulin-like-growth-factor-1 receptor containing the insulin receptor C-terminal domain. Comparison with the tyrosine kinase activities of the insulin and insulin-like-growth-factor-1 receptors using a cell-free system.

    PubMed

    Mothe, I; Tartare, S; Kowalski-Chauvel, A; Kaliman, P; Van Obberghen, E; Ballotti, R

    1995-03-15

    In a previous study, we showed that a chimeric insulin-like-growth-factor-1 (IGF-1) receptor, with the beta subunit C-terminal part of the insulin receptor was more efficient in stimulating glycogen synthesis and p44mapk activity compared to the wild-type IFG-1 receptor [Tartare, S., Mothe, I., Kowalski-Chauvel, A., Breittmayer, J.-P., Ballotti, R. & Van Obberghen, E. (1994) J. Biol. Chem. 269, 11449-11455]. These data indicate that the receptor C-terminal domain plays an important role in the transmission of biological effects. To understand the molecular basis of the differences in receptor specificity, we studied the characteristics of insulin, IGF-1 and chimeric receptor tyrosine kinase activities in a cell-free system. We found that, compared to wild-type insulin and IGF-1 receptors, the chimeric receptor showed a decrease in (a) autophosphorylation, (b) tyrosine kinase activity towards insulin receptor substrate-1 and the insulin receptor-(1142-1158)-peptide, and (c) the ability to activate phosphatidylinositol 3-kinase. However, for all the effects measured in a cell-free system, the chimeric receptor displayed an increased response to IGF-1 compared to the native IGF-1 receptor. Concerning the cation dependence of the tyrosine kinase activity, we showed that, at 10 mM Mg2+, the ligand-stimulated phosphorylation of poly(Glu80Tyr20) by both insulin receptor and chimeric receptor was increased by Mn2+. Conversely at 50 mM Mg2+, the chimeric receptor behaved like the IGF-1 receptor, since the presence of Mn2+ decreased the stimulatory effect of IGF-1 on their kinase activity. Furthermore, the Km of the chimeric receptor for ATP was increased compared to the wild-type receptors. These data demonstrate that the replacement of the C-terminal tail of the IGF-1 receptor by that of the insulin receptor has changed the receptor characteristics studied in a cell-free system. Our findings indicate that the C-terminal domain of the insulin receptor beta subunit plays a

  20. In vitro activation of a transcription factor by gamma interferon requires a membrane-associated tyrosine kinase and is mimicked by vanadate.

    PubMed Central

    Igarashi, K; David, M; Larner, A C; Finbloom, D S

    1993-01-01

    Gamma interferon (IFN-gamma) activates the formation of a DNA-binding protein complex (FcRF gamma) that recognizes the gamma response region (GRR) of the promoter for the human high-affinity Fc gamma receptor. In a membrane-enriched fraction prepared from human peripheral blood monocytes, IFN-gamma activation of FcRF gamma occurred within 1 min and was ATP dependent. Activation of FcRF gamma required a tyrosine kinase activity, and recognition of the GRR sequence by FcRF gamma could be abrogated by treatment with a tyrosine-specific protein phosphatase. Treatment of cells with vanadate alone resulted in the formation of FcRF gamma without the need for IFN-gamma. UV cross-linking and antibody competition experiments demonstrated that the FcRF gamma complex was composed of at least two components: the 91-kDa protein of the IFN-alpha-induced transcription complex ISGF3 and a 43-kDa component that bound directly to the GRR. Therefore, specificity for IFN-induced transcriptional activation of early response genes requires at least two events: (i) ligand-induced activation of membrane-associated protein by tyrosine phosphorylation and (ii) formation of a complex composed of an activated membrane protein(s) and a sequence-specific DNA-binding component. Images PMID:8321205

  1. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    SciTech Connect

    Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  2. Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

    PubMed

    Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

    2016-07-01

    Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

  3. MERTK receptor tyrosine kinase is a therapeutic target in melanoma

    PubMed Central

    Schlegel, Jennifer; Sambade, Maria J.; Sather, Susan; Moschos, Stergios J.; Tan, Aik-Choon; Winges, Amanda; DeRyckere, Deborah; Carson, Craig C.; Trembath, Dimitri G.; Tentler, John J.; Eckhardt, S. Gail; Kuan, Pei-Fen; Hamilton, Ronald L.; Duncan, Lyn M.; Miller, C. Ryan; Nikolaishvili-Feinberg, Nana; Midkiff, Bentley R.; Liu, Jing; Zhang, Weihe; Yang, Chao; Wang, Xiaodong; Frye, Stephen V.; Earp, H. Shelton; Shields, Janiel M.; Graham, Douglas K.

    2013-01-01

    Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies. PMID:23585477

  4. Targeting Angiogenesis in Colorectal Cancer: Tyrosine Kinase Inhibitors.

    PubMed

    Kircher, Sheetal Mehta; Nimeiri, Halla S; Benson, Al B

    2016-01-01

    Colorectal cancer is commonly diagnosed throughout the world, and treatment options have greatly expanded over the last 2 decades. Targeting angiogenesis has been a major focus of study in a variety of malignancy types. Targeting angiogenesis has been achieved by several mechanisms in colorectal cancer, including use of antiangiogenic small molecule tyrosine kinase inhibitors (TKIs). There have been many attempts and failures to prove efficacy of TKIs in the treatment of colorectal cancer including sorafenib, sunitinib, vatalanib, and tivozanib. Regorafenib was the first TKI to demonstrate efficacy and is an orally active inhibitor of angiogenic (including the vascular endothelial growth factor receptors 1, 2, and 3), stromal, and oncogenic receptor tyrosine kinases. There are ongoing investigations of both regorafenib and ninetanib; however, there remains a critical need to better understand novel combinations with TKIs that could prove more efficacious than available options. PMID:27341596

  5. Eph-mediated tyrosine phosphorylation of citron kinase controls abscission.

    PubMed

    Jungas, Thomas; Perchey, Renaud T; Fawal, Mohamad; Callot, Caroline; Froment, Carine; Burlet-Schiltz, Odile; Besson, Arnaud; Davy, Alice

    2016-08-29

    Cytokinesis is the last step of cell division, culminating in the physical separation of daughter cells at the end of mitosis. Cytokinesis is a tightly regulated process that until recently was mostly viewed as a cell-autonomous event. Here, we investigated the role of Ephrin/Eph signaling, a well-known local cell-to-cell communication pathway, in cell division. We show that activation of Eph signaling in vitro leads to multinucleation and polyploidy, and we demonstrate that this is caused by alteration of the ultimate step of cytokinesis, abscission. Control of abscission requires Eph kinase activity, and Src and citron kinase (CitK) are downstream effectors in the Eph-induced signal transduction cascade. CitK is phosphorylated on tyrosines in neural progenitors in vivo, and Src kinase directly phosphorylates CitK. We have identified the specific tyrosine residues of CitK that are phosphorylated and show that tyrosine phosphorylation of CitK impairs cytokinesis. Finally, we show that, similar to CitK, Ephrin/Eph signaling controls neuronal ploidy in the developing neocortex. Our study indicates that CitK integrates intracellular and extracellular signals provided by the local environment to coordinate completion of cytokinesis. PMID:27551053

  6. Multiple receptor tyrosine kinase activation attenuates therapeutic efficacy of the fibroblast growth factor receptor 2 inhibitor AZD4547 in FGFR2 amplified gastric cancer

    PubMed Central

    Zhang, Zhe; Liu, Xinyang; Wu, Zheng; Geng, Ruixuan; Ge, Xiaoxiao; Dai, Congqi; Liu, Rujiao; Zhang, Qunling; Li, Wenhua; Li, Jin

    2015-01-01

    Fibroblast growth factor receptor 2 (FGFR2)-targeted therapy has attracted considerable attention as novel anticancer agents in gastric cancer (GC). However, intrinsic or acquired drug resistance has emerged as a major challenge to their clinical use. In this study, we demonstrated that several receptor tyrosine kinase (RTK), including EGFR, HER3 and MET, activations contributed to AZD4547 (a selective FGFR2 inhibitor) hyposensitivity in FGFR2 amplified GC cells. The rescue effect was abrogated by inhibiting these RTKs with their targeted tyrosine kinase inhibitors (TKIs). In addition, synergy in growth inhibition was observed when the GC cells were treated with a combination of AZD4547 and cetuximab (an EGFR monoclonal antibody) both in vitro and in vivo. More importantly, tissue microarray analysis revealed that these resistance-conferring RTKs were highly expressed in FGFR2 positive GC patients. Taken together, these observations demonstrated RTKs including EGFR, HER3 and MET activations as novel mechanisms of hyposensitivity to AZD4547. It will be clinically valuable to investigate the involvement of RTK-mediated signaling in intrinsicor acquired resistance to FGFR2 TKIs in GC. A combination targeted therapeutic strategy may be recommended for treating FGFR2 amplified GC patients with these RTK activations. PMID:25576915

  7. TTT-3002 is a novel FLT3 tyrosine kinase inhibitor with activity against FLT3-associated leukemias in vitro and in vivo

    PubMed Central

    Ma, Hayley; Nguyen, Bao; Li, Li; Greenblatt, Sarah; Williams, Allen; Zhao, Ming; Levis, Mark; Rudek, Michelle; Duffield, Amy

    2014-01-01

    More than 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FMS-like tyrosine kinase-3 (FLT3). The most common type, internal tandem duplication (ITD), confers poor prognosis. We report for the first time on TTT-3002, a tyrosine kinase inhibitor (TKI) that is one of the most potent FLT3 inhibitors discovered to date. Studies using human FLT3/ITD mutant leukemia cell lines revealed the half maximal inhibitory concentration (IC50) for inhibiting FLT3 autophosphorylation is from 100 to 250 pM. The proliferation IC50 for TTT-3002 in these same cells was from 490 to 920 pM. TTT-3002 also showed potent activity when tested against the most frequently occurring FLT3-activating point mutation, FLT3/D835Y, against which many current TKIs are ineffective. These findings were validated in vivo by using mouse models of FLT3-associated AML. Survival and tumor burden of mice in several FLT3/ITD transplantation models is significantly improved by administration of TTT-3002 via oral dosing. Finally, we demonstrated that TTT-3002 is cytotoxic to leukemic blasts isolated from FLT3/ITD-expressing AML patients, while displaying minimal toxicity to normal hematopoietic stem/progenitor cells from healthy blood and bone marrow donors. Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI in the treatment of FLT3-mutant AML. PMID:24408321

  8. Heat stress activates the yeast high-osmolarity glycerol mitogen-activated protein kinase pathway, and protein tyrosine phosphatases are essential under heat stress.

    PubMed

    Winkler, Astrid; Arkind, Christopher; Mattison, Christopher P; Burkholder, Anne; Knoche, Kathryn; Ota, Irene

    2002-04-01

    The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress. In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1. The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses. The possible function of Hog1 activation during heat stress was examined, and it was found that the hog1 delta strain does not recover as rapidly from heat stress as well as the wild type. It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress. First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation. Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways. PMID:12455951

  9. Heat Stress Activates the Yeast High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway, and Protein Tyrosine Phosphatases Are Essential under Heat Stress

    PubMed Central

    Winkler, Astrid; Arkind, Christopher; Mattison, Christopher P.; Burkholder, Anne; Knoche, Kathryn; Ota, Irene

    2002-01-01

    The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress. In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1. The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses. The possible function of Hog1 activation during heat stress was examined, and it was found that the hog1Δ strain does not recover as rapidly from heat stress as well as the wild type. It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress. First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation. Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways. PMID:12455951

  10. Transformation of erythroid progenitors by viral and cellular tyrosine kinases.

    PubMed

    Beug, H; Schroeder, C; Wessely, O; Deiner, E; Meyer, S; Ischenko, I D; Hayman, M J

    1995-08-01

    Recently, two different normal avian erythroid progenitors were described. They differ in the receptor tyrosine kinases they express and in their ability to undergo self-renewal in culture. A common progenitor, termed stem cell factor (SCF) progenitor, expresses the receptor for avian SCF c-Kit, and undergoes short-term self-renewal when grown in the presence of avian SCF. A second progenitor, referred to as SCF/transforming growth factor-alpha progenitor, coexpresses c-Kit and the avian epidermal growth factor receptor homologue c-ErbB. These progenitors undergo sustained self-renewal when grown in the presence of transforming growth factor-alpha plus estradiol. The phenotype of the normal SCF/transforming growth factor-alpha progenitors closely corresponded to that of erythroid cells transformed by the tyrosine kinase oncogenes v-erbB or v-sea. This suggested that these cells, but not the SCF progenitors, would be the target cells for erythroblast transformation by these oncogenes. However, we demonstrate that both progenitor cells can be transformed by the v-erbB and v-sea oncogenes and also by the ligand-activated proto-oncogene product c-ErbB. We conclude that the target cell specificity of certain tyrosine kinase oncoproteins for erythroid cells is a reflection of their ability to provide signals for self-renewal that normally emanate from the endogenous c-ErbB protein. PMID:8547228

  11. Receptor tyrosine kinase targeting in multicellular spheroids.

    PubMed

    Breslin, Susan; O'Driscoll, Lorraine

    2015-01-01

    While growing cells as a monolayer is the traditional method for cell culture, the incorporation of multicellular spheroids into experimental design is becoming increasingly popular. This is due to the understanding that cells grown as spheroids tend to replicate the in vivo situation more reliably than monolayer cells. Thus, the use of multicellular spheroids may be more clinically relevant than monolayer cell cultures. Here, we describe methods for multicellular 3D spheroid generation that may be used to provide samples for receptor tyrosine kinase (and other protein) detection. Methods described include the forced-floating poly-HEMA method, the hanging-drop method, and the use of ECM to form multicellular 3D spheroids. PMID:25319898

  12. Tyrosine kinase inhibitors and the thyroid.

    PubMed

    Sherman, Steven I

    2009-12-01

    Protein tyrosine kinase inhibitors (TKIs) have emerged as significant targets for novel cancer therapies. For patients with differentiated or medullary carcinomas unresponsive to conventional treatments, multiple novel therapies primarily targeting angiogenesis have entered clinical trials. Partial response rates up to 30% have been reported in single-agent studies, but prolonged disease stabilisation is more commonly seen. The most successful agents target the vascular endothelial growth factor receptors. Sorafenib and sunitinib have had promising preliminary results reported and are being used selectively for patients who do not qualify for clinical trials. Treatment for patients with metastatic or advanced thyroid carcinoma now emphasises clinical trial opportunities for novel agents with considerable promise. Adverse effects on thyroid function and thyroid hormone metabolism have also been seen with several TKIs, necessitating prospective thyroid function testing for all patients starting therapy. PMID:19942148

  13. Antibodies directed against receptor tyrosine kinases

    PubMed Central

    FAUVEL, Bénédicte; Yasri, Aziz

    2014-01-01

    Approximately 30 therapeutic monoclonal antibodies have already been approved for cancers and inflammatory diseases, and monoclonal antibodies continue to be one of the fastest growing classes of therapeutic molecules. Because aberrant signaling by receptor tyrosine kinases (RTKs) is a commonly observed factor in cancer, most of the subclasses of RTKs are being extensively studied as potential targets for treating malignancies. The first two RTKs that have been targeted by antibody therapy, with five currently marketed antibodies, are the growth factor receptors EGFR and HER2. However, due to systemic side effects, refractory patients and the development of drug resistance, these treatments are being challenged by emerging therapeutics. This review examines current monoclonal antibody therapies against RTKs. After an analysis of agents that have already been approved, we present an analysis of antibodies in clinical development that target RTKs. Finally, we highlight promising RTKs that are emerging as new oncological targets for antibody-based therapy. PMID:24859229

  14. Using "On/Off" (19)F NMR/Magnetic Resonance Imaging Signals to Sense Tyrosine Kinase/Phosphatase Activity in Vitro and in Cell Lysates.

    PubMed

    Zheng, Zhen; Sun, Hongbin; Hu, Chen; Li, Gongyu; Liu, Xiaomei; Chen, Peiyao; Cui, Yusi; Liu, Jing; Wang, Junfeng; Liang, Gaolin

    2016-03-15

    Tyrosine kinase and phosphatase are two important, antagonistic enzymes in organisms. Development of noninvasive approach for sensing their activity with high spatial and temporal resolution remains challenging. Herein, we rationally designed a hydrogelator Nap-Phe-Phe(CF3)-Glu-Tyr-Ile-OH (1a) whose supramolecular hydrogel (i.e., Gel 1a) can be subjected to tyrosine kinase-directed disassembly, and its phosphate precursor Nap-Phe-Phe(CF3)-Glu-Tyr(H2PO3)-Ile-OH (1b), which can be subjected to alkaline phosphatase (ALP)-instructed self-assembly to form supramolecular hydrogel Gel 1b, respectively. Mechanic properties and internal fibrous networks of the hydrogels were characterized with rheology and cryo transmission electron microscopy (cryo-TEM). Disassembly/self-assembly of their corresponding supramolecular hydrogels conferring respective "On/Off" (19)F NMR/MRI signals were employed to sense the activity of these two important enzymes in vitro and in cell lysates for the first time. We anticipate that our new (19)F NMR/magnetic resonance imaging (MRI) method would facilitate pharmaceutical researchers to screen new inhibitors for these two enzymes without steric hindrance. PMID:26901415

  15. Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase.

    PubMed Central

    Conway, A M; Rakhit, S; Pyne, S; Pyne, N J

    1999-01-01

    The mechanism used by the platelet-derived growth factor receptor (PDGFR) to activate the mitogen-activated- protein-kinase (p42/p44 MAPK) pathway was investigated in cultured airway smooth muscle (ASM) cells. We have found that pertussis toxin (PTX, which was used to inactivate the heterotrimeric G-protein Gi) induced an approx. 40-50% decrease in the activation of c-Src and p42/p44 MAPK by PDGF. An essential role for c-Src was confirmed using the c-Src inhibitor, PP1, which abolished p42/p44 MAPK activation (PP1 and PTX were without effect on PDGFR tyrosine phosphorylation). Furthermore, the PTX-dependent decrease in c-Src and p42/p44 MAPK activation appeared correlated. These findings suggest that the PDGFR can utilize the PTX-sensitive G-protein, Gi, to regulate c-Src and subsequent p42/p44 MAPK activation. Phosphoinositide 3-kinase (PI3K) has been shown by others to be involved in p42/p44 MAPK activation. This is confirmed here by experiments which showed that PI3K inhibitors (wortmannin and LY294002) reduced the activation of p42/p44 MAPK by PDGF. PI3K activity was increased in Grb-2 immunoprecipitates from PDGF-stimulated cells and was decreased by pretreating these cells with PTX. These findings show that Gi might also promote Grb-2-PI3K complex formation and that Grb-2 may be a site at which PI3K is integrated into the p42/p44 MAPK cascade. In conclusion, our results demonstrate that Gi enables the PDGFR to signal more efficiently to p42/p44 MAPK, and this appears to be achieved through the regulation of c-Src and Grb-2/PI3K, which are intermediates in the p42/p44 MAPK cascade. PMID:9882612

  16. The role of AXL and the in vitro activity of the receptor tyrosine kinase inhibitor BGB324 in Ewing sarcoma

    PubMed Central

    Fleuren, Emmy D.G.; Hillebrandt-Roeffen, Melissa H.S.; Flucke, Uta E.; te Loo, D. Maroeska W.M.; Boerman, Otto C.; van der Graaf, Winette T.A.; Versleijen-Jonkers, Yvonne M.H.

    2014-01-01

    New targets for Ewing sarcoma (ES) patients are urgently needed. Therefore, we investigated the expression and genetic aberrations of the oncogenic receptor tyrosine kinase (RTK) AXL in ES and determined the efficacy of AXL targeting on cell viability and migration. First, AXL and Gas6 (ligand) mRNA expression was determined by RT-PCR on 29 ES samples. Low, medium and high AXL mRNA expression was observed in 31% (n = 9), 48% (n = 14) and 21% (n = 6) of samples. Gas6 was abundantly present in all specimens. We next tested AXL protein expression immunohistochemically in 36 tumors (primary, post-chemotherapy, metastasized and relapsed samples) from 25 ES patients. Low, medium and high AXL protein expression was observed in 17% (n = 6), 19% (n = 7) and 36% (n = 13) of samples. In primary tumors (n = 15), high AXL expression correlated significantly with a worse overall survival compared to patients with lower expression (61 vs. 194 months, p = 0.026). No genetic aberrations were detected in the AXL RTK domain (n = 29). The AXL-inhibitor BGB324 affected viability (IC50 0.79–2.13 μmol/L) and migratory potential of all tested ES cell lines in vitro (n = 5–6). BGB324 chemosensitized chemotherapy-resistant ES-4 cells to vincristine and doxorubicin. These data suggest that AXL is a potential novel, druggable therapeutic target in ES. PMID:25528764

  17. Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

    PubMed Central

    Wiedłocha, A; Falnes, P O; Rapak, A; Muñoz, R; Klingenberg, O; Olsnes, S

    1996-01-01

    U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell. PMID:8524304

  18. Splice Variants of the Dual Specificity Tyrosine Phosphorylation-regulated Kinase 4 (DYRK4) Differ in Their Subcellular Localization and Catalytic Activity*

    PubMed Central

    Papadopoulos, Chrisovalantis; Arato, Krisztina; Lilienthal, Eva; Zerweck, Johannes; Schutkowski, Mike; Chatain, Nicolas; Müller-Newen, Gerhard; Becker, Walter; de la Luna, Susana

    2011-01-01

    Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P −3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs. PMID:21127067

  19. Mapping of sites on the Src family protein tyrosine kinases p55blk, p59fyn, and p56lyn which interact with the effector molecules phospholipase C-gamma 2, microtubule-associated protein kinase, GTPase-activating protein, and phosphatidylinositol 3-kinase.

    PubMed Central

    Pleiman, C M; Clark, M R; Gauen, L K; Winitz, S; Coggeshall, K M; Johnson, G L; Shaw, A S; Cambier, J C

    1993-01-01

    Engagement of the B-cell antigen receptor complex induces immediate activation of receptor-associated Src family tyrosine kinases including p55blk, p59fyn, p53/56lyn, and perhaps p56lck, and this response is accompanied by tyrosine phosphorylation of distinct cellular substrates. These kinases act directly or indirectly to phosphorylate and/or activate effector proteins including p42 (microtubule-associated protein kinase) (MAPK), phospholipases C-gamma 1 (PLC gamma 1) and C-gamma 2 (PLC gamma 2), phosphatidylinositol 3-kinase (PI 3-K), and p21ras-GTPase-activating protein (GAP). Although coimmunoprecipitation results indicate that the Src family protein tyrosine kinases interact physically with some of these effector molecules, the molecular basis of this interaction has not been established. Here, we show that three distinct sites mediate the interaction of these kinases with effectors. The amino-terminal 27 residues of the unique domain of p56lyn mediate association with PLC gamma 2, MAPK, and GAP. Binding to PI 3-K is mediated through the Src homology 3 (SH3) domains of the Src family kinases. Relatively small proportions of cellular PI 3-K, PLC gamma 2, MAPK, and GAP, presumably those which are tyrosine phosphorylated, bind to the SH2 domains of these kinases. Comparative analysis of binding activities of Blk, Lyn, and Fyn shows that these kinases differ in their abilities to associate with MAPK and PI 3-K, suggesting that they may preferentially bind and subsequently phosphorylate distinct sets of downstream effector molecules in vivo. Fast protein liquid chromatography Mono Q column-fractionated MAPK maintains the ability to bind bacterially expressed Lyn, suggesting that the two kinases may interact directly. Images PMID:8395016

  20. Bosutinib: a novel second-generation tyrosine kinase inhibitor.

    PubMed

    Isfort, Susanne; Keller-v Amsberg, Gunhild; Schafhausen, Philippe; Koschmieder, Steffen; Brümmendorf, Tim H

    2014-01-01

    Bosutinib (SKI-606) is a 4-anilino-3-quinoline carbonitrile, which acts as a dual inhibitor of Src and ABL kinases. In addition, the BCR-ABL fusion gene product, a constitutively activated tyrosine kinase which is crucial for the development of chronic myeloid leukemia (CML), is highly sensitive to bosutinib. Interestingly, distinctly lower concentrations of bosutinib are required to ablate BCR-ABL phosphorylation when compared to the first-generation tyrosine kinase inhibitor imatinib (IM). Bosutinib is a potent inhibitor of CML cell proliferation in vitro and has demonstrated promising activity in CML patients resistant or intolerant to IM as well as in newly diagnosed patients with chronic phase CML (CML-CP). Remarkably, bosutinib has been found to be capable of overcoming the majority of IM-resistant BCR-ABL mutations. Bosutinib has the potency to induce deep and fast responses in second- and third-/fourth-line treatment, and as a consequence, the drug has recently been licensed for patients previously treated with one or more tyrosine kinase inhibitor(s) and for whom imatinib, nilotinib, and dasatinib are not considered appropriate treatment options. Due to its potency and differing toxicity profile, it promises to be a good therapeutic option for a defined cohort of patients. The most common side effects are gastrointestinal with most of the patients suffering from nausea, vomiting, or diarrhea. For the most part, these gastrointestinal symptoms occur early after treatment initiation, are manageable, and often self-limiting. Continuous monitoring of liver enzymes upon treatment initiation is necessary during bosutinib treatment. In addition to CML treatment, bosutinib has shown some efficacy in selected patients suffering from advanced-stage solid tumors. In conclusion, bosutinib is a promising novel small molecule inhibitor approved now for targeted therapy of CML and in clinical development for other malignancies. PMID:24756786

  1. Monitoring Conformational Changes in the Receptor Tyrosine Kinase EGFR.

    PubMed

    Becker, Christian; Öcal, Sinan; Nguyen, Hoang D; Phan, Trang; Keul, Marina; Simard, Jeffrey R; Rauh, Daniel

    2016-06-01

    The receptor tyrosine kinase EGFR is regulated by complex conformational changes, and this conformational control is disturbed in certain types of cancer. Many ligands are known to bind EGFR in its active conformation, thereby preventing ATP from binding. Only a few ligands are known to stabilize EGFR in its inactive conformation, thus providing novel strategies for perturbing EGFR activity. We report a direct binding assay that enables the identification of novel ligands that bind to and stabilize the inactive conformation of EGFR. PMID:26991964

  2. Proline-rich tyrosine kinase 2 downregulates peroxisome proliferator-activated receptor gamma to promote hypoxia-induced pulmonary artery smooth muscle cell proliferation.

    PubMed

    Bijli, Kaiser M; Kang, Bum-Yong; Sutliff, Roy L; Hart, C Michael

    2016-06-01

    Hypoxia stimulates pulmonary hypertension (PH), in part by increasing the proliferation of human pulmonary artery smooth muscle cells (HPASMCs) via sustained activation of mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2 (ERK 1/2), and nuclear factor-kappa B (NF-κB); elevated expression of NADPH oxidase 4 (Nox4); and downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) levels. However, the upstream mediators that control these responses remain largely unknown. We hypothesized that proline-rich tyrosine kinase 2 (Pyk2) plays a critical role in the mechanism of hypoxia-induced HPASMC proliferation. To test this hypothesis, HPASMCs were exposed to normoxia or hypoxia (1% O2) for 72 hours. Hypoxia activated Pyk2 (detected as Tyr402 phosphorylation), and inhibition of Pyk2 with small interfering RNA (siRNA) or tyrphostin A9 attenuated hypoxia-induced HPASMC proliferation. Pyk2 inhibition attenuated ERK 1/2 activation as early as 24 hours after the onset of hypoxia, suggesting a proximal role for Pyk2 in this response. Pyk2 inhibition also attenuated hypoxia-induced NF-κB activation, reduced HPASMC PPARγ messenger RNA levels and activity, and increased NF-κB-mediated Nox4 levels. The siRNA-mediated PPARγ knockdown enhanced Pyk2 activation, whereas PPARγ overexpression reduced Pyk2 activation in HPASMCs, confirming a reciprocal relationship between Pyk2 and PPARγ. Pyk2 depletion also attenuated hypoxia-induced NF-κB p65 activation and reduced PPARγ protein levels in human pulmonary artery endothelial cells. These in vitro findings suggest that Pyk2 plays a central role in the proliferative phenotype of pulmonary vascular wall cells under hypoxic conditions. Coupled with recent reports that hypoxia-induced PH is attenuated in Pyk2 knockout mice, these findings suggest that Pyk2 may represent a novel therapeutic target in PH. PMID:27252847

  3. Proline-rich tyrosine kinase 2 downregulates peroxisome proliferator–activated receptor gamma to promote hypoxia-induced pulmonary artery smooth muscle cell proliferation

    PubMed Central

    2016-01-01

    Abstract Hypoxia stimulates pulmonary hypertension (PH), in part by increasing the proliferation of human pulmonary artery smooth muscle cells (HPASMCs) via sustained activation of mitogen-activated protein kinase, extracellular signal–regulated kinases 1 and 2 (ERK 1/2), and nuclear factor-kappa B (NF-κB); elevated expression of NADPH oxidase 4 (Nox4); and downregulation of peroxisome proliferator–activated receptor gamma (PPARγ) levels. However, the upstream mediators that control these responses remain largely unknown. We hypothesized that proline-rich tyrosine kinase 2 (Pyk2) plays a critical role in the mechanism of hypoxia-induced HPASMC proliferation. To test this hypothesis, HPASMCs were exposed to normoxia or hypoxia (1% O2) for 72 hours. Hypoxia activated Pyk2 (detected as Tyr402 phosphorylation), and inhibition of Pyk2 with small interfering RNA (siRNA) or tyrphostin A9 attenuated hypoxia-induced HPASMC proliferation. Pyk2 inhibition attenuated ERK 1/2 activation as early as 24 hours after the onset of hypoxia, suggesting a proximal role for Pyk2 in this response. Pyk2 inhibition also attenuated hypoxia-induced NF-κB activation, reduced HPASMC PPARγ messenger RNA levels and activity, and increased NF-κB-mediated Nox4 levels. The siRNA-mediated PPARγ knockdown enhanced Pyk2 activation, whereas PPARγ overexpression reduced Pyk2 activation in HPASMCs, confirming a reciprocal relationship between Pyk2 and PPARγ. Pyk2 depletion also attenuated hypoxia-induced NF-κB p65 activation and reduced PPARγ protein levels in human pulmonary artery endothelial cells. These in vitro findings suggest that Pyk2 plays a central role in the proliferative phenotype of pulmonary vascular wall cells under hypoxic conditions. Coupled with recent reports that hypoxia-induced PH is attenuated in Pyk2 knockout mice, these findings suggest that Pyk2 may represent a novel therapeutic target in PH. PMID:27252847

  4. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ravikumar, Vaishnavi; Dobrinic, Paula; Macek, Boris; Franjevic, Damjan; Noirot-Gros, Marie-Francoise; Mijakovic, Ivan

    2014-01-01

    Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells. PMID:25278935

  5. Tyrosine kinase inhibitors: New class of antimalarials on the horizon?

    PubMed

    Pathak, Vrushali; Colah, Roshan; Ghosh, Kanjaksha

    2015-08-01

    Development of the antimalarial drug resistant strains has currently become a major public health challenge. There is an urgent need to develop new antimalarial drugs. Tyrosine kinase inhibitors (TKIs) are receiving increasing attention as anticancer therapy. It has revolutionarised the management of CML to say the least. TKIs are also increasingly being implicated in complicated but vital life cycle of malaria parasite. Hence we tested two commonly used but different classes of TKIs (imatinib and sorafenib) in-vitro for their antimalarial activity and possible synergistic activity with existing antimalarial drug. Antimalarial activity was tested with the help of modified WHO microtest technique in-vitro for five different Plasmodium falciparum laboratory strains (3D7, Dd2, 7G8, MRC2, PKL9). Imatinib and sorafenib showed a promising antimalarial activity with all the strains. These compounds caused dose dependent inhibition of parasite maturation. The isobologram analysis of the interactions of these TKIs with standard antimalarial drug, artesunate revealed distinct patterns of synergism, additivity and antagonism at different ratios. Imatinib showed worthwhile synergism with artesunate indicating imatinib and other tyrosine kinase inhibitors may have significant antimalarial activity and can be used in combination therapy. PMID:26142327

  6. [Side effect management of tyrosine kinase inhibitors in urology : Hypertension].

    PubMed

    Sikic, D; Meidenbauer, N; Lieb, V; Keck, B

    2016-07-01

    Tyrosine kinase inhibitors like sunitinib, sorafenib, pazopanib or axintinib are regarded the standard of care in the systemic therapy of metastatic renal cell carcinoma. However, the many side effects associated with this therapy pose challenges for the treating physician and the patient. This review offers an overview of the classification and the treatment of hypertension, which is one of the major side effects induced by all tyrosine kinase inhibitors, in order to improve treatment efficacy and patient compliance. PMID:27146871

  7. Pulmonary adenocarcinoma in situ: analyses of a large series with reference to smoking, driver mutations, and receptor tyrosine kinase pathway activation.

    PubMed

    Sato, Seijiro; Motoi, Noriko; Hiramatsu, Miyako; Miyauchi, Eisaku; Ono, Hiroshi; Saito, Yuichi; Nagano, Hiroko; Ninomiya, Hironori; Inamura, Kentaro; Uehara, Hirofumi; Mun, Mingyon; Sakao, Yukinori; Okumura, Sakae; Tsuchida, Masanori; Ishikawa, Yuichi

    2015-07-01

    Lung adenocarcinomas in situ (AISs) often occur in individuals who have never smoked, although smoking is one of the main causes of lung cancer. To characterize AIS and, in particular, determine how AIS might be related to smoking, we collected a large number of AIS cases and examined clinicopathologic features, EGFR and KRAS mutation status, and activation status of receptor tyrosine kinase downstream signal pathways, including pAkt, pERK, and pStat3, using immunohistochemistry. We identified 110 AISs (36 smokers and 74 nonsmokers) among 1549 adenocarcinomas resected surgically during 1995 to 2010. Between the AIS of smokers and nonsmokers, only the sex ratio was significantly different; all the other clinicopathologic factors including TTF-1 and driver mutations were not significantly different: EGFR and KRAS mutation rates (smokers:nonsmokers) were 61:58 (%) (P=0.7) and 6.1:1.4 (%) (P=0.2), respectively, whereas, in invasive adenocarcinomas, the rates were 41:69 (%) (P<0.001) and 9.4:2.3 (%) (P<0.04), respectively. For pAkt and pERK, around 40% to 50% of AISs were positive, and for pStat3, >80% were positive, with no significant differences between smokers and nonsmokers with AIS. Mucinous AIS (n=8) rarely harbored KRAS mutations and expressed significantly less pStat3 (P<0.001) than nonmucinous AIS. Taken together, AIS occurs predominantly in female individuals and nonsmokers. However, characteristics of AIS arising in smokers and nonsmokers were similar in terms of cell lineage, driver mutations, and receptor tyrosine kinase pathway activation. Our results suggest that smoking is not a major cause of AIS. Rather, smoking may play a role in progression of AIS to invasive adenocarcinoma with AIS features. PMID:25970685

  8. The Tyrosine Kinase Activity of c-Src Regulates Actin Dynamics and Organization of Podosomes in Osteoclasts

    PubMed Central

    Destaing, Olivier; Sanjay, Archana; Itzstein, Cecile; Horne, William C.; Toomre, Derek

    2008-01-01

    Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src−/− OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src−/− OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src−/− podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src−/− OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src−/− OCLs by Src inhibitors or by expressing dominant-negative SrcK295M induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization. PMID:17978100

  9. Molecular Basis of the Interaction of the Human Protein Tyrosine Phosphatase Non-receptor Type 4 (PTPN4) with the Mitogen-activated Protein Kinase p38γ.

    PubMed

    Maisonneuve, Pierre; Caillet-Saguy, Célia; Vaney, Marie-Christine; Bibi-Zainab, Edoo; Sawyer, Kristi; Raynal, Bertrand; Haouz, Ahmed; Delepierre, Muriel; Lafon, Monique; Cordier, Florence; Wolff, Nicolas

    2016-08-01

    The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction. PMID:27246854

  10. Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2.

    PubMed

    Rose, David M; Liu, Shouchun; Woodside, Darren G; Han, Jaewon; Schlaepfer, David D; Ginsberg, Mark H

    2003-06-15

    Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1). PMID:12794117

  11. Receptor tyrosine kinase inhibitors: Are they real tumor killers?

    PubMed

    Gaumann, Andreas K A; Kiefer, Friedemann; Alfer, Joachim; Lang, Sven A; Geissler, Edward K; Breier, Georg

    2016-02-01

    Inhibiting tumor growth by targeting the tumor vasculature was first proposed by Judah Folkman almost 40 years ago. Since then, different approaches and numerous drugs and agents have been developed to achieve this goal, either with the aim of inhibiting tumor neoangiogenesis or normalizing the tumor vasculature. Among the most promising therapeutic targets are receptor tyrosine kinases (RTKs), some of which are predominantly expressed on tumor endothelial cells, although they are sometimes also present on tumor cells. The majority of RTK inhibitors investigated over the past two decades competes with ATP at the active site of the kinase and therefore block the phosphorylation of intracellular targets. Some of these drugs have been approved for therapy, whereas others are still in clinical trials. Here, we discuss the scientific basis, current status, problems and future prospects of RTK inhibition in anti-tumor therapy. PMID:25716346

  12. Spleen tyrosine kinase suppresses osteoblastic differentiation through MAPK and PKCα.

    PubMed

    Yoshida, Kiyoshi; Higuchi, Chikahisa; Nakura, Akio; Yoshikawa, Hideki

    2011-08-12

    Spleen tyrosine kinase (Syk) is a non-receptor protein kinase present in abundance in a wide range of hematopoietic cells. Syk reportedly plays a crucial role in immune signaling in B cells and cells bearing Fcγ-activation receptors. The role of syk in osteoblastic differentiation has not been well elucidated. We report herein the role of syk in osteoblastic differentiation. We investigated the effects of two syk inhibitors on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. Expression of syk was detected in these two cell lines. Two syk inhibitors stimulated mRNA expression of osteoblastic markers (ALP, Runx2, Osterix). Mineralization of extracellular matrix was also promoted by treatment with syk inhibitors. Knockdown of Syk caused increased mRNA expression of osteoblastic markers. In addition, syk inhibitor and knockdown of Syk suppressed phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase Cα (PKCα). Our results indicate that syk might regulate osteoblastic differentiation through MAPK and PKCα. PMID:21782794

  13. De novo design of VEGFR-2 tyrosine kinase inhibitors based on a linked-fragment approach.

    PubMed

    Liu, Yi-Zhou; Wang, Xiao-Li; Wang, Xin-Ying; Yu, Ri-Lei; Liu, Dong-Qing; Kang, Cong-Min

    2016-09-01

    Vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitors have been demonstrated to possess substantial antitumor activity. VEGFR-2 tyrosine kinase inhibitors are crucial for development of antitumor drugs. Based on the crystal structure of VEGFR-2 tyrosine kinase, a linked-fragment strategy was employed to design novel VEGFR-2 tyrosine kinase inhibitors, and 1000 compounds were generated in this process. Absorption, distribution, metabolism, excretion and toxicity (ADMET) were used to screen the 1000 compounds, and 59 compounds were acceptable. Scaffold hopping was then used for further screening, and only four compounds were obtained in this way. Then, the binding energy of the four molecules to VEGFR-2 tyrosine kinase was calculated using molecular docking, and their values were found to be lower than that of Sorafenib. Finally, molecular dynamics simulations were performed on the complex of the compound with the lowest binding energy with VEGFR-2 tyrosine kinase, and the binding model was analyzed. At the end, four chemical entities with novel structures were obtained, and were suggested for experimental testing in future studies. PMID:27558799

  14. Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

    PubMed Central

    Hitosugi, Taro; Fan, Jun; Chung, Tae-Wook; Lythgoe, Katherine; Wang, Xu; Xie, Jianxin; Ge, Qingyuan; Gu, Ting-Lei; Polakiewicz, Roberto D.; Roesel, Johannes L.; Chen, Zhuo (Georgia); Boggon, Titus J.; Lonial, Sagar; Fu, Haian; Khuri, Fadlo R.; Kang, Sumin; Chen, Jing

    2011-01-01

    SUMMARY Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate, and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth. PMID:22195962

  15. The kinase c-Src and the phosphatase TC45 coordinately regulate c-Fos tyrosine phosphorylation and c-Fos phospholipid synthesis activation capacity.

    PubMed

    Ferrero, G O; Velazquez, F N; Caputto, B L

    2012-07-12

    Our previous work showed that in T98G cells, a human glioblastoma multiforme-derived cell line, the association of c-Fos to the endoplasmic reticulum (ER) and consequently, the capacity of c-Fos to activate phospholipid synthesis, is regulated by the phosphorylation state of tyrosine (tyr) residues #10 and #30 of c-Fos. The small amount of c-Fos present in quiescent cells is tyr-phosphorylated, is dissociated from the ER membranes and does not activate phospholipid synthesis. However, on induction of the cell to re-enter growth, c-Fos expression is rapidly induced, it is found dephosphorylated, associated to ER membranes and activating phospholipid synthesis (Portal et al., 2007). Herein, using in vivo and in vitro experimental strategies, we show that the kinase c-Src is capable of phosphorylating tyr residues of c-Fos whereas the phosphatase TC45 T-cell protein-tyr phosphatase (TC-PTP) dephosphorylates them, thus enabling c-Fos/ER association and activation of phospholipid synthesis. Results also suggest that the regulation of the phosphorylation/dephosphorylation cycle of c-Fos occurs at the TC-PTP level: induction of cells to re-enter growth promotes the translocation of TC45 from a nuclear to a cytoplasmic location concomitant with its activation. Activated TC45 in its turn promotes dephosphorylation of pre-formed c-Fos, enabling cells to rapidly activate phospholipid synthesis to respond to its growth demands. PMID:22105363

  16. c-MYC Generates Repair Errors via Increased Transcription of Alternative-NHEJ Factors, LIG3 and PARP1, in Tyrosine Kinase-activated Leukemias

    PubMed Central

    Muvarak, Nidal; Kelley, Shannon; Robert, Carine; Baer, Maria R.; Perrotti, Danilo; Gambacorti-Passerini, Carlo; Civin, Curt; Scheibner, Kara; Rassool, Feyruz

    2015-01-01

    Leukemias expressing the constitutively activated tyrosine kinases (TKs) BCR-ABL1 and FLT3/ITD activate signaling pathways that increase genomic instability through generation of reactive oxygen species (ROS), DNA double-strand breaks (DSBs) and error-prone repair. The non-homologous end-joining (NHEJ) pathway is a major pathway for DSB repair and is highly aberrant in TK-activated-leukemias; an alternative form of NHEJ (ALT-NHEJ) predominates, evidenced by increased expression of DNA ligase IIIα (LIG3) and poly (ADP-ribose) polymerase (PARP1), increased frequency of large genomic deletions, and repair using DNA sequence microhomologies. This study, for the first time, demonstrates that the TK target c-MYC plays a role in transcriptional activation and subsequent expression of LIG3 and PARP1 and contributes to the increased error-prone repair observed in TK-activated leukemias. c-MYC negatively regulates microRNAs miR-150 and miR-22 which demonstrate an inverse correlation with LIG3 and PARP1 expression in primary and cultured leukemia cells and chronic myelogenous leukemia (CML) human patient samples. Notably, inhibition of c-MYC and overexpression of miR-150 and -22 decreases ALT-NHEJ activity. Thus, BCR-ABL1 or FLT3/ITD induces c-MYC expression leads to genomic instability via augmented expression of ALT-NHEJ repair factors that generate repair errors. PMID:25828893

  17. Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis

    PubMed Central

    Liu, Feng; Zhuang, Shougang

    2016-01-01

    Renal fibrosis can be induced in different renal diseases, but ultimately progresses to end stage renal disease. Although the pathophysiologic process of renal fibrosis have not been fully elucidated, it is characterized by glomerulosclerosis and/or tubular interstitial fibrosis, and is believed to be caused by the proliferation of renal inherent cells, including glomerular epithelial cells, mesangial cells, and endothelial cells, along with defective kidney repair, renal interstitial fibroblasts activation, and extracellular matrix deposition. Receptor tyrosine kinases (RTKs) regulate a variety of cell physiological processes, including metabolism, growth, differentiation, and survival. Many studies from in vitro and animal models have provided evidence that RTKs play important roles in the pathogenic process of renal fibrosis. It is also showed that tyrosine kinases inhibitors (TKIs) have anti-fibrotic effects in basic research and clinical trials. In this review, we summarize the evidence for involvement of specific RTKs in renal fibrosis process and the employment of TKIs as a therapeutic approach for renal fibrosis. PMID:27331812

  18. Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis.

    PubMed

    Liu, Feng; Zhuang, Shougang

    2016-01-01

    Renal fibrosis can be induced in different renal diseases, but ultimately progresses to end stage renal disease. Although the pathophysiologic process of renal fibrosis have not been fully elucidated, it is characterized by glomerulosclerosis and/or tubular interstitial fibrosis, and is believed to be caused by the proliferation of renal inherent cells, including glomerular epithelial cells, mesangial cells, and endothelial cells, along with defective kidney repair, renal interstitial fibroblasts activation, and extracellular matrix deposition. Receptor tyrosine kinases (RTKs) regulate a variety of cell physiological processes, including metabolism, growth, differentiation, and survival. Many studies from in vitro and animal models have provided evidence that RTKs play important roles in the pathogenic process of renal fibrosis. It is also showed that tyrosine kinases inhibitors (TKIs) have anti-fibrotic effects in basic research and clinical trials. In this review, we summarize the evidence for involvement of specific RTKs in renal fibrosis process and the employment of TKIs as a therapeutic approach for renal fibrosis. PMID:27331812

  19. Activation of muscle-specific receptor tyrosine kinase and binding to dystroglycan are regulated by alternative mRNA splicing of agrin.

    PubMed

    Scotton, Patrick; Bleckmann, Dorothee; Stebler, Michael; Sciandra, Francesca; Brancaccio, Andrea; Meier, Thomas; Stetefeld, Jörg; Ruegg, Markus A

    2006-12-01

    Agrin induces the aggregation of postsynaptic proteins at the neuromuscular junction (NMJ). This activity requires the receptor-tyrosine kinase MuSK. Agrin isoforms differ in short amino acid stretches at two sites, called A and B, that are localized in the two most C-terminal laminin G (LG) domains. Importantly, agrin isoforms greatly differ in their activities of inducing MuSK phosphorylation and of binding to alpha-dystroglycan. By using site-directed mutagenesis, we characterized the amino acids important for these activities of agrin. We find that the conserved tripeptide asparagineglutamate-isoleucine in the eight-amino acid long insert at the B-site is necessary and sufficient for full MuSK phosphorylation activity. However, even if all eight amino acids were replaced by alanines, this agrin mutant still has significantly higher MuSK phosphorylation activity than the splice version lacking any insert. We also show that binding to alpha-dystroglycan requires at least two LG domains and that amino acid inserts at the A and the B splice sites negatively affect binding. PMID:17012237

  20. Proline-rich tyrosine kinase 2 via enhancing signal transducer and activator of transcription 3-dependent cJun expression mediates retinal neovascularization

    PubMed Central

    Kumar, Raj; Singh, Nikhlesh K.; Rao, Gadiparthi N.

    2016-01-01

    Despite the involvement of proline-rich tyrosine kinase 2 (Pyk2) in endothelial cell angiogenic responses, its role in pathological retinal angiogenesis is not known. In the present study, we show that vascular endothelial growth factor A (VEGFA) induces Pyk2 activation in mediating human retinal microvascular endothelial cell (HRMVEC) migration, sprouting and tube formation. Downstream to Pyk2, VEGFA induced signal transducer and activator of transcription 3 (STAT3) activation and cJun expression in the modulation of HRMVEC migration, sprouting and tube formation. Consistent with these observations, hypoxia induced activation of Pyk2-STAT3-cJun signaling axis and siRNA-mediated downregulation of Pyk2, STAT3 or cJun levels substantially inhibited hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization. Together, these observations suggest that activation of Pyk2-mediated STAT3-cJun signaling is required for VEGFA-induced HRMVEC migration, sprouting and tube formation in vitro and hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization in vivo. PMID:27210483

  1. Regulation of therapeutic resistance in cancers by receptor tyrosine kinases

    PubMed Central

    Chen, Mei-Kuang; Hung, Mien-Chie

    2016-01-01

    In response to DNA damage lesions due to cellular stress, DNA damage response (DDR) pathways are activated to promote cell survival and genetic stability or unrepaired lesion-induced cell death. Current cancer treatments predominantly utilize DNA damaging agents, such as irradiation and chemotherapy drugs, to inhibit cancer cell proliferation and induce cell death through the activation of DDR. However, a portion of cancer patients is reported to develop therapeutic resistance to these DDR-inducing agents. One significant resistance mechanism in cancer cells is oncogenic kinase overexpression, which promotes cell survival by enhancing DNA damage repair pathways and evading cell cycle arrest. Among the oncogenic kinases, overexpression of receptor tyrosine kinases (RTKs) is reported in many of solid tumors, and numerous clinical trials targeting RTKs are currently in progress. As the emerging trend in cancer treatment combines DNA damaging agents and RTK inhibitors, it is important to understand the substrates of RTKs relative to the DDR pathways. In addition, alteration of RTK expression and their phosphorylated substrates can serve as biomarkers to stratify patients for combination therapies. In this review, we summarize the deleterious effects of RTKs on the DDR pathways and the emerging biomarkers for personalized therapy. PMID:27186434

  2. Mechanism of dual specificity kinase activity of DYRK1A.

    PubMed

    Walte, Agnes; Rüben, Katharina; Birner-Gruenberger, Ruth; Preisinger, Christian; Bamberg-Lemper, Simone; Hilz, Nikolaus; Bracher, Franz; Becker, Walter

    2013-09-01

    The function of many protein kinases is controlled by the phosphorylation of a critical tyrosine residue in the activation loop. Dual specificity tyrosine-phosphorylation-regulated kinases (DYRKs) autophosphorylate on this tyrosine residue but phosphorylate substrates on aliphatic amino acids. This study addresses the mechanism of dual specificity kinase activity in DYRK1A and related kinases. Tyrosine autophosphorylation of DYRK1A occurred rapidly during in vitro translation and did not depend on the non-catalytic domains or other proteins. Expression in bacteria as well as in mammalian cells revealed that tyrosine kinase activity of DYRK1A is not restricted to the co-translational autophosphorylation in the activation loop. Moreover, mature DYRK1A was still capable of tyrosine autophosphorylation. Point mutants of DYRK1A and DYRK2 lacking the activation loop tyrosine showed enhanced tyrosine kinase activity. A series of structurally diverse DYRK1A inhibitors was used to pharmacologically distinguish different conformational states of the catalytic domain that are hypothesized to account for the dual specificity kinase activity. All tested compounds inhibited substrate phosphorylation with higher potency than autophosphorylation but none of the tested inhibitors differentially inhibited threonine and tyrosine kinase activity. Finally, the related cyclin-dependent kinase-like kinases (CLKs), which lack the activation loop tyrosine, autophosphorylated on tyrosine both in vitro and in living cells. We propose a model of DYRK autoactivation in which tyrosine autophosphorylation in the activation loop stabilizes a conformation of the catalytic domain with enhanced serine/threonine kinase activity without disabling tyrosine phosphorylation. The mechanism of dual specificity kinase activity probably applies to related serine/threonine kinases that depend on tyrosine autophosphorylation for maturation. PMID:23809146

  3. Metalloprotease-disintegrin ADAM 12 binds to the SH3 domain of Src and activates Src tyrosine kinase in C2C12 cells.

    PubMed Central

    Kang, Q; Cao, Y; Zolkiewska, A

    2000-01-01

    ADAM 12, a member of the ADAM (protein containing a disintegrin and metalloprotease) family of metalloprotease-disintegrins, has been implicated in the differentiation and fusion of skeletal myoblasts, and its expression is dramatically up-regulated in many cancer cells. While the extracellular portion of ADAM 12 contains an active metalloprotease and a cell-adhesion domain, the function of the cytoplasmic portion is much less clear. In this paper, we show that the cytoplasmic tail of ADAM 12 mediates interactions with the non-receptor protein tyrosine kinase Src. The interaction is direct, specific, and involves the N-terminal proline-rich region in the cytoplasmic tail of ADAM 12 and the Src homology 3 (SH3) domain of Src. ADAM 12 and Src co-immunoprecipitate from transfected C2C12 cells, suggesting that the two proteins form a complex in vivo. Co-expression of Src and ADAM 12, but not ADAM 9, in C2C12 cells results in activation of the recombinant Src. Moreover, endogenous ADAM 12 associates with and activates endogenous Src in differentiating C2C12 cells. These results indicate that ADAM 12 may mediate adhesion-induced signalling during myoblast differentiation. PMID:11104699

  4. In situ autoradiography and ligand-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor I receptors in prepancreatic chicken embryos.

    PubMed Central

    Girbau, M; Bassas, L; Alemany, J; de Pablo, F

    1989-01-01

    We previously reported specific cross-linking of 125I-labeled insulin and 125I-labeled insulin-like growth factor I (IGF-I) to the alpha subunit of their respective receptors in chicken embryos of 20 somites and older. To achieve adequate sensitivity and localize spatially the receptors in younger embryos, we adapted an autoradiographic technique using whole-mounted chicken blastoderms. Insulin receptors and IGF-I receptors were expressed and could be localized as early as gastrulation, before the first somite is formed. Relative density was analyzed by a computer-assisted image system, revealing overall slightly higher binding of IGF-I than of insulin. Structures rich in both types of receptors were predominantly of ectodermal origin: Hensen's node in gastrulating embryos and neural folds, neural tube and optic vesicles during neurulation. The signal transduction capability of the receptors in early organogenesis was assessed by their ability to phosphorylate the exogenous substrate poly(Glu80Tyr20). Ligand-dependent tyrosine phosphorylation was demonstrable with both insulin and IGF-I in glycoprotein-enriched preparations from embryos at days 2 through 6 of embryogenesis. There was a developmentally regulated change in ligand-dependent tyrosine kinase activity, with a sharp increase from day 2 to day 4, in contrast with a small increase in the ligand binding. Binding of 125I-labeled IGF-I was, with the solubilized receptors, severalfold higher than binding of 125I-labeled insulin. However, the insulin-dependent phosphorylation was as high as the IGF-I-dependent phosphorylation at each developmental stage. Images PMID:2548191

  5. Co-expression of protein tyrosine kinases EGFR-2 and PDGFRβ with protein tyrosine phosphatase 1B in Pichia pastoris.

    PubMed

    Tu, Pham Ngoc; Wang, Yamin; Cai, Menghao; Zhou, Xiangshan; Zhang, Yuanxing

    2014-02-28

    The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Coexpression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and PDGFRβ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and PDGFRβ fusion proteins were purified by Ni(2+) affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and PDGFRβ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics. PMID:24248091

  6. Global Analysis of Human Nonreceptor Tyrosine Kinase Specificity Using High-Density Peptide Microarrays

    PubMed Central

    2015-01-01

    Protein kinases phosphorylate substrates in the context of specific phosphorylation site sequence motifs. The knowledge of the specific sequences that are recognized by kinases is useful for mapping sites of phosphorylation in protein substrates and facilitates the generation of model substrates to monitor kinase activity. Here, we have adapted a positional scanning peptide library method to a microarray format that is suitable for the rapid determination of phosphorylation site motifs for tyrosine kinases. Peptide mixtures were immobilized on glass slides through a layer of a tyrosine-free Y33F mutant avidin to facilitate the analysis of phosphorylation by radiolabel assay. A microarray analysis provided qualitatively similar results in comparison with the solution phase peptide library “macroarray” method. However, much smaller quantities of kinases were required to phosphorylate peptides on the microarrays, which thus enabled a proteome scale analysis of kinase specificity. We illustrated this capability by microarray profiling more than 80% of the human nonreceptor tyrosine kinases (NRTKs). Microarray results were used to generate a universal NRTK substrate set of 11 consensus peptides for in vitro kinase assays. Several substrates were highly specific for their cognate kinases, which should facilitate their incorporation into kinase-selective biosensors. PMID:25164267

  7. Regulatory Interactions between a Bacterial Tyrosine Kinase and Its Cognate Phosphatase*

    PubMed Central

    Temel, Deniz B.; Dutta, Kaushik; Alphonse, Sébastien; Nourikyan, Julien; Grangeasse, Christophe; Ghose, Ranajeet

    2013-01-01

    The cyclic process of autophosphorylation of the C-terminal tyrosine cluster (YC) of a bacterial tyrosine kinase and its subsequent dephosphorylation following interactions with a counteracting tyrosine phosphatase regulates diverse physiological processes, including the biosynthesis and export of polysaccharides responsible for the formation of biofilms or virulence-determining capsules. We provide here the first detailed insight into this hitherto uncharacterized regulatory interaction at residue-specific resolution using Escherichia coli Wzc, a canonical bacterial tyrosine kinase, and its opposing tyrosine phosphatase, Wzb. The phosphatase Wzb utilizes a surface distal to the catalytic elements of the kinase, Wzc, to dock onto its catalytic domain (WzcCD). WzcCD binds in a largely YC-independent fashion near the Wzb catalytic site, inducing allosteric changes therein. YC dephosphorylation is proximity-mediated and reliant on the elevated concentration of phosphorylated YC near the Wzb active site resulting from WzcCD docking. Wzb principally recognizes the phosphate of its phosphotyrosine substrate and further stabilizes the tyrosine moiety through ring stacking interactions with a conserved active site tyrosine. PMID:23543749

  8. Cell-free expression of functional receptor tyrosine kinases

    PubMed Central

    He, Wei; Scharadin, Tiffany M.; Saldana, Matthew; Gellner, Candice; Hoang-Phou, Steven; Takanishi, Christina; Hura, Gregory L.; Tainer, John A; Carraway III, Kermit L.; Henderson, Paul T.; Coleman, Matthew A.

    2015-01-01

    Receptor tyrosine kinases (RTKs) play critical roles in physiological and pathological processes, and are important anticancer drug targets. In vitro mechanistic and drug discovery studies of full-length RTKs require protein that is both fully functional and free from contaminating proteins. Here we describe a rapid cell-free and detergent-free co-translation method for producing full-length and functional ERBB2 and EGFR receptor tyrosine kinases supported by water-soluble apolipoprotein A-I based nanolipoprotein particles. PMID:26274523

  9. Small activating ribonucleic acid reverses tyrosine kinase inhibitor resistance in epidermal growth factor receptor‐mutant lung cancer by increasing the expression of phosphatase and tensin homolog

    PubMed Central

    Li, Meng; Peng, Zhongmin; Ren, Wangang

    2016-01-01

    Background Epidermal growth factor receptor‐tyrosine kinase inhibitors (TKI‐EGFRs) present a new prospect for the treatment of lung cancer. However, in clinical application, the majority of patients become TKI resistant within a year. More and more studies have shown that a loss of phosphatase and tensin homolog (PTEN) expression is associated with TKI resistance. An alternative method of upregulating PTEN expression may reverse TKI resistance. Methods We designed five candidate small activating ribonucleic acids (saRNAs) to target PTEN, and transfected them into H‐157 cells to screen out functional saRNA. We used reverse transcriptase‐polymerase chain reaction and Western blot to evaluate the effect of saRNA to PTEN expression. We then analyzed the growth and apoptosis of cells transfected with saRNA under the treatment of TKI to investigate whether saRNAs can reverse TKI resistance by upregulating PTEN expression. Results The functional saRNA we designed could upregulate PTEN expression. The H‐157 cells transfected with saRNA grew slower in the presence of TKI drugs than the cells that were not transfected with saRNA. The apoptosis rate was also obviously higher. Conclusions Our study proves that loss of PTEN expression is an important mechanism of TKI resistance. It is possible to control TKI resistance by upregulating PTEN expression using RNA activation technology. PMID:27385992

  10. Acquired resistance mechanisms to tyrosine kinase inhibitors in lung cancer with activating epidermal growth factor receptor mutation--diversity, ductility, and destiny.

    PubMed

    Suda, Kenichi; Mizuuchi, Hiroshi; Maehara, Yoshihiko; Mitsudomi, Tetsuya

    2012-12-01

    Lung cancers that harbor somatic activating mutations in the gene for the epidermal growth factor receptor (EGFR) depend on mutant EGFR for their proliferation and survival; therefore, lung cancer patients with EGFR mutations often dramatically respond to orally available EGFR tyrosine kinase inhibitors (TKIs). However, emergence of acquired resistance is virtually inevitable, thus limiting improvement in patient outcomes. To elucidate and overcome this acquired resistance, multidisciplinary basic and clinical investigational approaches have been applied, using in vitro cell line models or samples obtained from lung cancer patients treated with EGFR-TKIs. These efforts have revealed several acquired resistance mechanisms and candidates, including EGFR secondary mutations (T790M and other rare mutations), MET amplification, PTEN downregulation, CRKL amplification, high-level HGF expression, FAS-NFκB pathway activation, epithelial-mesenchymal transition, and conversion to small cell lung cancer. Interestingly, cancer cells harbor potential destiny and ductility together in acquiring resistance to EGFR-TKIs, as shown in in vitro acquired resistance models. Molecular mechanisms of "reversible EGFR-TKI tolerance" that occur in early phase EGFR-TKI exposure have been identified in cell line models. Furthermore, others have reported molecular markers that can predict response to EGFR-TKIs in clinical settings. Deeper understanding of acquired resistance mechanisms to EGFR-TKIs, followed by the development of molecular target drugs that can overcome the resistance, might turn this fatal disease into a chronic disorder. PMID:22736441

  11. Angiotensin II-Induced Migration of Vascular Smooth Muscle Cells Is Mediated by p38 Mitogen-Activated Protein Kinase-Activated c-Src Through Spleen Tyrosine Kinase and Epidermal Growth Factor Receptor Transactivation

    PubMed Central

    Mugabe, Benon E.; Yaghini, Fariborz A.; Song, Chi Young; Buharalioglu, Cuneyt K.; Waters, Christopher M.

    2010-01-01

    Angiotensin II (Ang II) stimulates protein synthesis by activating spleen tyrosine kinase (Syk) and DNA synthesis through epidermal growth factor receptor (EGFR) transactivation in vascular smooth muscle cells (VSMCs). This study was conducted to determine whether Syk mediates Ang II-induced migration of aortic VSMCs using a scratch wound approach. Treatment with Ang II (200 nM) for 24 h increased VSMC migration by 1.56 ± 0.14-fold. Ang II-induced VSMC migration and Syk phosphorylation as determined by Western blot analysis were minimized by the Syk inhibitor piceatannol (10 μM) and by transfecting VSMCs with dominant-negative but not wild-type Syk plasmid. Ang II-induced VSMC migration and Syk phosphorylation were attenuated by inhibitors of c-Src [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2)], p38 mitogen-activated protein kinase (MAPK) [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB202190)], and extracellular signal-regulated kinase (ERK) 1/2 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126)]. SB202190 attenuated p38 MAPK and c-Src but not ERK1/2 phosphorylation, indicating that p38 MAPK acts upstream of c-Src and Syk. The c-Src inhibitor PP2 attenuated Syk and ERK1/2 phosphorylation, suggesting that c-Src acts upstream of Syk and ERK1/2. Ang II- and epidermal growth factor (EGF)-induced VSMC migration and EGFR phosphorylation were inhibited by the EGFR blocker 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) (2 μM). Neither the Syk inhibitor piceatannol nor the dominant-negative Syk mutant altered EGF-induced cell migration or Ang II- and EGF-induced EGFR phosphorylation. The c-Src inhibitor PP2 diminished EGF-induced VSMC migration and EGFR, ERK1/2, and p38 MAPK phosphorylation. The ERK1/2 inhibitor U0126 (10 μM) attenuated EGF-induced cell migration and ERK1/2 but not EGFR phosphorylation. These data suggest that Ang II stimulates VSMC migration via p38 MAPK-activated c-Src through

  12. Angiotensin II-induced migration of vascular smooth muscle cells is mediated by p38 mitogen-activated protein kinase-activated c-Src through spleen tyrosine kinase and epidermal growth factor receptor transactivation.

    PubMed

    Mugabe, Benon E; Yaghini, Fariborz A; Song, Chi Young; Buharalioglu, Cuneyt K; Waters, Christopher M; Malik, Kafait U

    2010-01-01

    Angiotensin II (Ang II) stimulates protein synthesis by activating spleen tyrosine kinase (Syk) and DNA synthesis through epidermal growth factor receptor (EGFR) transactivation in vascular smooth muscle cells (VSMCs). This study was conducted to determine whether Syk mediates Ang II-induced migration of aortic VSMCs using a scratch wound approach. Treatment with Ang II (200 nM) for 24 h increased VSMC migration by 1.56 +/- 0.14-fold. Ang II-induced VSMC migration and Syk phosphorylation as determined by Western blot analysis were minimized by the Syk inhibitor piceatannol (10 microM) and by transfecting VSMCs with dominant-negative but not wild-type Syk plasmid. Ang II-induced VSMC migration and Syk phosphorylation were attenuated by inhibitors of c-Src [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2)], p38 mitogen-activated protein kinase (MAPK) [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB202190)], and extracellular signal-regulated kinase (ERK) 1/2 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126)]. SB202190 attenuated p38 MAPK and c-Src but not ERK1/2 phosphorylation, indicating that p38 MAPK acts upstream of c-Src and Syk. The c-Src inhibitor PP2 attenuated Syk and ERK1/2 phosphorylation, suggesting that c-Src acts upstream of Syk and ERK1/2. Ang II- and epidermal growth factor (EGF)-induced VSMC migration and EGFR phosphorylation were inhibited by the EGFR blocker 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) (2 microM). Neither the Syk inhibitor piceatannol nor the dominant-negative Syk mutant altered EGF-induced cell migration or Ang II- and EGF-induced EGFR phosphorylation. The c-Src inhibitor PP2 diminished EGF-induced VSMC migration and EGFR, ERK1/2, and p38 MAPK phosphorylation. The ERK1/2 inhibitor U0126 (10 microM) attenuated EGF-induced cell migration and ERK1/2 but not EGFR phosphorylation. These data suggest that Ang II stimulates VSMC migration via p38 MAPK-activated c

  13. Identification and analysis of a novel protein-tyrosine kinase from bovine thymus

    SciTech Connect

    Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

    1986-05-01

    A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn/sup 2 +/ and (..gamma..-/sup 32/P)ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes.

  14. Curcumin suppresses Janus kinase-STAT inflammatory signaling through activation of Src homology 2 domain-containing tyrosine phosphatase 2 in brain microglia.

    PubMed

    Kim, Hee Young; Park, Eun Jung; Joe, Eun-Hye; Jou, Ilo

    2003-12-01

    Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that the inhibitory action of curcumin on Janus kinase (JAK)-STAT signaling can contribute to its anti-inflammatory activity in the brain. In both rat primary microglia and murine BV2 microglial cells, curcumin effectively suppressed the ganglioside-, LPS-, or IFN-gamma-stimulated induction of cyclooxygenase-2 and inducible NO synthase, important enzymes that mediate inflammatory processes. These anti-inflammatory effects appear to be due, at least in part, to the suppression of the JAK-STAT inflammatory signaling cascade. Curcumin markedly inhibited the phosphorylation of STAT1 and 3 as well as JAK1 and 2 in microglia activated with gangliosides, LPS, or IFN-gamma. Curcumin consistently suppressed not only NF binding to IFN-gamma-activated sequence/IFN-stimulated regulatory element, but also the expression of inflammation-associated genes, including ICAM-1 and monocyte chemoattractant protein 1, whose promoters contain STAT-binding elements. We further show that activation of Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-2, a negative regulator of JAK activity, is likely to be one of the mechanisms underlying the curcumin-mediated inhibition of JAK-STAT signaling. Treatment of microglial cells with curcumin led to an increase in phosphorylation and association with JAK1/2 of SHP-2, which inhibit the initiation of JAK-STAT inflammatory signaling in activated microglia. Taken together, these data suggest curcumin suppresses JAK-STAT signaling via activation of SHP-2, thus attenuating inflammatory response of brain microglial cells. PMID:14634121

  15. Analysis of receptor tyrosine kinase internalization using flow cytometry.

    PubMed

    Li, Ning; Hill, Kristen S; Elferink, Lisa A

    2008-01-01

    The internalization of activated receptor tyrosine kinases (RTKs) by endocytosis and their subsequent down regulation in lysosomes plays a critical role in regulating the duration and intensity of downstream signaling events. Uncoupling of the RTK cMet from ligand-induced degradation was recently shown to correlate with sustained receptor signaling and increased cell tumorigenicity, suggesting that the corruption of these endocytic mechanisms could contribute to increased cMet signaling in metastatic cancers. To understand how cMet signaling for normal cell growth is controlled by endocytosis and how these mechanisms are dysregulated in metastatic cancers, we developed flow cytometry-based assays to examine cMet internalization. PMID:19066037

  16. Mechanisms of resistance to EGFR tyrosine kinase inhibitors

    PubMed Central

    Huang, Lihua; Fu, Liwu

    2015-01-01

    Since the discovery that non-small cell lung cancer (NSCLC) is driven by epidermal growth factor receptor (EGFR) mutations, the EGFR tyrosine kinase inhibitors (EGFR-TKIs, e.g., gefitinib and elrotinib) have been effectively used for clinical treatment. However, patients eventually develop drug resistance. Resistance to EGFR-TKIs is inevitable due to various mechanisms, such as the secondary mutation (T790M), activation of alternative pathways (c-Met, HGF, AXL), aberrance of the downstream pathways (K-RAS mutations, loss of PTEN), impairment of the EGFR-TKIs-mediated apoptosis pathway (BCL2-like 11/BIM deletion polymorphism), histologic transformation, ATP binding cassette (ABC) transporter effusion, etc. Here we review and summarize the known resistant mechanisms to EGFR-TKIs and provide potential targets for development of new therapeutic strategies. PMID:26579470

  17. AG490 and PF431396 Sensitive Tyrosine Kinase Control the Population Heterogeneity of Basal STAT1 Activity in Ube1l Deficient Cells

    PubMed Central

    Now, Hesung; Yoo, Joo-Yeon

    2016-01-01

    A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that Ube1l−/− MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in Ube1l knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity. PMID:27427993

  18. Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression.

    PubMed

    Kotani, H; Ebi, H; Kitai, H; Nanjo, S; Kita, K; Huynh, T G; Ooi, A; Faber, A C; Mino-Kenudson, M; Yano, S

    2016-07-01

    Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR

  19. Intracerebroventricular administration of ouabain, a Na/K-ATPase inhibitor, activates tyrosine hydroxylase through extracellular signal-regulated kinase in rat striatum.

    PubMed

    Yu, Hyun Sook; Kim, Se Hyun; Park, Hong Geun; Kim, Yong Sik; Ahn, Yong Min

    2011-11-01

    Alteration in dopamine neurotransmission has been reported to be involved in the mania of bipolar disorder. Tyrosine hydroxylase (TH) is the rate-limiting enzyme that is crucial for dopamine biosynthesis, and its activity is tightly regulated by phosphorylation at multiple N-terminal serine residues. Previously, we have reported that intracerebroventricular (ICV) injection of ouabain, a selective Na/K-ATPase inhibitor, induces hyperactivity in rats that mimics manic symptoms related to the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2), which plays crucial roles in the modulation of TH phosphorylation. In this study, we investigated the effects of ICV injection of ouabain on TH phosphorylation in rat striatum and the involvement of ERK1/2 in ouabain-induced TH activation. ICV ouabain induced an acute dose-dependent increase in locomotor activity and in TH phosphorylation in rat striatum. TH phosphorylation at Ser19 was significantly increased with 100, 500, and 1000μM ouabain, and phosphorylation at Ser31 and Ser40 was significantly increased with 500 and 1000μM. We also found that ICV pretreatment with U0126, a specific MEK1/2 inhibitor, attenuated the 1000μM ouabain-induced increase in TH phosphorylation at Ser19, Ser31, and Ser40, as well as the hyperactivity of rats. Moreover, the increased phosphorylation of TH (Ser19, Ser31, and Ser40) was maintained until 8h after single administration ouabain was accompanied by increased phosphorylation of ERK1/2 (Thr202/Tyr204) and p90RSK (Thr359/Ser363). These findings imply that TH activation of the ERK1/2 signal pathway could play an important role in ouabain-induced hyperactivity of rats, a mania model. PMID:21871514

  20. Ligand-independent activation of the arylhydrocarbon receptor by ETK (Bmx) tyrosine kinase helps MCF10AT1 breast cancer cells to survive in an apoptosis-inducing environment.

    PubMed

    Fujisawa, Yasuko; Li, Wen; Wu, Dalei; Wong, Patrick; Vogel, Christoph; Dong, Bin; Kung, Hsing-Jien; Matsumura, Fumio

    2011-10-01

    It has been reported that the arylhydrocarbon receptor (AHR) is overexpressed in certain types of breast tumors. However, so far no concrete evidence has been provided yet as to why and how the overexpressed AHR in those cancer cells is functionally activated without exogenous ligands. Here we show that the AHR was functionally activated when estrogen receptor-negative, AHR overexpressing MCF10AT1 human breast cancer cells (designated P20E) were subjected to serum starvation. Transfection of cells with ETK-KQ, a plasmid for kinase-dead epithelial and endothelial tyrosine kinase (ETK), attenuated this AHR activation. Artificial over-expression of ETK in P20E cells through transfection with wild-type ETK plasmid (ETK-wt) caused up-regulation of cytochrome P4501a1 (CYP1A1; a marker of functional activation of AHR). Furthermore, ablation of ETK expression by a specific antisense oligonucleotide or AG879, a specific inhibitor of ETK kinase suppressed activation of AHR induced by omeprazole, a strong ligand-independent activator of AHR. Activation of ETK in those cells conferred them resistance to UVB- as well as doxorubicin-induced apoptosis, both of which were reversed by ETK-KQ. Together, these findings support our conclusion that ETK is the tyrosine kinase responsible for the functional activation of the AHR in these mammary epithelial cells. PMID:21861773

  1. Old Tyrosine Kinase Inhibitors and Newcomers in Gastrointestinal Cancer Treatment.

    PubMed

    Erika, Giordani; Federica, Zoratto; Martina, Strudel; Anselmo, Papa; Luigi, Rossi; Marina, Minozzi; Davide, Caruso; Eleonora, Zaccarelli; Monica, Verrico; Silverio, Tomao

    2016-01-01

    Gastrointestinal cancer treatment is based more on molecular biology that has provided increasing knowledge about cancer pathogenesis on which targeted therapy is being developed. Precisely, targeted therapy is defined as a "type of treatment that uses drugs, such as monoclonal antibodies or tyrosine kinase inhibitors, to identify and attack specific cancer cells". Nowadays, the United States Food and Drug Administration has approved many targeted therapies for gastrointestinal cancer treatment, as many are in various phases of development as well. In a previous review we discussed the main monoclonal antibodies used and studied in gastrointestinal cancer. In addition to monoclonal antibodies, tyrosine kinase inhibitors represent another class of targeted therapy and following the approval of imatinib for gastrointestinal stromal tumours, other tyrosine kinase inhibitors have been approved for gastrointestinal cancers treatment such as sunitinib, regoragenib, sorafenib and erlotinib. Moving forward, the purpose of this review is to focus on the efficacy data of main tyrosine kinase inhibitors commonly used in the personalized treatment of each gastrointestinal tumour and to provide a comprehensive overview about experimental targeted therapies ongoing in this setting. PMID:26278713

  2. ACK1/TNK2 Tyrosine Kinase: Molecular Signaling and Evolving Role in Cancers

    PubMed Central

    Mahajan, Kiran; Mahajan, Nupam P.

    2014-01-01

    Deregulated tyrosine kinase signaling alters cellular homeostasis to drive cancer progression. The emergence of a non-receptor tyrosine kinase, ACK1 as an oncogenic kinase, has uncovered novel mechanisms by which tyrosine kinase signaling promotes cancer progression. While early studies focused on ACK1 (also known as activated Cdc42-associated kinase 1 or TNK2) as a cytosolic effecter of activated transmembrane receptor tyrosine kinases (RTKs), wherein it shuttles between the cytosol and the nucleus to rapidly transduce extracellular signals from the RTKs to the intracellular effectors, recent data unfold a new aspect of its functionality as an epigenetic regulator. ACK1 interacts with the Estrogen Receptor (ER)/histone demethylase KDM3A (JHDM2a) complex, modifies KDM3A by tyrosine phosphorylation to regulate transcriptional outcome at HOXA1 locus to promote the growth of tamoxifen-resistant breast cancer. It is also well established that ACK1 regulates the activity of Androgen Receptor (AR) by tyrosine phosphorylation to fuel the growth of hormone-refractory prostate cancers. Further, recent explosion in genomic sequencing has revealed recurrent ACK1 gene amplification and somatic mutations in a variety of human malignancies, providing a molecular basis for its role in neoplastic transformation. In this review, we will discuss the various facets of ACK1 signaling, including its newly uncovered epigenetic regulator function, which enables cells to bypass the blockade to major survival pathways to promote resistance to standard cancer treatments. Not surprisingly, cancer cells appear to acquire an `addiction’ to ACK1 mediated survival, particularly under stress conditions, such as growth factor deprivation or genotoxic insults or hormone deprivation. With the accelerated development of potent and selective ACK1 inhibitors, targeted treatment for cancers harboring aberrant ACK1 activity may soon become a clinical reality. PMID:25347744

  3. Matrix metalloproteinase-2 and -9 are induced differently by metal nanoparticles in human monocytes: The role of oxidative stress and protein tyrosine kinase activation

    PubMed Central

    Wan, Rong; Mo, Yiqun; Zhang, Xing; Chien, Sufan; Tollerud, David J.; Zhang, Qunwei

    2009-01-01

    Recently, many studies have shown that nanoparticles can translocate from the lungs to the circulatory system. As a particulate foreign body, nanoparticles could induce host responses such as reactive oxygen species (ROS) generation, inflammatory cytokine and matrix metalloproteinase (MMP) release which play a major role in tissue destruction and remodeling. However, the direct effects of nanoparticles on leukocytes, especially monocytes, are still unclear. The objective of the present study was to compare the ability of Nano-Co and Nano-TiO2 to cause alteration of transcription and activity of MMPs and to explore possible mechanisms. We hypothesized that non-toxic doses of some transition metal nanoparticles stimulate an imbalance of MMP/TIMP that cause MMP production that may contribute to their health effects. To test this hypothesis, U937 cells were treated with Nano-Co and Nano-TiO2 and cytotoxic effects and ROS generation were measured. The alteration of MMP-2 and MMP-9 expression and activity of MMP-2 and MMP-9 after exposure to these metal nanoparticles were subsequently determined. To investigate the potential signaling pathways involved in the Nano-Co-induced MMP activation, the ROS scavengers or inhibitors, AP-1 inhibitor, and protein tyrosine kinase (PTK) inhibitors were also used to pre-treat U937 cells. Our results demonstrated that exposure of U937 cells to Nano-Co, but not to Nano-TiO2, at a dose that does not cause cytotoxicity, resulted in ROS generation and up-regulation of MMP-2 and MMP-9 mRNA expression.. Our results also showed dose- and time-related increases in pro-MMP-2 and pro-MMP-9 gelatinolytic activities in conditioned media after exposure of U937 cells to Nano-Co, but not to Nano-TiO2. Nano-Co-induced pro-MMP-2 and pro-MMP-9 activity increases were inhibited by pre-treatment with ROS scavengers or inhibitors. We also demonstrated dose- and time-related decreases in tissue inhibitors of metalloproteinases 2 (TIMP-2) in U937 cells after

  4. Matrix metalloproteinase-2 and -9 are induced differently by metal nanoparticles in human monocytes: The role of oxidative stress and protein tyrosine kinase activation

    SciTech Connect

    Wan Rong; Mo Yiqun; Zhang Xing; Chien Sufan; Tollerud, David J.; Zhang Qunwei

    2008-12-01

    Recently, many studies have shown that nanoparticles can translocate from the lungs to the circulatory system. As a particulate foreign body, nanoparticles could induce host responses such as reactive oxygen species (ROS) generation, inflammatory cytokine and matrix metalloproteinase (MMP) release which play a major role in tissue destruction and remodeling. However, the direct effects of nanoparticles on leukocytes, especially monocytes, are still unclear. The objective of the present study was to compare the ability of Nano-Co and Nano-TiO{sub 2} to cause alteration of transcription and activity of MMPs and to explore possible mechanisms. We hypothesized that non-toxic doses of some transition metal nanoparticles stimulate an imbalance of MMP/TIMP that cause MMP production that may contribute to their health effects. To test this hypothesis, U937 cells were treated with Nano-Co and Nano-TiO{sub 2} and cytotoxic effects and ROS generation were measured. The alteration of MMP-2 and MMP-9 expression and activity of MMP-2 and MMP-9 after exposure to these metal nanoparticles were subsequently determined. To investigate the potential signaling pathways involved in the Nano-Co-induced MMP activation, the ROS scavengers or inhibitors, AP-1 inhibitor, and protein tyrosine kinase (PTK) inhibitors were also used to pre-treat U937 cells. Our results demonstrated that exposure of U937 cells to Nano-Co, but not to Nano-TiO{sub 2}, at a dose that does not cause cytotoxicity, resulted in ROS generation and up-regulation of MMP-2 and MMP-9 mRNA expression{sub ..} Our results also showed dose- and time-related increases in pro-MMP-2 and pro-MMP-9 gelatinolytic activities in conditioned media after exposure of U937 cells to Nano-Co, but not to Nano-TiO{sub 2}. Nano-Co-induced pro-MMP-2 and pro-MMP-9 activity increases were inhibited by pre-treatment with ROS scavengers or inhibitors. We also demonstrated dose- and time-related decreases in tissue inhibitors of metalloproteinases 2

  5. The EGFR Family: Not So Prototypical Receptor Tyrosine Kinases

    PubMed Central

    Lemmon, Mark A.; Schlessinger, Joseph; Ferguson, Kathryn M.

    2014-01-01

    The epidermal growth factor receptor (EGFR) was among the first receptor tyrosine kinases (RTKs) for which ligand binding was studied and for which the importance of ligand-induced dimerization was established. As a result, EGFR and its relatives have frequently been termed “prototypical” RTKs. Many years of mechanistic studies, however, have revealed that—far from being prototypical—the EGFR family is quite unique. As we discuss in this review, the EGFR family uses a distinctive “receptor-mediated” dimerization mechanism, with ligand binding inducing a dramatic conformational change that exposes a dimerization arm. Intracellular kinase domain regulation in this family is also unique, being driven by allosteric changes induced by asymmetric dimer formation rather than the more typical activation-loop phosphorylation. EGFR family members also distinguish themselves from other RTKs in having an intracellular juxtamembrane (JM) domain that activates (rather than autoinhibits) the receptor and a very large carboxy-terminal tail that contains autophosphorylation sites and serves an autoregulatory function. We discuss recent advances in mechanistic aspects of all of these components of EGFR family members, attempting to integrate them into a view of how RTKs in this important class are regulated at the cell surface. PMID:24691965

  6. Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

    SciTech Connect

    Nakayama, Yuji; Kawana, Akiko; Igarashi, Asae; Yamaguchi, Naoto . E-mail: nyama@p.chiba-u.ac.jp

    2006-07-15

    Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to {approx}200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation.

  7. The Tyrosine Kinase c-Src Specifically Binds to the Active Integrin αIIbβ3 to Initiate Outside-in Signaling in Platelets*

    PubMed Central

    Wu, Yibing; Span, Lisa M.; Nygren, Patrik; Zhu, Hua; Moore, David T.; Cheng, Hong; Roder, Heinrich; DeGrado, William F.; Bennett, Joel S.

    2015-01-01

    It is currently believed that inactive tyrosine kinase c-Src in platelets binds to the cytoplasmic tail of the β3 integrin subunit via its SH3 domain. Although a recent NMR study supports this contention, it is likely that such binding would be precluded in inactive c-Src because an auto-inhibitory linker physically occludes the β3 tail binding site. Accordingly, we have re-examined c-Src binding to β3 by immunoprecipitation as well as NMR spectroscopy. In unstimulated platelets, we detected little to no interaction between c-Src and β3. Following platelet activation, however, c-Src was co-immunoprecipitated with β3 in a time-dependent manner and underwent progressive activation as well. We then measured chemical shift perturbations in the 15N-labeled SH3 domain induced by the C-terminal β3 tail peptide NITYRGT and found that the peptide interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the β1 cytoplasmic tail induced only small chemical shift perturbations on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the β3 peptide to the RT- loop. Under these conditions, the β3 peptide induced chemical shift perturbations similar to the negative control. We conclude that the primary interaction of c-Src with the β3 tail occurs in its activated state and at a site that overlaps with PPII binding site in its SH3 domain. Interactions of inactive c-Src with β3 are weak and insensitive to β3 tail mutations. PMID:25947380

  8. Antigen receptor signaling: integration of protein tyrosine kinase functions.

    PubMed

    Tamir, I; Cambier, J C

    1998-09-17

    Antigen receptors on T and B cells function to transduce signals leading to a variety of biologic responses minimally including antigen receptor editing, apoptotic death, developmental progression, cell activation, proliferation and survival. The response to antigen depends upon antigen affinity and valence, involvement of coreceptors in signaling and differentiative stage of the responding cell. The requirement that these receptors integrate signals that drive an array of responses may explain their evolved structural complexity. Antigen receptors are composed of multiple subunits compartmentalized to provide antigen recognition and signal transduction function. In lieu of on-board enzymatic activity these receptors rely on associated Protein Tyrosine Kinases (PTKs) for their signaling function. By aggregating the receptors, and hence their appended PTKs, antigens induce PTK transphosphorylation, activating them to phosphorylate the receptor within conserved motifs termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in transducer subunits. The tyrosyl phosphorylated ITAMs then interact with Src Homology 2 (SH2) domains within the PTKs leading to their further activation. As receptor phosphorylation is amplified, other effectors, such as Shc, dock by virtue of SH2 binding, and serve, in-turn, as substrates for these PTKs. This sequence of events not only provides a signal amplification mechanism by combining multiple consecutive steps with positive feedback, but also allows for signal diversification by differential recruitment of effectors that provide access to distinct parallel downstream signaling pathways. The subject of antigen receptor signaling has been recently reviewed in depth (DeFranco, 1997; Kurosaki, 1997). Here we discuss the biochemical basis of antigen receptor signal transduction, using the B cell receptor (BCR) as a paradigm, with specific emphasis on the involved PTKs. We review several specific mechanisms by which responses

  9. The role of tyrosine kinases in systemic lupus erythematosus and their potential as therapeutic targets

    PubMed Central

    Shao, Wen-Hai; Cohen, Philip L

    2014-01-01

    The autoimmune disease systemic lupus erythematosus is characterized by loss of tolerance to nuclear antigens. Breakdown of tolerance is associated with alterations in T-cell and B-cell receptor signal transduction, including increased protein phosphorylation that may underlie pathogenesis and explain the characteristic hyperactivity of T and B cells and other immune cells in active disease. Tyrosine kinases play a central role in signaling processes in cells known to be important in the pathogenesis of autoimmune diseases. Considerable progress has been made in understanding the function of tyrosine kinases in immune cell signaling pathways. In this review, we will summarize the function of tyrosine kinases and their novel inhibitors from studies made in animal lupus models and systemic lupus erythematosus patients. PMID:24678775

  10. Identification of novel inhibitors of BCR-ABL tyrosine kinase via virtual screening.

    PubMed

    Peng, Hui; Huang, Niu; Qi, Jing; Xie, Ping; Xu, Chen; Wang, Jianxiang; Yang, Chunzheng

    2003-11-01

    Inhibition of BCR-ABL tyrosine kinase activity has shown to be essential for the treatment of chronic myelogenous leukemia (CML). However, drug resistance has quickly arisen in recent clinical trials for STI571 (Gleevec), which is the first approved drug of CML by inhibiting ABL tyrosine kinase. It is desirable to develop new types of ABL tyrosine kinase inhibitors that may overcome this drug resistance problem. Here we present the discovery of novel inhibitors targeted at the catalytic domain of ABL tyrosine kinase by using three-dimensional database searching techniques. From a database containing 200,000 commercially available compounds, the top 1000 compounds with the best DOCK energy score were selected and subjected to structural diversity and drug likeness analysis, 15 compounds were submitted for biological assay. Eight out of the 15 showed inhibitory activity against K562 cells with IC(50) value ranging from 10 to 200 microM. Two promising compounds showed inhibition in further ABL tyrosine phosphorylation assay. It is anticipated that those two compounds can serve as lead compounds for further drug design and optimization. PMID:14552760

  11. Reduction of brain barrier tight junctional proteins by lead exposure: role of activation of nonreceptor tyrosine kinase Src via chaperon GRP78.

    PubMed

    Song, Han; Zheng, Gang; Shen, Xue-Feng; Liu, Xin-Qin; Luo, Wen-Jing; Chen, Jing-Yuan

    2014-04-01

    Lead (Pb) has long been recognized as a neurodevelopmental toxin. Developing blood-brain barrier (BBB) is known to be a target of Pb neurotoxicity; however, the underlying mechanisms are still unclear. Recent evidence suggests that intracellular nonreceptor protein tyrosine kinase Src regulates tight junctional proteins (TJPs). This study was designed to investigate whether Pb acted on the Src-mediated cascade event leading to an altered TJP expression at BBB. Rats aged 20-22 days were exposed to Pb in drinking water (0, 100, 200, and 300 ppm Pb) for eight weeks. Electron microscopic and Western blot analyses revealed a severe leakage of BBB and significantly decreased expressions of TJP occludin and ZO-1. When cultured brain endothelial RBE4 cells were exposed to 10μM Pb for 24 h, expressions of phosphor-Src and an upstream regulator GRP78 were significantly increased by 6.42-fold and 8.29-fold (p < 0.01), respectively. Inactivation of Src pathway by a Src-specific inhibitor reversed Pb-induced downregulation of occludin, but not ZO-1; small interfering RNA knockdown of GRP78 attenuated Pb-induced Src phosphorylation and occludin reduction. Furthermore, Pb exposure caused redistribution of GRP78 from endoplasmic reticulum to cytosol and toward cell member. However, the data from immunoneutralization studies did not show the involvement of cell-surface GRP78 in regulating Src phosphorylation upon Pb exposure, suggesting that the cytosolic GRP78, rather than cell-surface GRP78, was responsible to Pb-induced Src activation and ensuing occludin reduction. Taken together, this study provides the evidence of a novel linkage of GRP78, Src activation to downregulation of occludin, and BBB disruption during Pb exposure. PMID:24431214

  12. Antitumor activity of S116836, a novel tyrosine kinase inhibitor, against imatinib-resistant FIP1L1-PDGFRα-expressing cells

    PubMed Central

    Ding, Ke; Zhang, Zhang; Wang, Deping; Pan, Jingxuan

    2014-01-01

    The FIP1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRα) fusion oncogene is the driver factor in a subset of patients with hypereosinophilic syndrome (HES)/chronic eosinophilic leukemia (CEL). Most FIP1L1-PDGFRα-positive patients respond well to the tyrosine kinase inhibitor (TKI) imatinib. Resistance to imatinib in HES/CEL has been described mainly due to the T674I mutation in FIP1L1-PDGFRα, which is homologous to the imatinib-resistant T315I mutation in BCR-ABL. Development of novel TKIs is imperative to overcome resistance to imatinib. We synthesized S116836, a novel TKI. In this study, we evaluated the antitumor activity of S116836 in FIP1L1-PDGFRα-expressing cells. The results showed that S116836 potently inhibited PDGFRα and its downstream signaling molecules such as STAT3, AKT, and Erk1/2. S116836 effectively inhibited the growth of the WT and T674I FIP1L1-PDGFRα-expressing neoplastic cells in vitro and in nude mouse xenografts. Moreover, S116836 induced intrinsic pathway of apoptosis as well as the death receptor pathway, coincided with up-regulation of the proapoptotic BH3-only protein Bim-EL through the Erk1/2 pathway. In conclusion, S116836 is active against WT and T674I FIP1L1-PDGFRα-expressing cells, and may be a prospective agent for the treatment of HES/CEL. PMID:25431951

  13. Ligand-independent tyrosine kinase signalling in RTH 149 trout hepatoma cells: comparison among heavy metals and pro-oxidants.

    PubMed

    Burlando, Bruno; Magnelli, Valeria; Panfoli, Isabella; Berti, Elena; Viarengo, Aldo

    2003-01-01

    Tyrosine phosphorylation depends on the activity of receptor and non-receptor tyrosine kinases and promote cell growth, differentiation and apoptosis. Different stressors are known to stimulate tyrosine kinase activities and this could explain a wide spectrum of effects that these agents produce on different organisms. We studied the effects of heavy metals and pro-oxidants on tyrosine kinase signalling in trout hepatoma cells (RTH 149) by Western immunoblotting. Use of antiphosphotyrosine showed that Hg(2+) and Cu(2+)in the microM range, and H(2)O(2) in the mM range, induced tyrosine phosphorylation. The effect of Cu(2+)was prevented by pre-incubation with genistein, while those of Hg(2+)and H(2)O(2) were only decreased, probably due to tyrosine kinase stimulation coupled to phosphatase inhibition. Phosphospecific antibodies against the three types of MAPKs showed that ERK is activated by heavy metals only, while p38 and SAPK/JNK are activated by H(2)O(2), Hg(2+), and Cu(2+) plus low H(2)O(2). Cell pre-incubation with p38 inhibitors indicated that ERK activation by H(2)O(2) is prevented by concomitant activation of p38. Phosphospecific STAT antibodies revealed activation by H(2)O(2) only. In conclusion, fish cell exposure to heavy metals and pro-oxidants produce specific tyrosine kinase responses, involving cross talk and redox modulatory effects. PMID:12876385

  14. Transformation and scattering activities of the receptor tyrosine kinase RON/Stk in rodent fibroblasts and lack of regulation by the jaagsiekte sheep retrovirus receptor, Hyal2

    PubMed Central

    Miller, A Dusty; Van Hoeven, Neal S; Liu, Shan-Lu

    2004-01-01

    Background The envelope (Env) protein of jaagsiekte sheep retrovirus (JSRV) can transform cells in culture and is likely to be the main factor responsible for lung cancer induction by JSRV in animals. A recent report indicates that the epithelial-cell transforming activity of JSRV Env depends on activation of the cell-surface receptor tyrosine kinase Mst1r (called RON for the human and Stk for the rodent orthologs). In the immortalized line of human epithelial cells used (BEAS-2B cells), the virus receptor Hyal2 was found to bind to and suppress the activity of RON. When Env was expressed it bound to Hyal2 causing its degradation, release of RON activity from Hyal2 suppression, and activation of pathways resulting in cell transformation. Methods Due to difficulty with reproducibility of the transformation assay in BEAS-2B cells, we have used more tractable rodent fibroblast models to further study Hyal2 modulation of RON/Stk transforming activity and potential effects of Hyal2 on RON/Stk activation by its natural ligand, macrophage stimulating protein (MSP). Results We did not detect transformation of NIH 3T3 cells by plasmids expressing RON or Stk, but did detect transformation of 208F rat fibroblasts by these plasmids at a very low rate. We were able to isolate 208F cell clones that expressed RON or Stk and that showed changes in morphology indicative of transformation. The parental 208F cells did not respond to MSP but 208F cells expressing RON or Stk showed obvious increases in scattering/transformation in response to MSP. Human Hyal2 had no effect on the basal or MSP-induced phenotypes of RON-expressing 208F cells, and human, mouse or rat Hyal2 had no effect on the basal or MSP-induced phenotypes of Stk-expressing 208F cells. Conclusions We have shown that RON or Stk expression in 208F rat fibroblasts results in a transformed phenotype that is enhanced by addition of the natural ligand for these proteins, MSP. Hyal2 does not directly modulate the basal or MSP

  15. SAFETY AND ACTIVITY OF TEMSIROLIMUS AND BEVACIZUMAB IN PATIENTS WITH ADVANCED RENAL CELL CARCINOMA PREVIOUSLY TREATED WITH TYROSINE KINASE INHIBITORS: A PHASE 2 CONSORTIUM STUDY

    PubMed Central

    Merchan, Jaime R.; Qin, Rui; Pitot, Henry; Picus, Joel; Liu, Glenn; Fitch, Tom; Maples, William J.; Flynn, Patrick J.; Fruth, Briant F.; Erlichman, Charles

    2015-01-01

    Purpose Bevacizumab or Temsirolimus regimens have clinical activity in the first line treatment of advanced renal cell carcinoma (RCC). This phase I/II trial was conducted to determine the safety of combining both agents and its efficacy in RCC patients who progressed on at least one prior anti-VEGF receptor tyrosine kinase inhibitor (RTKI) agent. Methods In the phase I portion, eligible patients were treated with Temsirolimus (25 mg IV weekly) and escalating doses of IV Bevacizumab (level 1=5mg/kg; level 2=10 mg/kg) every other week. The primary endpoint for the phase II portion (RTKI resistant patients) was the 6-month progression free rate. Secondary endpoints were response rate, toxicity evaluation, PFS and OS. Results MTD was not reached at the maximum dose administered in 12 phase I patients. Forty evaluable patients were treated with the phase II recommended dose (Temsirolimus 25 mg IV weekly and Bevacizumab 10 mg/kg IV every two weeks). The 6-month progression free rate was 40% (16/40 pts). Median PFS was 5.9 (4-7.8) months, and median OS was 20.6 (11.5-23.7) months. Partial response/stable/progressive disease were seen in 23%/63%/14% of patients. Most common grade 3-4 AEs included fatigue (17.8%), hypertriglyceridemia (11.1%), stomatitis (8.9%), proteinuria (8.9%), abdominal pain (6.7%), and anemia (6.7%). Baseline levels of serum sFLT-1 and VEGF-A were inversely correlated with PFS and OS, respectively. Conclusions Temsirolimus and Bevacizumab is a feasible combination in patients with advanced RCC previously exposed to oral anti-VEGF agents. The safety and efficacy results warrant further confirmatory studies in this patient population. PMID:25556030

  16. Atomistic Insights into Regulatory Mechanisms of the HER2 Tyrosine Kinase Domain: A Molecular Dynamics Study

    PubMed Central

    Telesco, Shannon E.; Radhakrishnan, Ravi

    2009-01-01

    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 αC 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. PMID:19289058

  17. Novel morpholin-3-one fused quinazoline derivatives as EGFR tyrosine kinase inhibitors.

    PubMed

    Qin, Xuemei; Lv, Yongjuan; Liu, Peng; Li, Zhipeng; Hu, Liming; Zeng, Chengchu; Yang, Leifu

    2016-03-15

    A series of novel morpholin-3-one-fused quinazoline derivatives were designed, synthesized and evaluated as EGFR tyrosine kinase inhibitors. Nineteen compounds showed significant inhibitory activities against EGFR(wt) kinase (IC50<1 μM). Compound a8 demonstrated the most potent inhibitory activity toward EGFR(wt) (IC50=53.1 nM). Compound a7 and a8 showed excellent inhibitory activities against mutant EGFR(T790M/L858R) and strong antiproliferative activity against H358 and A549 cell lines. Finally, molecular docking studies were performed to predict the possible binding mode of the target compounds. It is believed that this work would be very useful for designing a new series of tyrosine kinase inhibitors targeting EGFR. PMID:26879314

  18. Tyrosine kinase inhibitors (TKIs) in human and pet tumours with special reference to breast cancer: a comparative review.

    PubMed

    Ranieri, Girolamo; Pantaleo, Marianna; Piccinno, Mariagrazia; Roncetti, Maria; Mutinati, Maddalena; Marech, Ilaria; Patruno, Rosa; Rizzo, Annalisa; Sciorsci, Raffaele Luigi

    2013-11-01

    Tyrosine kinase receptors (TKRs) play a key role in tumour cell proliferation and survival since they are involved in endothelial cell activation leading to tumour neoangiogenesis. In particular, vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptor (PDGFR), stem cell factor receptor (c-KitR), and colony-stimulating factor 1 (CSF-1) are overexpressed or constitutively activated in human and pet malignancies. A variety of small molecule inhibitors targeting specific tyrosine kinases (known as tyrosine kinase inhibitors or TKIs) have recently been approved, or are under investigation, for the treatment of human cancer. TKI application in animal cancer is however relatively recent. This review aims to illustrate the major aspects of tyrosine kinase dysfunctions, with special regard to human and animal cancer of the mammary gland, providing an update on the background of the anti-angiogenic and anti-neoplastic properties of TKIs in human and veterinary cancer. PMID:23768779

  19. Cheminfomatic-based Drug Discovery of Human Tyrosine Kinase Inhibitors.

    PubMed

    Reid, Terry-Elinor; Fortunak, Joseph M; Wutoh, Anthony; Simon Wang, Xiang

    2016-01-01

    Receptor Tyrosine Kinases (RTKs) are essential components for regulating cell-cell signaling and communication events in cell growth, proliferation, differentiation, survival and metabolism. Deregulation of RTKs and their associated signaling pathways can lead to a wide variety of human diseases such as immunodeficiency, diabetes, arterosclerosis, psoriasis and cancer. Thus RTKs have become one of the most important drug targets families in recent decade. Pharmaceutical companies have dedicated their research efforts towards the discovery of small-molecule inhibitors of RTKs, many of which had been approved by the U.S. Food and Drug Administration (US FDA) or are currently in clinical trials. The great successes in the development of small-molecule inhibitors of RTKs are largely attributed to the use of modern cheminformatic approaches to identifying lead scaffolds. Those include the quantitative structure-activity relationship (QSAR) modeling, as well as the structure-, and ligand-based pharmacophore modeling techniques in this case. Herein we inspected the literature thoroughly in an effort to conduct a comparative analysis of major findings regarding the essential structure-activity relationships (SARs)/pharmacophore features of known active RTK inhibitors, most of which were collected from cheminformatic modeling approaches. PMID:26369823

  20. Computer-aided identification of EGFR tyrosine kinase inhibitors using ginsenosides from Panax ginseng.

    PubMed

    Sathishkumar, Natarajan; Karpagam, Veerappan; Sathiyamoorthy, Subramaniyam; Woo, Min Jin; Kim, Yeon-Ju; Yang, Deok-Chun

    2013-07-01

    Natural products have served as structural resources in the history of drug discovery for cancer therapy. Among these natural products, Korean Panax ginseng serves as a potential anti-cancer medicinal plant. To determine the anti-cancer activities of Korean P. ginseng active compounds, we performed pharmacophore-based virtual screening and molecular docking studies on EGFR (epidermal growth factor receptor) tyrosine kinase domain. The EGFR family tyrosine kinase receptor is a cell surface receptor that regulates diverse biological processes including cell proliferation, differentiation, survival, and apoptosis. Over expression of EGFR tyrosine kinase domain associated with the development and progression of numerous human cancers. In our study, we developed the best pharmacophore model (Hypo1) using a diverse training set and validated by Fischer's randomization, a test set, and a decoy set. The best validated model was employed in the virtual screening of P. ginseng compound database. Further, chosen molecules were evaluated by applying ADMET screening and molecular docking studies. Finally, 14 compounds were obtained based on binding affinity scores and interactions with protein active site residues. These final lead compounds from P. ginseng can be used in the designing of new EGFR tyrosine kinase inhibitors. PMID:23668355

  1. Tailoring Tyrosine Kinase Inhibitors to Fit the Lung Cancer Genome

    PubMed Central

    Looyenga, Brendan D; Cherni, Irene; MacKeigan, Jeffrey P; Weiss, Glen J

    2011-01-01

    Tyrosine kinase inhibitors (TKIs) have been in use as cancer therapeutics for nearly a decade, and their utility in targeting specific malignancies with defined genetic lesions has proven to be remarkably effective. Recent efforts to characterize the spectrum of genetic lesions found in non-small cell lung carcinoma (NSCLC) have provided important insights into the molecular basis of this disease and have also revealed a wide array of tyrosine kinases that might be effectively targeted for rationally designed therapies. The findings of these studies, however, also provide a cautionary tale about the limitations of single-agent therapies, which fail to account for the genetic heterogeneity and pathway redundancy that characterize advanced NSCLC. Emergence of drug resistance mechanisms to specific TKIs, such as gefitinib and erlotinib, suggests that more sophisticated chemotherapeutic paradigms that target multiple pathways at the same time will be required to effectively treat this disease. PMID:21461169

  2. Targeting kinases with anilinopyrimidines: discovery of N-phenyl-N’-[4-(pyrimidin-4-ylamino)phenyl]urea derivatives as selective inhibitors of class III receptor tyrosine kinase subfamily

    PubMed Central

    Gandin, Valentina; Ferrarese, Alessandro; Dalla Via, Martina; Marzano, Cristina; Chilin, Adriana; Marzaro, Giovanni

    2015-01-01

    Kinase inhibitors are attractive drugs/drug candidates for the treatment of cancer. The most recent literature has highlighted the importance of multi target kinase inhibitors, although a correct balance between specificity and non-specificity is required. In this view, the discovery of multi-tyrosine kinase inhibitors with subfamily selectivity is a challenging goal. Herein we present the synthesis and the preliminary kinase profiling of a set of novel 4-anilinopyrimidines. Among the synthesized compounds, the N-phenyl-N’-[4-(pyrimidin-4-ylamino)phenyl]urea derivatives selectively targeted some members of class III receptor tyrosine kinase family. Starting from the structure of hit compound 19 we synthesized a further compound with an improved affinity toward the class III receptor tyrosine kinase members and endowed with a promising antitumor activity both in vitro and in vivo in a murine solid tumor model. Molecular modeling simulations were used in order to rationalize the behavior of the title compounds. PMID:26568452

  3. Intracellular Ca2+ stores modulate SOCCs and NMDA receptors via tyrosine kinases in rat hippocampal neurons.

    PubMed

    Koss, David J; Riedel, Gernot; Platt, Bettina

    2009-07-01

    The regulation of intracellular Ca(2+) signalling by phosphorylation processes remains poorly defined, particularly with regards to tyrosine phosphorylation. Evidence from non-excitable cells implicates tyrosine phosphorylation in the activation of so-called store-operated Ca(2+) channels (SOCCs), but their involvement in neuronal Ca(2+) signalling is still elusive. In the present study, we determined the role of protein tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs) in the coupling between intracellular Ca(2+) stores and SOCCs in neonatal rat hippocampal neurons by Fura-2 Ca(2+) imaging. An early Ca(2+) response from intracellular stores was triggered with thapsigargin, and followed by a secondary plasma membrane Ca(2+) response. This phase was blocked by the non-specific Ca(2+) channel blocker NiCl and the SOCC blocker, 2-aminoethoxydiphenyl borate (2-APB). Interestingly, two structurally distinct PTK inhibitors, genistein and AG126, also inhibited this secondary response. Application of the PTP inhibitor sodium orthovanadate (OV) also activated a sustained and tyrosine kinase dependent Ca(2+) response, blocked by NiCl and 2-APB. In addition, OV resulted in a Ca(2+) store dependent enhancement of NMDA responses, corresponding to, and occluding the signalling pathway for group I metabotropic glutamate receptors (mGluRs). This study provides first evidence for tyrosine based phospho-regulation of SOCCs and NMDA signalling in neurons. PMID:19423160

  4. A conserved isoleucine maintains the inactive state of Bruton’s tyrosine kinase

    PubMed Central

    Boyken, Scott E.; Chopra, Nikita; Xie, Qian; Joseph, Raji E.; Wales, Thomas E.; Fulton, D. Bruce; Engen, John R.; Jernigan, Robert L.; Andreotti, Amy H.

    2014-01-01

    Despite high homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamic simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state. The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest this isoleucine side chain in the Tec family kinases acts as a ‘wedge’ that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain. PMID:25193673

  5. Role of Tyrosine Kinase Inhibitors in Indolent and Other Mature B-Cell Neoplasms

    PubMed Central

    Kutsch, Nadine; Marks, Reinhard; Ratei, Richard; Held, Thomas K; Schmidt-Hieber, Martin

    2015-01-01

    Targeting tyrosine kinases represents a highly specific treatment approach for different malignancies. This also includes non-Hodgkin lymphoma since it is well known that these enzymes are frequently involved in the lymphomagenesis. Hereby, tyrosine kinases might either be dysregulated intrinsically or be activated within signal transduction pathways leading to tumor survival and growth. Among others, Bruton’s tyrosine kinase (Btk) is of particular interest as a potential therapeutic target. Btk is stimulated by B-cell receptor signaling and activates different transcription factors such as nuclear factor κB. The Btk inhibitor ibrutinib has been approved for the treatment of chronic lymphocytic leukemia and mantle-cell lymphoma recently. Numerous clinical trials evaluating this agent in different combinations (eg, with rituximab or classical chemotherapeutic agents) as a treatment option for aggressive and indolent lymphoma are under way. Here, we summarize the role of tyrosine kinase inhibitors in the treatment of indolent and other non-Hodgkin lymphomas (eg, mantle-cell lymphoma). PMID:26327780

  6. Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma.

    PubMed

    Patwardhan, Parag P; Ivy, Kathryn S; Musi, Elgilda; de Stanchina, Elisa; Schwartz, Gary K

    2016-01-26

    Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10-18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma. PMID:26675259

  7. Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma

    PubMed Central

    Musi, Elgilda; de Stanchina, Elisa; Schwartz, Gary K.

    2016-01-01

    Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10–18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma. PMID:26675259

  8. Bruton's tyrosine kinase inhibitors for the treatment of rheumatoid arthritis.

    PubMed

    Whang, Jennifer A; Chang, Betty Y

    2014-08-01

    The function and role of Bruton's tyrosine kinase (BTK) in human B cell development was demonstrated by its association with X-linked agammaglobulinemia (XLA) manifested by a substantial reduction in immunoglobulins and B cells. BTK has a crucial role in pre-B cell receptor (BCR) and BCR signaling during normal B cell development and activation. Aberrant BCR signaling is associated with autoimmune diseases, such as rheumatoid arthritis (RA). In addition, BTK is also expressed in myeloid cell populations, including monocytes, macrophages, neutrophils and mast cells. These innate cells infiltrate the synovial cavity and produce inflammatory cytokines, aggravating arthritic symptoms. In myeloid cell populations, BTK functions downstream of the Fcγ receptors (FcγR) and Fcɛ receptors (FcɛR). In the absence of BTK, FcR-mediated functions, such as cytokine production, are impaired. In addition, Xid mice, which have a mutation in BTK, have decreased susceptibility to developing collagen-induced arthritis (CIA). Given that BTK is involved in multiple signaling pathways downstream of the BCR and FcR, it is an attractive therapeutic target for RA. PMID:24721226

  9. Role of Receptor Tyrosine Kinases and Their Ligands in Glioblastoma

    PubMed Central

    Carrasco-García, Estefanía; Saceda, Miguel; Martínez-Lacaci, Isabel

    2014-01-01

    Glioblastoma multiforme is the most frequent, aggressive and fatal type of brain tumor. Glioblastomas are characterized by their infiltrating nature, high proliferation rate and resistance to chemotherapy and radiation. Recently, oncologic therapy experienced a rapid evolution towards “targeted therapy,” which is the employment of drugs directed against particular targets that play essential roles in proliferation, survival and invasiveness of cancer cells. A number of molecules involved in signal transduction pathways are used as molecular targets for the treatment of various tumors. In fact, inhibitors of these molecules have already entered the clinic or are undergoing clinical trials. Cellular receptors are clear examples of such targets and in the case of glioblastoma multiforme, some of these receptors and their ligands have become relevant. In this review, the importance of glioblastoma multiforme in signaling pathways initiated by extracellular tyrosine kinase receptors such as EGFR, PDGFR and IGF-1R will be discussed. We will describe their ligands, family members, structure, activation mechanism, downstream molecules, as well as the interaction among these pathways. Lastly, we will provide an up-to-date review of the current targeted therapies in cancer, in particular glioblastoma that employ inhibitors of these pathways and their benefits. PMID:24709958

  10. The Role of Tyrosine Kinase Receptors in Peritoneal Fibrosis.

    PubMed

    Wang, Li; Zhuang, Shougang

    2015-01-01

    Peritoneal dialysis (PD) is a modality for treatment of patients with end-stage renal disease (ESRD) that depends on the structural and functional integrity of the peritoneal membrane. However, long-term PD can lead to morphological and functional changes in the peritoneum; in particular, peritoneal fibrosis has become one of the most common complications that ultimately results in ultrafiltration failure (UFF) and discontinuation of PD. Several factors and mechanisms such as inflammation and overproduction of transforming growth factor-β1 have been implicated in the development of peritoneal fibrosis, but there is no effective therapy to prevent or delay this process. Recent studies have shown that activation of multiple receptor tyrosine kinases (RTKs) is associated with the development and progression of tissue fibrosis in various organs, and there are also reports indicating the involvement of some RTKs in peritoneal fibrosis. This review will describe the role and mechanisms of RTKs in peritoneal fibrosis and discuss the possibility of using them as therapeutic targets for prevention and treatment of this complication. PMID:26450477

  11. Getting Syk: Spleen Tyrosine Kinase as a Therapeutic Target

    PubMed Central

    Geahlen, Robert L.

    2014-01-01

    Syk is a cytoplasmic protein-tyrosine kinase well known for its ability to couple immune cell receptors to intracellular signaling pathways that regulate cellular responses to extracellular antigens and antigen-immunoglobulin complexes of particular importance to the initiation of inflammatory responses. Thus, Syk is an attractive target for therapeutic kinase inhibitors designed to ameliorate symptoms and consequences of acute and chronic inflammation. Its more recently recognized role as a promoter of cell survival in numerous cancer cell types ranging from leukemia to retinoblastoma has attracted considerable interest as a target for a new generation of anticancer drugs. This review discusses the biological processes in which Syk participates that have made this kinase such a compelling drug target. PMID:24975478

  12. Activation of phosphatidylinositol 3-kinase by insulin.

    PubMed Central

    Ruderman, N B; Kapeller, R; White, M F; Cantley, L C

    1990-01-01

    Insulin action appears to require the protein-tyrosine kinase domain of the beta subunit of the insulin receptor. Despite this, the identities and biochemical functions of the cellular targets of this tyrosine kinase are unknown. A phosphatidylinositol 3-kinase (PI 3-kinase) that phosphorylates the D-3 position of the inositol ring associates with several protein-tyrosine kinases. Here we report that PI 3-kinase activity is immunoprecipitated from insulin-stimulated CHO cells by antiphosphotyrosine and anti-insulin receptor antibodies. Insulin as low as 0.3 nM increased immunoprecipitable PI 3-kinase activity within 1 min. Increases in activity were much greater in CHO cells expressing the human insulin receptor (100,000 receptors per cell) than in control CHO cells (2000 receptors per cell). During insulin stimulation, various lipid products of the PI 3-kinase either appeared or increased in quantity in intact cells, suggesting that the appearance of immunoprecipitable PI 3-kinase reflects an increase in its activity in vivo. These results indicate that insulin at physiological concentrations regulates the PI 3-kinase and suggest that this regulation involves a physical association between the insulin receptor and the PI 3-kinase and tyrosyl phosphorylation. Images PMID:2154747

  13. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase* ♦

    PubMed Central

    Fan, Gaofeng; Aleem, Saadat; Yang, Ming; Miller, W. Todd; Tonks, Nicholas K.

    2015-01-01

    Despite significant evidence to the contrary, the view that phosphatases are “nonspecific” still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as “erasers” that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of “nonspecific phosphatases.” We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. PMID:25897081

  14. Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.

    PubMed Central

    Rausch, O; Marshall, C J

    1997-01-01

    The receptor for granulocyte colony-stimulating factor (G-CSF) can mediate differentiation and proliferation of hemopoietic cells. A proliferative signal is associated with activation of the ERK mitogen-activated protein kinase (MAPK) pathway. To determine whether other MAPK pathways are activated by G-CSF signalling, we have investigated activation of JNK/SAPK in cells proliferating in response to G-CSF. Here we show that G-CSF and interleukin-3 activate JNK/SAPK in two hemopoietic cell lines. The region of the G-CSF receptor required for G-CSF-induced JNK/SAPK activation is located within the C-terminal 68 amino acids of the cytoplasmic domain, which contains Tyr 763. Mutation of Tyr 763 to Phe completely blocks JNK/SAPK activation. However, the C-terminal 68 amino acids are not required for ERK2 activation. We show that activation of JNK/SAPK, like that of ERK2, is dependent on Ras but that higher levels of Ras-GTP are associated with activation of JNK/SAPK than with activation of ERK2. Two separate functional regions of the G-CSF receptor contribute to activation of Ras. The Y763F mutation reduces G-CSF-induced Ras activation from 30 to 35% Ras-GTP to 10 to 13% Ras-GTP. Low levels of Ras activation (10 to 13% Ras-GTP), which are sufficient for ERK2 activation, require only the 100 membrane-proximal amino acids. High levels of Ras-GTP provided by expression of oncogenic Ras are not sufficient to activate JNK/SAPK. An additional signal, also mediated by Tyr 763, is required for activation of JNK/SAPK. PMID:9032244

  15. Involvement of the Tyrosine Kinase Fer in Cell Adhesion

    PubMed Central

    Rosato, Roberto; Veltmaat, Jacqueline M.; Groffen, John; Heisterkamp, Nora

    1998-01-01

    The Fer protein belongs to the fes/fps family of nontransmembrane receptor tyrosine kinases. Lack of success in attempts to establish a permanent cell line overexpressing it at significant levels suggested a strong negative selection against too much Fer protein and pointed to a critical cellular function for Fer. Using a tetracycline-regulatable expression system, overexpression of Fer in embryonic fibroblasts was shown to evoke a massive rounding up, and the subsequent detachment of the cells from the substratum, which eventually led to cell death. Induction of Fer expression coincided with increased complex formation between Fer and the cadherin/src-associated substrate p120cas and elevated tyrosine phosphorylation of p120cas. β-Catenin also exhibited clearly increased phosphotyrosine levels, and Fer and β-catenin were found to be in complex. Significantly, although the levels of α-catenin, β-catenin, and E-cadherin were unaffected by Fer overexpression, decreased amounts of α-catenin and β-catenin were coimmunoprecipitated with E-cadherin, demonstrating a dissolution of adherens junction complexes. A concomitant decrease in levels of phosphotyrosine in the focal adhesion-associated protein p130 was also observed. Together, these results provide a mechanism for explaining the phenotype of cells overexpressing Fer and indicate that the Fer tyrosine kinase has a function in the regulation of cell-cell adhesion. PMID:9742093

  16. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

  17. Photoprotective and antioxidant effects of Rhubarb: inhibitory action on tyrosinase and tyrosine kinase activities and TNF-α, IL-1α and α-MSH production in human melanocytes

    PubMed Central

    2013-01-01

    Background Exposure to ultraviolet (UV) radiation causes various forms of acute and chronic skin damage, including immunosuppression, inflammation, premature aging and photodamage. Furthermore, it induces the generation of reactive oxygen species, produces proinflammatory cytokines and melanocyte-stimulating hormone (MSH) and increases tyrosinase activity. The aim of this study was to evaluate the potential photoprotective effects of Rheum rhaponticum L. rhizome extract on human UV-stimulated melanocytes. Methods The effects of Rheum rhaponticum rhizome extract on tyrosine kinase activity, and on interleukin-1α (IL-1α), tumour necrosis factor α (TNF-α), and α-MSH production in human epidermal melanocytes were evaluated under UV-stimulated and non-stimulated conditions. Antioxidant activity was evaluated by lipid peroxidation and 1,1-dyphenyl-2-picryl-hydrazyl (DPPH) assays, while anti-tyrosinase activity was evaluated by the mushroom tyrosinase method. Results Rheum rhaponticum L. rhizome extract showed in vitro antioxidant properties against lipid peroxidation, free radical scavenging and anti-tyrosinase activities, and inhibited the production of IL-1α, TNF-α, α-MSH, and tyrosine kinase activity in melanocytes subjected to UV radiation. Conclusions These results support the inclusion of Rheum rhaponticum L. rhizome extract into cosmetic, sunscreen and skin care products for the prevention or reduction of photodamage. PMID:23445687

  18. Design and synthesis of constrained analogs of LCRF-0004 as potent RON tyrosine kinase inhibitors.

    PubMed

    Raeppel, Stéphane L; Therrien, Eric; Raeppel, Franck

    2015-09-01

    New fused bicyclic lactam head groups as rigidified analogs of thieno[3,2-b]pyridine-based kinase inhibitor LCRF-0004 were designed and synthesized. Depending on the functionalities and the size of these bicyclic head groups, potent inhibitors of RON tyrosine kinase with various level of selectivity against c-Met tyrosine kinase were obtained. PMID:26112445

  19. TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

    SciTech Connect

    Li, Feifei; Jiang, Yinan; Zheng, Qiping; Yang, Xiaoming; Wang, Siying

    2011-01-07

    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 involved 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.

  20. Tyrosine kinase receptors as molecular targets In pheochromocytomas and paragangliomas

    PubMed Central

    Cassol, Clarissa A.; Winer, Daniel; Liu, Wei; Guo, Miao; Ezzat, Shereen; Asa, Sylvia L.

    2016-01-01

    Pheochromocytomas and paragangliomas are neuroendocrine tumors shown to be responsive to multi-targeted tyrosine kinase inhibitor treatment. Despite growing knowledge regarding their genetic basis, the ability to predict behavior in these tumors remains challenging. There is also limited knowledge of their tyrosine kinase receptor expression and whether the clinical response observed to the tyrosine kinase inhibitor Sunitinib relates only to its anti-angiogenic properties or also due to a direct effect on tumor cells. To answer these questions, an in vitro model of sunitinib treatment of a pheochromocytoma cell line was created. Sunitinib targets (VEGFRs, PDGFRs, C-KIT), FGFRs and cell cycle regulatory proteins were investigated in human tissue microarrays. SDHB immunohistochemistry was used as a surrogate marker for the presence of succinate dehydrogenase mutations. The FGFR4 G388R SNP was also investigated. Sunitinib treatment in vitro decreases cell proliferation mainly by targeting cell cycle, DNA metabolism, and cell organization genes. FGFR1, -2 and -4, VEGFR2, PDGFRα and p16 were overexpressed in primary human pheochromocytomas and paragangliomas. Discordant results were observed for VEGFR1, p27 and p21 (overexpressed in paragangliomas but underexpressed in pheochromoctyomas); PDGFRβ, Rb and Cyclin D1 (overexpressed in paragangliomas only) and FGFR3 (overexpressed in pheochromocytomas and underexpressed in paragangliomas). Low expression of C-KIT, p53, Aurora Kinase A and B was observed. Nuclear FGFR2 expression was associated with increased risk of metastasis (odds ratio [OR]=7.61; p=0.008), as was membranous PDGFRα (OR= 13.71, p=0.015), membranous VEGFR1 (OR=8.01; p=0.037), nuclear MIB1 (OR=1.26, p=0.008) and cytoplasmic p27 (OR=1.037, p=0.030). FGFR3, VEGFR2 and C-KIT levels were associated with decreased risk of metastasis. We provide new insights into the mechanistic actions of sunitinib in pheochromoctyomas and paragangliomas and support current

  1. Expression, purification and preliminary crystallographic studies on the catalytic region of the nonreceptor tyrosine kinase Fes

    SciTech Connect

    Gnemmi, Ilaria; Scotti, Claudia; Cappelletti, Donata; Canonico, Pier Luigi; Condorelli, Fabrizio; Rosano, Camillo

    2007-01-01

    The catalytic domain of human Fes tyrosine kinase has been cloned, expressed, purified and crystallized. The proto-oncogene tyrosine protein kinase c-fps/fes encodes a structurally unique protein (Fes) of the nonreceptor protein-tyrosine kinase (PTK) family. Its expression has been demonstrated in myeloid haematopoietic cells, vascular endothelial cells and in neurons. In human-derived and murine-derived cell lines, the activated form of this kinase can induce cellular transformation; moreover, it has been shown that Fes is involved in the regulation of cell–cell and cell–matrix interactions mediated by adherens junctions and focal adhesions. The N-terminus of Fes contains the FCH (Fps/Fes/Fer/CIP4 homology) domain, which is unique to the Fes/Fer kinase family. It is followed by three coiled-coil domains and an SH2 (Src-homology 2) domain. The catalytic region (Fes-CR) is located at the C-terminus of the protein. The successful expression, purification and crystallization of the catalytic part of Fes (Fes-CR) are described.

  2. Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle. Role of endothelial differentiation gene 1, c-Src tyrosine kinase and phosphoinositide 3-kinase.

    PubMed Central

    Rakhit, S; Conway, A M; Tate, R; Bower, T; Pyne, N J; Pyne, S

    1999-01-01

    We report here that cultured airway smooth muscle cells contain transcripts of endothelial differentiation gene 1 (EDG-1), a prototypical orphan Gi-coupled receptor whose natural ligand is sphingosine 1-phosphate (S1P). This is consistent with data that showed that S1P activated both c-Src and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) in a pertussis toxin (PTX)-sensitive manner in these cells. An essential role for c-Src was confirmed by using the c-Src inhibitor, PP1, which markedly decreased p42/p44 MAPK activation. We have also shown that phosphoinositide 3-kinase (PI-3K) inhibitors (wortmannin and LY294002) decreased p42/p44 MAPK activation. An essential role for PI-3K was supported by experiments that showed that PI-3K activity was increased in Grb-2 immunoprecipitates from S1P-stimulated cells. Significantly, Grb-2 associated PI-3K activity was decreased by pretreatment of cells with PTX. Finally, we have shown that the co-stimulation of cells with platelet-derived growth factor (PDGF) and S1P (which failed to stimulate DNA synthesis) elicited a larger p42/p44 MAPK activation over a 30 min stimulation compared with each agonist alone. This was associated with a S1P-dependent increase in PDGF-stimulated DNA synthesis. These results demonstrate that S1P activates c-Src and Grb-2-PI-3K (intermediates in the p42/p44 MAPK cascade) via a PTX-sensitive mechanism. This action of S1P is consistent with the stimulation of EDG-1 receptors. S1P might also function as a co-mitogen with PDGF, producing a more robust activation of a common permissive signal transduction pathway linked to DNA synthesis. PMID:10051434

  3. 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

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

    2013-04-16

    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 nonreceptor 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

  4. Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis.

    PubMed

    Dixelius, J; Larsson, H; Sasaki, T; Holmqvist, K; Lu, L; Engström, A; Timpl, R; Welsh, M; Claesson-Welsh, L

    2000-06-01

    Endostatin, which corresponds to the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth factor-2 (FGF-2)-induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant R158/270A, lacking heparin-binding ability. Endostatin was internalized by endothelial cells, but not by mouse fibroblasts. Treatment of murine brain endothelial (IBE) cells with endostatin reduced the proportion of cells in S phase, whereas growth-arrested IBE cells in collagen gels treated with endostatin displayed enhanced tubular morphogenesis. IBE cells overexpressing Shb, an adaptor protein implicated in angiostatin-induced apoptosis, displayed elevated apoptosis and decreased tubular morphogenesis in collagen gels in response to endostatin when added together with FGF-2. Induction of apoptosis was dependent on the heparin-binding ability of endostatin and the expression of Shb with a functional Src homology 2 (SH2)-domain. Endostatin treatment for 10 minutes or 24 hours induced tyrosine phosphorylation of Shb and formation of multiprotein complexes. An Shb SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein in endostatin-treated cells. The 125-kd component either contained intrinsic tyrosine kinase activity or occurred in complex with a tyrosine kinase. In conclusion, our data show that endostatin induces tyrosine kinase activity and enhanced apoptosis in FGF-treated endothelial cells. PMID:10828022

  5. Agonistic TAM-163 antibody targeting tyrosine kinase receptor-B

    PubMed Central

    Vugmeyster, Yulia; Rohde, Cynthia; Perreault, Mylene; Gimeno, Ruth E.; Singh, Pratap

    2013-01-01

    TAM-163, an agonist monoclonal antibody targeting tyrosine receptor kinase-B (TrkB), is currently being investigated as a potential body weight modulatory agent in humans. To support the selection of the dose range for the first-in-human (FIH) trial of TAM-163, we conducted a mechanistic analysis of the pharmacokinetic (PK) and pharmacodynamic (PD) data (e.g., body weight gain) obtained in lean cynomolgus and obese rhesus monkeys following single doses ranging from 0.3 to 60 mg/kg. A target-mediated drug disposition (TMDD) model was used to describe the observed nonlinear PK and Emax approach was used to describe the observed dose-dependent PD effect. The TMDD model development was supported by the experimental determination of the binding affinity constant (9.4 nM) and internalization rate of the drug-target complex (2.08 h−1). These mechanistic analyses enabled linking of exposure, target (TrkB) coverage, and pharmacological activity (e.g., PD) in monkeys, and indicated that ≥ 38% target coverage (time-average) was required to achieve significant body weight gain in monkeys. Based on the scaling of the TMDD model from monkeys to humans and assuming similar relationship between the target coverage and pharmacological activity between monkey and humans, subcutaneous (SC) doses of 1 and 15 mg/kg in humans were projected to be the minimally and the fully pharmacologically active doses, respectively. Based on the minimal anticipated biological effect level (MABEL) approach for starting dose selection, the dose of 0.05 mg/kg (3 mg for a 60 kg human) SC was recommended as the starting dose for FIH trials, because at this dose level < 10% target coverage was projected at Cmax (and all other time points). This study illustrates a rational mechanistic approach for the selection of FIH dose range for a therapeutic protein with a complex model of action. PMID:23529133

  6. Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization

    PubMed Central

    Kwon, Annie; Byrne, Dominic P.; Ferries, Samantha; Ruan, Zheng; Hanold, Laura E.; Katiyar, Samiksha; Kennedy, Eileen J.; Eyers, Patrick A.; Kannan, Natarajan

    2016-01-01

    Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they

  7. Neuroblastoma Tyrosine Kinase Signaling Networks Involve FYN and LYN in Endosomes and Lipid Rafts

    PubMed Central

    Guo, Ailan; Stokes, Matthew P.; Kuehn, Emily D.; George, Lynn; Comb, Michael; Grimes, Mark L.

    2015-01-01

    Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma. PMID:25884760

  8. Genomic organization of Bruton`s tyrosine kinase

    SciTech Connect

    Rohrer, J.; Conley, M.E.

    1994-09-01

    Bruton`s tyrosine kinase (Btk), is a nonreceptor tyrosine kinase that has been identified as the defective gene in X-linked agammaglobulinemia (XLA). XLA patients have profound hypogammaglobulinemia and markedly reduced numbers of B cells while their T cell and phagocyte numbers remain normal. To determine the genomic organization of Btk, intron/exon borders were identified by sequencing cosmid DNA using cDNA primers. Nineteen exons spanning 37 kb of genomic DNA were identified. All the intron/exon splice junctions followed the GT/AG rule. The translational ATG start codon was in exon 2 which was 6 kb downstream of exon 1. Exon 19, 519 bp in length and 3.8 kb distal to exon 18, was the largest exon and included the 450 bp of the 3{prime} untranslated region. Exons 6 through 18 formed the largest cluster of exons with no intron being longer than 1550 bp. There was no apparent correlation between the exon boundaries of Btk and the functional domains of the protein or the exon boundaries of src, the nonreceptor protein tyrosine kinase prototype. The region 500 bp upstream of the presumed transcriptional start site was sequenced and found to have a G+C content of 52%. No TATA-type promoter elements in the -20 bp to -30 bp region were identified. However, at position -48 bp, a TGTGAA motif was found that bears some similarity to the TATA box. This sequence was preceded by a perfect inverted CCAAT box at position -90 bp. Three retinoic acid binding sites were also identified at positions -50 bp, -83 bp and -197 bp. Defining the genomic structure of Btk will permit us to identify regulatory elements in this gene and to identify mutations in genomic DNA of patients with XLA.

  9. Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition.

    PubMed

    Yen, Hsin-Yung; Liu, Ying-Chih; Chen, Nai-Yu; Tsai, Chia-Feng; Wang, Yi-Ting; Chen, Yu-Ju; Hsu, Tsui-Ling; Yang, Pan-Chyr; Wong, Chi-Huey

    2015-06-01

    Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKI-sensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKI-resistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKI-resistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation. PMID:25971727

  10. WEE1 Tyrosine Kinase, A Novel Epigenetic Modifier

    PubMed Central

    Mahajan, Kiran; Mahajan, Nupam P.

    2013-01-01

    The cell cycle requires cells to duplicate their chromatin, DNA, and histones, while retaining a subset of epigenetic marks, in a highly coordinated manner. The WEE1 kinase was identified as an important regulator during S phase, preventing entry into mitosis until DNA replication has been completed. Interestingly, WEE1 has also emerged as a key player in regulating histone synthesis. It phosphorylates histone H2B at tyrosine 37 in the nucleosomes found upstream of the histone gene cluster, which suppresses histone transcription in late S phase. These observations highlight a dual role for WEE1 as both a mitotic gatekeeper and a surveyor of chromatin synthesis, providing a direct link between epigenetics and cell cycle progression. Importantly, this link has implications for the design of novel epigenetic inhibitors targeting cancers that display elevated expression of this kinase. PMID:23537585

  11. MAP-kinase activity necessary for TGFbeta1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397.

    PubMed

    Hayashida, Tomoko; Wu, Ming-Hua; Pierce, Amy; Poncelet, Anne-Christine; Varga, John; Schnaper, H William

    2007-12-01

    The signals mediating transforming growth factor beta (TGFbeta)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFbeta-stimulated, Smad-mediated collagen production by human kidney mesangial cells, and that ERK MAP kinase activity optimizes collagen expression and enhances phosphorylation of the Smad3 linker region. Furthermore, we showed that disrupting cytoskeletal integrity decreases type I collagen production. Focal adhesion kinase (FAK, PTK2) activity could integrate these findings. Adhesion-dependent FAK Y397 phosphorylation was detected basally, whereas FAK Y925 phosphorylation was TGFbeta1-dependent. By immunocytochemistry, TGFbeta1 stimulated the merging of phosphorylated FAK with the ends of thickening stress fibers. Cells cultured on poly-L-lysine (pLL) to promote integrin-independent attachment spread less than those on control substrate and failed to demonstrate focal adhesion (FA) engagement with F-actin. FAK Y397 phosphorylation and ERK activity were also decreased under these conditions. In cells with decreased FAK Y397 phosphorylation from either plating on pLL or overexpressing a FAK Y397F point mutant, serine phosphorylation of the Smad linker region, but not of the C-terminus, was reduced. Y397F and Y925F FAK point mutants inhibited TGFbeta-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFbeta-stimulated collagen-promoter activity. The inhibition by the Y397F mutant or by culture on pLL was prevented by co-transfection of constitutively active ERK MAP kinase kinase (MEK), suggesting that FAK Y397 phosphorylation promotes collagen expression via ERK MAP kinase activity. Finally, Y397 FAK phosphorylation, and both C-terminal and linker-region Smad3 phosphorylation were detected in murine TGFbeta-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFbeta-induced responses to regulate collagen production. PMID:18032789

  12. Growth inhibition by tyrosine kinase inhibitors in mesothelioma cell lines.

    PubMed

    Nutt, Joyce E; O'Toole, Kieran; Gonzalez, David; Lunec, John

    2009-06-01

    Clinical outcome following chemotherapy for malignant pleural mesothelioma is poor and improvements are needed. This preclinical study investigates the effect of five tyrosine kinase inhibitors (PTK787, ZD6474, ZD1839, SU6668 and SU11248) on the growth of three mesothelioma cell lines (NCI H226, NCI H28 and MSTO 211H), the presence of growth factor receptors and inhibition of their downstream signalling pathways. GI50 values were determined: ZD6474 and SU11248, mainly VEGFR2 inhibitors, gave the lowest GI50 across all cell lines (3.5-6.9 microM) whereas ZD1839 gave a GI50 in this range only in H28 cells. All cell lines were positive for EGFR, but only H226 cells were positive for VEGFR2 by Western blotting. ZD6474 and ZD1839 inhibited EGF-induced phosphorylation of EGFR, AKT and ERK, whereas VEGF-induced phosphorylation of VEGFR2 was completely inhibited with 0.1 microM SU11248. VEGFR2 was detected in tumour samples by immunohistochemistry. VEGFR2 tyrosine kinase inhibitors warrant further investigation in mesothelioma. PMID:19318229

  13. 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

  14. Nitric oxide reversibly inhibits the epidermal growth factor receptor tyrosine kinase.

    PubMed Central

    Estrada, C; Gómez, C; Martín-Nieto, J; De Frutos, T; Jiménez, A; Villalobo, A

    1997-01-01

    Although it has been demonstrated that NO inhibits the proliferation of different cell types, the mechanisms of its anti-mitotic action are not well understood. In this work we have studied the possible interaction of NO with the epidermal growth factor receptor (EGFR), using transfected fibroblasts which overexpress the human EGFR. The NO donors S-nitroso-N-acetylpenicillamine (SNAP), 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) and N-¿4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl¿propane -1, 3-diamine (DETA-NO) inhibited DNA synthesis of fibroblasts growing in the presence of fetal calf serum, epidermal growth factor (EGF) or EGF plus insulin, as assessed by [methyl-3H]thymidine incorporation. Neither 8-bromo-cGMP nor the cGMP-phosphodiesterase inhibitor zaprinast mimicked this effect, suggesting that NO is unlikely to inhibit cell proliferation via a cGMP-dependent pathway. SNAP, DEA-NO and DETA-NO also inhibited the transphosphorylation of the EGFR and its tyrosine kinase activity toward the exogenous substrate poly-l-(Glu-Tyr), as measured in permeabilized cells using [gamma-32P]ATP as phosphate donor. In contrast, 3-[morpholinosydnonimine hydrochloride] (SIN-1), a peroxynitrite-forming compound, did not significantly inhibit either DNA synthesis or the EGFR tyrosine kinase activity. The inhibitory action of DEA-NO on the EGFR tyrosine kinase was prevented by haemoglobin, an NO scavenger, but not by superoxide dismutase, and was reversed by dithiothreitol. The binding of EGF to its receptor was unaffected by DEA-NO. The inhibitory action of DEA-NO on the EGF-dependent transphosphorylation of the receptor was also demonstrated in intact cells by immunoblot analysis using an anti-phosphotyrosine antibody. Taken together, these results suggest that NO, but not peroxynitrite, inhibits in a reversible manner the EGFR tyrosine kinase activity by S-nitrosylation of the receptor. PMID:9291107

  15. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    SciTech Connect

    Okabe, Seiichi Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-06-07

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

  16. Bruton tyrosine kinase inhibition in chronic lymphocytic leukemia.

    PubMed

    Maddocks, Kami; Jones, Jeffrey A

    2016-04-01

    Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and remains incurable outside of the setting of allogeneic stem cell transplant. While the standard therapy for both initial and relapsed CLL has traditionally included monoclonal antibody therapy in combination with chemotherapy, there are patients with high-risk disease features including unmutated IgVH, del(11q22) and del(17p13) that are associated with poor overall responses to these therapies with short time to relapse and shortened overall survival. Additionally, many of these therapies have a high rate of infectious toxicity in a population already at increased risk. Targeting the B-cell receptor (BCR) signaling pathway has emerged as a promising therapeutic advance in a variety of B-cell malignancies, including CLL. Bruton agammaglobulinemia tyrosine kinase (Btk) is a tyrosine kinase in the BCR pathway critical to the survival of both normal and malignant B cells and inhibition of this kinase has shown to block the progression of CLL. Ibrutinib, a first in class oral inhibitor of Btk, has shown promise as a very effective agent in the treatment of CLL-in both relapsed and upfront therapy, alone and in combination with other therapies, and in patients of all-risk disease-which has led to its approval in relapsed CLL and as frontline therapy in patients with the high-risk del(17p13) disease. Several studies are ongoing to evaluate the efficacy and safety of ibrutinib in combination with chemotherapy as frontline treatment for CLL and investigation into newer-generation Btk inhibitors is also underway. PMID:27040703

  17. Epidermal growth factor receptor tyrosine kinase inhibitors for non-small cell lung cancer

    PubMed Central

    Asami, Kazuhiro; Atagi, Shinji

    2014-01-01

    First-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib and erlotinib, have proven to be highly effective agents for advanced non-small cell lung cancer (NSCLC) in patients harboring an activating EGFR mutation such as the exon 19 deletion mutation and L858R. Although those reversible small molecular targeted agents provide a significant response and survival benefit, all responders eventually acquire resistance. Second-generation EGFR-targeting agents, such as irreversible EGFR/HER2 tyrosine kinase inhibitors and pan-HER TKIs, may improve survival further and be useful for patients who acquired resistance to first-generation EGFR-TKIs. This review discusses novel therapeutic strategies for EGFR-mutated advanced NSCLC using first- and second-generation EGFR-TKIs. PMID:25302168

  18. A combination of tyrosine kinase inhibitors, crizotinib and dasatinib for the treatment of glioblastoma multiforme

    PubMed Central

    Nehoff, Hayley; Parayath, Neha N.; McConnell, Melanie J.; Taurin, Sebastien; Greish, Khaled

    2015-01-01

    Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Despite the advances in surgery, radiotherapy and chemotherapy, patient survival averages only 14.6 months. In most GBM tumors, tyrosine kinases show increased activity and/or expression and actively contribute to the development, recurrence and onset of treatment resistance; making their inhibition an appealing therapeutic strategy. We compared the cytotoxicity of 12 tyrosine kinase inhibitors in vitro. A combination of crizotinib and dasatinib emerged as the most cytotoxic across established and primary human GBM cell lines. The combination treatment induced apoptotic cell death and polyploidy. Furthermore, the combination treatment led to the altered expression and localization of several tyrosine kinase receptors such as Met and EGFR and downstream effectors as such as SRC. Furthermore, the combination treatment reduced the migration and invasion of GBM cells and prevented endothelial cell tube formation in vitro. Overall, our study demonstrated the broad specificity of a combination of crizotinib and dasatinib across multiple GBM cell lines. These findings provide insight into the development of alternative therapy for the treatment of GBM. PMID:26517812

  19. QSAR and molecular docking studies on oxindole derivatives as VEGFR-2 tyrosine kinase inhibitors.

    PubMed

    Kang, Cong-Min; Liu, Dong-Qing; Zhao, Xu-Hao; Dai, Ying-Jie; Cheng, Jia-Gao; Lv, Ying-Tao

    2016-01-01

    The three-dimensional quantitative structure-activity relationships (3D-QSAR) were established for 30 oxindole derivatives as vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitors by using comparative molecular field analysis (CoMFA) and comparative similarity indices analysis comparative molecular similarity indices analysis (CoMSIA) techniques. With the CoMFA model, the cross-validated value (q(2)) was 0.777, the non-cross-validated value (R(2)) was 0.987, and the external cross-validated value ([Formula: see text]) was 0.72. And with the CoMSIA model, the corresponding q(2), R(2) and [Formula: see text] values were 0.710, 0.988 and 0.78, respectively. Docking studies were employed to bind the inhibitors into the active site to determine the probable binding conformation. The binding mode obtained by molecular docking was in good agreement with the 3D-QSAR results. Based on the QSAR models and the docking binding mode, a set of new VEGFR-2 tyrosine kinase inhibitors were designed, which showed excellent predicting inhibiting potencies. The result revealed that both QSAR models have good predictive capability to guide the design and structural modification of homologic compounds. It is also helpful for further research and development of new VEGFR-2 tyrosine kinase inhibitors. PMID:26416217

  20. A systematic analysis of the resistance and sensitivity of HER2YVMA receptor tyrosine kinase mutant to tyrosine kinase inhibitors in HER2-positive lung cancer.

    PubMed

    Shen, Xiaokun; Chen, Beibei; Ma, Zhaosheng; Xie, Bojian; Cao, Xinguang; Yang, Tiejun; Zhao, Yuzhou; Qin, Jianjun; Li, Jicheng; Cao, Feilin; Chen, Xiaobing

    2016-01-01

    Human epidermal growth factor receptor 2 (HER2) has become a well-established target for the treatment of HER2-positive lung cancer. However, a frequently observed in-frame mutation that inserts amino acid quadruplex Tyr776-Val777-Met778-Ala779 at G776 (G776(YVMA)) in HER2 kinase domain can cause drug resistance and sensitivity, largely limiting the application of reversible tyrosine kinase inhibitors in lung cancer therapy. A systematic investigation of the intermolecular interactions between the HER2(YVMA) mutant and clinical small-molecule inhibitors would help to establish a complete picture of drug response to HER2 G776(YVMA) insertion in lung cancer, and to design new tyrosine kinase inhibitors with high potency and selectivity to target the lung cancer-related HER2(YVMA) mutant. Here, we combined homology modeling, ligand grafting, structure minimization, molecular simulation and binding affinity analysis to profile a number of tyrosine kinase inhibitors against the G776(YVMA) insertion in HER2. It is found that the insertion is far away from HER2 active pocket and thus cannot contact inhibitor ligand directly. However, the insertion is expected to induce marked allosteric effect on some regions around the pocket, including A-loop and hinges connecting between the N- and C-lobes of HER2 kinase domain, which may exert indirect influence to inhibitor binding. Most investigated inhibitors exhibit weak binding strength to both wild-type and mutant HER2, which can be attributed to steric hindrance that impairs ligand compatibility with HER2 active pocket. However, the cognate inhibitor lapatinib and the non-cognate inhibitor bosutinib were predicted to have low affinity for wild-type HER2 but high affinity for HER2(YVMA) mutant, which was confirmed by subsequent kinase assay experiments; the inhibitory potencies of bosutinib against wild-type and mutant HER2 were determined to be IC(50) > 1000 and =27 nM, respectively, suggesting that the bosutinib might be

  1. Spleen tyrosine kinase regulates mammary epithelial cell proliferation in mammary glands of dairy cows.

    PubMed

    Hou, Xiaoming; Lin, Lin; Xing, Weinan; Yang, Yang; Duan, Xiaoyu; Li, Qingzhang; Gao, Xuejun; Lin, Ye

    2016-05-01

    Spleen tyrosine kinase (SYK) is a nonreceptor tyrosine kinase that has been considered a hematopoietic cell-specific signal transducer involved in cell proliferation and differentiation. However, the role of SYK in normal mammary gland is still poorly understood. Here we show that SYK is expressed in mammary glands of dairy cows. Expression of SYK was higher in dry period mammary tissues than in lactating mammary tissues. Knockdown and overexpression of SYK affected dairy cow mammary epithelial cell proliferation as well as the expression of signal molecules involved in proliferation, including protein kinase B (PKB, also known as AKT1), p42/44 mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription 5 (STAT5). Dual-luciferase reporter assay showed that SYK increased the transcriptional activity of the AKT1 promoter, and cis-elements within the AKT1 promoter region from -439 to -84 bp mediated this regulation. These results suggest that SYK affects mammary epithelial cell proliferation by activating AKT1 at the transcriptional level in mammary glands of dairy cows, which is important for the mammary remodeling process in dry cows as well as for increasing persistency of lactation in lactating cows. PMID:26947307

  2. Beyond trastuzumab: small molecule tyrosine kinase inhibitors in HER-2-positive breast cancer.

    PubMed

    Roy, Vivek; Perez, Edith A

    2009-11-01

    HER-2 is a transmembrane, tyrosine kinase (TK) receptor whose overexpression is associated with adverse prognosis in breast cancer. The biological effects of HER-2 are mediated by kinase activity causing phosphorylation of tyrosine residues in the cytoplasmic domain of the receptor molecule, leading to activation of downstream growth-promoting pathways. Antibody-mediated inhibition by trastuzumab as well as TK inhibition are clinically effective anti-HER-2 strategies. Kinase inhibitors offer some potential therapeutic advantages over antibody-based therapies. Being small molecules, TK inhibitors (TKIs) have oral bioavailability and ability to cross the blood-brain barrier. Because of their different mode of action, TKIs may be able to overcome some of the mechanisms of trastuzumab resistance. Preclinical, and limited clinical data also suggest that TKIs and trastuzumab have synergistic activity. Lapatinib is the only TKI available for clinical use at present, but several molecules with anti-HER-2 activity have been identified and are undergoing evaluation. These differ in the spectrum of kinases that they inhibit, potency of HER-2 inhibition, pharmacokinetic properties, and toxicity profiles, and are at various stages of clinical development. In this article we summarize selected HER-2 TKIs approved for clinical use or in development for which clinical data are available. PMID:19887469

  3. Purification, characterization, and crystallization of membrane bound Escherichia coli tyrosine kinase.

    PubMed

    Chesterman, Chelsy; Jia, Zongchao

    2016-09-01

    Escherichia coli tyrosine kinase (Etk) is a membrane bound kinase in gram-negative bacteria that regulates the export of capsular polysaccharides (CPS). The molecular mechanism behind CPS regulation remains unclear, despite access to a crystal structure of the cytoplasmic kinase domain of Etk. In this study, an efficient protocol to produce full length Etk solubilized in n-dodecyl-β-d-maltoside has been established with high yield. We have determined that detergent solubilized Etk retains kinase activity, but the protein is prone to aggregation, degradation, and has been unsuccessful in protein crystallization trials. In response, we designed and characterized truncations of Etk that do not aggregate and have led to successful crystallization experiments. In this article, we discuss our optimized expression and purification protocol for Etk, the design of Etk protein truncations, and the behavior of Etk during purification in a range of stabilizing detergents. These efforts have successfully produced protein suitable for crystallization. Our results will be a useful guide for future structural and functional studies of the bacterial tyrosine kinase family. PMID:26363120

  4. Evidence that simulated microgravity may alter the vascular nonreceptor tyrosine kinase second messenger pathway

    NASA Technical Reports Server (NTRS)

    Kahwaji, C. I.; Sheibani, S.; Han, S.; Siu, W. O.; Kaka, A. H.; Fathy, T. M.; el-Abbadi, N. H.; Purdy, R. E.

    2000-01-01

    Simulated microgravity (hind limb unweighting; HU) reduces maximal contractile capacity to norepinephrine (NE) but not 5-hydroxytryptamine (5-HT) in the rat abdominal aorta of male Wistar rats. Our earlier study showed that voltage-operated calcium channels, the MAPK pathway [1], and vasoconstrictive prostaglandins contribute to the NE-induced contraction of control (C) but not HU, aorta rings. Genistein, a general tyrosine kinase inhibitor, caused a significant reduction in vascular contractility in C but not HU arteries. The present study explored the role of protein kinase C (PKC) and extracellular receptor-activated kinase 1 and 2 (ERK1/2) in the HU-induced vascular hyporesponsiveness to NE. Microgravity was simulated in Wistar rats by 20 day HU. The abdominal aorta was removed from control and HU rats, cut into 3 mm rings, and mounted in tissue baths to measure isometric contraction. Protein levels were determined using Western blot analysis. PD98059, a selective MAPKK inhibitor, caused a marked inhibition of NE-induced contraction in both C and HU arteries. Calphostin C, a PKC inhibitor, completely abolished the contractile response to NE in both C and HU tissues. Phosphorylated (activated) ERK1/2 protein mass was greater in C, compared to HU, aortas, and was reduced by genistein only in C tissues. MAPK total protein levels in the rat aorta were increased in the HU-treated, compared to C, animals. These results indicate that PKC represents an early transduction step in the contractile response to NE in the rat abdominal aorta. That inhibition of the step immediately before activation of MAPK reduced contraction in both C and HU tissues, while general tyrosine kinase inhibition with genistein blocked only the control responses, suggests that a nonreceptor tyrosine kinase may be involved in HU-induced vascular hyporesponsiveness to NE.

  5. Targeting of DNA molecules, BSA/c-Met tyrosine kinase receptors and anti-proliferative activity of bis(terpyridine)copper(ii) complexes.

    PubMed

    Mahendiran, Dharmasivam; Kumar, Raju Senthil; Viswanathan, Vijayan; Velmurugan, Devadasan; Rahiman, Aziz Kalilur

    2016-05-01

    A series of homoleptic bis(terpyridine)copper(ii) complexes of the type [Cu(L(1-5))2]Cl2 (), where L(1-5) = 4'-(4-substituted)-2,2':6',2''-terpyridines, have been synthesized and characterized. The molecular structure of complex was confirmed by the single crystal XRD technique, and the geometry of the complexes is best described as distorted octahedral. Structural parameters from the crystallographic and DFT studies are in good agreement with each other. The small HOMO-LUMO energy gap supports bioefficacy of the complexes. DNA binding studies show high intrinsic binding constant values 1.53 ± 0.15, 1.62 ± 0.08 and 3.09 ± 0.12 × 10(5) M(-1) for complexes , and , respectively, with intercalative mode of binding to CT-DNA. The binding results were further supported by molecular docking studies. The experimental results indicate that the interaction between the complexes and BSA protein involves a static quenching mechanism. The molecular docking studies with c-Met tyrosine kinase receptors show hydrophobic and π-π interactions. All the complexes bring about hydroxyl radical mediated DNA cleavage in the presence of H2O2. In vitro cytotoxicities of the complexes () were tested against three cancerous cell lines, namely human breast adenocarcinoma (MCF-7), epithelioma (Hep-2) and cervical (HeLa) cell lines, and one non-tumorigenic human dermal fibroblast (NHDF) cell line by MTT reduction assay. The morphological assessment data obtained using Hoechst 33258 staining revealed that complex induces apoptosis much more effectively than the other complexes. PMID:27063595

  6. Autoregulation by the Juxtamembrane Region of the Human Ephrin Receptor Tyrosine Kinase A3 (EphA3)

    SciTech Connect

    Davis, Tara L.; Walker, John R.; Loppnau, Peter; Butler-Cole, Christine; Allali-Hassani, Abdellah; Dhe-Paganon, Sirano

    2008-07-08

    Ephrin receptors (Eph) affect cell shape and movement, unlike other receptor tyrosine kinases that directly affect proliferative pathways. The kinase domain of EphA3 is activated by ephrin binding and receptor oligomerization. This activation is associated with two tyrosines in the juxtamembrane region; these tyrosines are sites of autophosphorylation and interact with the active site of the kinase to modulate activity. This allosteric event has important implications both in terms of understanding signal transduction pathways mediated by Eph kinases as well as discovering specific therapeutic ligands for receptor kinases. In order to provide further details of the molecular mechanism through which the unphosphorylated juxtamemebrane region blocks catalysis, we studied wild-type and site-specific mutants in detail. High-resolution structures of multiple states of EphA3 kinase with and without the juxtamembrane segment allowed us to map the coupled pathway of residues that connect the juxtamembrane segment, the activation loop, and the catalytic residues of the kinase domain. This highly conserved set of residues likely delineates a molecular recognition pathway for most of the Eph RTKs, helping to characterize the dynamic nature of these physiologically important enzymes.

  7. Molecular cloning of a docking protein, BRDG1, that acts downstream of the Tec tyrosine kinase

    PubMed Central

    Ohya, Ken-ichi; Kajigaya, Sachiko; Kitanaka, Akira; Yoshida, Koji; Miyazato, Akira; Yamashita, Yoshihiro; Yamanaka, Takeo; Ikeda, Uichi; Shimada, Kazuyuki; Ozawa, Keiya; Mano, Hiroyuki

    1999-01-01

    Tec, Btk, Itk, Bmx, and Txk constitute the Tec family of protein tyrosine kinases (PTKs), a family with the distinct feature of containing a pleckstrin homology (PH) domain. Tec acts in signaling pathways triggered by the B cell antigen receptor (BCR), cytokine receptors, integrins, and receptor-type PTKs. Although upstream regulators of Tec family kinases are relatively well characterized, little is known of the downstream effectors of these enzymes. The yeast two-hybrid system has identified several proteins that interact with the kinase domain of Tec, one of which is now revealed to be a previously unknown docking protein termed BRDG1 (BCR downstream signaling 1). BRDG1 contains a proline-rich motif, a PH domain, and multiple tyrosine residues that are potential target sites for Src homology 2 domains. In 293 cells expressing recombinant BRDG1 and various PTKs, Tec and Pyk2, but not Btk, Bmx, Lyn, Syk, or c-Abl, induced marked phosphorylation of BRDG1 on tyrosine residues. BRDG1 was also phosphorylated by Tec directly in vitro. Efficient phosphorylation of BRDG1 by Tec required the PH and SH2 domains as well as the kinase domain of the latter. Furthermore, BRDG1 was shown to participate in a positive feedback loop by increasing the activity of Tec. BRDG1 transcripts are abundant in the human B cell line Ramos, and the endogenous protein underwent tyrosine phosphorylation in response to BCR stimulation. BRDG1 thus appears to function as a docking protein acting downstream of Tec in BCR signaling. PMID:10518561

  8. Tyrosine kinase receptor alteration of renal vasoconstriction in rats is sex- and age-related.

    PubMed

    Passmore, John C; Fleming, John T; Tyagi, Suresh C; Falcone, Jeff C

    2012-10-01

    Male rat renal blood vessels undergo reduced contraction to norepinephrine with aging. There is a greater renal vascular impairment in male compared with female rats. We investigated specific tyrosine kinase receptor inhibition of renal interlobar artery responsiveness to phenylephrine in male and female rats at specifically designated ages. Vessels from young male rats contracted much less to phenylephrine when the vessels were pretreated with the tyrosine kinase inhibitors Lavendustin A, HNMPA-(AM)₃, or AG1478. Vessels from adult female rats pretreated with Lavendustin A showed no difference in contraction from control, but did demonstrate a slightly reduced contraction when pretreated with AG1478. Middle-aged male rat vessels treated with Lavendustin A demonstrated no inhibition, but the insulin and epidermal growth factor receptor (EGFR) antagonists both induced a decline in contraction. Vessels from aged male rats demonstrated no effect related to the 3 pretreatments. Middle-aged and aged female rats pretreated with any inhibitor demonstrated no inhibitor-dependent alterations. We conclude that maximum contraction of interlobar arteries from adult male rats is reduced when tyrosine kinase receptor activity is reduced. Female rats demonstrated much less inhibitor-related change of contraction. PMID:22724583

  9. Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors.

    PubMed

    Ji, Hongbin; Zhao, Xiaojun; Yuza, Yuki; Shimamura, Takeshi; Li, Danan; Protopopov, Alexei; Jung, Boonim L; McNamara, Kate; Xia, Huili; Glatt, Karen A; Thomas, Roman K; Sasaki, Hidefumi; Horner, James W; Eck, Michael; Mitchell, Albert; Sun, Yangping; Al-Hashem, Ruqayyah; Bronson, Roderick T; Rabindran, Sridhar K; Discafani, Carolyn M; Maher, Elizabeth; Shapiro, Geoffrey I; Meyerson, Matthew; Wong, Kwok-Kin

    2006-05-16

    The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2-7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation. PMID:16672372

  10. Tyrosine Kinase Receptor Landscape in Lung Cancer: Therapeutical Implications.

    PubMed

    Quintanal-Villalonga, A; Paz-Ares, Luis; Ferrer, Irene; Molina-Pinelo, S

    2016-01-01

    Lung cancer is a heterogeneous disease responsible for the most cases of cancer-related deaths. The majority of patients are clinically diagnosed at advanced stages, with a poor survival rate. For this reason, the identification of oncodrivers and novel biomarkers is decisive for the future clinical management of this pathology. The rise of high throughput technologies popularly referred to as "omics" has accelerated the discovery of new biomarkers and drivers for this pathology. Within them, tyrosine kinase receptors (TKRs) have proven to be of importance as diagnostic, prognostic, and predictive tools and, due to their molecular nature, as therapeutic targets. Along this review, the role of TKRs in the different lung cancer histologies, research on improvement of anti-TKR therapy, and the current approaches to manage anti-TKR resistance will be discussed. PMID:27528792

  11. Recent developments in receptor tyrosine kinases targeted anticancer therapy

    PubMed Central

    Raval, Samir H.; Singh, Ratn D.; Joshi, Dilip V.; Patel, Hitesh B.; Mody, Shailesh K.

    2016-01-01

    Novel concepts and understanding of receptors lead to discoveries and optimization of many small molecules and antibodies as anti-cancerous drugs. Receptor tyrosine kinases (RTKs) are such a promising class of receptors under the investigation in past three decades. RTKs are one of the essential mediators of cell signaling mechanism for various cellular processes. Transformations such as overexpression, dysregulation, or mutations of RTKs may result into malignancy, and thus are an important target for anticancer therapy. Numerous subfamilies of RTKs, such as epidermal growth factor receptor, vascular endothelial growth factor receptor, fibroblast growth factor receptors, insulin-like growth factor receptor, and hepatocyte growth factor receptor, have been being investigated in recent years as target for anticancer therapy. The present review focuses several small molecules drugs as well as monoclonal antibodies targeting aforesaid subfamilies either approved or under investigation to treat the various cancers. PMID:27051190

  12. Receptor Tyrosine Kinases: Molecular Switches Regulating CNS Axon Regeneration

    PubMed Central

    Vigneswara, Vasanthy; Kundi, Sarina; Ahmed, Zubair

    2012-01-01

    The poor or lack of injured adult central nervous system (CNS) axon regeneration results in devastating consequences and poor functional recovery. The interplay between the intrinsic and extrinsic factors contributes to robust inhibition of axon regeneration of injured CNS neurons. The insufficient or lack of trophic support for injured neurons is considered as one of the major obstacles contributing to their failure to survive and regrow their axons after injury. In the CNS, many of the signalling pathways associated with neuronal survival and axon regeneration are regulated by several classes of receptor tyrosine kinases (RTK) that respond to a variety of ligands. This paper highlights and summarises the most relevant recent findings pertinent to different classes of the RTK family of molecules, with a particular focus on elucidating their role in CNS axon regeneration. PMID:22848811

  13. Tyrosine Kinase Receptor Landscape in Lung Cancer: Therapeutical Implications

    PubMed Central

    Quintanal-Villalonga, A.; Paz-Ares, Luis

    2016-01-01

    Lung cancer is a heterogeneous disease responsible for the most cases of cancer-related deaths. The majority of patients are clinically diagnosed at advanced stages, with a poor survival rate. For this reason, the identification of oncodrivers and novel biomarkers is decisive for the future clinical management of this pathology. The rise of high throughput technologies popularly referred to as “omics” has accelerated the discovery of new biomarkers and drivers for this pathology. Within them, tyrosine kinase receptors (TKRs) have proven to be of importance as diagnostic, prognostic, and predictive tools and, due to their molecular nature, as therapeutic targets. Along this review, the role of TKRs in the different lung cancer histologies, research on improvement of anti-TKR therapy, and the current approaches to manage anti-TKR resistance will be discussed. PMID:27528792

  14. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

    PubMed

    Chen, Ying-Nan P; LaMarche, Matthew J; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G; Dobson, Jason R; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J; Sellers, William R; Stams, Travis; Fortin, Pascal D

    2016-07-01

    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 phosphatase. 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 neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. 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. PMID:27362227

  15. A Novel Triazolopyridine-Based Spleen Tyrosine Kinase Inhibitor That Arrests Joint Inflammation

    PubMed Central

    Ferguson, Gregory D.; Delgado, Mercedes; Plantevin-Krenitsky, Veronique; Jensen-Pergakes, Kristen; Bates, R. J.; Torres, Sanaa; Celeridad, Maria; Brown, Heather; Burnett, Kelven; Nadolny, Lisa; Tehrani, Lida; Packard, Garrick; Pagarigan, Barbra; Haelewyn, Jason; Nguyen, Trish; Xu, Li; Tang, Yang; Hickman, Matthew; Baculi, Frans; Pierce, Steven; Miyazawa, Keiji; Jackson, Pilgrim; Chamberlain, Philip; LeBrun, Laurie; Xie, Weilin; Bennett, Brydon; Blease, Kate

    2016-01-01

    Autoantibodies and the immunoreceptors to which they bind can contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Spleen Tyrosine Kinase (Syk) is a non-receptor tyrosine kinase with a central role in immunoreceptor (FcR) signaling and immune cell functionality. Syk kinase inhibitors have activity in antibody-dependent immune cell activation assays, in preclinical models of arthritis, and have progressed into clinical trials for RA and other autoimmune diseases. Here we describe the characterization of a novel triazolopyridine-based Syk kinase inhibitor, CC-509. This compound is a potent inhibitor of purified Syk enzyme, FcR-dependent and FcR-independent signaling in primary immune cells, and basophil activation in human whole blood. CC-509 is moderately selective across the kinome and against other non-kinase enzymes or receptors. Importantly, CC-509 was optimized away from and has modest activity against cellular KDR and Jak2, kinases that when inhibited in a preclinical and clinical setting may promote hypertension and neutropenia, respectively. In addition, CC-509 is orally bioavailable and displays dose-dependent efficacy in two rodent models of immune-inflammatory disease. In passive cutaneous anaphylaxis (PCA), CC-509 significantly inhibited skin edema. Moreover, CC-509 significantly reduced paw swelling and the tissue levels of pro-inflammatory cytokines RANTES and MIP-1α in the collagen-induced arthritis (CIA) model. In summary, CC-509 is a potent, moderately selective, and efficacious inhibitor of Syk that has a differentiated profile when compared to other Syk compounds that have progressed into the clinic for RA. PMID:26756335

  16. Phosphorylation of Mycobacterium tuberculosis protein tyrosine kinase A PtkA by Ser/Thr protein kinases.

    PubMed

    Zhou, Peifu; Wong, Dennis; Li, Wu; Xie, Jianping; Av-Gay, Yossef

    2015-11-13

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has inflicted about one third of mankind and claims millions of deaths worldwide annually. Signalling plays an important role in Mtb pathogenesis and persistence, and thus represents attractive resource for drug target candidates. Here, we show that protein tyrosine kinase A (PtkA) can be phosphorylated by Mtb endogenous eukaryotic-like Ser/Thr protein kinases (eSTPKs). Kinase assays showed that PknA, PknD, PknF, and PknK can phosphorylate PtkA in dose- and time-dependent manner. Enzyme kinetics suggests that PknA has the highest affinity and enzymatic efficiency towards PtkA. Furthermore, protein-protein interaction assay in surrogate host showed that PtkA interacts with multi-eSTPKs in vivo, including PknA. Lastly, we show that PtkA phosphorylation by eSTPKs occurs on threonine residues and may effect tyrosine phosphorylation levels and thus PtkA activity in vitro. These results demonstrate that PtkA can serve as a substrate to many eSTPKs and suggests that's its activity can be regulated. PMID:26417687

  17. Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics.

    PubMed

    Ferrando, Isabel Martinez; Chaerkady, Raghothama; Zhong, Jun; Molina, Henrik; Jacob, Harrys K C; Herbst-Robinson, Katie; Dancy, Beverley M; Katju, Vikram; Bose, Ron; Zhang, Jin; Pandey, Akhilesh; Cole, Philip A

    2012-08-01

    The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of the substrates of c-Src remains a major challenge, because there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable isotope labeling of amino acids in cell culture-based quantitation allowed the detection of 97 nonredundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of

  18. Grb2, a Double-Edged Sword of Receptor Tyrosine Kinase Signaling

    PubMed Central

    Belov, Artur A.; Mohammadi, Moosa

    2013-01-01

    Receptor tyrosine kinases (RTKs) exhibit basal tyrosine phosphorylation and activity in the absence of ligand stimulation, which has been attributed to the “leaky” nature of tyrosine kinase autoinhibition and stochastic collisions of receptors in the membrane bilayer. This basal phosphorylation does not produce a signal of sufficient amplitude and intensity to manifest in a biological response and hence is considered to be a passive, futile process that does not have any biological function. This paradigm has now been challenged by a study showing that the basal phosphorylation of RTKs is a physiologically relevant process that is actively inhibited by the intracellular adaptor protein growth factor receptor-bound 2 (Grb2) and serves to “prime” receptors for a rapid response to ligand stimulation. Grb2 is conventionally known for playing positive roles in RTK signaling. The discovery of a negative regulatory role for Grb2 reveals that this adaptor acts as a double-edged sword in the regulation of RTK signaling. PMID:23131845

  19. Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

    PubMed

    Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

    2016-04-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

  20. The role of non-receptor protein tyrosine kinases in the excitotoxicity induced by the overactivation of NMDA receptors.

    PubMed

    Sun, Yongjun; Chen, You; Zhan, Liying; Zhang, Linan; Hu, Jie; Gao, Zibin

    2016-04-01

    Protein tyrosine phosphorylation is one of the primary modes of regulation of N-methyl-d-aspartate (NMDA) receptors. The non-receptor tyrosine kinases are one of the two types of protein tyrosine kinases that are involved in this process. The overactivation of NMDA receptors is a primary reason for neuron death following cerebral ischemia. Many studies have illustrated the important role of non-receptor tyrosine kinases in ischemia insults. This review introduces the roles of Src, Fyn, focal adhesion kinase, and proline-rich tyrosine kinase 2 in the excitotoxicity induced by the overactivation of NMDA receptors following cerebral ischemia. PMID:26540220

  1. Cucurbitacin I Inhibits Rac1 Activation in Breast Cancer Cells by a Reactive Oxygen Species-Mediated Mechanism and Independently of Janus Tyrosine Kinase 2 and P-Rex1

    PubMed Central

    Lopez-Haber, Cynthia

    2013-01-01

    The small GTPase Rac1 has been widely implicated in mammary tumorigenesis and metastasis. Previous studies established that stimulation of ErbB receptors in breast cancer cells activates Rac1 and enhances motility via the Rac-guanine nucleotide exchange factor P-Rex1. As the Janus tyrosine kinase 2 (Jak2)/signal transducer and activator of transcription 3 (Stat3) pathway has been shown to be functionally associated with ErbB receptors, we asked if this pathway could mediate P-Rex1/Rac1 activation in response to ErbB ligands. Here we found that the anticancer agent cucurbitacin I, a Jak2 inhibitor, reduced the activation of Rac1 and motility in response to the ErbB3 ligand heregulin in breast cancer cells. However, Rac1 activation was not affected by Jak2 or Stat3 RNA interference, suggesting that the effect of cucurbitacin I occurs through a Jak2-independent mechanism. Cucurbitacin I also failed to affect the activation of P-Rex1 by heregulin. Subsequent analysis revealed that cucurbitacin I strongly activates RhoA and the Rho effector Rho kinase (ROCK) in breast cancer cells and induces the formation of stress fibers. Interestingly, disruption of the RhoA-ROCK pathway prevented the inhibitory effect of cucurbitacin I on Rac1 activation by heregulin. Lastly, we found that RhoA activation by cucurbitacin I is mediated by reactive oxygen species (ROS). The ROS scavenger N-acetyl l-cysteine and the mitochondrial antioxidant Mito-TEMPO rescued the inhibitory effect of cucurbitacin I on Rac1 activation. In conclusion, these results indicate that ErbB-driven Rac1 activation in breast cancer cells proceeds independently of the Jak2 pathway. Moreover, they established that the inhibitory effect of cucurbitacin I on Rac1 activity involves the alteration of the balance between Rho and Rac. PMID:23478800

  2. p120 Catenin-Associated Fer and Fyn Tyrosine Kinases Regulate β-Catenin Tyr-142 Phosphorylation and β-Catenin-α-Catenin Interaction

    PubMed Central

    Piedra, Jose; Miravet, Susana; Castaño, Julio; Pálmer, Héctor G.; Heisterkamp, Nora; García de Herreros, Antonio; Duñach, Mireia

    2003-01-01

    β-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and α-catenin. We show here that interaction of β-catenin with α-catenin is regulated by the phosphorylation of β-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases. Transfection of these kinases to epithelial cells disrupted the association between both catenins. We have also examined whether these kinases are involved in the regulation of this interaction by K-ras. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little α-catenin-β-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of β-catenin-α-catenin interaction. PMID:12640114

  3. Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function

    PubMed Central

    Ko, Han Seok; Lee, Yunjong; Shin, Joo-Ho; Karuppagounder, Senthilkumar S.; Gadad, Bharathi Shrikanth; Koleske, Anthony J.; Pletnikova, Olga; Troncoso, Juan C.; Dawson, Valina L.; Dawson, Ted M.

    2010-01-01

    Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkin's ubiquitin E3 ligase activity and protective function. c-Abl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571’s protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD. PMID:20823226

  4. A Myxococcus xanthus Bacterial Tyrosine Kinase, BtkA, Is Required for the Formation of Mature Spores▿

    PubMed Central

    Kimura, Yoshio; Yamashita, Shinji; Mori, Yumi; Kitajima, Yuki; Takegawa, Kaoru

    2011-01-01

    A Myxococcus xanthus cytoplasmic bacterial tyrosine kinase, BtkA, showed phosphorylation activity in the presence of Exo. Phosphorylated BtkA was expressed late after starvation induction and early after glycerol induction. The btkA mutant was unable to complete maturation to heat- and sonication-resistant spores under both starvation- and glycerol-induced developmental conditions. PMID:21840977

  5. Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization

    SciTech Connect

    Takahashi, Akinori; Obata, Yuuki; Fukumoto, Yasunori; Nakayama, Yuji; Kasahara, Kousuke; Kuga, Takahisa; Higashiyama, Yukihiro; Saito, Takashi; Yokoyama, Kazunari K.; Yamaguchi, Naoto

    2009-04-15

    Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G{sub 1} phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

  6. Structural Basis for Autoinhibition of c-Abl Tyrosine Kinase

    SciTech Connect

    Nagar, Bhushan; Hantschel, Oliver; Young, Matthew A.; Scheffzek,Klaus; Veach, Darren; Bornmann, William; Clarkson, Bayard; Superti-Furga,Giulio; Kuriyan, John

    2003-03-21

    c-Abl is normally regulated by an autoinhibitory mechanism, the disruption of which leads to chronic myelogenous leukemia. The details of this mechanism have been elusive because c-Abl lacks aphosphotyrosine residue that triggers the assembly of the autoinhibited form of the closely related Src kinases by internally engaging the SH2 domain. Crystal structures of c-Abl show that the N-terminal myristoyl modification of c-Abl 1b binds to the kinase domain and induces conformational changes that allow the SH2 and SH3 domains to dock onto it. Autoinhibited c-Abl forms an assembly that is strikingly similar to that of inactive Src kinases but with specific differences that explain the differential ability of the drug STI-571/Gleevec/imatinib (STI-571)to inhibit the catalytic activity of Abl, but not that of c-Src.

  7. The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

    PubMed Central

    Roh, Kyung-Ho; Lillemeier, Björn F.; Wang, Feng; Davis, Mark M.

    2015-01-01

    CD4 molecules on the surface of T lymphocytes greatly augment the sensitivity and activation process of these cells, but how it functions is not fully understood. Here we studied the spatial organization of CD4, and its relationship to T-cell antigen receptor (TCR) and the active form of Src kinase p56lck (Lck) using single and dual-color photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). In nonactivated T cells, CD4 molecules are clustered in small protein islands, as are TCR and Lck. By dual-color imaging, we find that CD4, TCR, and Lck are localized in their separate clusters with limited interactions in the interfaces between them. Upon T-cell activation, the TCR and CD4 begin clustering together, developing into microclusters, and undergo a larger scale redistribution to form supramolecluar activation clusters (SMACs). CD4 and Lck localize in the inner TCR region of the SMAC, but this redistribution of disparate cluster structures results in enhanced segregation from each other. In nonactivated cells these preclustered structures and the limited interactions between them may serve to limit spontaneous and random activation events. However, the small sizes of these island structures also ensure large interfacial surfaces for potential interactions and signal amplification when activation is initiated. In the later activation stages, the increasingly larger clusters and their segregation from each other reduce the interfacial surfaces and could have a dampening effect. These highly differentiated spatial distributions of TCR, CD4, and Lck and their changes during activation suggest that there is a more complex hierarchy than previously thought. PMID:25829544

  8. Oral administration of Bruton's tyrosine kinase inhibitors impairs GPVI-mediated platelet function.

    PubMed

    Rigg, Rachel A; Aslan, Joseph E; Healy, Laura D; Wallisch, Michael; Thierheimer, Marisa L D; Loren, Cassandra P; Pang, Jiaqing; Hinds, Monica T; Gruber, András; McCarty, Owen J T

    2016-03-01

    The Tec family kinase Bruton's tyrosine kinase (Btk) plays an important signaling role downstream of immunoreceptor tyrosine-based activation motifs in hematopoietic cells. Mutations in Btk are involved in impaired B-cell maturation in X-linked agammaglobulinemia, and Btk has been investigated for its role in platelet activation via activation of the effector protein phospholipase Cγ2 downstream of the platelet membrane glycoprotein VI (GPVI). Because of its role in hematopoietic cell signaling, Btk has become a target in the treatment of chronic lymphocytic leukemia and mantle cell lymphoma; the covalent Btk inhibitor ibrutinib was recently approved by the US Food and Drug Administration for treatment of these conditions. Antihemostatic events have been reported in some patients taking ibrutinib, although the mechanism of these events remains unknown. We sought to determine the effects of Btk inhibition on platelet function in a series of in vitro studies of platelet activation, spreading, and aggregation. Our results show that irreversible inhibition of Btk with two ibrutinib analogs in vitro decreased human platelet activation, phosphorylation of Btk, P-selectin exposure, spreading on fibrinogen, and aggregation under shear flow conditions. Short-term studies of ibrutinib analogs administered in vivo also showed abrogation of platelet aggregation in vitro, but without measurable effects on plasma clotting times or on bleeding in vivo. Taken together, our results suggest that inhibition of Btk significantly decreased GPVI-mediated platelet activation, spreading, and aggregation in vitro; however, prolonged bleeding was not observed in a model of bleeding. PMID:26659727

  9. Have adjuvant tyrosine kinase inhibitors lost their shine?

    PubMed Central

    Sabari, Joshua K.

    2016-01-01

    Despite broad advances in molecularly targeted therapies, lung cancer remains the leading cause of cancer related mortality in the United States. Epidermal growth factor receptor (EGFR) mutations occur in approximately 17% of advanced non-small cell lung cancer (NSCLC) in the US population. The remarkable efficacy of small-molecule EGFR tyrosine kinase inhibitors (TKIs) in this unique subset of patients has revolutionized the therapeutic approach to lung cancer. The success of these agents in the metastatic setting leads to the logical question of what role these drugs may have in the adjuvant setting for patients with earlier stage disease. RADIANT, an international randomized, double-blind, placebo controlled phase III study in patients with completely resected stage IB to IIIA NSLC whose tumors expressed EGFR by IHC and EGFR amplification by FISH, attempted to answer the question of whether erlotinib would improve disease free survival and overall survival in the adjuvant setting. While RADIANT does not conclude for or against adjuvant use of EGFR-TKIs, all data points towards benefit in a selected population. As clinicians, we must continue to enroll to potentially practice changing therapeutic neoadjuvant and adjuvant chemotherapy studies internationally. PMID:27568486

  10. Have adjuvant tyrosine kinase inhibitors lost their shine?

    PubMed

    Sabari, Joshua K; Chaft, Jamie E

    2016-08-01

    Despite broad advances in molecularly targeted therapies, lung cancer remains the leading cause of cancer related mortality in the United States. Epidermal growth factor receptor (EGFR) mutations occur in approximately 17% of advanced non-small cell lung cancer (NSCLC) in the US population. The remarkable efficacy of small-molecule EGFR tyrosine kinase inhibitors (TKIs) in this unique subset of patients has revolutionized the therapeutic approach to lung cancer. The success of these agents in the metastatic setting leads to the logical question of what role these drugs may have in the adjuvant setting for patients with earlier stage disease. RADIANT, an international randomized, double-blind, placebo controlled phase III study in patients with completely resected stage IB to IIIA NSLC whose tumors expressed EGFR by IHC and EGFR amplification by FISH, attempted to answer the question of whether erlotinib would improve disease free survival and overall survival in the adjuvant setting. While RADIANT does not conclude for or against adjuvant use of EGFR-TKIs, all data points towards benefit in a selected population. As clinicians, we must continue to enroll to potentially practice changing therapeutic neoadjuvant and adjuvant chemotherapy studies internationally. PMID:27568486

  11. Mutations in Bruton's tyrosine kinase impair IgA responses.

    PubMed

    Mitsuiki, Noriko; Yang, Xi; Bartol, Sophinus J W; Grosserichter-Wagener, Christina; Kosaka, Yoshiyuki; Takada, Hidetoshi; Imai, Kohsuke; Kanegane, Hirokazu; Mizutani, Shuki; van der Burg, Mirjam; van Zelm, Menno C; Ohara, Osamu; Morio, Tomohiro

    2015-03-01

    X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by mutations in Bruton's tyrosine kinase (BTK), and is characterized by markedly decreased numbers of blood B cells and an absence of all immunoglobulin isotypes. We performed whole exome sequencing in a male pediatric patient with dysgammaglobulinemia with IgA deficiency. Genetic analysis revealed a BTK missense mutation (Thr316Ala). To investigate whether a BTK mutation underlay this antibody deficiency with marked decrease of IgA in this patient, we performed functional analyses of B cells and phagocytes, and molecular analyses of somatic hypermutation and class switch recombination. The BTK missense mutation resulted in B cells with reduced BTK and high IgM expression. Equal proportions of CD19(low) and CD19(normal) fractions were observed, and both included naïve and memory B cells. Calcium influx and phospholipase Cγ2 phosphorylation upon IgM stimulation were marginally impaired in CD19(low), but not in CD19(+) B cells. Similar to XLA patients, IgA transcripts showed low SHM levels, whereas IgG transcripts were hardly affected. Our analyses suggest that the BTK mutation likely underlies the disease in this case, and that hypomorphic BTK mutations can result in normal circulating B cell numbers, but specifically impair IgA responses. PMID:25589397

  12. Studying N-linked glycosylation of receptor tyrosine kinases.

    PubMed

    Itkonen, Harri M; Mills, Ian G

    2015-01-01

    Metabolic alterations have been identified as a frequent event in cancer. This is often associated with increased flux through glycolysis, and also a secondary pathway to glycolysis, hexosamine biosynthetic pathway (HBP). HBP provides substrate for N-linked glycosylation, which occurs in the endoplasmic reticulum and the Golgi apparatus. N-linked glycosylation supports protein folding and correct sorting of proteins to plasma membrane and secretion. This process generates complex glycoforms, which can be recognized by other proteins and glycosylation of receptor tyrosine kinases (RTK) can also regulate their plasma-membrane retention time. Of special interest for experimental biologists, plants produce proteins, termed lectins, which bind with high specificity to glyco-conjugates. For the purposes of molecular biology, plant lectins can be conjugated to different moieties, such as agarose beads, which enable precipitation of specifically glycosylated proteins. In this chapter, we describe in detail how to perform pull-down experiments with commercially available lectins to identify changes in the glycosylation of RTKs. PMID:25319893

  13. Bruton's tyrosine kinase inhibitors in chronic lymphocytic leukemia and lymphoma.

    PubMed

    Varma, Gaurav; Johnson, Tyler P; Advani, Ranjana H

    2016-07-01

    The development of Bruton's tyrosine kinase (BTK) inhibitors and their introduction into clinical practice represent a major advance in the treatment of chronic lymphocytic leukemia (CLL) and other B-cell lymphomas. Although ibrutinib is the only BTK inhibitor that has been approved by the US Food and Drug Administration, several others are under investigation. Ibrutinib is currently approved for use in relapsed/refractory CLL, CLL with 17p deletion (del[17p]), relapsed or refractory mantle cell lymphoma, and Waldenström macroglobulinemia. Although it is clear that ibrutinib has altered treatment paradigms and outcomes in these diseases, several questions remain regarding (1) its role in frontline vs salvage therapy; (2) its use as a single agent vs in combination with biologic agents, other small molecules, or traditional chemoimmunotherapy; (3) the optimal duration of treatment; and (4) the treatment of patients who cannot tolerate or have disease resistant to ibrutinib. Because sparse clinical data are available on other BTK inhibitors, it is unclear at present whether their clinical efficacy and toxicity will differ from those of ibrutinib. PMID:27379948

  14. Emerging Role of Tyrosine Kinases as Drugable Targets in Cancer

    PubMed Central

    Na, Il-Kang; le Coutre, Philipp

    2015-01-01

    Tyrosine kinases (TKs) play a significant role in cancerogenesis and cancer cell function. Initial developments in this field go back to the early 80s, but the success story really started with the selective BCR-ABL inhibitor, imatinib. Owing to the cancer-driving role of BCR-ABL in chronic myeloid leukemia (CML), excellent response rates lead to fast FDA approval in both the first and second treatments of CML patients. Since then, numerous TKs were identified. TK inhibitors have been developed accordingly, and technology to test for ideal drug–target interactions has profoundly improved. By now, medical oncologists and hematologists struggle to have a pool of potential TK inhibitors, where the most efficient one could be picked out to treat a specific cancer patient, which might also help overcome the occurring resistance mechanisms against TK inhibitors. Whether disease eradication can be achieved via single or sequential TK inhibitor treatment(s) needs to be tested in the present and in the future. PMID:26401097

  15. Proteolytic cleavage, trafficking, and functions of nuclear receptor tyrosine kinases.

    PubMed

    Chen, Mei-Kuang; Hung, Mien-Chie

    2015-10-01

    Intracellular localization has been reported for over three-quarters of receptor tyrosine kinase (RTK) families in response to environmental stimuli. Internalized RTK may bind to non-canonical substrates and affect various cellular processes. Many of the intracellular RTKs exist as fragmented forms that are generated by γ-secretase cleavage of the full-length receptor, shedding, alternative splicing, or alternative translation initiation. Soluble RTK fragments are stabilized and intracellularly transported into subcellular compartments, such as the nucleus, by binding to chaperone or transcription factors, while membrane-bound RTKs (full-length or truncated) are transported from the plasma membrane to the ER through the well-established Rab- or clathrin adaptor protein-coated vesicle retrograde trafficking pathways. Subsequent nuclear transport of membrane-bound RTK may occur via two pathways, INFS or INTERNET, with the former characterized by release of receptors from the ER into the cytosol and the latter characterized by release of membrane-bound receptor from the ER into the nucleoplasm through the inner nuclear membrane. Although most non-canonical intracellular RTK signaling is related to transcriptional regulation, there may be other functions that have yet to be discovered. In this review, we summarize the proteolytic processing, intracellular trafficking and nuclear functions of RTKs, and discuss how they promote cancer progression, and their clinical implications. PMID:26096795

  16. Management of tyrosine kinase inhibitor resistance in lung cancer with EGFR mutation

    PubMed Central

    Becker, Kevin; Xu, Yiqing

    2014-01-01

    The identification of driver mutations and drugs that inhibit their activity has been a major therapeutic advance for patients with advanced lung adenocarcinoma. Unfortunately, the success of these drugs is limited by the universal development of resistance. Treatment failure can result from inadequate drug exposure or selection of resistant malignant clones. Clinically distinct mechanisms of disease progression have been identified and can inform treatment decisions. Investigations into the biochemical mechanisms of tyrosine kinase inhibitor resistance may provide additional therapeutic targets by which the efficacy of targeted therapy can be improved. PMID:25302160

  17. Regulation of Connexin43 Function and Expression by Tyrosine Kinase 2.

    PubMed

    Li, Hanjun; Spagnol, Gaelle; Zheng, Li; Stauch, Kelly L; Sorgen, Paul L

    2016-07-22

    Connexin43 (Cx43) assembly and degradation, the regulation of electrical and metabolic coupling, as well as modulating the interaction with other proteins, involve phosphorylation. Here, we identified and characterized the biological significance of a novel tyrosine kinase that phosphorylates Cx43, tyrosine kinase 2 (Tyk2). Activation of Tyk2 led to a decrease in Cx43 gap junction communication by increasing the turnover rate of Cx43 from the plasma membrane. Tyk2 directly phosphorylated Cx43 residues Tyr-247 and Tyr-265, leading to indirect phosphorylation on residues Ser-279/Ser-282 (MAPK) and Ser-368 (PKC). Although this phosphorylation pattern is similar to what has been observed following Src activation, the response caused by Tyk2 occurred when Src was inactive in NRK cells. Knockdown of Tyk2 at the permissive temperature (active v-Src) in LA-25 cells decreased Cx43 phosphorylation, indicating that although activation of Tyk2 and v-Src leads to phosphorylation of the same Cx43CT residues, they are not identical in level at each site. Additionally, angiotensin II activation of Tyk2 increased the intracellular protein level of Cx43 via STAT3. These findings indicate that, like Src, Tyk2 can also inhibit gap junction communication by phosphorylating Cx43. PMID:27235399

  18. The TAM family: phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer.

    PubMed

    Graham, Douglas K; DeRyckere, Deborah; Davies, Kurtis D; Earp, H Shelton

    2014-12-01

    The TYRO3, AXL (also known as UFO) and MERTK (TAM) family of receptor tyrosine kinases (RTKs) are aberrantly expressed in multiple haematological and epithelial malignancies. Rather than functioning as oncogenic drivers, their induction in tumour cells predominately promotes survival, chemoresistance and motility. The unique mode of maximal activation of this RTK family requires an extracellular lipid–protein complex. For example, the protein ligand, growth arrest-specific protein 6 (GAS6), binds to phosphatidylserine (PtdSer) that is externalized on apoptotic cell membranes, which activates MERTK on macrophages. This triggers engulfment of apoptotic material and subsequent anti-inflammatory macrophage polarization. In tumours, autocrine and paracrine ligands and apoptotic cells are abundant, which provide a survival signal to the tumour cell and favour an anti-inflammatory, immunosuppressive microenvironment. Thus, TAM kinase inhibition could stimulate antitumour immunity, reduce tumour cell survival, enhance chemosensitivity and diminish metastatic potential. PMID:25568918

  19. Endocytosis and the Src family of non-receptor tyrosine kinases

    PubMed Central

    Reinecke, James; Caplan, Steve

    2014-01-01

    The regulated intracellular transport of nutrient, adhesion, and growth factor receptors is crucial for maintaining cell and tissue homeostasis. Endocytosis, or endocytic membrane trafficking, involves the steps of intracellular transport that include, but are not limited to: internalization from the plasma membrane, sorting in early endosomes, transport to late endosomes/lysosomes followed by degradation, and/or recycling back to the plasma membrane via tubular recycling endosomes. In addition to regulating the localization of transmembrane receptor proteins, the endocytic pathway also controls the localization of non-receptor molecules. The non-receptor tyrosine kinase c-Src (Src), and its closely related family members Yes and Fyn, represent three proteins whose localization and signaling activities are tightly regulated by endocytic trafficking. Here, we provide a brief overview of endocytosis, Src function and its biochemical regulation. We will then concentrate on recent advances in understanding how Src intracellular localization is regulated and how its subcellular localization ultimately dictates downstream functioning. Since Src kinases are hyperactive in many cancers, it is essential to decipher the spatiotemporal regulation of this important family of tyrosine kinases. PMID:25372749

  20. Protein tyrosine kinase 6 mediates TNFα-induced endothelial barrier dysfunction

    PubMed Central

    Haines, RJ; Beard, RS; Wu, MH

    2014-01-01

    A key event in the progression of systemic inflammation resulting from severe trauma or shock involves microvascular hyperpermeability, which leads to excessive plasma fluid and proteins accumulating in extravascular space resulting in tissue edema. The precise molecular mechanism of the hyperpermeability response is not completely understood. Protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) is a non-receptor tyrosine kinase related to Src-family proteins. Although it has also been shown that PTK6 participates in regulating epithelial barrier function, the role of PTK6 in endothelial barrier function has not been reported. In this study, we hypothesized that PTK6 is 1) expressed in vascular endothelial cells, and 2) contributes to vascular endothelial hyperpermeability in response to TNFα. Results showed that PTK6 was detected in mouse endothelial cells at the level of protein and mRNA. In addition, PTK6 knockdown attenuated TNFα induced decrease in endothelial barrier function as measured by electric cell-substrate impedance sensing (ECIS) and in vitro transwell albumin-flux assays. Furthermore, we showed that TNFα treatment of endothelial cells increased active PTK6 association with p120-catenin at endothelial cell-cell junctions. Further analysis using immunocytochemistry and immunoprecipitation demonstrated that PTK6 knockdown attenuated TNFα induced VE-cadherin internalization as well as promoting its association with p120-catenin. Our study demonstrates a novel role of PTK6 in mediating endothelial barrier dysfunction. PMID:25446122

  1. Intersecting roles of protein tyrosine kinase and calcium signaling during fertilization.

    PubMed

    Kinsey, William H

    2013-01-01

    The oocyte is a highly specialized cell that must respond to fertilization with a preprogrammed series of signal transduction events that establish a block to polyspermy, trigger resumption of the cell cycle and execution of a developmental program. The fertilization-induced calcium transient is a key signal that initiates the process of oocyte activation and studies over the last several years have examined the signaling pathways that act upstream and downstream of this calcium transient. Protein tyrosine kinase signaling was found to be an important component of the upstream pathways that stimulated calcium release at fertilization in oocytes from animals that fertilize externally, but a similar pathway has not been found in mammals which fertilize internally. The following review will examine the diversity of signaling in oocytes from marine invertebrates, amphibians, fish and mammals in an attempt to understand the basis for the observed differences. In addition to the pathways upstream of the fertilization-induced calcium transient, recent studies are beginning to unravel the role of protein tyrosine kinase signaling downstream of the calcium transient. The PYK2 kinase was found to respond to fertilization in the zebrafish system and seems to represent a novel component of the response of the oocyte to fertilization. The potential impact of impaired PTK signaling in oocyte quality will also be discussed. PMID:23201334

  2. Management of Chronic Myeloid Leukemia Patients Resistant to Tyrosine Kinase Inhibitors Treatment

    PubMed Central

    Wieczorek, Agnieszka; Uharek, Lutz

    2015-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with a characteristic chromosomal translocation called the Philadelphia chromosome. This oncogene is generated by the fusion of breakpoint cluster region (BCR) and Abelson leukemia virus (ABL) genes and encodes a novel fusion gene translating into a protein with constitutive tyrosine kinase activity. The discovery and introduction of tyrosine kinase inhibitors (TKIs) irreversibly changed the landscape of CML treatment, leading to dramatic improvement in long-term survival rates. The majority of patients with CML in the chronic phase have a life expectancy comparable with that of healthy age-matched individuals. Although an enormous therapeutic improvement has been accomplished, there are still some unresolved issues in the treatment of patients with CML. One of the most important problems is based on the fact that TKIs can efficiently target proliferating mature cells but do not eradicate leukemic stem cells, allowing persistence of the malignant clone. Owing to the resistance mechanisms arising during the course of the disease, treatment with most of the approved BCR-ABL1 TKIs may become ineffective in a proportion of patients. This article highlights the different molecular mechanisms of acquired resistance being developed during treatment with TKIs as well as the pharmacological strategies to overcome it. Moreover, it gives an overview of novel drugs and therapies that are aiming in overcoming drug resistance, loss of response, and kinase domain mutations. PMID:26917943

  3. Serine/Threonine/Tyrosine Protein Kinase Phosphorylates Oleosin, a Regulator of Lipid Metabolic Functions1[OA

    PubMed Central

    Parthibane, Velayoudame; Iyappan, Ramachandiran; Vijayakumar, Anitha; Venkateshwari, Varadarajan; Rajasekharan, Ram

    2012-01-01

    Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A2 activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A2 activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca2+ inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation. PMID:22434039

  4. SPE-8, a protein-tyrosine kinase, localizes to the spermatid cell membrane through interaction with other members of the SPE-8 group spermatid activation signaling pathway in C. elegans

    PubMed Central

    2014-01-01

    Background The SPE-8 group gene products transduce the signal for spermatid activation initiated by extracellular zinc in C. elegans. Mutations in the spe-8 group genes result in hermaphrodite-derived spermatids that cannot activate to crawling spermatozoa, although spermatids from mutant males activate through a pathway induced by extracellular TRY-5 protease present in male seminal fluid. Results Here, we identify SPE-8 as a member of a large family of sperm-expressed non-receptor-like protein-tyrosine kinases. A rescuing SPE-8::GFP translational fusion reporter localizes to the plasma membrane in all spermatogenic cells from the primary spermatocyte stage through spermatids. Once spermatids become activated to spermatozoa, the reporter moves from the plasma membrane to the cytoplasm. Mutations in the spe-8 group genes spe-12, spe-19, and spe-27 disrupt localization of the reporter to the plasma membrane, while localization appears near normal in a spe-29 mutant background. Conclusions These results suggest that the SPE-8 group proteins form a functional complex localized at the plasma membrane, and that SPE-8 is correctly positioned only when all members of the SPE-8 group are present, with the possible exception of SPE-29. Further, SPE-8 is released from the membrane when the activation signal is transduced into the spermatid. PMID:25022984

  5. Role of tyrosine-kinase inhibitors in myeloproliferative neoplasms: comparative lessons learned.

    PubMed

    Pinilla-Ibarz, Javier; Sweet, Kendra L; Corrales-Yepez, Gabriela M; Komrokji, Rami S

    2016-01-01

    An important pathogenetic distinction in the classification of myeloproliferative neoplasms (MPNs) is the presence or absence of the BCR-ABL fusion gene, which encodes a unique oncogenic tyrosine kinase. The BCR-ABL fusion, caused by the formation of the Philadelphia chromosome (Ph) through translocation, constitutes the disease-initiating event in chronic myeloid leukemia. The development of successive BCR-ABL-targeted tyrosine-kinase inhibitors has led to greatly improved outcomes in patients with chronic myeloid leukemia, including high rates of complete hematologic, cytogenetic, and molecular responses. Such levels of treatment success have long been elusive for patients with Ph-negative MPNs, because of the difficulties in identifying specific driver proteins suitable as drug targets. However, in recent years an improved understanding of the complex pathobiology of classic Ph-negative MPNs, characterized by variable, overlapping multimutation profiles, has prompted the development of better and more broadly targeted (to pathway rather than protein) treatment options, particularly JAK inhibitors. In classic Ph-negative MPNs, overactivation of JAK-dependent signaling pathways is a central pathogenic mechanism, and mutually exclusive mutations in JAK2, MPL, and CALR linked to aberrant JAK activation are now recognized as key drivers of disease progression in myelofibrosis (MF). In clinical trials, the JAK1/JAK2 inhibitor ruxolitinib - the first therapy approved for MF worldwide - improved disease-related splenomegaly and symptoms independent of JAK2 (V617F) mutational status, and prolonged survival compared with placebo or standard therapy in patients with advanced MF. In separate trials, ruxolitinib also provided comprehensive hematologic control in patients with another Ph-negative MPN - polycythemia vera. However, complete cytogenetic or molecular responses with JAK inhibitors alone are normally not observed, underscoring the need for novel combination

  6. Attenuation of serum inducibility of immediate early genes by oncoproteins in tyrosine kinase signaling pathways.

    PubMed Central

    Yu, C L; Prochownik, E V; Imperiale, M J; Jove, R

    1993-01-01

    Immediate early genes involved in controlling cell proliferation are rapidly and transiently induced following stimulation of susceptible cells with serum. To study how oncoproteins regulate immediate early genes, we examined serum inducibility of these genes in cells transformed by various oncoproteins. We found that induction of the immediate early gene, c-fos, by serum stimulation was markedly attenuated in four independent cell lines stably transformed by the v-Src tyrosine kinase. Cells chronically transformed by other oncoproteins implicated in tyrosine kinase signaling pathways, including v-Sis, v-Ras, and v-Raf, showed the same pattern of attenuation. In contrast, serum inducibility of c-fos was not attenuated in cells transformed by simian virus 40, which is thought to transform cells through a different pathway. Cell cycle analyses showed that proliferation of these transformed cell lines could be arrested effectively in 0.1% serum, demonstrating that the attenuation was not simply due to continuous cycling of transformed cells after serum deprivation. Moreover, serum inducibility of other immediate early genes, including c-jun, junB, egr-1, and NGFI-B, also was strikingly attenuated by these same oncoproteins. Nuclear run-on transcription assays established that this attenuation of serum inducibility occurred at the transcriptional level. Finally, flow cytometric analysis demonstrated that serum-starved v-Src-transformed cells were viable and able to progress into S phase of the cell cycle after serum stimulation, even though the induction of immediate early genes was greatly attenuated in these cells. Our results suggest that activation of immediate early genes is repressed by chronic stimulation of tyrosine kinase signaling pathways in transformed cells. Images PMID:8384301

  7. KSHV-TK is a tyrosine kinase that disrupts focal adhesions and induces Rho-mediated cell contraction

    PubMed Central

    Gill, Michael B; Turner, Rachel; Stevenson, Philip G; Way, Michael

    2015-01-01

    Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma-associated herpesvirus (KSHV) is an extremely inefficient nucleoside kinase, when compared to TKs from related herpesviruses. We now show that KSHV-TK, in contrast to HSV1-TK, associates with the actin cytoskeleton and induces extensive cell contraction followed by membrane blebbing. These dramatic changes in cell morphology depend on the auto-phosphorylation of tyrosines 65, 85 and 120 in the N-terminus of KSHV-TK. Phosphorylation of tyrosines 65/85 and 120 results in an interaction with Crk family proteins and the p85 regulatory subunit of PI3-Kinase, respectively. The interaction of Crk with KSHV-TK leads to tyrosine phoshorylation of this cellular adaptor. Auto-phosphorylation of KSHV-TK also induces a loss of FAK and paxillin from focal adhesions, resulting in activation of RhoA-ROCK signalling to myosin II and cell contraction. In the absence of FAK or paxillin, KSHV-TK has no effect on focal adhesion integrity or cell morphology. Our observations demonstrate that by acting as a tyrosine kinase, KSHV-TK modulates signalling and cell morphology. PMID:25471072

  8. A Novel Isoform of the B Cell Tyrosine Kinase BTK Protects Breast Cancer Cells from Apoptosis

    PubMed Central

    Eifert, Cheryl; Wang, Xianhui; Kokabee, Leila; Kourtidis, Antonis; Jain, Ritu; Gerdes, Michael J.; Conklin, Douglas S.

    2016-01-01

    Tyrosine kinases orchestrate key cellular signaling pathways and their dysregulation is often associated with cellular transformation. Several recent cases in which inhibitors of tyrosine kinases have been successfully used as anticancer agents have underscored the importance of this class of proteins in the development of targeted cancer therapies. We have carried out a large-scale loss-of-function analysis of the human tyrosine kinases using RNA interference to identify novel survival factors for breast cancer cells. In addition to kinases with known roles in breast and other cancers, we identified several kinases that were previously unknown to be required for breast cancer cell survival. The most surprising of these was the cytosolic, nonreceptor tyrosine kinase, Bruton’s tyrosine kinase (BTK), which has been extensively studied in B cell development. Down regulation of this protein with RNAi or inhibition with pharmacological inhibitors causes apoptosis; overexpression inhibits apoptosis induced by Doxorubicin in breast cancer cells. Our results surprisingly show that BTK is expressed in several breast cancer cell lines and tumors. The predominant form of BTK found in tumor cells is transcribed from an alternative promoter and results in a protein with an amino-terminal extension. This alternate form of BTK is expressed at significantly higher levels in tumorigenic breast cells than in normal breast cells. Since this protein is a survival factor for these cells, it represents both a potential marker and novel therapeutic target for breast cancer. PMID:23913792

  9. Oxido-reductive regulation of vascular remodeling by receptor tyrosine kinase ROS1

    PubMed Central

    Ali, Ziad A.; de Jesus Perez, Vinicio; Yuan, Ke; Orcholski, Mark; Pan, Stephen; Qi, Wei; Chopra, Gaurav; Adams, Christopher; Kojima, Yoko; Leeper, Nicholas J.; Qu, Xiumei; Zaleta-Rivera, Kathia; Kato, Kimihiko; Yamada, Yoshiji; Oguri, Mitsutoshi; Kuchinsky, Allan; Hazen, Stanley L.; Jukema, J. Wouter; Ganesh, Santhi K.; Nabel, Elizabeth G.; Channon, Keith; Leon, Martin B.; Charest, Alain; Quertermous, Thomas; Ashley, Euan A.

    2014-01-01

    Angioplasty and stenting is the primary treatment for flow-limiting atherosclerosis; however, this strategy is limited by pathological vascular remodeling. Using a systems approach, we identified a role for the network hub gene glutathione peroxidase-1 (GPX1) in pathological remodeling following human blood vessel stenting. Constitutive deletion of Gpx1 in atherosclerotic mice recapitulated this phenotype of increased vascular smooth muscle cell (VSMC) proliferation and plaque formation. In an independent patient cohort, gene variant pair analysis identified an interaction of GPX1 with the orphan protooncogene receptor tyrosine kinase ROS1. A meta-analysis of the only genome-wide association studies of human neointima-induced in-stent stenosis confirmed the association of the ROS1 variant with pathological remodeling. Decreased GPX1 expression in atherosclerotic mice led to reductive stress via a time-dependent increase in glutathione, corresponding to phosphorylation of the ROS1 kinase activation site Y2274. Loss of GPX1 function was associated with both oxidative and reductive stress, the latter driving ROS1 activity via s-glutathiolation of critical residues of the ROS1 tyrosine phosphatase SHP-2. ROS1 inhibition with crizotinib and deglutathiolation of SHP-2 abolished GPX1-mediated increases in VSMC proliferation while leaving endothelialization intact. Our results indicate that GPX1-dependent alterations in oxido-reductive stress promote ROS1 activation and mediate vascular remodeling. PMID:25401476

  10. Receptor Tyrosine Kinase Signaling: Regulating Neural Crest Development One Phosphate at a Time

    PubMed Central

    Fantauzzo, Katherine A.; Soriano, Philippe

    2015-01-01

    Receptor tyrosine kinases (RTKs) bind to a subset of growth factors on the surface of cells and elicit responses with broad roles in developmental and postnatal cellular processes. Receptors in this subclass consist of an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular domain harboring a catalytic tyrosine kinase and regulatory sequences that are phosphorylated either by the receptor itself or various interacting proteins. Once activated, RTKs bind signaling molecules and recruit effector proteins to mediate downstream cellular responses through various intracellular signaling pathways. In this chapter, we will highlight the role of a subset of RTK families in regulating the activity of neural crest cells (NCCs) and the development of their derivatives in mammalian systems. NCCs are migratory, multipotent cells that can be subdivided into four axial populations, cranial, cardiac, vagal and trunk. These cells migrate throughout the vertebrate embryo along defined pathways and give rise to unique cell types and structures. Interestingly, individual RTK families often have specific functions in a subpopulation of NCCs that contribute to the diversity of these cells and their derivatives in the mammalian embryo. We will additionally discuss current methods used to investigate RTK signaling, including genetic, biochemical, large-scale proteomic and biosensor approaches, which can be applied to study intracellular signaling pathways active downstream of this receptor subclass during NCC development. PMID:25662260

  11. A novel calmodulin-β-PIX interaction and its implication in receptor tyrosine kinase regulation.

    PubMed

    Singh, Vinay K; Munro, Kim; Jia, Zongchao

    2012-09-01

    Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates numerous cellular processes, primarily in response to calcium flux. We have identified and characterized a novel interaction between CaM and β-p21-activated kinase interacting exchange factor (β-PIX), a putative guanine exchange factor implicated in cell signaling, using affinity pull-down assays, co-immunoprecipitation, co-localization and circular dichroism studies. Fluorescence-based titration and isothermal titration calorimetry experiments revealed a Ca(2+)-dependent binding mechanism (K(D)≤10μM). Further, we show that CaM participates in a multi-protein complex involving β-PIX and E3 ubiquitin ligase c-Cbl (casitas B-cell lymphoma), which may play a critical role in receptor tyrosine kinase regulation and downstream signaling. PMID:22588125

  12. Increasing the Receptor Tyrosine Kinase EphB2 Prevents Amyloid-β-induced Depletion of Cell Surface Glutamate Receptors by a Mechanism That Requires the PDZ-binding Motif of EphB2 and Neuronal Activity*

    PubMed Central

    Miyamoto, Takashi; Kim, Daniel; Knox, Joseph A.; Johnson, Erik; Mucke, Lennart

    2016-01-01

    Diverse lines of evidence suggest that amyloid-β (Aβ) peptides causally contribute to the pathogenesis of Alzheimer disease (AD), the most frequent neurodegenerative disorder. However, the mechanisms by which Aβ impairs neuronal functions remain to be fully elucidated. Previous studies showed that soluble Aβ oligomers interfere with synaptic functions by depleting NMDA-type glutamate receptors (NMDARs) from the neuronal surface and that overexpression of the receptor tyrosine kinase EphB2 can counteract this process. Through pharmacological treatments and biochemical analyses of primary neuronal cultures expressing wild-type or mutant forms of EphB2, we demonstrate that this protective effect of EphB2 depends on its PDZ-binding motif and the presence of neuronal activity but not on its kinase activity. We further present evidence that the protective effect of EphB2 may be mediated by the AMPA-type glutamate receptor subunit GluA2, which can become associated with the PDZ-binding motif of EphB2 through PDZ domain-containing proteins and can promote the retention of NMDARs in the membrane. In addition, we show that the Aβ-induced depletion of surface NMDARs does not depend on several factors that have been implicated in the pathogenesis of Aβ-induced neuronal dysfunction, including aberrant neuronal activity, tau, prion protein (PrPC), and EphB2 itself. Thus, although EphB2 does not appear to be directly involved in the Aβ-induced depletion of NMDARs, increasing its expression may counteract this pathogenic process through a neuronal activity- and PDZ-dependent regulation of AMPA-type glutamate receptors. PMID:26589795

  13. Tyrosine Kinase Inhibitors in Ph+ Chronic Myeloid Leukemia Therapy: a Review.

    PubMed

    Shah, Krupa; Parikh, Sonia; Rawal, Rakesh

    2016-01-01

    Chronic myeloid leukaemia (CML) is a clonal myeloproliferative hematopoietic stem cell disorder. Deregulated BCRABL fusion tyrosine kinase activity is the main cause of CML disease pathogenesis, making BCRABL an ideal target for inhibition. Current tyrosine kinase inhibitors (TKIs) designed to inhibit BCRABL oncoprotein activity, have completely transformed the prognosis of CML. Interruption of TKI treatment leads to minimal residual disease reside (MRD), thought to reside in TKIinsensitive leukaemia stem cells which remain a potential reservoir for disease relapse. This highlights the need to develop new therapeutic strategies for CML either as small molecule master TKIs or phytopharmaceuticals derived from nature to achieve chronic molecular remission. This review outlines the past, present and future therapeutic approaches for CML including coverage of relevant mechanisms, whether ABL dependent or independent, and epigenetic factors responsible for developing resistance against TKIs. Appearance of mutant clones along the course of therapy either preexisting or induced due to therapy is still a challenge for the clinician. A proposed invitro model of generating colony forming units from CML stem cells derived from diagnostic samples seems to be achievable in the era of high throughput technology which can take care of single cell genomic profiling. PMID:27509925

  14. Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

    PubMed Central

    Christoph, Sandra; Lee-Sherick, Alisa B.; Sather, Susan; DeRyckere, Deborah; Graham, Douglas K.

    2013-01-01

    Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds. PMID:24084362

  15. Completing the structural family portrait of the human EphB tyrosine kinase domains

    PubMed Central

    Overman, Ross C; Debreczeni, Judit E; Truman, Caroline M; McAlister, Mark S; Attwood, Teresa K

    2014-01-01

    The EphB receptors have key roles in cell morphology, adhesion, migration and invasion, and their aberrant action has been linked with the development and progression of many different tumor types. Their conflicting expression patterns in cancer tissues, combined with their high sequence and structural identity, present interesting challenges to those seeking to develop selective therapeutic molecules targeting this large receptor family. Here, we present the first structure of the EphB1 tyrosine kinase domain determined by X-ray crystallography to 2.5Å. Our comparative crystalisation analysis of the human EphB family kinases has also yielded new crystal forms of the human EphB2 and EphB4 catalytic domains. Unable to crystallize the wild-type EphB3 kinase domain, we used rational engineering (based on our new structures of EphB1, EphB2, and EphB4) to identify a single point mutation which facilitated its crystallization and structure determination to 2.2 Å. This mutation also improved the soluble recombinant yield of this kinase within Escherichia coli, and increased both its intrinsic stability and catalytic turnover, without affecting its ligand-binding profile. The partial ordering of the activation loop in the EphB3 structure alludes to a potential cis-phosphorylation mechanism for the EphB kinases. With the kinase domain structures of all four catalytically competent human EphB receptors now determined, a picture begins to emerge of possible opportunities to produce EphB isozyme-selective kinase inhibitors for mechanistic studies and therapeutic applications. PMID:24677421

  16. Inhibition of human neutrophil responses by alpha-cyano-3,4-dihydroxythiocinnamamide; a protein-tyrosine kinase inhibitor.

    PubMed Central

    Dryden, P.; Duronio, V.; Martin, L.; Hudson, A. T.; Salari, H.

    1992-01-01

    1. Activation of neutrophils results in increased tyrosine phosphorylation of several proteins that may have important roles in receptor/effector coupling. In this study, the effect of a protein tyrosine kinase inhibitor on receptor-mediated neutrophil activation by platelet-activating factor (PAF), leukotriene, B4 (LTB4) and N-formylmethionylleucylphenylalanine (FMLP) is investigated. 2. alpha-Cyano-3,4-dihydroxythiocinnamamide dose-dependently inhibited intracellular calcium release and superoxide generation from human neutrophils activated by 1 microM LTB4, PAF, and FMLP. 3. In the presence of cytochalasin B, FMLP stimulated elastase release from neutrophils was also inhibited to unstimulated levels by 5 min pretreatment with alpha-cyano-3,4-dihydroxythiocinnamamide. 4. The inhibitory action of alpha-cyano-3,4-dihydroxythiocinnamamide was found to be at or upstream of phospholipase C activation, blocking both phosphatidylinositol hydrolysis and protein kinase C activation. alpha-Cyano-3,4-dihydroxythiocinnamamide did not affect agonist receptor binding sites or receptor affinity in neutrophils. 5. Immunoblot analysis demonstrated the tyrosine phosphorylation of proteins of 41, 56, 66, and 104 kDa in neutrophils treated with agonists. Treatment of neutrophils with alpha-cyano-3,4-dihydroxythiocinnamamide prior to stimulation with chemoattractants reduced tyrosine phosphorylation of the above phosphoproteins. 6. These results indicate that alpha-cyano-3,4-dihydroxythiocinnamamide might be a useful agent in characterizing the essential proteins and biochemical pathways that regulate neutrophil activation. PMID:1504749

  17. JAK2 Tyrosine Kinase Phosphorylates and Is Negatively Regulated by Centrosomal Protein Ninein

    PubMed Central

    Jay, Jennifer; Hammer, Alan; Nestor-Kalinoski, Andrea

    2014-01-01

    JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin- and interferon gamma (IFN-γ)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways. PMID:25332239

  18. Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

    SciTech Connect

    Kim, Rae-Kwon; Yoon, Chang-Hwan; Hyun, Kyung-Hwan; Lee, Hyejin; An, Sungkwan; Park, Myung-Jin; Kim, Min-Jung; Lee, Su-Jae

    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 this 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

  19. A computational study of the phosphorylation mechanism of the insulin receptor tyrosine kinase.

    PubMed

    Zhou, Baojing; Wong, Chung F

    2009-04-30

    Although various groups have studied the phosphorylation mechanism of the insulin receptor tyrosine kinase (IRK), an unanimous picture has not yet emerged. In this work, we performed a computational study to gain further insights. We first built a structural model of the reactant complex with the guide of several crystal structures and previous computational studies of the cyclic AMP-dependent protein kinase. We then optimized the structure by performing geometry optimization using a quantum mechanical model containing nearly 300 atoms. A reaction path was then traced between the reactant and the product by using a multiple coordinate-driven method. The calculations mapped out a sequence of structural changes depicting the conversion of the reactant to the product. Analysis of the structural changes revealed the formation of a dissociative transition state and the involvement of a proton transfer from the hydroxyl group of the tyrosyl residue of the peptide substrate to a conserved aspartate in the active site of the enzyme. The proton transfer began well before the transition state was reached and finished only shortly before the product was completely formed. In addition, the formation of a hydrogen bonding network among Arg1136, Asp1132, the gamma-phosphate of ATP, and the tyrosine residue of the substrate appeared to hold the latter two in a near-attack position for reaction. The model estimated a reaction barrier of 14 kcal/mol, semiquantitatively in accord with experiment. PMID:19334696

  20. The discovery of novel vascular endothelial growth factor receptor tyrosine kinases inhibitors: pharmacophore modeling, virtual screening and docking studies.

    PubMed

    Yu, Hui; Wang, Zhanli; Zhang, Liangren; Zhang, Jufeng; Huang, Qian

    2007-03-01

    We have applied pharmacophore generation, database searching and docking methodologies to discover new structures for the design of vascular endothelial growth factor receptors, the tyrosine kinase insert domain-containing receptor kinase inhibitors. The chemical function based pharmacophore models were built for kinase insert domain-containing receptor kinase inhibitors from a set of 10 known inhibitors using the algorithm HipHop, which is implemented in the CATALYST software. The highest scoring HipHop model consists of four features: one hydrophobic, one hydrogen bond acceptor, one hydrogen bond donor and one ring aromatic function. Using the algorithm CatShape within CATALYST, the bound conformation of 4-amino-furo [2, 3-d] pyrimidine binding to kinase insert domain-containing receptor kinase was used to generate a shape query. A merged shape and hypothesis query that is in an appropriate alignment was then built. The combined shape and hypothesis model was used as a query to search Maybridge database for other potential lead compounds. A total of 39 compounds were retrieved as hits. The hits obtained were docked into kinase insert domain-containing receptor kinase active site. One novel potential lead was proposed based on CATALYST fit value, LigandFit docking scores, and examination of how the hit retain key interactions known to be required for kinase binding. This compound inhibited vascular endothelial growth factor stimulated kinase insert domain-containing receptor phosphorylation in human umbilical vein endothelial cells. PMID:17441906

  1. Identification of Nuclear Protein Targets for Six Leukemogenic Tyrosine Kinases Governed by Post-Translational Regulation

    PubMed Central

    Pierce, Andrew; Williamson, Andrew; Jaworska, Ewa; Griffiths, John R.; Taylor, Sam; Walker, Michael; O’Dea, Mark Aspinall; Spooncer, Elaine; Unwin, Richard D.; Poolman, Toryn; Ray, David; Whetton, Anthony D.

    2012-01-01

    Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases. PMID:22745689

  2. The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis

    PubMed Central

    Gerwig, Jan; Kiley, Taryn B.; Gunka, Katrin; Stanley-Wall, Nicola

    2014-01-01

    The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA. PMID:24493247

  3. The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis.

    PubMed

    Gerwig, Jan; Kiley, Taryn B; Gunka, Katrin; Stanley-Wall, Nicola; Stülke, Jörg

    2014-04-01

    The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA. PMID:24493247

  4. Tyrosine kinase chimeras for antigen-selective T-body therapy.

    PubMed

    Eshhar; Fitzer-Attas

    1998-04-01

    Protein tyrosine kinases (PTKs) transmit activation signals in almost every cell type, including immune effector cells. The aberrant or constitutive activation of PTKs can often cause neoplastic transformation. The use of chimeric receptors based on PTKs may enable us to elucidate the signaling pathways of normal immune cells and other cell types, and the abnormal events that can lead to malignant transformation. In this review, we focus on antigen specific chimeric PTKs in which antibody-derived scFv are joined to the Syk family of PTKs. These chimeric receptors yielded reagents that can selectively redirect immune effector cells and specifically activate them to produce cytokines or lyse their target. The advantages of using such PTK-based chimeras to redirect lymphocytes to tumor targets and their potential as an immunotherapeutic approach to malignant disease is discussed. PMID:10837624

  5. The SYK tyrosine kinase: a crucial player in diverse biological functions

    PubMed Central

    Mócsai, Attila; Ruland, Jürgen; Tybulewicz, Victor L. J.

    2016-01-01

    Spleen tyrosine kinase (SYK) has been known to relay adaptive immune receptor signalling. However, recent reports indicate that SYK also mediates other, unexpectedly diverse biological functions including cellular adhesion, innate immune recognition, osteoclast maturation, platelet activation and vascular development. SYK is activated by C-type lectins and integrins, and activates novel targets including the CARD9/CARMA1–BCL10–MALT1 pathway and the NLRP3 inflammasome. Drosophila studies indicate evolutionary ancient origin of SYK-mediated signalling. Moreover, SYK has a crucial role in autoimmune diseases and haematological malignancies. This Review summarizes our current understanding of SYK functions and the translation of this knowledge for therapeutic purposes. PMID:20467426

  6. Salmonella induces SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling pathways in commercial and wild-type turkey leukocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on d...

  7. Damnacanthal inhibits the NF-κB/RIP-2/caspase-1 signal pathway by inhibiting p56lck tyrosine kinase.

    PubMed

    Kim, Min-Ho; Jeong, Hyun-Ja

    2014-10-01

    Damnacanthal is a major constituent of Morinda citrifolia L. (noni) and exhibits anti-cancer and anti-inflammatory activities. However, the effects of damnacanthal on allergic diseases have not been determined. In this study, we investigated the effect of damnacanthal on mast cell-mediated allergic inflammatory responses. Damnacanthal significantly and dose-dependently inhibited compound 48/80-induced systemic anaphylactic shock, histamine release and intracellular calcium levels. In particular, IgE-mediated passive cutaneous anaphylaxis was significantly inhibited by the oral administration of damnacanthal. In addition, we report for the first time that p56lck tyrosine kinase was expressed in phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-stimulated mast cells. Furthermore, damnacanthal inhibited the up-regulation of p56lck tyrosine kinase activity by PMACI and repressed PMACI-induced histidine decarboxylase expression and activity. Damnacanthal also inhibited PMACI-induced interleukin (IL)-1β, IL-6 and tumor necrosis factor-α expressions by suppressing nuclear factor-kappa B (NF-κB) activation and suppressed the activation of caspase-1 and the expression of receptor interacting protein-2. This study shows damnacanthal inhibits the NF-κB/receptor-interacting protein-2/caspase-1 signal pathway by inhibiting p56lck tyrosine kinase and suggests that damnacanthal has potential for the treatment of mast cell-mediated allergic disorders. PMID:25139491

  8. Production of recombinant insulin-like androgenic gland hormones from three decapod species: In vitro testicular phosphorylation and activation of a newly identified tyrosine kinase receptor from the Eastern spiny lobster, Sagmariasus verreauxi.

    PubMed

    Aizen, Joseph; Chandler, Jennifer C; Fitzgibbon, Quinn P; Sagi, Amir; Battaglene, Stephen C; Elizur, Abigail; Ventura, Tomer

    2016-04-01

    In crustaceans the insulin-like androgenic gland hormone (IAG) is responsible for male sexual differentiation. To date, the biochemical pathways through which IAG exerts its effects are poorly understood and could be elucidated through the production of a functional recombinant IAG (rIAG). We have successfully expressed glycosylated, biologically active IAG using the Pichia pastoris yeast expression system. We co-expressed recombinant single-chain precursor molecules consisting of the B and A chains (the mature hormone) tethered by a flexible linker, producing rIAGs of the following commercially important species: Eastern spiny lobster Sagmariasus verreauxi (Sv), redclaw crayfish Cherax quadricarinatus (Cq) and giant freshwater prawn Macrobrachium rosenbergii (Mr). We then tested the biological activity of each, through the ability to increase phosphorylation in the testis; both Sv and Cq rIAGs significantly elevated phosphorylation specific to their species, and in a dose-dependent manner. Mr rIAG was tested on Macrobrachium australiense (Ma), eliciting a similar response. Moreover, using bioinformatics analyses of the de novo assembled spiny lobster transcriptome, we identified a spiny lobster tyrosine kinase insulin receptor (Sv-TKIR). We validated this discovery with a receptor activation assay in COS-7 cells expressing Sv-TKIR, using a reporter SRE-LUC system designed for RTKs, with each of the rIAG proteins acting as the activation ligand. Using recombinant proteins, we aim to develop specific tools to control sexual development through the administration of IAG within the critical sexual differentiation time window. The biologically active rIAGs generated might facilitate commercially feasible solutions for the long sought techniques for sex-change induction and monosex population culture in crustaceans and shed new light on the physiological mode of action of IAG in crustaceans. PMID:26883686

  9. Targeting the Spleen Tyrosine Kinase with Fostamatinib as a Strategy Against Waldenström’s Macroglobulinemia

    PubMed Central

    Kuiatse, Isere; Baladandayuthapani, Veerabhadran; Lin, Heather Y.; Thomas, Sheeba K.; Bjorklund, Chad C.; Weber, Donna M.; Wang, Michael; Shah, Jatin J.; Zhang, Xing-Ding; Jones, Richard J.; Ansell, Stephen M.; Yang, Guang; Treon, Steven P.; Orlowski, Robert Z.

    2015-01-01

    Purpose Waldenström’s macroglobulinemia (WM) is a lymphoproliferative disorder characterized by good initial responses to standard therapeutics, but only a minority of patients achieve complete remissions, and most inevitably relapse, indicating a need for novel agents. B-cell receptor signaling has been linked to clonal evolution in WM, and Spleen tyrosine kinase (Syk) is over-expressed in primary cells, suggesting that it could be a novel and rational target. Experimental Design We studied the impact of the Syk inhibitor fostamatinib on BCWM.1 and MWCL-1 Waldenstrom’s-derived cell lines both in vitro and in vivo, as well as on primary patient cells. Results In WM-derived cell lines, fostamatinib induced a time- and dose-dependent reduction in viability, associated with activation of apoptosis. At the molecular level, fostamatinib reduced activation of Syk and Bruton’s tyrosine kinase, and also downstream signaling through mitogen activated protein kinase (MAPK) kinase (MEK), p44/42 MAPK, and protein kinase B/Akt. As a single agent, fostamatinib induced tumor growth delay in an in vivo model of WM, and reduced viability of primary WM cells, along with inhibition of p44/42 MAPK signaling. Finally, fostamatinib in combination with other agents, including dexamethasone, bortezomib, and rituximab, showed enhanced activity. Conclusions Taken together, these data support the translation of approaches targeting Syk with fostamatinib to the clinic for patients with relapsed, and possibly even newly diagnosed Waldenstrom’s. PMID:25748087

  10. Phosphoinositide 3-kinase and Bruton's tyrosine kinase regulate overlapping sets of genes in B lymphocytes

    PubMed Central

    Fruman, David A.; Ferl, Gregory Z.; An, Sam S.; Donahue, Amber C.; Satterthwaite, Anne B.; Witte, Owen N.

    2002-01-01

    Bruton's tyrosine kinase (Btk) acts downstream of phosphoinositide 3-kinase (PI3K) in a pathway required for B cell receptor (BCR)-dependent proliferation. We used DNA microarrays to determine what fraction of genes this pathway influences and to investigate whether PI3K and Btk mediate distinct gene regulation events. As complete loss-of-function mutations in PI3K and Btk alter B cell subpopulations and may cause compensatory changes in gene expression, we used B cells with partial loss of function in either PI3K or Btk. Only about 5% of the BCR-dependent gene expression changes were significantly affected by reduced PI3K or Btk. The results indicate that PI3K and Btk share target genes, and that PI3K influences additional genes independently of Btk. These data are consistent with PI3K acting through Btk and other effectors to regulate expression of a critical subset of BCR target genes that determine effective entry into the cell cycle. PMID:11756681

  11. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.

    PubMed

    González-Fernández, Lauro; Macías-García, Beatriz; Loux, Shavahn C; Varner, Dickson D; Hinrichs, Katrin

    2013-06-01

    Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways. PMID:23595906

  12. Chromosomal location of the Syk and ZAP-70 tyrosine kinase genes in mice and humans

    SciTech Connect

    Ku, G.; Malissen, B.; Mattei, M.G.

    1994-12-31

    Several protein tyrosine kinases (PTKs), which may be grouped into two structurally different families, have been implicated in antigen receptor proximal signaling. Blk, Fyn, Lck, Lyn, and Yes belong to the Src-family kinases, whereas the spleen tyrosine kinase (Syk) and the CD3-{zeta}-associated PTK (ZAP-70) define a new one, the Syk family. These kinases differ from the Src-family kinases in that they are non-myristylated cytoplasmic polypeptides composed of two N-terminal Src homology-2 (SH2) domains and a C-terminal catalytic domain. ZAP-70 appears to be expressed exclusively in T cells and NK cells, whereas Syk is preferentially expressed in B cells, T cells, and myeloid cells. 15 refs., 2 figs.

  13. Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition.

    PubMed

    Shi, Xiarong; Sousa, Leiliane P; Mandel-Bausch, Elizabeth M; Tome, Francisco; Reshetnyak, Andrey V; Hadari, Yaron; Schlessinger, Joseph; Lax, Irit

    2016-08-16

    Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants. PMID:27482095

  14. Lemur Tyrosine Kinase 2, a novel target in prostate cancer therapy.

    PubMed

    Shah, Kalpit; Bradbury, Neil A

    2015-06-10

    Progression from early forms of prostate cancer to castration-resistant disease is associated with an increase in signal transduction activity. The majority of castration-resistance cancers persist in the expression of the androgen receptor (AR), as well as androgen-dependent genes. The AR is regulated not only by it associated steroid hormone, but also by manifold regulatory and signaling molecules, including several kinases. We undertook evaluation of the role of Lemur Tyrosine Kinase 2 (LMTK2) in modulating AR activity, as several Genome Wide Association Studies (GWAS) have shown a marked association of LMTK2 activity with the development of prostate cancer. We confirm that not only is LMTK2 mRNA reduced in prostate cancer tissue, but also LMTK2 protein levels are markedly diminished. Knockdown of LMTK2 protein in prostate cell lines greatly increased the transcription of androgen-responsive genes. In addition, LMTK2 knockdown led to an increase in prostate cancer stem cell populations in LNCaP cells, indicative of increased tumorogenicity. Using multiple approaches, we also demonstrate that LMTK2 interacts with the AR, thus putting LMTK2 as a component of a signaling complex modulating AR activity. Our finding that LMTK2 is a negative regulator of AR activity defines a novel cellular pathway for activation of AR-responsive genes in castrate resistant-prostate cancer. Moreover, pharmacologic manipulation of LMTK2 activity will provide a novel therapeutic target for more effective treatments for patients with castrate-resistant prostate cancer. PMID:26008968

  15. The ROR2 tyrosine kinase receptor regulates dendritic spine morphogenesis in hippocampal neurons.

    PubMed

    Alfaro, Iván E; Varela-Nallar, Lorena; Varas-Godoy, Manuel; Inestrosa, Nibaldo C

    2015-07-01

    Wnt signaling regulates synaptic development and function and contributes to the fine-tuning of the molecular and morphological differentiation of synapses. We have shown previously that Wnt5a activates non-canonical Wnt signaling to stimulate postsynaptic differentiation in excitatory hippocampal neurons promoting the clustering of the postsynaptic scaffold protein PSD-95 and the development of dendritic spines. At least three different kinds of Wnt receptors have been associated with Wnt5a signaling: seven trans-membrane Frizzled receptors and the tyrosine kinase receptors Ryk and ROR2. We report here that ROR2 is distributed in the dendrites of hippocampal neurons in close proximity to synaptic contacts and it is contained in dendritic spine protrusions. We demonstrate that ROR2 is necessary to maintain dendritic spine number and morphological distribution in cultured hippocampal neurons. ROR2 overexpression increased dendritic spine growth without affecting the density of dendritic spine protrusions in a form dependent on its extracellular Wnt binding cysteine rich domain (CRD) and kinase domain. Overexpression of dominant negative ROR2 lacking the extracellular CRD decreased spine density and the proportion of mushroom like spines, while ROR2 lacking the C-terminal and active kinase domains only affected spine morphology. Our results indicate a crucial role of the ROR2 in the formation and maturation of the postsynaptic dendritic spines in hippocampal neurons. PMID:26003414

  16. A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells

    PubMed Central

    Chakedis, Jeffery; French, Randall; Babicky, Michele; Jaquish, Dawn; Howard, Haleigh; Mose, Evangeline; Lam, Raymond; Holman, Patrick; Miyamoto, Jaclyn; Walterscheid, Zakk; Lowy, Andrew M.

    2015-01-01

    The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription (IPT) domain. The splice variant is detected in 73% of pancreatic adenocarcinoma patient derived xenografts and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirms translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing (HPNE) cells activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target. PMID:26477314

  17. A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells.

    PubMed

    Chakedis, J; French, R; Babicky, M; Jaquish, D; Howard, H; Mose, E; Lam, R; Holman, P; Miyamoto, J; Walterscheid, Z; Lowy, A M

    2016-06-23

    The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription domain. The splice variant is detected in 73% of xenografts derived from pancreatic adenocarcinoma patients and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirm translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing cells, activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target. PMID:26477314

  18. Activation of the IGF1R pathway potentially mediates acquired resistance to mutant-selective 3rd-generation EGF receptor tyrosine kinase inhibitors in advanced non-small cell lung cancer.

    PubMed

    Park, Ji Hyun; Choi, Yun Jung; Kim, Seon Ye; Lee, Jung-Eun; Sung, Ki Jung; Park, Sojung; Kim, Woo Sung; Song, Joon Seon; Choi, Chang-Min; Sung, Young Hoon; Rho, Jin Kyung; Lee, Jae Cheol

    2016-04-19

    Mutant-selective, 3rd-generation EGFR-TKIs were recently developed to control lung cancer cells harboring T790M-mediated resistance. However, the development of resistance to these novel drugs seems inevitable. Thus, we investigated the mechanism of acquired resistance to the mutant-selective EGFR-TKI WZ4002. We established five WZ4002-resistant cells, derived from cells harboring both EGFR and T790M mutations by long-term exposure to increasing doses of WZ4002. Compared with the parental cells, all resistant cells showed 10-100-folds higher resistance to WZ4002, as well as cross-resistance to other mutant-selective inhibitors. Among them, three resistant cells (HCC827/WR, PC-9/WR and H1975/WR) showed dependency on EGFR signaling, but two other cells (PC-9/GR/WR and PC-9/ER/WR) were not. Notably, insulin-like growth factor-1 receptor (IGF1R) was aberrantly activated in PC-9/GR/WR cells in phospho-receptor tyrosine kinase array, consistently accompanied by loss of IGF binding protein-3 (IGFBP3). Down-regulation of IGF1R by shRNA, as well as inhibition of IGF1R activity either by AG-1024 (a small molecule IGF1R inhibitor) or BI 836845 (a monoclonal anti-IGF1/2 blocking antibody), restored the sensitivity to WZ4002 both in vitro and xenograft. Taken together, these results suggest that activation of the IGF1R pathway associated with IGFBP3 loss can induce an acquired resistance to the mutant-selective EGFR-TKI, WZ4002. Therefore, a combined therapy of IGF1R inhibitors and mutant-selective EGFR-TKIs might be a viable treatment strategy for overcoming acquired resistance. PMID:26980747

  19. Activation of the IGF1R pathway potentially mediates acquired resistance to mutant-selective 3rd-generation EGF receptor tyrosine kinase inhibitors in advanced non-small cell lung cancer

    PubMed Central

    Park, Ji Hyun; Choi, Yun Jung; Kim, Seon Ye; Lee, Jung-Eun; Sung, Ki Jung; Park, Sojung; Kim, Woo Sung; Song, Joon Seon; Choi, Chang-Min; Sung, Young Hoon; Rho, Jin Kyung; Lee, Jae Cheol

    2016-01-01

    Mutant-selective, 3rd-generation EGFR-TKIs were recently developed to control lung cancer cells harboring T790M-mediated resistance. However, the development of resistance to these novel drugs seems inevitable. Thus, we investigated the mechanism of acquired resistance to the mutant-selective EGFR-TKI WZ4002. We established five WZ4002-resistant cells, derived from cells harboring both EGFR and T790M mutations by long-term exposure to increasing doses of WZ4002. Compared with the parental cells, all resistant cells showed 10–100-folds higher resistance to WZ4002, as well as cross-resistance to other mutant-selective inhibitors. Among them, three resistant cells (HCC827/WR, PC-9/WR and H1975/WR) showed dependency on EGFR signaling, but two other cells (PC-9/GR/WR and PC-9/ER/WR) were not. Notably, insulin-like growth factor-1 receptor (IGF1R) was aberrantly activated in PC-9/GR/WR cells in phospho-receptor tyrosine kinase array, consistently accompanied by loss of IGF binding protein-3 (IGFBP3). Down-regulation of IGF1R by shRNA, as well as inhibition of IGF1R activity either by AG-1024 (a small molecule IGF1R inhibitor) or BI 836845 (a monoclonal anti-IGF1/2 blocking antibody), restored the sensitivity to WZ4002 both in vitro and xenograft. Taken together, these results suggest that activation of the IGF1R pathway associated with IGFBP3 loss can induce an acquired resistance to the mutant-selective EGFR-TKI, WZ4002. Therefore, a combined therapy of IGF1R inhibitors and mutant-selective EGFR-TKIs might be a viable treatment strategy for overcoming acquired resistance. PMID:26980747

  20. Using ovality to predict nonmutagenic, orally efficacious pyridazine amides as cell specific spleen tyrosine kinase inhibitors.

    PubMed

    Lucas, Matthew C; Bhagirath, Niala; Chiao, Eric; Goldstein, David M; Hermann, Johannes C; Hsu, Pei-Yuan; Kirchner, Stephan; Kennedy-Smith, Joshua J; Kuglstatter, Andreas; Lukacs, Christine; Menke, John; Niu, Linghao; Padilla, Fernando; Peng, Ying; Polonchuk, Liudmila; Railkar, Aruna; Slade, Michelle; Soth, Michael; Xu, Daigen; Yadava, Preeti; Yee, Calvin; Zhou, Mingyan; Liao, Cheng

    2014-03-27

    Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented. PMID:24520947

  1. The Fer tyrosine kinase regulates interactions of Rho GDP-Dissociation Inhibitor α with the small GTPase Rac

    PubMed Central

    2010-01-01

    Background RhoGDI proteins are important regulators of the small GTPase Rac, because they shuttle Rac from the cytoplasm to membranes and also protect Rac from activation, deactivation and degradation. How the binding and release of Rac from RhoGDI is regulated is not precisely understood. Results We report that the non-receptor tyrosine kinase Fer is able to phosphorylate RhoGDIα and form a direct protein complex with it. This interaction is mediated by the C-terminal end of RhoGDIα. Activation of Fer by reactive oxygen species caused increased phosphorylation of RhoGDIα and pervanadate treatment further augmented this. Tyrosine phosphorylation of RhoGDIα by Fer prevented subsequent binding of Rac to RhoGDIα, but once a RhoGDIα-Rac complex was formed, the Fer kinase was not able to cause Rac release through tyrosine phosphorylation of preformed RhoGDIα-Rac complexes. Conclusions These results identify tyrosine phosphorylation of RhoGDIα by Fer as a mechanism to regulate binding of RhoGDIα to Rac. PMID:21122136

  2. Bruton’s Tyrosine Kinase (BTK)-dependent immune cell crosstalk drives pancreas cancer

    PubMed Central

    Gunderson, Andrew J.; Kaneda, Megan M.; Tsujikawa, Takahiro; Nguyen, Abraham V.; Affara, Nesrine I.; Ruffell, Brian; Gorjestani, Sara; Liudahl, Shannon M.; Truitt, Morgan; Olson, Peter; Kim, Grace; Hanahan, Douglas; Tempero, Margaret A.; Sheppard, Brett; Irving, Bryan; Chang, Betty Y.; Varner, Judith A.; Coussens, Lisa M.

    2015-01-01

    Pancreas ductal adenocarcinoma (PDAC) has one of the worst five-year survival rates of all solid tumors, and thus new treatment strategies are urgently needed. Here we report that targeting Bruton’s Tyrosine Kinase (BTK), a key B cell and macrophage kinase, restores T cell-dependent anti-tumor immune responses, thereby inhibiting PDAC growth and improving responsiveness to standard-of-care chemotherapy (CTX). We report that PDAC tumor growth depends on crosstalk between B cells and FcRγ+ tumor-associated macrophages, resulting in TH2-type macrophage programming via BTK activation in a phosphatidylinositide 3-kinase (PI3K)γ-dependent manner. Treatment of PDAC-bearing mice with the BTK inhibitor PCI32765 (ibrutinib) or by PI3Kγ inhibition reprogrammed macrophages toward a TH1 phenotype that fostered CD8+ T cell cytotoxicity, and suppressed PDAC growth, indicating that BTK signaling mediates PDAC immunosuppression. These data indicate that pharmacological inhibition of BTK in PDAC can reactivate adaptive immune responses, presenting a new therapeutic modality for this devastating tumor type. PMID:26715645

  3. Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion

    PubMed Central

    Davy, Alice; Gale, Nicholas W.; Murray, Elizabeth W.; Klinghoffer, Richard A.; Soriano, Philippe; Feuerstein, Claude; Robbins, Stephen M.

    1999-01-01

    Eph receptor tyrosine kinases and their corresponding surface-bound ligands, the ephrins, provide cues to the migration of cells and growth cones during embryonic development. Here we show that ephrin-A5, which is attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidylinositol-anchor, induces compartmentalized signaling within a caveolae-like membrane microdomain when bound to the extracellular domain of its cognate Eph receptor. The physiological response induced by this signaling event is concomitant with a change in the cellular architecture and adhesion of the ephrin-A5-expressing cells and requires the activity of the Fyn protein tyrosine kinase. This study stresses the relevance of bidirectional signaling involving the ephrins and Eph receptors during brain development. PMID:10601038

  4. Clinical efficacy of second generation tyrosine kinase inhibitor and 5-azacytidine combination in chronic myelogenous leukaemia in myeloid blast crisis.

    PubMed

    Ghez, David; Micol, Jean-Baptiste; Pasquier, Florence; Auger, Nathalie; Saada, Véronique; Spentchian, Marc; Ianotto, Jean-Christophe; Bourhis, Jean-Henri; Bennaceur-Griscelli, Anelyse; Terré, Christine; Castaigne, Sylvie; Rigaudeau, Sophie; Rousselot, Philippe; de Botton, Stéphane

    2013-11-01

    Even in the tyrosine kinase inhibitors era, the prognosis of patients with chronic myeloid leukaemia in myeloid blast crisis remains dismal with few patients surviving longer than 6 months. Here we report the cases of 5 patients treated with the combination of 5-azacytidine and tyrosine kinase inhibitors for myeloid blast crisis CML. All patients achieved a complete haematological response including two with a complete cytogenetic and major molecular response. Two patients underwent an allogeneic stem cell transplantation. One died from relapse 34 months from diagnosis. The second is alive and free from disease at 11 months from diagnosis. The other 3 patients are still in complete haematological response after 15, 24 and 33 months of follow-up. These results suggest that the combination has a significant activity in myeloid blast crisis and may increase survival. PMID:23968731

  5. Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle.

    PubMed Central

    Watanabe, N; Broome, M; Hunter, T

    1995-01-01

    In higher eukaryotes, the cyclin-dependent kinases (CDKs) are negatively regulated by phosphorylation on threonine 14 (T14) and tyrosine 15 (Y15). In fission yeast, the Wee1 and mitosis inhibitory kinase 1 (Mik1) protein kinases phosphorylate Y15 in Cdc2. WEE1Hu is the only known protein kinase that can carry out this inhibitory phosphorylation on Y15 in higher eukaryotes. In the present study, we examined the endogenous products of WEE1Hu in human cells and found that the original WEE1Hu cDNA lacked 214 amino acids at the N-terminus. The predicted full-length protein has weak, but significant, similarity over its entire length with Mik1. Thus, we suggest that 'WEE1Hu' is a Mik1-related protein rather than a Wee1 homologue. When isolated in immunoprecipitates, the endogenous WEE1Hu phosphorylated several cyclin-associated CDKs on Y15. WEE1Hu activity increased during S and G2 phases in parallel with the level of protein. Its activity decreased at M phase when WEE1Hu became transiently hyperphosphorylated. In addition, a decrease in WEE1Hu protein level was observed at M/G1 phase. Apparently, the hyperphosphorylation and degradation in combination caused inactivation of WEE1Hu at M phase and the following G1 phase. These results suggest that the activity of WEE1Hu is regulated by phosphorylation and proteolytic degradation, and that WEE1Hu plays a role in inhibiting mitosis before M phase by phosphorylating cyclin B1-Cdc2. Images PMID:7743995

  6. A platinum-based hybrid drug design approach to circumvent acquired resistance to molecular targeted tyrosine kinase inhibitors

    NASA Astrophysics Data System (ADS)

    Wei, Yuming; Poon, Daniel C.; Fei, Rong; Lam, Amy S. M.; Au-Yeung, Steve C. F.; To, Kenneth K. W.

    2016-05-01

    Three molecular targeted tyrosine kinase inhibitors (TKI) were conjugated to classical platinum-based drugs with an aim to circumvent TKI resistance, predominately mediated by the emergence of secondary mutations on oncogenic kinases. The hybrids were found to maintain specificity towards the same oncogenic kinases as the original TKI. Importantly, they are remarkably less affected by TKI resistance, presumably due to their unique structure and the observed dual mechanism of anticancer activity (kinase inhibition and DNA damage). The study is also the first to report the application of a hybrid drug approach to switch TKIs from being efflux transporter substrates into non-substrates. TKIs cannot penetrate into the brain for treating metastases because of efflux transporters at the blood brain barrier. The hybrids were found to escape drug efflux and they accumulate more than the original TKI in the brain in BALB/c mice. Further development of the hybrid compounds is warranted.

  7. A platinum-based hybrid drug design approach to circumvent acquired resistance to molecular targeted tyrosine kinase inhibitors

    PubMed Central

    Wei, Yuming; Poon, Daniel C.; Fei, Rong; Lam, Amy S. M.; Au-Yeung, Steve C. F.; To, Kenneth K. W.

    2016-01-01

    Three molecular targeted tyrosine kinase inhibitors (TKI) were conjugated to classical platinum-based drugs with an aim to circumvent TKI resistance, predominately mediated by the emergence of secondary mutations on oncogenic kinases. The hybrids were found to maintain specificity towards the same oncogenic kinases as the original TKI. Importantly, they are remarkably less affected by TKI resistance, presumably due to their unique structure and the observed dual mechanism of anticancer activity (kinase inhibition and DNA damage). The study is also the first to report the application of a hybrid drug approach to switch TKIs from being efflux transporter substrates into non-substrates. TKIs cannot penetrate into the brain for treating metastases because of efflux transporters at the blood brain barrier. The hybrids were found to escape drug efflux and they accumulate more than the original TKI in the brain in BALB/c mice. Further development of the hybrid compounds is warranted. PMID:27150583

  8. The Complex of Ciliary Neurotrophic Factor-Ciliary Neurotrophic Factor Receptor α Up-Regulates Connexin43 and Intercellular Coupling in Astrocytes via the Janus Tyrosine Kinase/Signal Transducer and Activator of Transcription PathwayD⃞

    PubMed Central

    Ozog, Mark A.; Bernier, Suzanne M.; Bates, Dave C.; Chatterjee, Bishwanath; Lo, Cecilia W.; Naus, Christian C.G.

    2004-01-01

    Cytokines regulate numerous cell processes, including connexin expression and gap junctional coupling. In this study, we examined the effect of ciliary neurotrophic factor (CNTF) on connexin43 (Cx43) expression and intercellular coupling in astrocytes. Murine cortical astrocytes matured in vitro were treated with CNTF (20 ng/ml), soluble ciliary neurotrophic factor receptor α (CNTFRα) (200 ng/ml), or CNTF-CNTFRα. Although CNTF and CNTFRα alone had no effect on Cx43 expression, the heterodimer CNTF-CNTFRα significantly increased both Cx43 mRNA and protein levels. Cx43 immunostaining correlated with increased intercellular coupling as determined by dye transfer analysis. By using the pharmacological inhibitor α-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG490), the increase in Cx43 was found to be dependent on the Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Immunocytochemical analysis revealed that CNTF-CNTFRα treatment produced nuclear localization of phosphorylated STAT3, whereas CNTF treatment alone did not. Transient transfection of constructs containing various sequences of the Cx43 promoter tagged to a LacZ reporter into ROS 17/2.8 cells confirmed that the promoter region between -838 to -1693 was deemed necessary for CNTF-CNTFRα to induce heightened expression. CNTF-CNTFRα did not alter Cx30 mRNA levels, suggesting selectivity of CNTF-CNTFRα for connexin signaling. Together in the presence of soluble receptor, CNTF activates the JAK/STAT pathway leading to enhanced Cx43 expression and intercellular coupling. PMID:15342787

  9. Tyrosine kinase-mediated axial motility of basal cells revealed by intravital imaging

    PubMed Central

    Roy, Jeremy; Kim, Bongki; Hill, Eric; Visconti, Pablo; Krapf, Dario; Vinegoni, Claudio; Weissleder, Ralph; Brown, Dennis; Breton, Sylvie

    2016-01-01

    Epithelial cells are generally considered to be static relative to their neighbours. Basal cells in pseudostratified epithelia display a single long cytoplasmic process that can cross the tight junction barrier to reach the lumen. Using in vivo microscopy to visualize the epididymis, a model system for the study of pseudostratified epithelia, we report here the surprising discovery that these basal cell projections—which we call axiopodia—periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement, which we refer to as periodic axial motility (PAM), is controlled by c-Src and MEK1/2–ERK1/2. Therapeutic inhibition of tyrosine kinase activity induces a retraction of these projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment. PMID:26868824

  10. Proteome-wide dataset supporting functional study of tyrosine kinases in breast cancer

    PubMed Central

    Angelopoulos, Nicos; Stebbing, Justin; Xu, Yichen; Giamas, Georgios; Zhang, Hua

    2016-01-01

    Tyrosine kinases (TKs) play an essential role in regulating various cellular activities and dysregulation of TK signaling contributes to oncogenesis. However, less than half of the TKs have been thoroughly studied. Through a combined use of RNAi and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics, a global functional proteomic landscape of TKs in breast cancer was recently revealed highlighting a comprehensive and highly integrated signaling network regulated by TKs (Stebbing et al., 2015) [1]. We collate the enormous amount of the proteomic data in an open access platform, providing a valuable resource for studying the function of TKs in cancer and benefiting the science community. Here we present a detailed description related to this study (Stebbing et al., 2015) [1] and the raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier PXD002065. PMID:27054188

  11. Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer

    SciTech Connect

    Staquicini, Fernanda I.; Qian, Ming D.; Salameh, Ahmad; Dobroff, Andrey S.; Edwards, Julianna K.; Cimino, Daniel F.; Moeller, Benjamin J.; Kelly, Patrick; Nunez, Maria I.; Tang, Ximing; Liu, Diane D.; Lee, J. Jack; Hong, Waun Ki; Ferrara, Fortunato; Bradbury, Andrew R. M.; Lobb, Roy R.; Edelman, Martin J.; Sidman, Richard L.; Wistuba, Ignacio I.; Arap, Wadih; Pasqualini, Renata

    2015-03-20

    Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. In conclusion, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.

  12. Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer

    DOE PAGESBeta

    Staquicini, Fernanda I.; Qian, Ming D.; Salameh, Ahmad; Dobroff, Andrey S.; Edwards, Julianna K.; Cimino, Daniel F.; Moeller, Benjamin J.; Kelly, Patrick; Nunez, Maria I.; Tang, Ximing; et al

    2015-03-20

    Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. In conclusion, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lungmore » cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.« less

  13. Proteome-wide dataset supporting functional study of tyrosine kinases in breast cancer.

    PubMed

    Angelopoulos, Nicos; Stebbing, Justin; Xu, Yichen; Giamas, Georgios; Zhang, Hua

    2016-06-01

    Tyrosine kinases (TKs) play an essential role in regulating various cellular activities and dysregulation of TK signaling contributes to oncogenesis. However, less than half of the TKs have been thoroughly studied. Through a combined use of RNAi and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics, a global functional proteomic landscape of TKs in breast cancer was recently revealed highlighting a comprehensive and highly integrated signaling network regulated by TKs (Stebbing et al., 2015) [1]. We collate the enormous amount of the proteomic data in an open access platform, providing a valuable resource for studying the function of TKs in cancer and benefiting the science community. Here we present a detailed description related to this study (Stebbing et al., 2015) [1] and the raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier PXD002065. PMID:27054188

  14. Receptor Tyrosine Kinase EphA5 Is a Functional Molecular Target in Human Lung Cancer*

    PubMed Central

    Staquicini, Fernanda I.; Qian, Ming D.; Salameh, Ahmad; Dobroff, Andrey S.; Edwards, Julianna K.; Cimino, Daniel F.; Moeller, Benjamin J.; Kelly, Patrick; Nunez, Maria I.; Tang, Ximing; Liu, Diane D.; Lee, J. Jack; Hong, Waun Ki; Ferrara, Fortunato; Bradbury, Andrew R. M.; Lobb, Roy R.; Edelman, Martin J.; Sidman, Richard L.; Wistuba, Ignacio I.; Arap, Wadih; Pasqualini, Renata

    2015-01-01

    Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. Finally, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications. PMID:25623065

  15. TAM receptor tyrosine kinases: Expression, disease and oncogenesis in the central nervous system

    PubMed Central

    Pierce, Angela M.; Keating, Amy K.

    2014-01-01

    Receptor tyrosine kinases (RTKs) are cell surface proteins that tightly regulate a variety of downstream intra-cellular processes; ligand-receptor interactions result in cascades of signaling events leading to growth, proliferation, differentiation and migration. There are 58 described RTKs, which are further categorized into 20 different RTK families. When dysregulated or overexpressed, these RTKs are implicated in disordered growth, development, and oncogenesis. The TAM family of RTKs, consisting of Tyro3, Axl, and MerTK, is prominently expressed during the development and function of the central nervous system (CNS). Aberrant expression and dysregulated activation of TAM family members has been demonstrated in a variety of CNS-related disorders and diseases, including the most common but least treatable brain cancer in children and adults: glioblastoma multiforme. PMID:24184575

  16. Receptor tyrosine kinases: Characterisation, mechanism of action and therapeutic interests for bone cancers

    PubMed Central

    Ségaliny, Aude I.; Tellez-Gabriel, Marta; Heymann, Marie-Françoise; Heymann, Dominique

    2015-01-01

    Bone cancers are characterised by the development of tumour cells in bone sites, associated with a dysregulation of their environment. In the last two decades, numerous therapeutic strategies have been developed to target the cancer cells or tumour niche. As the crosstalk between these two entities is tightly controlled by the release of polypeptide mediators activating signalling pathways through several receptor tyrosine kinases (RTKs), RTK inhibitors have been designed. These inhibitors have shown exciting clinical impacts, such as imatinib mesylate, which has become a reference treatment for chronic myeloid leukaemia and gastrointestinal tumours. The present review gives an overview of the main molecular and functional characteristics of RTKs, and focuses on the clinical applications that are envisaged and already assessed for the treatment of bone sarcomas and bone metastases. PMID:26579483

  17. Tyrosine kinase-mediated axial motility of basal cells revealed by intravital imaging.

    PubMed

    Roy, Jeremy; Kim, Bongki; Hill, Eric; Visconti, Pablo; Krapf, Dario; Vinegoni, Claudio; Weissleder, Ralph; Brown, Dennis; Breton, Sylvie

    2016-01-01

    Epithelial cells are generally considered to be static relative to their neighbours. Basal cells in pseudostratified epithelia display a single long cytoplasmic process that can cross the tight junction barrier to reach the lumen. Using in vivo microscopy to visualize the epididymis, a model system for the study of pseudostratified epithelia, we report here the surprising discovery that these basal cell projections--which we call axiopodia--periodically extend and retract over time. We found that axiopodia extensions and retractions follow an oscillatory pattern. This movement, which we refer to as periodic axial motility (PAM), is controlled by c-Src and MEK1/2-ERK1/2. Therapeutic inhibition of tyrosine kinase activity induces a retraction of these projections. Such unexpected cell motility may reflect a novel mechanism by which specialized epithelial cells sample the luminal environment. PMID:26868824

  18. Subcellular localization of proline-rich tyrosine kinase 2 during oocyte fertilization and early-embryo development in mice.

    PubMed

    Meng, Xiao-Qian; Dai, Yuan-Yuan; Jing, Lai-Dong; Bai, Jing; Liu, Shu-Zhen; Zheng, Ke-Gang; Pan, Jie

    2016-08-25

    Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase, is a member of the focal adhesion kinase family and is highly expressed in oocytes. Using a combination of confocal microscopy and RNAi, we localized and studied the function of both Pyk2 and tyrosine-phosphorylated Pyk2 (p-Pyk2) during mouse oocyte fertilization and early embryo development. At the onset of fertilization, Pyk2 and p-Pyk2 were detected predominantly in sperm heads and the oocyte cytoplasm. Upon formation of male and female pronuclei, Pyk2 and its activated form leave the cytoplasm and accumulate in the two pronuclei. We detected Pyk2 in blastomere nuclei and found both Pyk2 and p-Pyk2 in the pre-blastula cytoplasm. Pyk2 and its activated form then disappeared from the blastula nuclei and localized to the perinuclear regions, where blastula cells come into contact with each other. Pyk2 knockdown via microinjection of siRNA into the zygote did not inhibit early embryo development. Our results suggest that Pyk2 plays multiple functional roles in mouse oocyte fertilization as well as throughout early embryo development. PMID:27086609

  19. Subcellular localization of proline-rich tyrosine kinase 2 during oocyte fertilization and early-embryo development in mice

    PubMed Central

    MENG, Xiao-qian; DAI, Yuan-yuan; JING, Lai-dong; BAI, Jing; LIU, Shu-zhen; ZHENG, Ke-gang; PAN, Jie

    2016-01-01

    Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase, is a member of the focal adhesion kinase family and is highly expressed in oocytes. Using a combination of confocal microscopy and RNAi, we localized and studied the function of both Pyk2 and tyrosine-phosphorylated Pyk2 (p-Pyk2) during mouse oocyte fertilization and early embryo development. At the onset of fertilization, Pyk2 and p-Pyk2 were detected predominantly in sperm heads and the oocyte cytoplasm. Upon formation of male and female pronuclei, Pyk2 and its activated form leave the cytoplasm and accumulate in the two pronuclei. We detected Pyk2 in blastomere nuclei and found both Pyk2 and p-Pyk2 in the pre-blastula cytoplasm. Pyk2 and its activated form then disappeared from the blastula nuclei and localized to the perinuclear regions, where blastula cells come into contact with each other. Pyk2 knockdown via microinjection of siRNA into the zygote did not inhibit early embryo development. Our results suggest that Pyk2 plays multiple functional roles in mouse oocyte fertilization as well as throughout early embryo development. PMID:27086609

  20. Targeting Bruton's tyrosine kinase signaling as an emerging therapeutic agent of B-cell malignancies

    PubMed Central

    XIA, BING; QU, FULIAN; YUAN, TIAN; ZHANG, YIZHUO

    2015-01-01

    It is becoming increasingly evident that B-cell receptor (BCR) signaling is central to the development and function of B cells. BCR signaling has emerged as a pivotal pathway and a key driver of numerous B-cell lymphomas. Disruption of BCR signaling can be lethal to malignant B cells. Recently, kinase inhibitors that target BCR signaling have induced notable clinical responses. These inhibitors include spleen tyrosine kinase, mammalian target of rapamycin, phosphoinositide 3′-kinase and Bruton's tyrosine kinase (BTK). Ibrutinib, an oral irreversible BTK inhibitor, has emerged as a promising targeted therapy for patients with B-cell malignancies. The present review discusses the current understanding of BTK-mediated BCR signaling in the biology and pathobiology of normal and malignant B cells, and the cellular interaction with the tumor microenvironment. The data on ibrutinib in the preclinical and clinical settings is also discussed, and perspectives for the future use of ibrutinib are outlined. PMID:26788133

  1. Role of tyrosine-kinase inhibitors in myeloproliferative neoplasms: comparative lessons learned

    PubMed Central

    Pinilla-Ibarz, Javier; Sweet, Kendra L; Corrales-Yepez, Gabriela M; Komrokji, Rami S

    2016-01-01

    An important pathogenetic distinction in the classification of myeloproliferative neoplasms (MPNs) is the presence or absence of the BCR–ABL fusion gene, which encodes a unique oncogenic tyrosine kinase. The BCR–ABL fusion, caused by the formation of the Philadelphia chromosome (Ph) through translocation, constitutes the disease-initiating event in chronic myeloid leukemia. The development of successive BCR–ABL-targeted tyrosine-kinase inhibitors has led to greatly improved outcomes in patients with chronic myeloid leukemia, including high rates of complete hematologic, cytogenetic, and molecular responses. Such levels of treatment success have long been elusive for patients with Ph-negative MPNs, because of the difficulties in identifying specific driver proteins suitable as drug targets. However, in recent years an improved understanding of the complex pathobiology of classic Ph-negative MPNs, characterized by variable, overlapping multimutation profiles, has prompted the development of better and more broadly targeted (to pathway rather than protein) treatment options, particularly JAK inhibitors. In classic Ph-negative MPNs, overactivation of JAK-dependent signaling pathways is a central pathogenic mechanism, and mutually exclusive mutations in JAK2, MPL, and CALR linked to aberrant JAK activation are now recognized as key drivers of disease progression in myelofibrosis (MF). In clinical trials, the JAK1/JAK2 inhibitor ruxolitinib – the first therapy approved for MF worldwide – improved disease-related splenomegaly and symptoms independent of JAK2V617F mutational status, and prolonged survival compared with placebo or standard therapy in patients with advanced MF. In separate trials, ruxolitinib also provided comprehensive hematologic control in patients with another Ph-negative MPN – polycythemia vera. However, complete cytogenetic or molecular responses with JAK inhibitors alone are normally not observed, underscoring the need for novel

  2. Functions of the Lyn tyrosine kinase in health and disease

    PubMed Central

    2012-01-01

    Abstract Src family kinases such as Lyn are important signaling intermediaries, relaying and modulating different inputs to regulate various outputs, such as proliferation, differentiation, apoptosis, migration and metabolism. Intriguingly, Lyn can mediate both positive and negative signaling processes within the same or different cellular contexts. This duality is exemplified by the B-cell defect in Lyn−/− mice in which Lyn is essential for negative regulation of the B-cell receptor; conversely, B-cells expressing a dominant active mutant of Lyn (Lynup/up) have elevated activities of positive regulators of the B-cell receptor due to this hyperactive kinase. Lyn has well-established functions in most haematopoietic cells, viz. progenitors via influencing c-kit signaling, through to mature cell receptor/integrin signaling, e.g. erythrocytes, platelets, mast cells and macrophages. Consequently, there is an important role for this kinase in regulating hematopoietic abnormalities. Lyn is an important regulator of autoimmune diseases such as asthma and psoriasis, due to its profound ability to influence immune cell signaling. Lyn has also been found to be important for maintaining the leukemic phenotype of many different liquid cancers including acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML) and B-cell lymphocytic leukaemia (BCLL). Lyn is also expressed in some solid tumors and here too it is establishing itself as a potential therapeutic target for prostate, glioblastoma, colon and more aggressive subtypes of breast cancer. Lay Abstract To relay information, a cell uses enzymes that put molecular markers on specific proteins so they interact with other proteins or move to specific parts of the cell to have particular functions. A protein called Lyn is one of these enzymes that regulate information transfer within cells to modulate cell growth, survival and movement. Depending on which type of cell and the source of the information input, Lyn can

  3. Markers of tyrosine kinase activity in eosinophilic esophagitis: A pilot study of the FIP1L1-PDGFRα fusion gene, pERK 1/2, and pSTAT5

    PubMed Central

    Dellon, Evan S.; Bower, Jacquelyn J.; Keku, Temitope O.; Chen, Xiaoxin; Miller, C. Ryan; Woosley, John T.; Orlando, Roy C.; Shaheen, Nicholas J.

    2011-01-01

    Background and Aims The pathogenesis of eosinophilic esophagitis (EoE) is incompletely understood. In certain eosinophilic diseases, activation of tyrosine kinase after fusion of the Fip1-like-1 and platelet-derived growth factor receptor-α genes (F-P fusion gene) mediates eosinophilia via downstream effectors such as extracellular-regulated kinase (ERK1/2) and signal transducers and activators of transcription (STAT5). This mechanism has not been examined in EoE. Our aim was to detect the F-P fusion gene, pERK1/2, and pSTAT5 in esophageal tissue from patients with EoE, gastroesophageal reflux disease (GERD), and normal controls. Materials and Methods We performed a cross-sectional pilot study comparing patients with steroid-responsive and steroid-refractory EoE, to GERD patients and normal controls. EoE cases were defined by consensus guidelines. Fluorescence in-situ hybridization (FISH) was performed to detect the F-P fusion gene and immunohistochemistry (IHC) was performed to detect pERK1/2 and pSTAT5 in esophageal biopsies. Results Twenty-nine subjects (median age 30 yrs (range 1–59); 16 male; 24 Caucasian) were included: 8 normal, 6 GERD, and 15 EoE (5 steroid-refractory). On FISH, 98%, 99%, and 99% of the nuclei in the normal, GERD, and EoE groups, respectively, were normal (p=0.42). On IHC, a median of 250, 277, and 479 nuclei/mm2 stained for pERK1/2 in the normal, GERD, and EoE groups, respectively (p=0.07); the refractory EoE patients had the highest degree pERK1/2 staining (846 nuclei/mm2; p=0.07). No trend was seen for pSTAT5. Conclusions The F-P fusion gene was not detected with increased frequency in EoE. Patients with EoE had a trend towards higher levels of pERK1/2, but not STAT5, in the esophageal epithelium, with highest levels in steroid-refractory EoE patients. PMID:21819482

  4. Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia

    PubMed Central

    Lee-Sherick, A B; Eisenman, K M; Sather, S; McGranahan, A; Armistead, P M; McGary, C S; Hunsucker, S A; Schlegel, J; Martinson, H; Cannon, C; Keating, A K; Earp, H S; Liang, X; DeRyckere, D; Graham, D K

    2013-01-01

    Acute myeloid leukemia (AML) continues to be extremely difficult to treat successfully, and the unacceptably low overall survival rates mandate that we assess new potential therapies to ameliorate poor clinical response to conventional therapy. Abnormal tyrosine kinase activation in AML has been associated with poor prognosis and provides strategic targets for novel therapy development. We found that Mer receptor tyrosine kinase was over-expressed in a majority of pediatric (29/36, 80%) and adult (10/10, 100%) primary AML patient blasts at the time of diagnosis, and 100% of patient samples at the time of relapse. Mer was also found to be expressed in 12 of 14 AML cell lines (86%). In contrast, normal bone marrow myeloid precursors expressed little to no Mer. Following AML cell line stimulation with Gas6, a Mer ligand, we observed activation of prosurvival and proliferative signaling pathways, including phosphorylation of ERK1/2, p38, MSK1, CREB, ATF1, AKT and STAT6. To assess the phenotypic role of Mer in AML, two independent short-hairpin RNA (shRNA) constructs were used to decrease Mer expression in the AML cell lines Nomo-1 and Kasumi-1. Reduction of Mer protein levels significantly increased rates of myeloblast apoptosis two to threefold in response to serum starvation. Furthermore, myeloblasts with knocked-down Mer demonstrated decreased colony formation by 67–87%, relative to control cell lines (P<0.01). NOD-SCID-gamma mice transplanted with Nomo-1 myeloblasts with reduced levels of Mer had a significant prolongation in survival compared with mice transplanted with the parental or control cell lines (median survival 17 days in parental and control cell lines, versus 32–36 days in Mer knockdown cell lines, P<0.0001). These data suggest a role for Mer in acute myeloid leukemogenesis and indicate that targeted inhibition of Mer may be an effective therapeutic strategy in pediatric and adult AML. PMID:23474756

  5. Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia.

    PubMed

    Lee-Sherick, A B; Eisenman, K M; Sather, S; McGranahan, A; Armistead, P M; McGary, C S; Hunsucker, S A; Schlegel, J; Martinson, H; Cannon, C; Keating, A K; Earp, H S; Liang, X; DeRyckere, D; Graham, D K

    2013-11-14

    Acute myeloid leukemia (AML) continues to be extremely difficult to treat successfully, and the unacceptably low overall survival rates mandate that we assess new potential therapies to ameliorate poor clinical response to conventional therapy. Abnormal tyrosine kinase activation in AML has been associated with poor prognosis and provides strategic targets for novel therapy development. We found that Mer receptor tyrosine kinase was over-expressed in a majority of pediatric (29/36, 80%) and adult (10/10, 100%) primary AML patient blasts at the time of diagnosis, and 100% of patient samples at the time of relapse. Mer was also found to be expressed in 12 of 14 AML cell lines (86%). In contrast, normal bone marrow myeloid precursors expressed little to no Mer. Following AML cell line stimulation with Gas6, a Mer ligand, we observed activation of prosurvival and proliferative signaling pathways, including phosphorylation of ERK1/2, p38, MSK1, CREB, ATF1, AKT and STAT6. To assess the phenotypic role of Mer in AML, two independent short-hairpin RNA (shRNA) constructs were used to decrease Mer expression in the AML cell lines Nomo-1 and Kasumi-1. Reduction of Mer protein levels significantly increased rates of myeloblast apoptosis two to threefold in response to serum starvation. Furthermore, myeloblasts with knocked-down Mer demonstrated decreased colony formation by 67-87%, relative to control cell lines (P<0.01). NOD-SCID-gamma mice transplanted with Nomo-1 myeloblasts with reduced levels of Mer had a significant prolongation in survival compared with mice transplanted with the parental or control cell lines (median survival 17 days in parental and control cell lines, versus 32-36 days in Mer knockdown cell lines, P<0.0001). These data suggest a role for Mer in acute myeloid leukemogenesis and indicate that targeted inhibition of Mer may be an effective therapeutic strategy in pediatric and adult AML. PMID:23474756

  6. Effects of tyrosine kinase inhibitors on the contractility of rat mesenteric resistance arteries.

    PubMed Central

    Toma, C; Jensen, P E; Prieto, D; Hughes, A; Mulvany, M J; Aalkjaer, C

    1995-01-01

    1. A pharmacological characterization of tyrosine kinase inhibitors (TKI) belonging to two distinct groups (competitors at the ATP-binding site and the substrate-binding site, respectively) was performed, based on their effects on the contractility of rat mesenteric arteries. 2. Both the ATP-site competitors (genistein and its inactive analogue, daidzein) and the substrate-site competitors (tyrphostins A-23, A-47 and the inactive analogue, A-1) reversibly inhibited noradrenaline (NA, (10 microM)) and KCl (125 mM) induced contractions, concentration-dependently. Genistein was slightly but significantly more potent than daidzein; the tyrphostins were all less potent than genistein, and there were no significant differences between the individual potencies. The tyrosine kinase substrate-site inhibitor bis-tyrphostin had no inhibitory effect. 3. Genistein, daidzein, A-23 and A-47 each suppressed the contraction induced by Ca2+ (1 microM) in alpha-toxin permeabilized arteries. A-1 and bis-tyrphostin had little or no effect on contraction of the permeabilized arteries. 4. Genistein was significantly more potent than daidzein with respect to inhibition of the contraction induced by 200 nM Ca2+ in the presence of NA (100 microM) and GTP (3 microM). The effect of A-23, A-47, A-1 and bis-tyrphostin was similar in permeabilized arteries activated with Ca2+ (200 nM) + NA (100 microM) + GTP (3 microM) and permeabilized arteries activated with 1 microM Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7620718

  7. Arg tyrosine kinase modulates TGF-β1 production in human renal tubular cells under high-glucose conditions.

    PubMed

    Torsello, Barbara; Bianchi, Cristina; Meregalli, Chiara; Di Stefano, Vitalba; Invernizzi, Lara; De Marco, Sofia; Bovo, Giorgio; Brivio, Rinaldo; Strada, Guido; Bombelli, Silvia; Perego, Roberto A

    2016-08-01

    Renal tubular cells are involved in the tubular interstitial fibrosis observed in diabetic nephropathy. It is debated whether epithelial-mesenchymal transition (EMT) affects tubular cells, which under high-glucose conditions overproduce transforming growth factor-β (TGF-β), a fibrogenic cytokine involved in interstitial fibrosis development. Our study investigated the involvement of non-receptor tyrosine kinase Arg (also called Abl2) in TGF-β production. Human primary tubular cell cultures exposed to high-glucose conditions were used. These cells showed an elongated morphology, stress fibers and vimentin increment but maintained most of the epithelial marker expression and distribution. In these cells exposed to high glucose, which overexpressed and secreted active TGF-β1, Arg protein and activity was downregulated. A further TGF-β1 increase was induced by Arg silencing with siRNA, as with the Arg tyrosine kinase inhibitor Imatinib. In the cells exposed to high glucose, reactive oxygen species (ROS)-dependent Arg kinase downregulation induced both RhoA activation, through p190RhoGAPA (also known as ARHGAP35) modulation, and proteasome activity inhibition. These data evidence a new specific involvement of Arg kinase into the regulation of TGF-β1 expression in tubular cells under high-glucose conditions and provide cues for new translational approaches in diabetic nephropathy. PMID:27298228

  8. FMS-like tyrosine kinase 3 (FLT3) inhibitors: Molecular docking and experimental studies.

    PubMed

    Mashkani, Baratali; Tanipour, Mohammad Hossein; Saadatmandzadeh, Mohammad; Ashman, Leonie K; Griffith, Renate

    2016-04-01

    Activating mutations in FMS-like tyrosine kinase 3 (FLT3) occur in 25% of acute lymphoid and 30% of acute myeloid leukaemia cases. Therefore, FLT3 is a potential therapeutic target for small molecule kinase inhibitors. In this study, protein-ligand interactions between FLT3 and kinase inhibitors (CEP701, PKC412, sunitinib, imatinib and dasatinib) were obtained through homology modelling and molecular docking. A cellular system for experimental testing of the inhibitors was also established by expressing wildtype and internal tandem duplication mutant FLT3 (FLT3-WT and FLT3-ITD) in FDC-P1 cells. Imatinib and dasatinib could not be docked into any of the FLT3 models, consistent with their lack of activity in the experimental assays. CEP701, PKC412 and sunitinib interacted with the ATP-binding pocket of FLT3, forming H-bonds with Cys694 and Glu692. Based on the EC50 values in the cell proliferation assay, CEP701 was the most potent inhibitor; sunitinib and PKC412 were ranked second and third, respectively. Sunitinib was the most selective inhibitor, followed by PKC421 and CEP701. The potency of sunitinib and to a lesser extent CEP701 in inhibition of FLT3 autophosphorylation was lower than the cell proliferation inhibition, indicating that inhibition of FLT3 downstream proteins may contribute to the cellular effects. It was shown in this study that the docking procedure was able to differentiate FLT3 inhibitors from ineffective compounds. Additionally, interaction with the phosphate binding region in the ATP-binding pocket increased potency at the cost of selectivity. These findings can be applied in designing highly effective and selective inhibitors for FLT3 and other related kinases. PMID:26896780

  9. Oestradiol stimulates tyrosine phosphorylation and hormone binding activity of its own receptor in a cell-free system.

    PubMed Central

    Auricchio, F; Migliaccio, A; Di Domenico, M; Nola, E

    1987-01-01

    Recent experiments have shown that calf uterus oestrogen receptor exists in a tyrosine-phosphorylated hormone binding form and in non-phosphorylated, non-hormone binding form. We report here that physiological concentrations of oestradiol in complex with the receptor stimulate the calf uterus receptor kinase that converts the non-hormone binding receptor into hormone binding receptor through phosphorylation of the receptor on tyrosine. The activity of this enzyme has been followed by reactivation of hormone binding sites and phosphorylation on tyrosine of calf uterus phosphatase-inactivated receptor. Phosphorylation of the receptor has been demonstrated by interaction of kinase 32P-phosphorylated proteins with anti-receptor antibody followed either by sucrose gradient centrifugation or SDS-PAGE of the immunoprecipitated proteins. Hormone stimulation of the kinase is inhibited by receptor occupancy of the anti-oestrogen tamoxifen. Oestradiol-receptor complex increases the affinity of the kinase for the dephosphorylated receptor. Findings of this report are consistent with the observation that several protein tyrosine kinases that are associated with peptide hormone receptors are stimulated by the binding of the hormone to the receptor. This is the first report on the activation of a tyrosine kinase by a steroid hormone. The finding that hormones can regulate their own receptor binding activity through a tyrosine kinase is also new. Images Fig. 2. Fig. 4. Fig. 5. PMID:3691476

  10. Human RON receptor tyrosine kinase induces complete epithelial-to-mesenchymal transition but causes cellular senescence

    SciTech Connect

    Cote, Marceline; Miller, A. Dusty; Liu, Shan-Lu . E-mail: shan-lu.liu@mcgill.ca

    2007-08-17

    The RON receptor tyrosine kinase is a member of the MET proto-oncogene family and is important for cell proliferation, differentiation, and cancer development. Here, we created a series of Madin-Darby canine kidney (MDCK) epithelial cell clones that express different levels of RON, and have investigated their biological properties. While low levels of RON correlated with little morphological change in MDCK cells, high levels of RON expression constitutively led to morphological scattering or complete and stabilized epithelial-to-mesenchymal transition (EMT). Unexpectedly, MDCK clones expressing higher levels of RON exhibited retarded proliferation and senescence, despite increased motility and invasiveness. RON was constitutively tyrosine-phosphorylated in MDCK cells expressing high levels of RON and undergoing EMT, and the MAPK signaling pathway was activated. This study reveals for the first time that RON alone is sufficient to induce complete and stabilized EMT in MDCK cells, and overexpression of RON does not cause cell transformation but rather induces cell cycle arrest and senescence, leading to impaired cell proliferation.

  11. Quantitative Profiling of Protein Tyrosine Kinases in Human Cancer Cell Lines by Multiplexed Parallel Reaction Monitoring Assays.

    PubMed

    Kim, Hye-Jung; Lin, De; Lee, Hyoung-Joo; Li, Ming; Liebler, Daniel C

    2016-02-01

    Protein tyrosine kinases (PTKs) play key roles in cellular signal transduction, cell cycle regulation, cell division, and cell differentiation. Dysregulation of PTK-activated pathways, often by receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. In this study, we have developed a parallel reaction monitoring-based assay for quantitative profiling of 83 PTKs. The assay detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We implemented the assay in four cell models: 1) a comparison of proliferating versus epidermal growth factor-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, and 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility (11-18) or acquired resistance (11-18R) to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We observed distinct PTK expression changes that were induced by stimuli, genomic features or drug resistance, which were consistent with previous reports. However, most of the measured expression differences were novel observations. For example, acquired resistance to erlotinib in the 11-18 cell model was associated not only with previously reported up-regulation of MET, but also with up-regulation of FLK2 and down-regulation of LYN and PTK7. Immunoblot analyses and shotgun proteomics data were highly consistent with parallel reaction monitoring data. Multiplexed parallel reaction monitoring assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations. Data are available through Proteome eXchange Accession PXD002706. PMID:26631510

  12. Structure­based drug design and AutoDock study of potential protein tyrosine kinase inhibitors.

    PubMed Central

    Ali, Hamed Ismail; Nagamatsu, Tomofumi; Akaho, Eiichi

    2011-01-01

    Different classes of compounds were investigated for their binding affinities into different protein tyrosine kinases (PTKs) employing a novel flexible ligand docking approach by using AutoDock 3.05 and 4. These compounds include many flavin analogs, which were developed in our group with varying degrees of cytotoxic activity (comparable or moderately superior to cisplatin and ara-c), and database selected analogs. They were docked onto twelve different families of PTKs retrieved from the Protein Data Bank. These proteins are representatives of plausible models of interactions with chemotherapeutic agents. A comparative study of the intact co-crystallized ligands of various types of PTKs was carried out. Results revealed that the new class of 5-deazapteridine and steroid hybrid compounds VIa,b, and d, and the vertical-type bispyridodipyrimidine with n-hexyl chain junction between its N-10 and N-10 atoms Xa, exhibited non-selective PTK binding capacities, with the lowest (Gb). On the other hand, 2-amino benzoic acid analog IIa, phenoxypyrido [3, 4-d]pyrimidine derivative IVc, tyrosine containing tripeptide Vd, and the one from Sumisho data base 831 are proposed to have selective PTK binding affinities to certain classes of tyrosine kinases, namely, HGFR (c-met), ZAP-70, insulin receptor kinase, EGFR, respectively. All These compounds of highest affinities were docked within the binding sites of PTKs with reasonable RMSD and 1-5 hydrogen bonds. PMID:21383902

  13. Bruton’s tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL)

    PubMed Central

    Woyach, Jennifer A.; Bojnik, Engin; Ruppert, Amy S.; Stefanovski, Matthew R.; Goettl, Virginia M.; Smucker, Kelly A.; Smith, Lisa L.; Dubovsky, Jason A.; Towns, William H.; MacMurray, Jessica; Harrington, Bonnie K.; Davis, Melanie E.; Gobessi, Stefania; Laurenti, Luca; Chang, Betty Y.; Buggy, Joseph J.; Efremov, Dimitar G.; Byrd, John C.

    2014-01-01

    Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Bruton’s tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eμ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL. PMID:24311722

  14. Approaching the active conformation of 1,3-diaminopyrimidine based covalent inhibitors of Bruton's tyrosine kinase for treatment of Rheumatoid arthritis.

    PubMed

    Huang, Zhenhua; Zhang, Qian; Yan, Lianzhong; Zhong, Guizhen; Zhang, Linqi; Tan, Xiaojuan; Wang, Yanli

    2016-04-15

    By applying conformational restrictions, we were able to discover highly potent 1,3-diaminopyrimidine based covalent inhibitors of BTK, such as 8a (IC50=3.76nM), and providing useful information of its active conformation. We are developing these novel small molecule covalent inhibitors of BTK toward oral agents for Rheumatoid arthritis. PMID:26976214

  15. Cell cycle-dependent nuclear accumulation of the p94fer tyrosine kinase is regulated by its NH2 terminus and is affected by kinase domain integrity and ATP binding.

    PubMed

    Ben-Dor, I; Bern, O; Tennenbaum, T; Nir, U

    1999-02-01

    p94fer and p51ferT are two tyrosine kinases that are encoded by differentially spliced transcripts of the FER locus in the mouse. The two tyrosine kinases share identical SH2 and kinase domains but differ in their NH2-terminal amino acid sequence. Unlike p94fer, the presence of which has been demonstrated in most mammalian cell lines analyzed, the expression of p51ferT is restricted to meiotic cells. Here, we show that the two related tyrosine kinases also differ in their subcellular localization profiles. Although p51ferT accumulates constitutively in the cell nucleus, p94fer is cytoplasmic in quiescent cells and enters the nucleus concomitantly with the onset of S phase. The nuclear translocation of the FER proteins is driven by a nuclear localization signal (NLS), which is located within the kinase domain of these enzymes. The functioning of that NLS depends on the integrity of the kinase domain but was not affected by inactivation of the kinase activity. The NH2 terminus of p94fer dictated the cell cycle-dependent functioning of the NLS of FER kinase. This process was governed by coiled-coil forming sequences that are present in the NH2 terminus of the kinase. The regulatory effect of the p94fer NH2-terminal sequences was not affected by kinase activity but was perturbed by mutations in the kinase domain ATP binding site. Ectopic expression of the constitutively nuclear p51ferT in CHO cells interfered with S-phase progression in these cells. This was not seen in p94fer-overexpressing cells. The FER tyrosine kinases seem, thus, to be regulated by novel mechanisms that direct their different subcellular distribution profiles and may, consequently, control their cellular functioning. PMID:10074905

  16. Suppression of VEGF-induced angiogenesis by the protein tyrosine kinase inhibitor, lavendustin A.

    PubMed Central

    Hu, D E; Fan, T P

    1995-01-01

    1. Vascular endothelial growth factor (VEGF) is a heparin-binding angiogenic factor which specifically acts on endothelial cells via distinct membrane-spanning tyrosine kinase receptors. Here we used the rat sponge implant model to test the hypothesis that the angiogenic activity of VEGF can be suppressed by protein tyrosine kinase (PTK) inhibitors. 2. Neovascular responses in subcutaneous sponge implants were determined by measurements of relative sponge blood flow by use of a 133Xe clearance technique, and confirmed by histological studies and morphometric analysis. 3. Daily local administration of 250 ng VEGF165 accelerated the rate of 133Xe clearance from the sponges and induced an intense neovascularisation. This VEGF165-induced angiogenesis was inhibited by daily co-administration of the selective PTK inhibitor, lavendustin A (10 micrograms), but not its negative control, lavendustin B (10 micrograms). Blood flow measurements and morphometric analysis of 8-day-old sponges showed that lavendustin A reduced the 133Xe clearance of VEGF165-treated sponges from 32.9 +/- 1.5% to 20.9 +/- 1.6% and the total fibrovascular growth area from 62.4 +/- 6.1% to 21.6 +/- 6.8% (n = 12, P < 0.05). 4. Co-injection of suramin (3 mg), an inhibitor of heparin-binding growth factors, also suppressed the VEGF165-elicited neovascular response. In contrast, neither lavendustin A nor suramin produced any effect on the basal sponge-induced angiogenesis. 5. When given alone, low doses of VEGF165 (25 ng) or basic fibroblast growth factor (bFGF; 10 ng) did not modify the basal sponge-induced neovascularisation.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 2 Figure 2 PMID:7533611

  17. Receptor Tyrosine Kinase Expression Predicts Response to Sunitinib in Breast Cancer

    PubMed Central

    Spanheimer, Philip M.; Lorenzen, Allison W.; De Andrade, James P.; Kulak, Mikhail V.; Carr, Jennifer C.; Woodfield, George W.; Sugg, Sonia L.; Weigel, Ronald J.

    2016-01-01

    Background Preliminary data indicate that tyrosine kinase inhibitors (TKIs) function through rearranged during transfection (RET) in breast cancer. However, TKIs are not specific and can block several receptor tyrosine kinases (RTKs). This study used cell lines and primary breast cancer specimens to determine factors associated with TKI response. Methods Proliferation was assessed after short interfering RNA knockdown with or without sunitinib in breast cancer cell lines by MTT (3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide). Breast cancer tissue and matched normal breast was obtained from 30 women with invasive breast carcinoma. Gene expression was assessed by reverse transcriptase-polymerase chain reaction. Fresh tissue was treated in vitro with sunitinib or control media for 30 min, and response was assessed by phosphorylation-specific western blot. Results The RTKs including epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR1-3), platelet-derived growth factor receptor (PDGFRa/b), and Kit were overexpressed in triple-negative breast tumors relative to HER2- and estrogen receptor-alpha (ERα)-positive tumors and normal breast tissue. Knockdown of EGFR reduced in vitro proliferation in MCF-7 and MDA-MB-231 but not in SKBR-3 or ZR-75-1 breast cancer cells. With the exception of RET, response to sunitinib was independent of RTK expression in all four cell lines. Both ERα-positive and low-EGFR-expressing tumors had an increased in vitro sunitinib response, as determined by alteration of Erk activation. Expression of other RTKs and additional clinical factors were not associated with response. Conclusion Triple-negative breast cancers overexpress RTKs but have decreased in vitro response to the TKI sunitinib. In addition to RET, TKIs that block EGFR may increase the therapeutic efficacy of TKIs in breast cancer. PMID:25971960

  18. Activation of a protein tyrosine phosphatase and inactivation of Raf-1 by somatostatin.

    PubMed Central

    Reardon, D B; Wood, S L; Brautigan, D L; Bell, G I; Dent, P; Sturgill, T W

    1996-01-01

    Human somatostatin receptor 3 ('hsstr3') was transiently expressed in NIH 3T3 cells stably transformed with Ha-Ras (G12V). Somatostatin activated a protein tyrosine phosphatase and inactivated the constitutively active, membrane-associated form of the Raf-1 serine kinase present in these cells in vivo and in vitro. PMID:8670047

  19. Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia

    PubMed Central

    Green, Alexa S.; Maciel, Thiago T.; Hospital, Marie-Anne; Yin, Chae; Mazed, Fetta; Townsend, Elizabeth C.; Pilorge, Sylvain; Lambert, Mireille; Paubelle, Etienne; Jacquel, Arnaud; Zylbersztejn, Florence; Decroocq, Justine; Poulain, Laury; Sujobert, Pierre; Jacque, Nathalie; Adam, Kevin; So, Jason C. C.; Kosmider, Olivier; Auberger, Patrick; Hermine, Olivier; Weinstock, David M.; Lacombe, Catherine; Mayeux, Patrick; Vanasse, Gary J.; Leung, Anskar Y.; Moura, Ivan C.; Bouscary, Didier; Tamburini, Jerome

    2015-01-01

    ABSTRACT Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD–induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD+ cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy. PMID:26601252

  20. JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors

    PubMed Central

    Gao, Sizhi P.; Chang, Qing; Mao, Ninghui; Daly, Laura A.; Vogel, Robert; Chan, Tyler; Liu, Shu Hui; Bournazou, Eirini; Schori, Erez; Zhang, Haiying; Brewer, Monica Red; Pao, William; Morris, Luc; Ladanyi, Marc; Arcila, Maria; Manova-Todorova, Katia; de Stanchina, Elisa; Norton, Larry; Levine, Ross L.; Altan-Bonnet, Gregoire; Solit, David; Zinda, Michael; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline F.

    2016-01-01

    Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non–small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells’ dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC. PMID:27025877

  1. Characterization of interactions and pharmacophore development for DFG-out inhibitors to RET tyrosine kinase.

    PubMed

    Gao, Chunxia; Grøtli, Morten; Eriksson, Leif A

    2015-07-01

    RET (rearranged during transfection) tyrosine kinase is a promising target for several human cancers. Abt-348, Birb-796, Motesanib and Sorafenib are DFG-out multi-kinase inhibitors that have been reported to inhibit RET activity with good IC50 values. Although the DFG-out conformation has attracted great interest in the design of type II inhibitors, the structural requirements for binding to the RET DFG-out conformation remains unclear. Herein, the DFG-out conformation of RET was determined by homology modelling, the four inhibitors were docked, and the binding modes investigated by molecular dynamics simulation. Binding free energies were calculated using the molecular mechanics/Poisson-Bolzmann surface area (MM/PBSA) method. The trends in predicted binding free affinities correlated well with experimental data and were used to explain the activity difference of the studied inhibitors. Per-residue energy decomposition analyses provided further information on specific interaction properties. Finally, we also conducted a detailed e-pharmacophore modelling of the different RET-inhibitor complexes, explaining the common and specific pharmacophore features of the different complexes. The results reported herein will be useful in future rational design of novel DFG-out RET inhibitors. PMID:26044359

  2. JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors.

    PubMed

    Gao, Sizhi P; Chang, Qing; Mao, Ninghui; Daly, Laura A; Vogel, Robert; Chan, Tyler; Liu, Shu Hui; Bournazou, Eirini; Schori, Erez; Zhang, Haiying; Red Brewer, Monica; Pao, William; Morris, Luc; Ladanyi, Marc; Arcila, Maria; Manova-Todorova, Katia; de Stanchina, Elisa; Norton, Larry; Levine, Ross L; Altan-Bonnet, Gregoire; Solit, David; Zinda, Michael; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline F

    2016-01-01

    Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non-small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells' dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC. PMID:27025877

  3. Imipramine protects retinal ganglion cells from oxidative stress through the tyrosine kinase receptor B signaling pathway

    PubMed Central

    Han, Ming-lei; Liu, Guo-hua; Guo, Jin; Yu, Shu-juan; Huang, Jing

    2016-01-01

    Retinal ganglion cell (RGC) degeneration is irreversible in glaucoma and tyrosine kinase receptor B (TrkB)-associated signaling pathways have been implicated in the process. In this study, we attempted to examine whether imipramine, a tricyclic antidepressant, may protect hydrogen peroxide (H2O2)-induced RGC degeneration through the activation of the TrkB pathway in RGC-5 cell lines. RGC-5 cell lines were pre-treated with imipramine 30 minutes before exposure to H2O2. Western blot assay showed that in H2O2 -damaged RGC-5 cells, imipramine activated TrkB pathways through extracellular signal-regulated protein kinase/TrkB phosphorylation. TUNEL staining assay also demonstrated that imipramine ameliorated H2O2 -induced apoptosis in RGC-5 cells. Finally, TrkB-IgG intervention was able to reverse the protective effect of imipramine on H2O2 -induced RGC-5 apoptosis. Imipramine therefore protects RGCs from oxidative stress-induced apoptosis through the TrkB signaling pathway. PMID:27127489

  4. Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2).

    PubMed

    Min, Xiaoshan; Ungureanu, Daniela; Maxwell, Sarah; Hammarén, Henrik; Thibault, Steve; Hillert, Ellin-Kristina; Ayres, Merrill; Greenfield, Brad; Eksterowicz, John; Gabel, Chris; Walker, Nigel; Silvennoinen, Olli; Wang, Zhulun

    2015-11-01

    JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) participates in signaling through cytokine receptors involved in immune responses and inflammation. JAKs are characterized by dual kinase domain: a tyrosine kinase domain (JH1) that is preceded by a pseudokinase domain (JH2). The majority of disease-associated mutations in JAKs map to JH2, demonstrating its central regulatory function. JH2s were considered catalytically inactive, but JAK2 JH2 was found to have low autoregulatory catalytic activity. Whether the other JAK JH2s share ATP binding and enzymatic activity has been unclear. Here we report the crystal structure of TYK2 JH2 in complex with adenosine 5'-O-(thiotriphosphate) (ATP-γS) and characterize its nucleotide binding by biochemical and biophysical methods. TYK2 JH2 did not show phosphotransfer activity, but it binds ATP and the nucleotide binding stabilizes the protein without inducing major conformational changes. Mutation of the JH2 ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling. The overall structural characteristics of TYK2 JH2 resemble JAK2 JH2, but distinct stabilizing molecular interactions around helix αAL in the activation loop provide a structural basis for differences in substrate access and catalytic activities among JAK family JH2s. The structural and biochemical data suggest that ATP binding is functionally important for both TYK2 and JAK2 JH2s, whereas the regulatory phosphorylation appears to be a unique property of JAK2. Finally, the co-crystal structure of TYK2 JH2 complexed with a small molecule inhibitor demonstrates that JH2 is accessible to ATP-competitive compounds, which offers novel approaches for targeting cytokine signaling as well as potential therapeutic applications. PMID:26359499

  5. Activation of signal transduction in platelets by the tyrosine phosphatase inhibitor pervanadate (vanadyl hydroperoxide).

    PubMed Central

    Pumiglia, K M; Lau, L F; Huang, C K; Burroughs, S; Feinstein, M B

    1992-01-01

    The protein tyrosine phosphatase (PTPase) inhibitor pervanadate (vanadyl hydroperoxide) stimulated protein tyrosine phosphorylation 29-fold more than did thrombin in intact and saponin-permeabilized platelets. Increased tyrosine phosphorylation preceded, or was coincident with, a fall in PtdIns(4,5)P2 levels, production of PtdIns(3,4)P2 and phosphatidic acid, mobilization of intracellular Ca2+, stimulation of protein kinase C-dependent protein phosphorylation, secretion of dense and alpha-granules, increased actin polymerization, shape change and aggregation which required fibrinogen and was mediated by increased surface expression of GPIIb-IIIa. The tyrosine kinase inhibitor RG 50864 totally prevented induction of tyrosine phosphorylation by pervanadate, as well as all other responses measured; in contrast, the inactive structural analogue, tyrphostin #1, had no effect. Dense-granule secretion induced by pervanadate required protein kinase C activity; however, aggregation and alpha-granule secretion were independent of protein kinase C. In saponin-permeabilized platelets pervanadate and thrombin stimulated phospholipase C activity by GTP-independent and GTP-dependent mechanisms respectively. We conclude that PTPases are important regulators of signal transduction in platelets. Images Fig. 1. Fig. 2. PMID:1530576

  6. Discovery and Evaluation of Clinical Candidate AZD3759, a Potent, Oral Active, Central Nervous System-Penetrant, Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor.

    PubMed

    Zeng, Qingbei; Wang, Jiabing; Cheng, Ziqiang; Chen, Kan; Johnström, Peter; Varnäs, Katarina; Li, David Yunzhi; Yang, Zhen Fan; Zhang, Xiaolin

    2015-10-22

    Recent reports suggest that an increasing number of patients with lung cancer, especially those with activating mutations of the epidermal growth factor receptor (EGFR), also present with brain metastases and leptomeningeal metastases. These patients have poor prognosis as there are no approved drugs for these indications. Available agents have poor efficacy for these patients even at well above their standard dose. Herein, we report the discovery of (4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl (2R)-2,4-dimethylpiperazine-1-carboxylate 1m (AZD3759), an investigational drug currently in Phase 1 clinical trial, which has excellent central nervous system penetration and which induces profound regression of brain metastases in a mouse model. PMID:26313252

  7. The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

    NASA Astrophysics Data System (ADS)

    Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

    2014-11-01

    The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

  8. Phosphotyrosine-dependent targeting of mitogen-activated protein kinase in differentiated contractile vascular cells.

    PubMed

    Khalil, R A; Menice, C B; Wang, C L; Morgan, K G

    1995-06-01

    Tyrosine phosphorylation has been linked to plasmalemmal targeting of src homology-2-containing proteins, activation of mitogen-activated protein (MAP) kinase, nuclear signaling, and proliferation of cultured cells. Significant tyrosine phosphorylation and MAP kinase activities have also been reported in differentiated cells, but the signaling role of tyrosine-phosphorylated MAP kinase in these cells is unclear. The spatial and temporal relation between phosphotyrosine and MAP kinase immunoreactivity was quantified in differentiated contractile vascular smooth muscle cells by using digital imaging microscopy. An initial association of MAP kinase with the plasmalemma required upstream protein kinase C activity but occurred in a tyrosine phosphorylation-independent manner. Subsequent to membrane association, a delayed redistribution of MAP kinase, colocalizing with the actin-binding protein caldesmon, occurred in a tyrosine phosphorylation-dependent manner. The apparent association of MAP kinase with the contractile proteins coincided with contractile activation. Thus, tyrosine phosphorylation appears to target MAP kinase to cytoskeletal proteins in contractile vascular cells. This targeting mechanism may determine the specific destination and thereby the specialized function of MAP kinase in other phenotypes. PMID:7538916

  9. A New Transgenic Mouse Model of Heart Failure and Cardiac Cachexia Raised by Sustained Activation of Met Tyrosine Kinase in the Heart

    PubMed Central

    Sala, Valentina; Gatti, Stefano; Gallo, Simona; Medico, Enzo; Cantarella, Daniela; Cimino, James; Ponzetto, Antonio; Crepaldi, Tiziana

    2016-01-01

    Among other diseases characterized by the onset of cachexia, congestive heart failure takes a place of relevance, considering the high prevalence of this pathology in most European countries and in the United States, and is undergoing a rapid increase in developing countries. Actually, only few models of cardiac cachexia exist. Difficulties in the recruitment and follow-up of clinical trials implicate that new reproducible and well-characterized animal models are pivotal in developing therapeutic strategies for cachexia. We generated a new model of cardiac cachexia: a transgenic mouse expressing Tpr-Met receptor, the activated form of c-Met receptor of hepatocyte growth factor, specifically in the heart. We showed that the cardiac-specific induction of Tpr-Met raises a cardiac hypertrophic remodelling, which progresses into concentric hypertrophy with concomitant increase in Gdf15 mRNA levels. Hypertrophy progresses to congestive heart failure with preserved ejection fraction, characterized by reduced body weight gain and food intake and skeletal muscle wasting. Prevention trial by suppressing Tpr-Met showed that loss of body weight could be prevented. Skeletal muscle wasting was also associated with altered gene expression profiling. We propose transgenic Tpr-Met mice as a new model of cardiac cachexia, which will constitute a powerful tool to understand such complex pathology and test new drugs/approaches at the preclinical level. PMID:27298830

  10. Alignment independent 3D-QSAR, quantum calculations and molecular docking of Mer specific tyrosine kinase inhibitors as anticancer drugs

    PubMed Central

    Shiri, Fereshteh; Pirhadi, Somayeh; Ghasemi, Jahan B.

    2015-01-01

    Mer receptor tyrosine kinase is a promising novel cancer therapeutic target in many human cancers, because abnormal activation of Mer has been implicated in survival signaling and chemoresistance. 3D-QSAR analyses based on alignment independent descriptors were performed on a series of 81 Mer specific tyrosine kinase inhibitors. The fractional factorial design (FFD) and the enhanced replacement method (ERM) were applied and tested as variable selection algorithms for the selection of optimal subsets of molecular descriptors from a much greater pool of such regression variables. The data set was split into 65 molecules as the training set and 16 compounds as the test set. All descriptors were generated by using the GRid INdependent descriptors (GRIND) approach. After variable selection, GRIND were correlated with activity values (pIC50) by PLS regression. Of the two applied variable selection methods, ERM had a noticeable improvement on the statistical parameters of PLS model, and yielded a q2 value of 0.77, an rpred2 of 0.94, and a low RMSEP value of 0.25. The GRIND information contents influencing the affinity on Mer specific tyrosine kinase were also confirmed by docking studies. In a quantum calculation study, the energy difference between HOMO and LUMO (gap) implied the high interaction of the most active molecule in the active site of the protein. In addition, the molecular electrostatic potential energy at DFT level confirmed results obtained from the molecular docking. The identified key features obtained from the molecular modeling, enabled us to design novel kinase inhibitors. PMID:27013913

  11. Severe toxicity of skin rash, fever and diarrhea associated with imatinib: case report and review of skin toxicities associated with tyrosine kinase inhibitors

    PubMed Central

    Huang, Xuan; Patel, Samir; Ahmed, Nasir; Seiter, Karen; Liu, Delong

    2008-01-01

    Chronic myeloid leukemia (CML) is characterized by a Philadelphia chromosome which contains an oncogene, bcr-abl. This oncogene encodes a tyrosine kinase which is constitutively activated. Imatinib, a tyrosine kinase inhibitor (TKI), has been widely used in the treatment of CML. Dasatinib and nilotinib were recently approved for the treatment of CML. Other TKIs, such as bosutinib, erlotinib, and sunitinib, are under study for the treatment of CML as well as other hematologic and solid malignancies. Skin rash has been reported as one of the most common side effects of the TKIs. Here we present a case of severe skin rash together with unusual symptoms of high fever and diarrhea induced by imatinib in a CML patient. The dermatologic toxicities from a variety of tyrosine kinase inhibitors are reviewed and general principles of management are also discussed. PMID:19920908

  12. The role of small adaptor proteins in the control of oncogenic signaling driven by tyrosine kinases in human cancer

    PubMed Central

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-01-01

    Protein phosphorylation on tyrosine (Tyr) residues has evolved as an important mechanism to coordinate cell communication in multicellular organisms. The importance of this process has been revealed by the discovery of the prominent oncogenic properties of tyrosine kinases (TK) upon deregulation of their physiological activities, often due to protein overexpression and/or somatic mutation. Recent reports suggest that TK oncogenic signaling is also under the control of small adaptor proteins. These cytosolic proteins lack intrinsic catalytic activity and signal by linking two functional members of a catalytic pathway. While most adaptors display positive regulatory functions, a small group of this family exerts negative regulatory functions by targeting several components of the TK signaling cascade. Here, we review how these less studied adaptor proteins negatively control TK activities and how their loss of function induces abnormal TK signaling, promoting tumor formation. We also discuss the therapeutic consequences of this novel regulatory mechanism in human oncology. PMID:26788993

  13. Deletion of the endothelial Bmx tyrosine kinase decreases tumor angiogenesis and growth.

    PubMed

    Holopainen, Tanja; López-Alpuche, Vanessa; Zheng, Wei; Heljasvaara, Ritva; Jones, Dennis; He, Yun; Tvorogov, Denis; D'Amico, Gabriela; Wiener, Zoltan; Andersson, Leif C; Pihlajaniemi, Taina; Min, Wang; Alitalo, Kari

    2012-07-15

    Bmx, [corrected] also known as Etk, is a member of the Tec family of nonreceptor tyrosine kinases. Bmx is expressed mainly in arterial endothelia and in myeloid hematopoietic cells. Bmx regulates ischemia-mediated arteriogenesis and lymphangiogenesis, but its role in tumor angiogenesis is not known. In this study, we characterized the function of Bmx in tumor growth using both Bmx knockout and transgenic mice. Isogenic colon, lung, and melanoma tumor xenotransplants showed reductions in growth and tumor angiogenesis in Bmx gene-deleted ((-/-)) mice, whereas developmental angiogenesis was not affected. In addition, growth of transgenic pancreatic islet carcinomas and intestinal adenomas was also slower in Bmx(-/-) mice. Knockout mice showed high levels of Bmx expression in endothelial cells of tumor-associated and peritumoral arteries. Moreover, endothelial cells lacking Bmx showed impaired phosphorylation of extracellular signal-regulated kinase (Erk) upon VEGF stimulation, indicating that Bmx contributes to the transduction of vascular endothelial growth factor signals. In transgenic mice overexpressing Bmx in epidermal keratinocytes, tumors induced by a two-stage chemical skin carcinogenesis treatment showed increased growth and angiogenesis. Our findings therefore indicate that Bmx activity contributes to tumor angiogenesis and growth. PMID:22593188

  14. EGFRvIII-mediated transactivation of receptor tyrosine kinases in glioma: mechanism and therapeutic implications.

    PubMed

    Greenall, S A; Donoghue, J F; Van Sinderen, M; Dubljevic, V; Budiman, S; Devlin, M; Street, I; Adams, T E; Johns, T G

    2015-10-01

    A truncation mutant of the epidermal growth factor receptor, EGFRvIII, is commonly expressed in glioma, an incurable brain cancer. EGFRvIII is tumorigenic, in part, through its transactivation of other receptor tyrosine kinases (RTKs). Preventing the effects of this transactivation could form part of an effective therapy for glioma; however, the mechanism by which the transactivation occurs is unknown. Focusing on the RTK MET, we show that MET transactivation in U87MG human glioma cells in vitro is proportional to EGFRvIII activity and involves MET heterodimerization associated with a focal adhesion kinase (FAK) scaffold. The transactivation of certain other RTKs was, however, independent of FAK. Simultaneously targeting EGFRvIII (with panitumumab) and the transactivated RTKs themselves (with motesanib) in an intracranial mouse model of glioma resulted in significantly greater survival than with either agent alone, indicating that cotargeting these RTKs has potent antitumor efficacy and providing a strategy for treating EGFRvIII-expressing gliomas, which are usually refractory to treatment. PMID:25659577

  15. Receptor tyrosine kinase expression of circulating tumor cells in small cell lung cancer

    PubMed Central

    Hamilton, Gerhard; Rath, Barbara; Klameth, Lukas; Hochmair, Maximilian

    2015-01-01

    Small cell lung cancer (SCLC) has a poor prognosis and is found disseminated at first presentation in the majority of cases. The cell biological mechanisms underlying metastasis and drug resistance are not clear. SCLC is characterized by high numbers of circulating tumor cells (CTCs) and we were able to expand several CTC lines ex vivo and to relate chitinase-3-like-1/YKL-40 (CHI3L1) as marker. Availability of expanded SCLC CTC cells allowed for a screening of receptor tyrosine kinases (RTKs) expressed. The metastatic CHI3L1-negative SCLC cell line SCLC26A, established from a pleural effusion was used for comparison. The CTC cell line BHGc10 was found to exhibit increased expression of RYK, AXL, Tie-1, Dtk, ROR1/2, several ephrins (Eph) and FGF/EGF receptors compared to SCLC26A. All of these RTKs have been associated with cell motility, invasion and poor prognosis in diverse cancer entities without knowledge of their association with CTCs. The identification of RYK, AXL and ROR1/2 as pseudokinases, lacking activity, seems to be related to the observed failure of RTK inhibitors in SCLC. These kinases are involved in the noncanonical WNT pathway and their expression in SCLC CTCs represents a cancer stem cell-like phenotype. PMID:26328272

  16. Clinical pharmacology of tyrosine kinase inhibitors becoming generic drugs: the regulatory perspective

    PubMed Central

    2014-01-01

    Over the last decades, billions have been spent and huge efforts have been taken in basic and clinical cancer research [CA Cancer J Clin 63: 11-30]. About a decade ago, the arms race between drugs and cancer cells reached a new level by introduction of tyrosine kinase inhibitors (TKI) into pharmacological anti-cancer therapy. According to their molecular mechanism of action, TKI in contrast to so-called “classic” or “conventional” cytostatics belong to the group of targeted cancer medicines, characterized by accurately fitting with biological structures (i.e. active centers of kinases). Numerous (partly orphan) indications are covered by this new class of substances. Approximately ten years after the first substances of this class of medicines were authorized, patent protection will end within the next years. The following article covers clinical meaning and regulatory status of anti-cancer TKI and gives an outlook to what is expected from the introduction of generic anti-cancer TKI. PMID:24502453

  17. Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

    PubMed Central

    Dransfield, I; Zagórska, A; Lew, E D; Michail, K; Lemke, G

    2015-01-01

    Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization. PMID:25695599

  18. The cardiotoxicity and myocyte damage caused by small molecule anticancer tyrosine kinase inhibitors is correlated with lack of target specificity

    SciTech Connect

    Hasinoff, Brian B.

    2010-04-15

    The use of the new anticancer tyrosine kinase inhibitors (TKI) has revolutionized the treatment of certain cancers. However, the use of some of these results in cardiotoxicity. Large-scale profiling data recently made available for the binding of 7 of the 9 FDA-approved tyrosine kinase inhibitors to a panel of 317 kinases has allowed us to correlate kinase inhibitor binding selectivity scores with TKI-induced damage to neonatal rat cardiac myocytes. The tyrosine kinase selectivity scores, but not the serine-threonine kinase scores, were highly correlated with the myocyte damaging effects of the TKIs. Additionally, we showed that damage to myocytes gave a good rank order correlation with clinical cardiotoxicity. Finally, strength of TKI binding to colony-stimulating factor 1 receptor (CSF1R) was highly correlated with myocyte damage, thus possibly implicating this kinase in contributing to TKI-induced cardiotoxicity.

  19. A chimeric tyrosine/tryptophan hydroxylase. The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity.

    PubMed

    Mockus, S M; Kumer, S C; Vrana, K E

    1997-08-01

    The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t1/2 of 84 min at 37 degrees C, TPH was much less stable (t1/2 = 28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by a t1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain. PMID:9356925

  20. Activating Mutations in PIK3CA Lead to Widespread Modulation of the Tyrosine Phosphoproteome

    PubMed Central

    Blair, Brian G.; Pinto, Sneha M.; Nirujogi, Raja S.; Jelinek, Christine A.; Malhotra, Radhika; Kim, Min-Sik; Park, Ben Ho; Pandey, Akhilesh

    2015-01-01

    The human oncogene PIK3CA is frequently mutated in human cancers. Two hotspot mutations in PIK3CA, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. We used quantitative mass spectrometry to profile the global phosphotyrosine proteome of isogenic knock-in cell lines containing these activating mutations, where we identified 824 unique phosphopeptides. Although it is well understood that these mutations result in hyperactivation of the serine/threonine kinase AKT, we found a surprisingly widespread modulation of tyrosine phosphorylation levels of proteins in the mutant cells. In the tyrosine kinome alone, 29 tyrosine kinases were altered in their phosphorylation status. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activation sites, indicating that these kinases and their downstream signaling pathways were activated. Our study demonstrates that there is frequent and unexpected cross-talk that occurs between tyrosine signaling pathways and serine/threonine signaling pathways activated by the canonical PI3K-AKT axis. PMID:26267517

  1. Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

    SciTech Connect

    Aoyama, Kazumasa; Yuki, Ryuzaburo; Horiike, Yasuyoshi; Kubota, Sho; Yamaguchi, Noritaka; Morii, Mariko; Ishibashi, Kenichi; Nakayama, Yuji; Kuga, Takahisa; Hashimoto, Yuuki; Tomonaga, Takeshi; Yamaguchi, Naoto

    2013-12-10

    The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus. - Highlights: • We show the involvement of c-Abl tyrosine kinase in nuclear actin dynamics. • Nuclear F-actin is formed by nuclear-localized c-Abl and its kinase-dead version. • The c-Abl actin-binding domain is prerequisite for nuclear F-actin formation. • Formation of long nuclear F-actin bundles requires nuclear c-Abl kinase activity. • We discuss a role for nuclear F-actin bundle formation in chromatin regulation.

  2. A mechanistic basis for converting a receptor tyrosine kinase agonist to an antagonist

    SciTech Connect

    Tolbert, W. David; Daugherty, Jennifer; Gao, ChongFeng; Xie, Qian; Miranti, Cindy; Gherardi, Ermanno; Vande Woude, George; Xu, H. Eric

    2010-03-08

    Hepatocyte growth factor (HGF) activates the Met receptor tyrosine kinase by binding and promoting receptor dimerization. Here we describe a mechanistic basis for designing Met antagonists based on NK1, a natural variant of HGF containing the N-terminal and the first kringle domain. Through detailed biochemical and structural analyses, we demonstrate that both mouse and human NK1 induce Met dimerization via a conserved NK1 dimer interface. Mutations designed to alter the NK1 dimer interface abolish its ability to promote Met dimerization but retain full Met-binding activity. Importantly, these NK1 mutants act as Met antagonists by inhibiting HGF-mediated cell scattering, proliferation, branching, and invasion. The ability to separate the Met-binding activity of NK1 from its Met dimerization activity thus provides a rational basis for designing Met antagonists. This strategy of antagonist design may be applicable for other growth factor receptors by selectively abolishing the receptor activation ability but not the receptor binding of the growth factors.

  3. Receptor tyrosine kinase (c-Kit) inhibitors: a potential therapeutic target in cancer cells.

    PubMed

    Abbaspour Babaei, Maryam; Kamalidehghan, Behnam; Saleem, Mohammad; Huri, Hasniza Zaman; Ahmadipour, Fatemeh

    2016-01-01

    c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c-Kit for future cancer therapy. In addition, it has c-Kit inhibitor drug properties and their functions have been listed in tables and demonstrated in schematic pictures. This review also has collected previous studies that targeted c-Kit as a novel strategy for cancer therapy. This paper further emphasizes the advantages of this approach, as well as the limitations that must be addressed in the future. Finally, although c-Kit is an attractive target for cancer therapy, based on the outcomes of treatment of patients with c-Kit inhibitors, it is unlikely that Kit inhibitors alone can lead to cure. It seems that c-Kit mutations alone are not sufficient for tumorogenesis, but do play a crucial role in cancer occurrence. PMID:27536065

  4. Receptor tyrosine kinase (c-Kit) inhibitors: a potential therapeutic target in cancer cells

    PubMed Central

    Abbaspour Babaei, Maryam; Kamalidehghan, Behnam; Saleem, Mohammad; Huri, Hasniza Zaman; Ahmadipour, Fatemeh

    2016-01-01

    c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c-Kit for future cancer therapy. In addition, it has c-Kit inhibitor drug properties and their functions have been listed in tables and demonstrated in schematic pictures. This review also has collected previous studies that targeted c-Kit as a novel strategy for cancer therapy. This paper further emphasizes the advantages of this approach, as well as the limitations that must be addressed in the future. Finally, although c-Kit is an attractive target for cancer therapy, based on the outcomes of treatment of patients with c-Kit inhibitors, it is unlikely that Kit inhibitors alone can lead to cure. It seems that c-Kit mutations alone are not sufficient for tumorogenesis, but do play a crucial role in cancer occurrence. PMID:27536065

  5. Syk protein tyrosine kinase involves PECAM-1 signaling through tandem immunotyrosine inhibitory motifs in human THP-1 macrophages.

    PubMed

    Wang, Junchen; Wu, Yanling; Hu, Hai; Wang, Weimin; Lu, Ying; Mao, Huiming; Liu, Xiaoqing; Liu, Zhongmin; Chen, Bing-guan

    2011-01-01

    Although recent evidence supports a functional relationship between platelet endothelial cell adhesion molecule (PECAM-1) and Syk tyrosine kinase, little is known about the interaction of Syk with PECAM-1. We report that down-regulation of Syk inhibits the spreading of human THP-1 macrophage cells. Moreover, our data indicate that Syk binds PECAM-1 through its immune tyrosine-based inhibitory motif (ITIM), and dual phosphorylation of the ITIM domain of PECAM-1 leads to activation of Syk. Our results indicate that the distance between the phosphotyrosines could be up to 22 amino acids in length, depending on the conformational flexibility, and that the dual ITIM tyrosine motifs of PECAM-1 facilitate immunoreceptor tyrosine-based activation motif-like signaling. The preferential binding of PECAM-1 to Src homology region 2 domain-containing phosphatase-2 or Syk may depend on their relative affinities, and could provide a mechanism by which signal transduction from PECAM-1 is internally regulated by both positive and negative signaling enzymes. PMID:22000807

  6. Resistance to the tyrosine kinase inhibitor axitinib is associated with increased glucose metabolism in pancreatic adenocarcinoma.

    PubMed

    Hudson, C D; Hagemann, T; Mather, S J; Avril, N

    2014-01-01

    Alterations in energy (glucose) metabolism are key events in the development and progression of cancer. In pancreatic adenocarcinoma (PDAC) cells, we investigated changes in glucose metabolism induced by resistance to the receptor tyrosine kinase inhibitor (RTKI) axitinib. Here, we show that human cell lines and mouse PDAC cell lines obtained from the spontaneous pancreatic cancer mouse model (Kras(G12D)Pdx1-cre) were sensitive to axitinib. The anti-proliferative effect was due to a G2/M block resulting in loss of 70-75% cell viability in the most sensitive PDAC cell line. However, a surviving sub-population showed a 2- to 3-fold increase in [C-14]deoxyglucose ([C-14]DG) uptake. This was sustained in axitinib-resistant cell lines, which were derived from parental PDAC. In addition to the axitinib-induced increase in [C-14]DG uptake, we observed a translocation of glucose transporter-1 (Glut-1) transporters from cytosolic pools to the cell surface membrane and a 2-fold increase in glycolysis rates measured by the extracellular acidification rate (ECAR). We demonstrated an axitinib-induced increase in phosphorylated Protein Kinase B (pAkt) and by blocking pAkt with a phosphatidylinositol-3 kinase (PI3K) inhibitor we reversed the Glut-1 translocation and restored sensitivity to axitinib treatment. Combination treatment with both axitinib and Akt inhibitor in parental pancreatic cell line resulted in a decrease in cell viability beyond that conferred by single therapy alone. Our study shows that PDAC resistance to axitinib results in increased glucose metabolism mediated by activated Akt. Combining axitinib and an Akt inhibitor may improve treatment in PDAC. PMID:24722285

  7. Identification of the flavoprotein of succinate dehydrogenase and aconitase as in vitro mitochondrial substrates of Fgr tyrosine kinase.

    PubMed

    Salvi, Mauro; Morrice, Nick A; Brunati, Anna Maria; Toninello, Antonio

    2007-12-11

    Overlooked until recently, mitochondrial protein phosphorylation is now emerging as a key post-translational mechanism in the regulation of mitochondrial functions. In particular, tyrosine phosphorylation represents a promising field to discover new mechanisms of bioenergetic regulation. Tyrosine kinases belonging to the Src kinase family have been observed in mitochondrial compartments, however their substrates are almost unknown. Here, we provide evidence that the flavoprotein of succinate dehydrogenase and aconitase are "in vitro" substrates of Fgr tyrosine kinase. Fgr phosphorylates flavoprotein of succinate dehydrogenase at Y535 and Y596 and aconitase at Y71, Y544 and Y665. The significance of these findings is discussed. PMID:17997986

  8. The human tyrosine kinase Kit and its gatekeeper mutant T670I, show different kinetic properties: Implications for drug design.

    PubMed

    Kissova, Miroslava; Maga, Giovanni; Crespan, Emmanuele

    2016-10-01

    The tyrosine kinase Kit, a receptor for Stem Cell Factor, is involved, among others, in processes associated to cell survival, proliferation and migration. Upon physiological conditions, the activity of Kit is tightly regulated. However, primary mutations that lead to its constitutive activation are the causal oncogenic driver of gastrointestinal stromal tumours (GISTs). GISTs are known to be refractory to conventional therapies but the introduction of Imatinib, a selective inhibitor of tyrosine kinases Abl and Kit, significantly ameliorated the treatment options of GISTs patients. However, the acquisition of secondary mutations renders Kit resistant towards all available drugs. Mutation involving gatekeeper residues (such as V654a and T670I) influence both the structure and the catalytic activity of the enzyme. Therefore, detailed knowledge of the enzymatic properties of the mutant forms, in comparison with the wild type enzyme, is an important pre-requisite for the rational development of specific inhibitors. In this paper we report a thorough kinetic analysis of the reaction catalyzed by the Kit kinase and its gatekeeper mutated form T670I. Our results revealed the different mechanisms of action of these two enzymes and may open a new avenue for the future design of specific Kit inhibitors. PMID:27527414

  9. Phosphorylation of synthetic peptides by a tyrosine protein kinase from the particulate fraction of a lymphoma cell line.

    PubMed Central

    Casnellie, J E; Harrison, M L; Pike, L J; Hellström, K E; Krebs, E G

    1982-01-01

    The particulate fraction from a lymphoma cell line, LSTRA, was found to contain an apparent high level of tyrosine protein kinase activity. When this fraction was incubated with [gamma-32P]ATP in the presence of 10 mM MnCl2, hydrolyzed, and assayed, 70--80% of the radioactivity recovered in phosphoamino acids was in phosphotyrosine. Gel electrophoresis of the proteins showed that a large portion of the 32P was in a single protein with a molecular weight of approximately 58,000. The phosphorylated residue in this protein was identified as phosphotyrosine. Detergent extracts of the particulate fraction from LSTRA cells contained both the Mr 58,000 protein and the enzyme responsible for its phosphorylation. These extracts were found to catalyze the phosphorylation of the tyrosine residue in the synthetic peptide, Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly, corresponding to the sequence around the tyrosine that is phosphorylated in pp60src; the Km for the peptide in this reaction was 5 mM. High-performance liquid chromatography was used to assay for this phosphorylation. A second peptide was synthesized that contained two additional arginine residues whose presence permitted the phosphorylation of the peptide to be measured by a simple assay using phosphocellulose paper. The Km for this peptide was 3--4 mM, indicating that the presence of the additional arginine residues did not alter the apparent affinity of the kinase for the peptide. Images PMID:6804939

  10. CBL controls a tyrosine kinase network involving AXL, SYK and LYN in nilotinib-resistant chronic myeloid leukaemia.

    PubMed

    Gioia, Romain; Trégoat, Claire; Dumas, Pierre-Yves; Lagarde, Valérie; Prouzet-Mauléon, Valérie; Desplat, Vanessa; Sirvent, Audrey; Praloran, Vincent; Lippert, Eric; Villacreces, Arnaud; Leconet, Wilhem; Robert, Bruno; Vigon, Isabelle; Roche, Serge; Mahon, François-Xavier; Pasquet, Jean-Max

    2015-09-01

    A tyrosine kinase network composed of the TAM receptor AXL and the cytoplasmic kinases LYN and SYK is involved in nilotinib-resistance of chronic myeloid leukaemia (CML) cells. Here, we show that the E3-ubiquitin ligase CBL down-regulation occurring during prolonged drug treatment plays a critical role in this process. Depletion of CBL in K562 cells increases AXL and LYN protein levels, promoting cell resistance to nilotinib. Conversely, forced expression of CBL in nilotinib-resistant K562 cells (K562-rn) dramatically reduces AXL and LYN expression and resensitizes K562-rn cells to nilotinib. A similar mechanism was found to operate in primary CML CD34(+) cells. Mechanistically, the E3-ligase CBL counteracts AXL/SYK signalling, promoting LYN transcription by controlling AXL protein stability. Surprisingly, the role of AXL in resistance was independent of its ligand GAS6 binding and its TK activity, in accordance with a scaffold activity for this receptor being involved in this cellular process. Collectively, our results demonstrate a pivotal role for CBL in the control of a tyrosine kinase network mediating resistance to nilotinib treatment in CML cells. PMID:25965880

  11. Crosstalk between G protein-coupled receptors (GPCRs) and tyrosine kinase receptor (TXR) in the heart after morphine withdrawal

    PubMed Central

    Almela, Pilar; García-Carmona, Juan-Antonio; Martínez-Laorden, Elena; Milanés, María-Victoria; Laorden, María-Luisa

    2013-01-01

    G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK) pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signaling, resulting in high expression of protein kinase (PK) A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK), one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR) can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH) at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies. PMID:24409147

  12. The receptor tyrosine kinase Pvr promotes tissue closure by coordinating corpse removal and epidermal zippering.

    PubMed

    Garlena, Rebecca A; Lennox, Ashley L; Baker, Lewis R; Parsons, Trish E; Weinberg, Seth M; Stronach, Beth E

    2015-10-01

    A leading cause of human birth defects is the incomplete fusion of tissues, often manifested in the palate, heart or neural tube. To investigate the molecular control of tissue fusion, embryonic dorsal closure and pupal thorax closure in Drosophila are useful experimental models. We find that Pvr mutants have defects in dorsal midline closure with incomplete amnioserosa internalization and epidermal zippering, as well as cardia bifida. These defects are relatively mild in comparison to those seen with other signaling mutants, such as in the JNK pathway, and we demonstrate that JNK signaling is not perturbed by altering Pvr receptor tyrosine kinase activity. Rather, modulation of Pvr levels in the ectoderm has an impact on PIP3 membrane accumulation, consistent with a link to PI3K signal transduction. Polarized PI3K activity influences protrusive activity from the epidermal leading edge and the protrusion area changes in accord with Pvr signaling intensity, providing a possible mechanism to explain Pvr mutant phenotypes. Tissue-specific rescue experiments indicate a partial requirement in epithelial tissue, but confirm the essential role of Pvr in hemocytes for embryonic survival. Taken together, we argue that inefficient removal of the internalizing amnioserosa tissue by mutant hemocytes coupled with impaired midline zippering of mutant epithelium creates a situation in some embryos whereby dorsal midline closure is incomplete. Based on these observations, we suggest that efferocytosis (corpse clearance) could contribute to proper tissue closure and thus might underlie some congenital birth defects. PMID:26293306

  13. Dysregulation of tyrosine kinases and use of imatinib in small animal practice.

    PubMed

    Bonkobara, Makoto

    2015-08-01

    Imatinib inhibits the activity of several tyrosine kinases, including BCR-ABL, KIT and platelet-derived growth factor receptor (PDGFR). Dysregulation of KIT is found in mast cell tumours (MCTs) and KIT is mutated in approximately 30% and 70% of canine and feline MCTs, respectively. KIT mutations have also been reported in canine and feline gastrointestinal stromal tumours (GISTs), canine acute myeloid leukaemia and canine melanoma. In addition, BCR-ABL and PDGFR mutations have been found in canine leukaemia and haemangiosarcoma, respectively. Imatinib has anti-tumour activity with tolerable toxicity towards a certain subset of MCTs in dogs and cats. Favourable clinical responses are likely to be associated with the presence of KIT mutation. Anti-tumour activity of imatinib has also been demonstrated in canine GISTs with a KIT mutation and in feline hypereosinophilic syndrome; however, to date only one of each of these cases has been reported. In conclusion, analysis of KIT mutations appears to provide valuable data for individual treatment with imatinib in dogs and cats. PMID:25592261

  14. β-Catenin is required for intrinsic but not extrinsic BCR-ABL1 kinase-independent resistance to tyrosine kinase inhibitors in chronic myeloid leukemia

    PubMed Central

    Eiring, Anna M.; Khorashad, Jamshid S.; Anderson, David J.; Yu, Fan; Redwine, Hannah M.; Mason, Clinton C.; Reynolds, Kimberly R.; Clair, Phillip M.; Gantz, Kevin C.; Zhang, Tian Y.; Pomicter, Anthony D.; Kraft, Ira L.; Bowler, Amber D.; Johnson, Kara; Mac Partlin, Mary; O’Hare, Thomas; Deininger, Michael W.

    2015-01-01

    Activation of nuclear β-catenin and expression of its transcriptional targets promotes chronic myeloid leukemia (CML) progression, tyrosine kinase inhibitor (TKI) resistance, and leukemic stem cell self-renewal. We report that nuclear β-catenin plays a role in leukemia cell-intrinsic but not -extrinsic BCR-ABL1 kinase-independent TKI resistance. Upon imatinib inhibition of BCR-ABL1 kinase activity, β-catenin expression was maintained in intrinsically resistant cells grown in suspension culture and sensitive cells cultured in direct contact (DC) with bone marrow (BM) stromal cells. Thus, TKI resistance uncouples β-catenin expression from BCR-ABL1 kinase activity. In β-catenin reporter assays, intrinsically resistant cells showed increased transcriptional activity versus parental TKI-sensitive controls, and this was associated with restored expression of β-catenin target genes. In contrast, DC with BM stromal cells promoted TKI resistance, but had little effects on Lef/Tcf reporter activity and no consistent effects on cytoplasmic β-catenin levels, arguing against a role for β-catenin in extrinsic TKI resistance. N-cadherin or H-cadherin blocking antibodies abrogated DC-based resistance despite increasing Lef/Tcf reporter activity, suggesting that factors other than β-catenin contribute to extrinsic, BM-derived TKI resistance. Our data indicate that, while nuclear β-catenin enhances survival of intrinsically TKI-resistant CML progenitors, it is not required for extrinsic resistance mediated by the BM microenvironment. PMID:26202934

  15. Tyrosine Kinase Inhibitors Induce Down-Regulation of c-Kit by Targeting the ATP Pocket

    PubMed Central

    Descarpentries, Clotilde; Frisan, Emilie; Adam, Kevin; Verdier, Frederique; Floquet, Célia; Dubreuil, Patrice; Lacombe, Catherine; Fontenay, Michaela; Mayeux, Patrick; Kosmider, Olivier

    2013-01-01

    The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket. PMID:23637779

  16. [Possibilities for inhibiting tumor-induced angiogenesis: results with multi-target tyrosine kinase inhibitors].

    PubMed

    Török, Szilvia; Döme, Balázs

    2012-03-01

    Functional blood vasculature is essential for tumor progression. The main signalization pathways that play a key role in the survival and growth of tumor vessels originate from the VEGF-, PDGF- and FGF tyrosine kinase receptors. In the past decade, significant results have been published on receptor tyrosine kinase inhibitors (RTKIs). In this paper, the mechanisms of action and the results so far available of experimental and clinical studies on multi-target antiangiogenic TKIs are discussed. On the one hand, notable achievements have been made recently and these drugs are already used in clinical practice in some patient populations. On the other hand, the optimal combination and dosage of these drugs, selection of the apropriate biomarker and better understanding of the conflicting role of PDGFR and FGFR signaling in angiogenesis remain future challenges. PMID:22403757

  17. How tyrosine kinase inhibitors impair metabolism and endocrine system function: a systematic updated review.

    PubMed

    Breccia, Massimo; Molica, Matteo; Alimena, Giuliana

    2014-12-01

    Tyrosine kinase inhibitors (TKIs) advent has deeply changed the outcome of chronic myeloid leukemia (CML) patients, with improved rates of response and overall survival. However, for this success some patients paid the price of a number of peculiar side effects, the so-called off-target side effects, specific for each one TKI. These effects are due to non-selective inhibition of other tyrosine kinase receptors, such as PDGFR, c-KIT, Src, VEGF. Consequences of this inhibition, some metabolic changes during the treatment with TKIs are reported. Aim of present review is to report metabolic changes and potential mechanisms involved in the pathogenesis related to imatinib, second (nilotinib and dasatinib) and third generation (bosutinib and ponatinib) TKIs. PMID:25449685

  18. Tyrosine Kinase Inhibitors and Vascular Toxicity: Impetus for a Classification System?

    PubMed

    Herrmann, Joerg

    2016-06-01

    The introduction of molecularly targeted therapies with tyrosine kinase inhibitors has revolutionized cancer therapy and has contributed to a steady decline in cancer-related mortality since the late 1990s. However, not only cardiac but also vascular toxicity has been reported for these agents, some as expected on-target effects (e.g., VEGF receptor inhibitors) and others as unanticipated events (e.g., BCR-Abl inhibitors). A sound understanding of these cardiovascular toxic effects is critical to advance mechanistic insight into vascular disease and clinical care. From a conceptual standpoint, there might be value in defining type I (permanent) and type II (transient) vascular toxicity. This review will focus on the tyrosine kinase inhibitors in current clinical use and their associated vascular side effects. PMID:27099141

  19. Taxol shares the ability of bacterial lipopolysaccharide to induce tyrosine phosphorylation of microtubule-associated protein kinase.

    PubMed

    Ding, A; Sanchez, E; Nathan, C F

    1993-11-15

    Microtubule-associated proteins may mediate the activation of macrophages by bacterial LPS. Three lines of evidence support this hypothesis: a) Taxol, a microtubule-binding diterpene, mimics the ability of LPS to induce cytokines and down-regulate receptors for TNF-alpha. In recombinant inbred mouse strains differing at the Lps gene, responsiveness to these effects of Taxol co-segregates with responsiveness to LPS. b) In vitro, LPS binds specifically to MT and preferentially to beta-tubulin. c) LPS activates microtubule-associated protein-2 kinase (MAPK). The present studies bring together and extend these lines of evidence. a) Taxol, like LPS, rapidly induces tyrosine phosphorylation of MAPK in mouse macrophages, and triggers MAPK to phosphorylate an exogenous substrate. b) Tyrosine phosphorylation of MAPK is an extremely rapid cellular response both to taxol and LPS. c) Macrophages from C3H/HeJ mice, which carry a defective Lps gene, fail to activate MAPK in response to taxol or LPS, although they activate MAPK in response to insulin or IFN-gamma. These results suggest that tyrosine phosphorylation of MAPK is among the earliest known response of macrophages to LPS. Taxol mimics LPS with respect to immediate MAPK activation, later transcriptional events, and the genetic control of both sets of responses. LPS and taxol thus appear to share an early step in a functionally important signal transduction pathway that may involve MT. PMID:7901279

  20. Identification of Fer tyrosine kinase localized on microtubules as a platelet endothelial cell adhesion molecule-1 phosphorylating kinase in vascular endothelial cells.

    PubMed

    Kogata, Naoko; Masuda, Michitaka; Kamioka, Yuji; Yamagishi, Akiko; Endo, Akira; Okada, Masato; Mochizuki, Naoki

    2003-09-01

    Platelet endothelial adhesion molecule-1 (PECA