Science.gov

Sample records for nonselective protein kinase

  1. A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP).

    PubMed

    Vainshtein, Inna; Silveria, Scott; Kaul, Poonam; Rouhani, Riaz; Eglen, Richard M; Wang, John

    2002-12-01

    A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta

  2. Stimulation of nonselective amino acid export by glutamine dumper proteins.

    PubMed

    Pratelli, Réjane; Voll, Lars M; Horst, Robin J; Frommer, Wolf B; Pilot, Guillaume

    2010-02-01

    Phloem and xylem transport of amino acids involves two steps: export from one cell type to the apoplasm, and subsequent import into adjacent cells. High-affinity import is mediated by proton/amino acid cotransporters, while the mechanism of export remains unclear. Enhanced expression of the plant-specific type I membrane protein Glutamine Dumper1 (GDU1) has previously been shown to induce the secretion of glutamine from hydathodes and increased amino acid content in leaf apoplasm and xylem sap. In this work, tolerance to low concentrations of amino acids and transport analyses using radiolabeled amino acids demonstrate that net amino acid uptake is reduced in the glutamine-secreting GDU1 overexpressor gdu1-1D. The net uptake rate of phenylalanine decreased over time, and amino acid net efflux was increased in gdu1-1D compared with the wild type, indicating increased amino acid export from cells. Independence of the export from proton gradients and ATP suggests that overexpression of GDU1 affects a passive export system. Each of the seven Arabidopsis (Arabidopsis thaliana) GDU genes led to similar phenotypes, including increased efflux of a wide spectrum of amino acids. Differences in expression profiles and functional properties suggested that the GDU genes fulfill different roles in roots, vasculature, and reproductive organs. Taken together, the GDUs appear to stimulate amino acid export by activating nonselective amino acid facilitators.

  3. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  4. Teaching resources. Protein kinases.

    PubMed

    Caplan, Avrom

    2005-02-22

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein kinases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the genomics and evolutionary relationships among kinases and then proceeds to describe the structure-function relationships of specific kinases, the molecular mechanisms underlying substrate specificity, and selected issues in regulation of kinase activity.

  5. Conserved herpesvirus protein kinases

    PubMed Central

    Gershburg, Edward; Pagano, Joseph S.

    2008-01-01

    Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task. PMID:17881303

  6. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  7. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  8. Neuronal migration and protein kinases

    PubMed Central

    Ohshima, Toshio

    2015-01-01

    The formation of the six-layered structure of the mammalian cortex via the inside-out pattern of neuronal migration is fundamental to neocortical functions. Extracellular cues such as Reelin induce intracellular signaling cascades through the protein phosphorylation. Migrating neurons also have intrinsic machineries to regulate cytoskeletal proteins and adhesion properties. Protein phosphorylation regulates these processes. Moreover, the balance between phosphorylation and dephosphorylation is modified by extracellular cues. Multipolar-bipolar transition, radial glia-guided locomotion and terminal translocation are critical steps of radial migration of cortical pyramidal neurons. Protein kinases such as Cyclin-dependent kinase 5 (Cdk5) and c-Jun N-terminal kinases (JNKs) involve these steps. In this review, I shall give an overview the roles of protein kinases in neuronal migration. PMID:25628530

  9. The protein kinase C family.

    PubMed

    Azzi, A; Boscoboinik, D; Hensey, C

    1992-09-15

    Protein kinase C represents a structurally homologous group of proteins similar in size, structure and mechanism of activation. They can modulate the biological function of proteins in a rapid and reversible manner. Protein kinase C participates in one of the major signal transduction systems triggered by the external stimulation of cells by various ligands including hormones, neurotransmitters and growth factors. Hydrolysis of membrane inositol phospholipids by phospholipase C or of phosphatidylcholine, generates sn-1,2-diacylglycerol, considered the physiological activator of this kinase. Other agents, such as arachidonic acid, participate in the activation of some of these proteins. Activation of protein kinase C by phorbol esters and related compounds is not physiological and may be responsible, at least in part, for their tumor-promoting activity. The cellular localization of the different calcium-activated protein kinases, their substrate and activator specificity are dissimilar and thus their role in signal transduction is unlike. A better understanding of the exact cellular function of the different protein kinase C isoenzymes requires the identification and characterization of their physiological substrates.

  10. CUL3 and protein kinases

    PubMed Central

    Metzger, Thibaud; Kleiss, Charlotte; Sumara, Izabela

    2013-01-01

    Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22.1 In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases. PMID:24067371

  11. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Linn, Anning

    1996-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK.

  12. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    PubMed Central

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

  13. Oncoprotein protein kinase

    DOEpatents

    Karin, M.; Hibi, M.; Lin, A.

    1997-02-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

  14. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2004-03-16

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  15. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Lin, Anning

    1999-11-30

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  16. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1998-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  17. Oncoprotein protein kinase

    DOEpatents

    Davis, Roger; Derijard, Benoit; Karin, Michael; Hibi, Masahiko; Lin, Anning

    2005-01-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  18. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  19. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  20. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2005-03-08

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  1. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  2. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  3. Casein kinase-mediated phosphorylation of serine 839 is necessary for basolateral localization of the Ca²⁺-activated non-selective cation channel TRPM4.

    PubMed

    Cerda, Oscar; Cáceres, Mónica; Park, Kang-Sik; Leiva-Salcedo, Elías; Romero, Aníbal; Varela, Diego; Trimmer, James S; Stutzin, Andrés

    2015-08-01

    Transient receptor potential melastatin-like 4 (TRPM4) is a Ca(2+)-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction, and allergic reactions. TRPM4 Ca(2+) sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-bisphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remains unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry) to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4.

  4. CASEIN KINASE-MEDIATED PHOSPHORYLATION OF SERINE 839 IS NECESSARY FOR BASOLATERAL LOCALIZATION OF THE Ca2+-ACTIVATED NON-SELECTIVE CATION CHANNEL TRPM4

    PubMed Central

    Cerda, Oscar; Cáceres, Mónica; Park, Kang-Sik; Leiva-Salcedo, Elías; Romero, Aníbal; Varela, Diego

    2014-01-01

    TRPM4 is a Ca2+-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction and allergic reactions. TRPM4 Ca2+ sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-biphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remain unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry), to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4. PMID:25231975

  5. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  6. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

    PubMed

    Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia*

    PubMed Central

    Roth Flach, Rachel J.; Danai, Laura V.; DiStefano, Marina T.; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B.; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K.; Bortell, Rita; Alonso, Laura C.; Czech, Michael P.

    2016-01-01

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo. After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. PMID:27226575

  8. Immunochemical characterization of rat brain protein kinase

    SciTech Connect

    Huang, K.P.; Huang, F.L.

    1986-11-05

    Polyclonal antibodies against rat brain protein kinase C (the Ca/sup 2 +//phospholipid-dependent enzyme) were raised in goat. These antibodies can neutralize completely the kinase activity in purified enzyme preparation as well as that in the crude homogenate. Immunoblot analysis of the purified and the crude protein kinase C preparations revealed a major immunoreactive band of 80 kDa. The antibodies also recognize the same enzyme from other rat tissues. Neuronal tissues (cerebral cortex, cerebellum, hypothalamus, and retina) and lymphoid organs (thymus and spleen) were found to be enriched in protein kinase C, whereas lung, kidney, liver, heart, and skeletal muscle contained relatively low amounts of this kinase. Limited proteolysis of the purified rat brain protein kinase C with trypsin results in an initial degradation of the kinase into two major fragments of 48 and 38 kDa. Both fragments are recognized by the antibodies. However, further digestion of the 48-kDa fragment to 45 kDa and the 38-kDa fragment to 33 kDa causes a loss of the immunoreactivity. Upon incubation of the cerebellar extract with Ca/sup 2 +/, the 48-kDa fragment was also identified as a major proteolytic product of protein kinase C. Proteolytic degradation of protein kinase C converts the Ca/sup 2 +//phospholipid-dependent kinase to an independent form without causing a large impairment of the binding of (/sup 3/H)phorbol 12,13-dibutyrate. The two major proteolytic fragments were separated by ion exchange chromatography and one of them (45-48 kDa) was identified as a protein kinase and the other (33-38 kDa) as a phorbol ester-binding protein. These results demonstrate that rat brain protein kinase C is composed of two functionally distinct units, namely, a protein kinase and a Ca/sup 2 +/-independent/phospholipid-dependent phorbol ester-binding protein.

  9. Pathway illuminated: visualizing protein kinase C signaling.

    PubMed

    Violin, Jonathan D; Newton, Alexandra C

    2003-12-01

    Protein kinase C has been at the center of cell signaling since the discovery 25 years ago that it transduces signals that promote phospholipid hydrolysis. In recent years, the use of genetically encoded fluorescent reporters has enabled studies of the regulation of protein kinase C signaling in living cells. Advances in imaging techniques have unveiled unprecedented detail of the signal processing mechanics of protein kinase C, from the second messengers calcium and diacylglycerol that regulate protein kinase C activity, to the locations and kinetics of different protein kinase C isozymes, to the spatial and temporal dynamics of substrate phosphorylation by this key enzyme. This review discusses how fluorescence imaging studies have illuminated the fidelity with which protein kinase C transduces rapidly changing extracellular information into intracellular phosphorylation signals.

  10. Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

    DTIC Science & Technology

    2014-10-01

    kinase . This grant proposal will explore the resistance to small molecule AKT protein kinase inhibitors mediated by the... molecule AKT protein kinase inhibitors is potentially mediated by the Pim-1 protein kinase , and that unique Pim protein kinase inhibitors that can in...application is essential for the development of this combined chemotherapeutic strategy. 15. SUBJECT TERMS Small Molecule AKT Inhibitors ,

  11. Protein kinase biochemistry and drug discovery.

    PubMed

    Schwartz, Phillip A; Murray, Brion W

    2011-12-01

    Protein kinases are fascinating biological catalysts with a rapidly expanding knowledge base, a growing appreciation in cell regulatory control, and an ascendant role in successful therapeutic intervention. To better understand protein kinases, the molecular underpinnings of phosphoryl group transfer, protein phosphorylation, and inhibitor interactions are examined. This analysis begins with a survey of phosphate group and phosphoprotein properties which provide context to the evolutionary selection of phosphorylation as a central mechanism for biological regulation of most cellular processes. Next, the kinetic and catalytic mechanisms of protein kinases are examined with respect to model aqueous systems to define the elements of catalysis. A brief structural biology overview further delves into the molecular basis of catalysis and regulation of catalytic activity. Concomitant with a prominent role in normal physiology, protein kinases have important roles in the disease state. To facilitate effective kinase drug discovery, classic and emerging approaches for characterizing kinase inhibitors are evaluated including biochemical assay design, inhibitor mechanism of action analysis, and proper kinetic treatment of irreversible inhibitors. As the resulting protein kinase inhibitors can modulate intended and unintended targets, profiling methods are discussed which can illuminate a more complete range of an inhibitor's biological activities to enable more meaningful cellular studies and more effective clinical studies. Taken as a whole, a wealth of protein kinase biochemistry knowledge is available, yet it is clear that a substantial extent of our understanding in this field remains to be discovered which should yield many new opportunities for therapeutic intervention.

  12. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  13. Cyclic-GMP-dependent protein kinase inhibits the Ras/Mitogen-activated protein kinase pathway.

    PubMed

    Suhasini, M; Li, H; Lohmann, S M; Boss, G R; Pilz, R B

    1998-12-01

    Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3', 5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by

  14. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  15. Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases*

    PubMed Central

    Lai, Shenshen; Safaei, Javad

    2016-01-01

    The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies. PMID:26742849

  16. Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel.

    PubMed

    González-Perrett, S; Kim, K; Ibarra, C; Damiano, A E; Zotta, E; Batelli, M; Harris, P C; Reisin, I L; Arnaout, M A; Cantiello, H F

    2001-01-30

    Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of unknown function, is a major cause of autosomal dominant polycystic kidney disease (ADPKD), whose manifestation entails the development of fluid-filled cysts in target organs. Here, we demonstrate that polycystin-2 is present in term human syncytiotrophoblast, where it behaves as a nonselective cation channel. Lipid bilayer reconstitution of polycystin-2-positive human syncytiotrophoblast apical membranes displayed a nonselective cation channel with multiple subconductance states, and a high perm-selectivity to Ca2+. This channel was inhibited by anti-polycystin-2 antibody, Ca2+, La3+, Gd3+, and the diuretic amiloride. Channel function by polycystin-2 was confirmed by patch-clamping experiments of polycystin-2 heterologously infected Sf9 insect cells. Further, purified insect cell-derived recombinant polycystin-2 and in vitro translated human polycystin-2 had similar ion channel activity. The polycystin-2 channel may be associated with fluid accumulation and/or ion transport regulation in target epithelia, including placenta. Dysregulation of this channel provides a mechanism for the onset and progression of ADPKD.

  17. Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel

    PubMed Central

    González-Perrett, Silvia; Kim, Keetae; Ibarra, Cristina; Damiano, Alicia E.; Zotta, Elsa; Batelli, Marisa; Harris, Peter C.; Reisin, Ignacio L.; Arnaout, M. Amin; Cantiello, Horacio F.

    2001-01-01

    Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of unknown function, is a major cause of autosomal dominant polycystic kidney disease (ADPKD), whose manifestation entails the development of fluid-filled cysts in target organs. Here, we demonstrate that polycystin-2 is present in term human syncytiotrophoblast, where it behaves as a nonselective cation channel. Lipid bilayer reconstitution of polycystin-2-positive human syncytiotrophoblast apical membranes displayed a nonselective cation channel with multiple subconductance states, and a high perm-selectivity to Ca2+. This channel was inhibited by anti-polycystin-2 antibody, Ca2+, La3+, Gd3+, and the diuretic amiloride. Channel function by polycystin-2 was confirmed by patch-clamping experiments of polycystin-2 heterologously infected Sf9 insect cells. Further, purified insect cell-derived recombinant polycystin-2 and in vitro translated human polycystin-2 had similar ion channel activity. The polycystin-2 channel may be associated with fluid accumulation and/or ion transport regulation in target epithelia, including placenta. Dysregulation of this channel provides a mechanism for the onset and progression of ADPKD. PMID:11252306

  18. Selective and Nonselective Cleavages in Positive and Negative CID of the Fragments Generated from In-Source Decay of Intact Proteins in MALDI-MS

    NASA Astrophysics Data System (ADS)

    Takayama, Mitsuo; Sekiya, Sadanori; Iimuro, Ryunosuke; Iwamoto, Shinichi; Tanaka, Koichi

    2014-01-01

    Selective and nonselective cleavages in ion trap low-energy collision-induced dissociation (CID) experiments of the fragments generated from in-source decay (ISD) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of intact proteins are described in both positive and negative ion modes. The MALDI-ISD spectra of the proteins demonstrate common, discontinuous, abundant c- and z'-ions originating from cleavage at the N-Cα bond of Xxx-Asp/Asn and Gly-Xxx residues in both positive- and negative-ion modes. The positive ion CID of the c- and z'-ions resulted in product ions originating from selective cleavage at Asp-Xxx, Glu-Xxx and Cys-Xxx residues. Nonselective cleavage product ions rationalized by the mechanism of a "mobile proton" are also observed in positive ion CID spectra. Negative ion CID of the ISD fragments results in complex product ions accompanied by the loss of neutrals from b-, c-, and y-ions. The most characteristic feature of negative ion CID is selective cleavage of the peptide bonds of acidic residues, Xxx-Asp/Glu/Cys. A definite influence of α-helix on the CID product ions was not obtained. However, the results from positive ion and negative ion CID of the MALDI-ISD fragments that may have long α-helical domains suggest that acidic residues in helix-free regions tend to degrade more than those in helical regions.

  19. Selective and nonselective cleavages in positive and negative CID of the fragments generated from in-source decay of intact proteins in MALDI-MS.

    PubMed

    Takayama, Mitsuo; Sekiya, Sadanori; Iimuro, Ryunosuke; Iwamoto, Shinichi; Tanaka, Koichi

    2014-01-01

    Selective and nonselective cleavages in ion trap low-energy collision-induced dissociation (CID) experiments of the fragments generated from in-source decay (ISD) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of intact proteins are described in both positive and negative ion modes. The MALDI-ISD spectra of the proteins demonstrate common, discontinuous, abundant c- and z'-ions originating from cleavage at the N-Cα bond of Xxx-Asp/Asn and Gly-Xxx residues in both positive- and negative-ion modes. The positive ion CID of the c- and z'-ions resulted in product ions originating from selective cleavage at Asp-Xxx, Glu-Xxx and Cys-Xxx residues. Nonselective cleavage product ions rationalized by the mechanism of a "mobile proton" are also observed in positive ion CID spectra. Negative ion CID of the ISD fragments results in complex product ions accompanied by the loss of neutrals from b-, c-, and y-ions. The most characteristic feature of negative ion CID is selective cleavage of the peptide bonds of acidic residues, Xxx-Asp/Glu/Cys. A definite influence of α-helix on the CID product ions was not obtained. However, the results from positive ion and negative ion CID of the MALDI-ISD fragments that may have long α-helical domains suggest that acidic residues in helix-free regions tend to degrade more than those in helical regions.

  20. Novel protein kinase C inhibitors: alpha-terthiophene derivatives.

    PubMed

    Kim, D S; Ashendel, C L; Zhou, Q; Chang, C T; Lee, E S; Chang, C J

    1998-10-06

    A series of alpha-terthiophene derivatives were prepared and their protein kinase C inhibitory activity were evaluated. The aldehyde derivatives were most potent inhibitors (IC50 < 1 microM). alpha-Terthiophene monoaldehyde was inactive in the inhibitions of protein kinase A, mitogen activated protein kinase and protein tyrosine kinase.

  1. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2004-10-12

    The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  2. Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase.

    PubMed

    Chen, L; Haider, K; Ponda, M; Cariappa, A; Rowitch, D; Pillai, S

    2001-06-15

    A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.

  3. Dynamics driven allostery in protein kinases

    PubMed Central

    Kornev, Alexandr P.; Taylor, Susan S.

    2015-01-01

    Protein kinases have very dynamic structures and their functionality strongly depends on their dynamic state. Active kinases reveal a dynamic pattern with residues clustering into semirigid communities that move in µs-ms timescale. Previously detected hydrophobic spines serve as connectors between communities. Communities do not follow the traditional subdomain structure of the kinase core or its secondary structure elements. Instead they are organized around main functional units. Integration of the communities depends on the assembly of the hydrophobic spine and phosphorylation of the activation loop. Single mutations can significantly disrupt the dynamic infrastructure and thereby interfere with long distance allosteric signaling that propagates throughout the whole molecule. Dynamics is proposed to be the underlying mechanism for allosteric regulation in protein kinases. PMID:26481499

  4. Oncoprotein protein kinase antibody kit

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  5. Non-degradative Ubiquitination of Protein Kinases

    PubMed Central

    Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

    2016-01-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

  6. Protein Kinase A: A Master Kinase of Granulosa Cell Differentiation

    PubMed Central

    Puri, Pawan; Little-Ihrig, Lynda; Chandran, Uma; Law, Nathan C.; Hunzicker-Dunn, Mary; Zeleznik, Anthony J.

    2016-01-01

    Activation of protein kinase A (PKA) by follicle stimulating hormone (FSH) transduces the signal that drives differentiation of ovarian granulosa cells (GCs). An unresolved question is whether PKA is sufficient to initiate the complex program of GC responses to FSH. We compared signaling pathways and gene expression profiles of GCs stimulated with FSH or expressing PKA-CQR, a constitutively active mutant of PKA. Both FSH and PKA-CQR stimulated the phosphorylation of proteins known to be involved in GC differentiation including CREB, ß-catenin, AKT, p42/44 MAPK, GAB2, GSK-3ß, FOXO1, and YAP. In contrast, FSH stimulated the phosphorylation of p38 MAP kinase but PKA-CQR did not. Microarray analysis revealed that 85% of transcripts that were up-regulated by FSH were increased to a comparable extent by PKA-CQR and of the transcripts that were down-regulated by FSH, 76% were also down-regulated by PKA-CQR. Transcripts regulated similarly by FSH and PKA-CQR are involved in steroidogenesis and differentiation, while transcripts more robustly up-regulated by PKA-CQR are involved in ovulation. Thus, PKA, under the conditions of our experimental approach appears to function as a master upstream kinase that is sufficient to initiate the complex pattern of intracellular signaling pathway and gene expression profiles that accompany GC differentiation. PMID:27324437

  7. AMP-activated protein kinase--an archetypal protein kinase cascade?

    PubMed

    Hardie, D G; MacKintosh, R W

    1992-10-01

    Mammalian AMP-activated protein kinase is the central component of a protein kinase cascade which inactivates three key enzymes involved in the synthesis or release of free fatty acids and cholesterol inside the cell. The kinase cascade is activated by elevation of AMP, and perhaps also by fatty acid and cholesterol metabolites. The system may fulfil a protective function, preventing damage caused by depletion of ATP or excessive intracellular release of free lipids, a type of stress response. Recent evidence suggests that it may have been in existence for at least a billion years, since a very similar protein kinase cascade is present in higher plants. This system therefore represents an early eukaryotic protein kinase cascade, which is unique in that it is regulated by intracellular metabolites rather than extracellular signals or cell cycle events.

  8. Problem-Solving Test: "In Vitro" Protein Kinase A Reaction

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Phosphorylation of proteins by protein kinases is an important mechanism in the regulation of protein activity. Among hundreds of protein kinases present in human cells, PKA, the first kinase discovered, belongs to the most important and best characterized group of these enzymes. The author presents an experiment that analyzes the "in vitro"…

  9. Problem-Solving Test: "In Vitro" Protein Kinase A Reaction

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Phosphorylation of proteins by protein kinases is an important mechanism in the regulation of protein activity. Among hundreds of protein kinases present in human cells, PKA, the first kinase discovered, belongs to the most important and best characterized group of these enzymes. The author presents an experiment that analyzes the "in vitro"…

  10. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

    The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis. PMID:25429354

  11. A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

    PubMed

    Luangwedchakarn, Voravich; Day, Noorbibi K; Hitchcock, Remi; Brown, Pam G; Lerner, Danica L; Rucker, Rajivi P; Cianciolo, George J; Good, Robert A; Haraguchi, Soichi

    2003-05-01

    CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

  12. Differential regulation of rice mitogen activated protein kinase kinase (MKK) by abiotic stress.

    PubMed

    Kumar, Kundan; Rao, Kudupudi Prabhakara; Sharma, Pallavi; Sinha, Alok Krishna

    2008-10-01

    Mitogen activated protein kinase cascade plays a crucial role in various biotic and abiotic stresses, hormones, cell division and developmental processes. MAP kinase kinase being integral part of this cascade performs an important function of integrating upstream signals to mitogen activated protein kinase for further appropriate cellular responses. We here report cloning of five MAP kinase kinase members from Oryza sativa indica cultivar var. Pusa Basmati 1, namely MAP kinase kinases 1, 3, 4, 6 and 10-2. All these members, except MKK10-2 possess fully canonical motif structures of MAP kinase kinase. The deduced amino acid sequence showed changes at certain position within japonica and indica variety of rice. Analysis of transcript regulation by quantitative real time PCR revealed that these five members are differentially regulated by cold, heat, salinity and drought stresses. MAP kinase kinases 4 and 6 are strongly regulated by cold and salt stresses while MAP kinase kinase 1 is regulated by salt and drought stresses. MAP kinase kinase 10-2 is regulated only by cold stress. The study provides the indication of involvement of specific MAP kinase kinase in different abiotic stress signaling and also possible cross talks that exist during the signaling processes.

  13. Crystal Structure of the Protein Kinase Domain of Yeast AMP-Activated Protein Kinase Snf1

    SciTech Connect

    Rudolph,M.; Amodeo, G.; Bai, Y.; Tong, L.

    2005-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator, and is an important target for drug development against diabetes, obesity, and other diseases. AMPK is a hetero-trimeric enzyme, with a catalytic ({alpha}) subunit, and two regulatory ({beta} and {gamma}) subunits. Here we report the crystal structure at 2.2 Angstrom resolution of the protein kinase domain (KD) of the catalytic subunit of yeast AMPK (commonly known as SNF1). The Snf1-KD structure shares strong similarity to other protein kinases, with a small N-terminal lobe and a large C-terminal lobe. Two negative surface patches in the structure may be important for the recognition of the substrates of this kinase.

  14. Heart failure-specific changes in protein kinase signalling.

    PubMed

    Lorenz, Kristina; Stathopoulou, Konstantina; Schmid, Evelyn; Eder, Petra; Cuello, Friederike

    2014-06-01

    Among the myriad of molecular alterations occurring in heart failure development, aggravation of the disease is often attributed to global or local changes in protein kinase activity, thus making protein kinases attractive targets for therapeutic intervention. Since protein kinases do not only have maladaptive roles, but also contribute to the physiological integrity of cells, it is a challenging task to circumvent undesired inhibition of protein kinase activity. Identification of posttranslational modifications and/or protein-protein interactions that are exclusively apparent under pathophysiological conditions provides exciting information for alternative non-kinase inhibitory treatment strategies that eliminate maladaptive functions of a protein kinase, but preserve the beneficial ones. Here, we focus on the disease-specific regulation of a number of protein kinases, namely, Ca(2+)/calmodulin-dependent protein kinase II isoform δ (CaMKIIδ), G protein-coupled receptor kinase 2 (GRK2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein kinase D (PKD) and protein kinase C isoform β2 (PKCβ2), which are embedded in complex signal transduction pathways implicated in heart failure development, and discuss potential avenues for novel treatment strategies to combat heart disease.

  15. A-kinase Anchoring Protein 79/150 Recruits Protein Kinase C to Phosphorylate Roundabout Receptors.

    PubMed

    Samelson, Bret K; Gore, Bryan B; Whiting, Jennifer L; Nygren, Patrick J; Purkey, Alicia M; Colledge, Marcie; Langeberg, Lorene K; Dell'Acqua, Mark L; Zweifel, Larry S; Scott, John D

    2015-05-29

    Anchoring proteins direct protein kinases and phosphoprotein phosphatases toward selected substrates to control the efficacy, context, and duration of neuronal phosphorylation events. The A-kinase anchoring protein AKAP79/150 interacts with protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (calcineurin) to modulate second messenger signaling events. In a mass spectrometry-based screen for additional AKAP79/150 binding partners, we have identified the Roundabout axonal guidance receptor Robo2 and its ligands Slit2 and Slit3. Biochemical and cellular approaches confirm that a linear sequence located in the cytoplasmic tail of Robo2 (residues 991-1070) interfaces directly with sites on the anchoring protein. Parallel studies show that AKAP79/150 interacts with the Robo3 receptor in a similar manner. Immunofluorescent staining detects overlapping expression patterns for murine AKAP150, Robo2, and Robo3 in a variety of brain regions, including hippocampal region CA1 and the islands of Calleja. In vitro kinase assays, peptide spot array mapping, and proximity ligation assay staining approaches establish that human AKAP79-anchored PKC selectively phosphorylates the Robo3.1 receptor subtype on serine 1330. These findings imply that anchored PKC locally modulates the phosphorylation status of Robo3.1 in brain regions governing learning and memory and reward. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Redox Regulation of the AMP-Activated Protein Kinase

    PubMed Central

    Han, Yingying; Wang, Qilong; Song, Ping; Zhu, Yi; Zou, Ming-Hui

    2010-01-01

    Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death. Objectives The aim of this study is to determine if AMP-activated protein kinase (AMPK), a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC). Methods Bovine aortic endothelial cells (BAEC) were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation. Results In BAEC, Berberine caused a dose- and time-dependent increase in the phosphorylation of AMPK at Thr172 and acetyl CoA carboxylase (ACC) at Ser79, a well characterized downstream target of AMPK. Concomitantly, Berberine increased peroxynitrite, a potent oxidant formed by simultaneous generation of superoxide and nitric oxide. Pre-incubation of BAEC with anti-oxidants markedly attenuated Berberine-enhanced phosphorylation of both AMPK and ACC. Consistently, adenoviral expression of superoxide dismutase and pretreatment of L-NG-Nitroarginine methyl ester (L-NAME; a non-selective NOS inhibitor) blunted Berberine-induced phosphorylation of AMPK. Furthermore, mitochondria-targeted tempol (mito-tempol) pretreatment or expression of uncoupling protein attenuated AMPK activation caused by Berberine. Depletion of mitochondria abolished the effects of Berberine on AMPK in EC. Finally, Berberine significantly increased the phosphorylation of LKB1 at Ser307 and gene silencing of LKB1 attenuated Berberine-enhanced AMPK Thr172 phosphorylation in BAEC. Conclusion Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells. PMID:21079763

  17. Protein Kinase C Isozyme in Mammary Carcinogenesis.

    DTIC Science & Technology

    1996-10-01

    11 A B Clone 72 Clone 34 AKAP 86 Clone 35H CInn 72 lonn 4 P 95 lone 351 9. 5J 4.4- 688 431 Clone 45 Clone 35F Clone 64 Annexln 1 Clone 45 Clone 35F...purified antibodies directed against a variety of PKC substrates and an A-kinase anchoring protein, AKAP 95. (B) PolyA+ mRNAs isolated from confluent cell

  18. Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

    PubMed

    Fujiwara, Yuya; Kawaguchi, Yoshinori; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2016-06-24

    Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKβ/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα.

  19. MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase.

    PubMed Central

    Nakielny, S; Cohen, P; Wu, J; Sturgill, T

    1992-01-01

    A 'MAP kinase activator' was purified several thousand-fold from insulin-stimulated rabbit skeletal muscle, which resembled the 'activator' from nerve growth factor-stimulated PC12 cells in that it could be inactivated by incubation with protein phosphatase 2A, but not by protein tyrosine phosphatases and its apparent molecular mass was 45-50 kDa. In the presence of MgATP, 'MAP kinase activator' converted the normal 'wild-type' 42 kDa MAP kinase from an inactive dephosphorylated form to the fully active diphosphorylated species. Phosphorylation occurred on the same threonine and tyrosine residues which are phosphorylated in vivo in response to growth factors or phorbol esters. A mutant MAP kinase produced by changing a lysine at the active centre to arginine was phosphorylated in an identical manner by the 'MAP kinase activator', but no activity was generated. The results demonstrate that 'MAP kinase activator' is a protein kinase (MAP kinase kinase) and not a protein that stimulates the autophosphorylation of MAP kinase. MAP kinase kinase is the first established example of a protein kinase that can phosphorylate an exogenous protein on threonine as well as tyrosine residues. Images PMID:1318193

  20. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    PubMed

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  1. Protein kinase activity of the insulin receptor.

    PubMed Central

    Gammeltoft, S; Van Obberghen, E

    1986-01-01

    exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS) Images Fig. 3. Fig. 5. PMID:3017297

  2. Real-time protein kinase assay.

    PubMed

    Sun, Hongye; Low, Karen E; Woo, Sam; Noble, Richard L; Graham, Ronald J; Connaughton, Sonia S; Gee, Melissa A; Lee, Linda G

    2005-04-01

    We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC(50) values of inhibitors for PKC betaII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma's LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained.

  3. Kinase Pathway Database: An Integrated Protein-Kinase and NLP-Based Protein-Interaction Resource

    PubMed Central

    Koike, Asako; Kobayashi, Yoshiyuki; Takagi, Toshihisa

    2003-01-01

    Protein kinases play a crucial role in the regulation of cellular functions. Various kinds of information about these molecules are important for understanding signaling pathways and organism characteristics. We have developed the Kinase Pathway Database, an integrated database involving major completely sequenced eukaryotes. It contains the classification of protein kinases and their functional conservation, ortholog tables among species, protein–protein, protein–gene, and protein–compound interaction data, domain information, and structural information. It also provides an automatic pathway graphic image interface. The protein, gene, and compound interactions are automatically extracted from abstracts for all genes and proteins by natural-language processing (NLP).The method of automatic extraction uses phrase patterns and the GENA protein, gene, and compound name dictionary, which was developed by our group. With this database, pathways are easily compared among species using data with more than 47,000 protein interactions and protein kinase ortholog tables. The database is available for querying and browsing at http://kinasedb.ontology.ims.u-tokyo.ac.jp/. PMID:12799355

  4. Protein Scaffolds in MAP Kinase Signalling

    PubMed Central

    Brown, Matthew D.; Sacks, David B.

    2009-01-01

    The mitogen-activated protein kinase (MAPK) pathway allows cells to interpret external signals and respond in an appropriate way. Diverse cellular functions, ranging from differentiation and proliferation to migration and inflammation, are regulated by MAPK signalling. Therefore, cells have developed mechanisms by which this single pathway modulates numerous cellular responses from a wide range of activating factors. This specificity is achieved by several mechanisms, including temporal and spatial control of MAPK signalling components. Key to this control are protein scaffolds, which are multidomain proteins that interact with components of the MAPK cascade in order to assemble signalling complexes. Studies conducted on different scaffolds, in different biological systems, have shown that scaffolds exert substantial control over MAPK signalling, influencing the signal intensity, time course and, importantly, the cellular responses. Protein scaffolds, therefore, are integral elements in the modulation of the MAPK network in fundamental physiological processes. PMID:19091303

  5. Endothelial Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Is Critical for Lymphatic Vascular Development and Function

    PubMed Central

    Guo, Chang-An; Danai, Laura V.; Yawe, Joseph C.; Gujja, Sharvari; Edwards, Yvonne J. K.

    2016-01-01

    The molecular mechanisms underlying lymphatic vascular development and function are not well understood. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and atherosclerosis. Here, we show that constitutive loss of EC Map4k4 in mice causes postnatal lethality due to chylothorax, suggesting that Map4k4 is required for normal lymphatic vascular function. Mice constitutively lacking EC Map4k4 displayed dilated lymphatic capillaries, insufficient lymphatic valves, and impaired lymphatic flow; furthermore, primary ECs derived from these animals displayed enhanced proliferation compared with controls. Yeast 2-hybrid analyses identified the Ras GTPase-activating protein Rasa1, a known regulator of lymphatic development and lymphatic endothelial cell fate, as a direct interacting partner for Map4k4. Map4k4 silencing in ECs enhanced basal Ras and extracellular signal-regulated kinase (Erk) activities, and primary ECs lacking Map4k4 displayed enhanced lymphatic EC marker expression. Taken together, these results reveal that EC Map4k4 is critical for lymphatic vascular development by regulating EC quiescence and lymphatic EC fate. PMID:27044870

  6. Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein*

    PubMed Central

    Hundsrucker, Christian; Skroblin, Philipp; Christian, Frank; Zenn, Hans-Michael; Popara, Viola; Joshi, Mangesh; Eichhorst, Jenny; Wiesner, Burkhard; Herberg, Friedrich W.; Reif, Bernd; Rosenthal, Walter; Klussmann, Enno

    2010-01-01

    A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways. PMID:20007971

  7. Photoinduced structural changes to protein kinase A

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.; Thomas, Robert J.; Brancaleon, Lorenzo

    2014-03-01

    The importance of porphyrins in organisms is underscored by the ubiquitous biological and biochemical functions that are mediated by these compounds and by their potential biomedical and biotechnological applications. Protoporphyrin IX (PPIX) is the precursor to heme and has biomedical applications such as its use as a photosensitizer in phototherapy and photodetection of cancer. Among other applications, our group has demonstrated that low-irradiance exposure to laser irradiation of PPIX, Fe-PPIX, or meso-tetrakis (4-sulfonatophenyl) porphyrin (TSPP) non-covalently docked to a protein causes conformational changes in the polypeptide. Such approach can have remarkable consequences in the study of protein structure/function relationship and can be used to prompt non-native protein properties. Therefore we have investigated protein kinase A (PKA), a more relevant protein model towards the photo-treatment of cancer. PKA's enzymatic functions are regulated by the presence of cyclic adenosine monophosphate for intracellular signal transduction involved in, among other things, stimulation of transcription, tumorigenesis in Carney complex and migration of breast carcinoma cells. Since phosphorylation is a necessary step in some cancers and inflammatory diseases, inhibiting the protein kinase, and therefore phosphorylation, may serve to treat these diseases. Changes in absorption, steady-state fluorescence, and fluorescence lifetime indicate: 1) both TSPP and PPIX non-covalently bind to PKA where they maintain photoreactivity; 2) absorptive photoproduct formation occurs only when PKA is bound to TSPP and irradiated; and 3) PKA undergoes secondary structural changes after irradiation with either porphyrin bound. These photoinduced changes could affect the protein's enzymatic and signaling capabilities.

  8. Non-selective cation channel-mediated Ca2+-entry and activation of Ca2+/calmodulin-dependent kinase II contribute to G2/M cell cycle arrest and survival of irradiated leukemia cells.

    PubMed

    Heise, Nicole; Palme, Daniela; Misovic, Milan; Koka, Saisudha; Rudner, Justine; Lang, Florian; Salih, Helmut R; Huber, Stephan M; Henke, Guido

    2010-01-01

    Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca(2+) signaling in the cell cycle control and survival of chronic myeloid leukemia (CML) cells subjected to ionizing radiation (IR). To this end, K562 erythroid leukemia cells were irradiated (0-10 Gy). Tumor survival was analyzed by clonogenic survival assay and cell cycle progression via flow cytometry. Plasma membrane cation conductance was assessed by patch-clamp whole-cell recording and the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 Ca(2+) imaging. Nuclear activity of Ca(2+)/calmodulin-dependent kinase II (CaMKII) was defined by Western blotting. In addition, the effect of IR (5 Gy) on the cation conductance of primary CML cells was determined. The results indicated that IR (10 Gy) induced a G(2)/M cell cycle arrest of K562 cells within 24 h post-irradiation (p.i.) and decreased the clonogenic survival to 0.5 % of that of the control cells. In K562 cells, G(2)/M cell cycle arrest was preceded by activation of TRPV5/6-like nonselective cation channels in the plasma membrane 1-5 h p.i., resulting in an elevated Ca(2+) entry as evident from fura-2 Ca(2+) imaging. Similarly, IR stimulated a Ca(2+)-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca(2+) entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G(2) phase was delayed upon buffering [Ca(2+)](i) with the Ca(2+) chelator BAPTA-AM or inhibiting CaMKII with KN93 (1 nM). In addition, KN93 decreased the clonogenic survival of irradiated cells but not of control cells. In conclusion, the data suggest that IR-stimulated cation channel activation, Ca(2+) entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.

  9. Protein Kinases in Mammary Gland Development and Carcinogenesis

    DTIC Science & Technology

    1999-09-01

    differ among CaM kinase family members include their subcellular localization , regulation by autophosphorylation, and regulation by other proteins. In...addition, CaM kinases have unique amino- and carboxyl- terminal domains that contribute to kinase-specific differences in subcellular localization ...chromosomal localization of Punc, a calcium/calmodulin-dependent protein kinase, (Submitted). 14. Hennings, H., Glick, A., Lowry, D., Krsmanovic, L

  10. Novel, potent and selective inhibitors of protein kinase C show oral anti-inflammatory activity.

    PubMed

    Nixon, J S; Bishop, J; Bradshaw, D; Davis, P D; Hill, C H; Elliott, L H; Kumar, H; Lawton, G; Lewis, E J; Mulqueen, M

    1991-01-01

    Clarification of the precise role of protein kinase C (PKC) in cellular functional responses has been hampered by a lack of potent, selective inhibitors. The structural lead provided by staurosporine, a potent but non-selective protein kinase (PK) inhibitor, was used to derive a series of bis(indolyl)maleimides of which the most potent, Ro 31-8425 (I50: PKC = 8 nM) showed 350-fold selectivity for PKC over cAMP-dependent protein kinase. Ro 31-8425 antagonised cellular processes triggered by phorbol esters (potent, specific PKC activators) and inhibited the allogeneic mixed lymphocyte reaction, suggesting a role for PKC in T-cell activation. Methylation of the primary amine in Ro 31-8425 produced an analogue. Ro 31-8830 which, when administered orally, produced a dose-dependent inhibition of a phorbol ester-induced paw oedema in mice (minimum effective dose = 15 mg/kg). Ro 31-8830 also selectively inhibited the secondary inflammation in a developing adjuvant arthritis model in the rat. The results presented here suggest that these selective inhibitors of PKC may have therapeutic value in the treatment of T-cell-mediated autoimmune diseases.

  11. Protein kinase C alpha-dependent phosphorylation of Golgi proteins.

    PubMed

    Radau, B; Otto, A; Müller, E C; Westermann, P

    2000-07-01

    Golgi-enriched membranes were phosphorylated in order to understand the mechanism for protein kinase C (PKC) regulation of exocytic vesicle formation at the trans-Golgi network. Two of the main PKC substrates were identified as MARCKS and Mac-MARCKS by two-dimensional electrophoresis (2-DE) and mass spectrometric sequencing. Annexin IV and profilin I, two other Golgi-associated proteins--although known as in vitro PKC substrates--were not phosphorylated in the Golgi-bound state.

  12. Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases).

    PubMed Central

    Verhey, S. D.; Gaiser, J. C.; Lomax, T. L.

    1993-01-01

    Using an in situ phosphorylation assay with zucchini (Cucurbita pepo L. cv Dark Green) seedling tissue, we have identified numerous polypeptides that are capable of acting as protein kinases. Total protein preparations from different organs contain different kinase profiles, but all are within the range of 55 to 70 kD. At least four kinases are associated with highly purified plasma membranes from etiolated zucchini hypocotyls. The major phosphorylated polypeptides from plasma membranes range in apparent molecular mass from 58 to 68 kD. The plasma membrane kinases are activated by micromolar concentrations of calcium and phosphorylate serine, and, to a lesser extent, threonine residues. These characteristics are similar to those of a soluble calcium-dependent protein kinase that has been purified to homogeneity from soybean suspension cultures. Three of the zucchini plasma membrane kinases share antigenic epitopes with the soluble soybean kinase. The presence of kinase activity at different apparent molecular masses may be indicative of separate kinases with similar characteristics. The zucchini hypocotyl protein kinases are not removed from plasma membrane vesicles by 0.5 M NaCl/5 mM ethylenediaminetetraacetate or by detergent concentrations below the critical micelle concentration of two types of detergent. This indicates that the plasma membrane protein kinases are tightly associated with the membrane in zucchini seedlings. PMID:12231949

  13. Phosphoregulators: Protein Kinases and Protein Phosphatases of Mouse

    PubMed Central

    Forrest, Alistair R.R.; Ravasi, Timothy; Taylor, Darrin; Huber, Thomas; Hume, David A.; Grimmond, Sean

    2003-01-01

    With the completion of the human and mouse genome sequences, the task now turns to identifying their encoded transcripts and assigning gene function. In this study, we have undertaken a computational approach to identify and classify all of the protein kinases and phosphatases present in the mouse gene complement. A nonredundant set of these sequences was produced by mining Ensembl gene predictions and publicly available cDNA sequences with a panel of InterPro domains. This approach identified 561 candidate protein kinases and 162 candidate protein phosphatases. This cohort was then analyzed using TribeMCL protein sequence similarity clustering followed by CLUSTALV alignment and hierarchical tree generation. This approach allowed us to (1) distinguish between true members of the protein kinase and phosphatase families and enzymes of related biochemistry, (2) determine the structure of the families, and (3) suggest functions for previously uncharacterized members. The classifications obtained by this approach were in good agreement with previous schemes and allowed us to demonstrate domain associations with a number of clusters. Finally, we comment on the complementary nature of cDNA and genome-based gene detection and the impact of the FANTOM2 transcriptome project. PMID:12819143

  14. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  15. A framework for classification of prokaryotic protein kinases.

    PubMed

    Tyagi, Nidhi; Anamika, Krishanpal; Srinivasan, Narayanaswamy

    2010-05-26

    Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular organization which indicates a degree of complexity and protein-protein interactions in the

  16. A Framework for Classification of Prokaryotic Protein Kinases

    PubMed Central

    Tyagi, Nidhi; Anamika, Krishanpal; Srinivasan, Narayanaswamy

    2010-01-01

    Background Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. Methodology/Principal Findings We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. Conclusion/Significance Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular organization which indicates a

  17. Genomic analysis of the eukaryotic protein kinase superfamily: a perspective

    PubMed Central

    Hanks, Steven K

    2003-01-01

    Protein kinases with a conserved catalytic domain make up one of the largest 'superfamilies' of eukaryotic proteins and play many key roles in biology and disease. Efforts to identify and classify all the members of the eukaryotic protein kinase superfamily have recently culminated in the mining of essentially complete human genome data. PMID:12734000

  18. The Roles of Protein Kinases in Learning and Memory

    ERIC Educational Resources Information Center

    Giese, Karl Peter; Mizuno, Keiko

    2013-01-01

    In the adult mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to enable learning and memory. Based on this information it appears that learning and memory-related kinases either impact on synaptic transmission by altering ion channel properties or ion channel density, or regulate…

  19. The Roles of Protein Kinases in Learning and Memory

    ERIC Educational Resources Information Center

    Giese, Karl Peter; Mizuno, Keiko

    2013-01-01

    In the adult mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to enable learning and memory. Based on this information it appears that learning and memory-related kinases either impact on synaptic transmission by altering ion channel properties or ion channel density, or regulate…

  20. Mitogen-activated protein kinase cascades in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

    Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

  1. The nuts and bolts of AGC protein kinases.

    PubMed

    Pearce, Laura R; Komander, David; Alessi, Dario R

    2010-01-01

    The AGC kinase subfamily of protein kinases contains 60 members, including PKA, PKG and PKC. The family comprises some intensely examined protein kinases (such as Akt, S6K, RSK, MSK, PDK1 and GRK) as well as many less well-studied enzymes (such as SGK, NDR, LATS, CRIK, SGK494, PRKX, PRKY and MAST). Research has shed new light onto the architecture and regulatory mechanisms of these kinases. In addition, AGC kinases mediate diverse and important cellular functions, and their mutation and/or dysregulation contributes to the pathogenesis of many human diseases, including cancer and diabetes.

  2. Protein kinase A contributes to the negative control of Snf1 protein kinase in Saccharomyces cerevisiae.

    PubMed

    Barrett, LaKisha; Orlova, Marianna; Maziarz, Marcin; Kuchin, Sergei

    2012-02-01

    Snf1 protein kinase regulates responses to glucose limitation and other stresses. Snf1 activation requires phosphorylation of its T-loop threonine by partially redundant upstream kinases (Sak1, Tos3, and Elm1). Under favorable conditions, Snf1 is turned off by Reg1-Glc7 protein phosphatase. The reg1 mutation causes increased Snf1 activation and slow growth. To identify new components of the Snf1 pathway, we searched for mutations that, like snf1, suppress reg1 for the slow-growth phenotype. In addition to mutations in genes encoding known pathway components (SNF1, SNF4, and SAK1), we recovered "fast" mutations, designated fst1 and fst2. Unusual morphology of the mutants in the Σ1278b strains employed here helped us identify fst1 and fst2 as mutations in the RasGAP genes IRA1 and IRA2. Cells lacking Ira1, Ira2, or Bcy1, the negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA), exhibited reduced Snf1 pathway activation. Conversely, Snf1 activation was elevated in cells lacking the Gpr1 sugar receptor, which contributes to PKA signaling. We show that the Snf1-activating kinase Sak1 is phosphorylated in vivo on a conserved serine (Ser1074) within an ideal PKA motif. However, this phosphorylation alone appears to play only a modest role in regulation, and Sak1 is not the only relevant target of the PKA pathway. Collectively, our results suggest that PKA, which integrates multiple regulatory inputs, could contribute to Snf1 regulation under various conditions via a complex mechanism. Our results also support the view that, like its mammalian counterpart, AMP-activated protein kinase (AMPK), yeast Snf1 participates in metabolic checkpoint control that coordinates growth with nutrient availability.

  3. KinasePhos: a web tool for identifying protein kinase-specific phosphorylation sites.

    PubMed

    Huang, Hsien-Da; Lee, Tzong-Yi; Tzeng, Shih-Wei; Horng, Jorng-Tzong

    2005-07-01

    KinasePhos is a novel web server for computationally identifying catalytic kinase-specific phosphorylation sites. The known phosphorylation sites from public domain data sources are categorized by their annotated protein kinases. Based on the profile hidden Markov model, computational models are learned from the kinase-specific groups of the phosphorylation sites. After evaluating the learned models, the model with highest accuracy was selected from each kinase-specific group, for use in a web-based prediction tool for identifying protein phosphorylation sites. Therefore, this work developed a kinase-specific phosphorylation site prediction tool with both high sensitivity and specificity. The prediction tool is freely available at http://KinasePhos.mbc.nctu.edu.tw/.

  4. Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID.

    PubMed

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês C R; Willige, Björn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

    2014-06-19

    The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the--in many cells--asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

  5. Understanding and exploiting substrate recognition by protein kinases.

    PubMed

    Turk, Benjamin E

    2008-02-01

    Protein kinases play a virtually universal role in cellular regulation and are emerging as an important class of new drug targets, yet the cellular functions of most human kinases largely remain obscure. Aspects of substrate recognition common to all kinases in the ATP nucleotide binding site have been exploited in the generation of analog-specific mutants for exploring kinase function and discovering novel protein substrates. Likewise, understanding interactions with the protein substrate, which differ substantially between kinases, can also help to identify substrates and to produce tools for studying kinase pathways, including fluorescent biosensors. Principles of kinase substrate recognition are particularly valuable in guiding bioinformatics and phosphoproteomics approaches that impact our understanding of signaling pathways and networks on a global scale.

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

    USDA-ARS?s Scientific Manuscript database

    Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

  7. Identification of intracellular receptor proteins for activated protein kinase C.

    PubMed Central

    Mochly-Rosen, D; Khaner, H; Lopez, J

    1991-01-01

    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC. Images PMID:1850844

  8. Structural investigation of protein kinase C inhibitors.

    PubMed

    Barak, D; Shibata, M; Rein, R

    1991-01-01

    The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

  9. Protein kinase C isozymes and addiction.

    PubMed

    Olive, M Foster; Messing, Robert O

    2004-04-01

    Protein kinase C (PKC) has long been recognized an important family of enzymes that regulate numerous aspects of neuronal signal transduction, neurotransmitter synthesis, release and reuptake, receptor and ion channel function, neuronal excitability, development, and gene expression. Much evidence has implicated PKCs in the effects of several drugs of abuse, and in behavioral responses to these drugs. The present review summarizes the effects of both acute and chronic exposure to various drugs of abuse on individual PKC isozymes in the brain. In addition, we summarize recent studies utilizing mice with targeted deletions of the genes for PKCgamma and PKCepsilon. These studies suggest that individual PKC isozymes play a role in the development of drug dependence and addiction.

  10. Structural investigation of protein kinase C inhibitors

    NASA Technical Reports Server (NTRS)

    Barak, D.; Shibata, M.; Rein, R.

    1991-01-01

    The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

  11. AKAP-Lbc nucleates a protein kinase D activation scaffold.

    PubMed

    Carnegie, Graeme K; Smith, F Donelson; McConnachie, George; Langeberg, Lorene K; Scott, John D

    2004-09-24

    The transmission of cellular signals often proceeds through multiprotein complexes where enzymes are positioned in proximity to their upstream activators and downstream substrates. In this report we demonstrate that the A-kinase anchoring protein AKAP-Lbc assembles an activation complex for the lipid-dependent enzyme protein kinase D (PKD). Using a combination of biochemical, enzymatic, and immunofluorescence techniques, we show that the anchoring protein contributes to PKD activation in two ways: it recruits an upstream kinase PKCeta and coordinates PKA phosphorylation events that release activated protein kinase D. Thus, AKAP-Lbc synchronizes PKA and PKC activities in a manner that leads to the activation of a third kinase. This configuration illustrates the utility of kinase anchoring as a mechanism to constrain the action of broad-spectrum enzymes.

  12. Rational Redesign of a Functional Protein Kinase-Substrate Interaction

    PubMed Central

    2017-01-01

    Eukaryotic protein kinases typically phosphorylate substrates in the context of specific sequence motifs, contributing to specificity essential for accurate signal transmission. Protein kinases recognize their target sequences through complementary interactions within the active site cleft. As a step toward the construction of orthogonal kinase signaling systems, we have re-engineered the protein kinase Pim1 to alter its phosphorylation consensus sequence. Residues in the Pim1 catalytic domain interacting directly with a critical arginine residue in the substrate were substituted to produce a kinase mutant that instead accommodates a hydrophobic residue. We then introduced a compensating mutation into a Pim1 substrate, the pro-apoptotic protein BAD, to reconstitute phosphorylation both in vitro and in living cells. Coexpression of the redesigned kinase with its substrate in cells protected them from apoptosis. Such orthogonal kinase–substrate pairs provide tools to probe the functional consequences of specific phosphorylation events in living cells and to design synthetic signaling pathways. PMID:28314095

  13. Prediction of 492 human protein kinase substrate specificities.

    PubMed

    Safaei, Javad; Maňuch, Ján; Gupta, Arvind; Stacho, Ladislav; Pelech, Steven

    2011-10-14

    Complex intracellular signaling networks monitor diverse environmental inputs to evoke appropriate and coordinated effector responses. Defective signal transduction underlies many pathologies, including cancer, diabetes, autoimmunity and about 400 other human diseases. Therefore, there is high impetus to define the composition and architecture of cellular communications networks in humans. The major components of intracellular signaling networks are protein kinases and protein phosphatases, which catalyze the reversible phosphorylation of proteins. Here, we have focused on identification of kinase-substrate interactions through prediction of the phosphorylation site specificity from knowledge of the primary amino acid sequence of the catalytic domain of each kinase. The presented method predicts 488 different kinase catalytic domain substrate specificity matrices in 478 typical and 4 atypical human kinases that rely on both positive and negative determinants for scoring individual phosphosites for their suitability as kinase substrates. This represents a marked advancement over existing methods such as those used in NetPhorest (179 kinases in 76 groups) and NetworKIN (123 kinases), which consider only positive determinants for kinase substrate prediction. Comparison of our predicted matrices with experimentally-derived matrices from about 9,000 known kinase-phosphosite substrate pairs revealed a high degree of concordance with the established preferences of about 150 well studied protein kinases. Furthermore for many of the better known kinases, the predicted optimal phosphosite sequences were more accurate than the consensus phosphosite sequences inferred by simple alignment of the phosphosites of known kinase substrates. Application of this improved kinase substrate prediction algorithm to the primary structures of over 23, 000 proteins encoded by the human genome has permitted the identification of about 650, 000 putative phosphosites, which are posted on the

  14. Leishmania MAP kinases--familiar proteins in an unusual context.

    PubMed

    Wiese, Martin

    2007-08-01

    Mitogen-activated protein kinases are well-known mediators of signal transduction of higher eukaryotes regulating important processes like proliferation, differentiation, stress response and apoptosis. In Leishmania, the typical three-tiered module of MAP kinase signal transduction pathways is present. However, typical activators like cell surface receptors and substrates such as RNA polymerase II transcription factors are missing. Here, I describe the set of 15 putative mitogen-activated protein kinases encoded in the Leishmania genome and discuss their potential function.

  15. Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald; Dardick, Chris; Canlas, Patrick; Xu, Xia; Gribskov, Michael; Kanrar, Siddhartha; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2006-04-01

    Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety-five percent of the TAP-tagged kinases were recovered. Fifty-six percent of the TAP-tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP-tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14-3-3 proteins or cyclins.

  16. Reusable amperometric biosensor for measuring protein tyrosine kinase activity.

    PubMed

    Wang, Chung-Liang; Wei, Lan-Yi; Yuan, Chiun-Jye; Hwang, Kuo Chu

    2012-01-17

    This work presents a simple, low-cost and reusable label-free method for detecting protein tyrosine kinase activity using a tyrosinase-based amperometric biosensor (tyrosine kinase biosensor). This method is based on the observation that phosphorylation can block the tyrosinase-catalyzed oxidation of tyrosine or tyrosyl residue in peptides. Therefore, the activity of p60c-src protein tyrosine kinase (Src) on the developed tyrosine kinase biosensor could be quickly determined when its specific peptide substrate, p60c-src substrate I, was used. The tyrosine kinase biosensor was highly sensitive to the activity of Src with a linear dynamic range of 1.9-237.6 U/mL and the lowest detection limit of 0.23 U/mL. Interestingly, the tyrosine kinase activity can be measured using the developed tyrosine kinase biosensor repetitively without regeneration. The inhibitory effect of various kinase inhibitors on the Src activity could be determined on the tyrosine kinase biosensor. Src-specific inhibitors, PP2 and Src inhibitor I, effectively suppressed Src activity, whereas PD153035, an inhibitor of the epidermal growth factor receptor, was ineffective. Staurosporine, a universal kinase inhibitor, inhibited Src activity in an ATP concentration-dependent manner. These results suggests that the activities of tyrosine kinases and their behaviors toward various reagents can be effectively measured using the developed tyrosine kinase biosensor.

  17. Diversity, classification and function of the plant protein kinase superfamily

    PubMed Central

    Lehti-Shiu, Melissa D.; Shiu, Shin-Han

    2012-01-01

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants. PMID:22889912

  18. Diversity, classification and function of the plant protein kinase superfamily.

    PubMed

    Lehti-Shiu, Melissa D; Shiu, Shin-Han

    2012-09-19

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants.

  19. Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway.

    PubMed

    Funahashi, Koji; Cao, Xia; Yamauchi, Masako; Kozaki, Yasuko; Ishiguro, Naoki; Kambe, Fukushi

    2009-01-01

    We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.

  20. Protein Kinase A Subunit α Catalytic and A Kinase Anchoring Protein 79 in Human Placental Mitochondria.

    PubMed

    Ma, Maggie Pui Chi; Thomson, Murray

    2012-01-01

    Components of protein phosphorylation signalling systems have been discovered in mitochondria and it has been proposed that these molecules modulate processes including oxidative phosphorylation, apoptosis and steroidogenesis. We used electrophoresis and Western blots probed with specific antibodies to protein kinase A α catalytic subunit (PKAα Cat) and A kinase anchoring protein of approximately 79 kDa molecular weight (AKAP79) to demonstrate the presence of these two proteins in human placental mitochondria. Heavy mitochondria characteristic of cytotrophoblast were separated from light mitochondria characteristic of syncytiotrophoblast by centrifugation. PKAα Cat and AKAP79 were present in both heavy and light mitochondria with no significant difference in concentration. Sucrose density gradient separation of submitochondrial fractions indicated PKAα Cat is located predominantly in the outer membrane whereas AKAP79 is present mainly in the contact site fractions. These data indicate that PKAα Cat is present in the cytoplasm, nucleus and mitochondria of placental cells. AKAP79 is also present in human placental mitochondria but there may be anchoring proteins other than AKAP79 responsible for fixing PKA to the outer membrane. PKA may play roles in mitochondrial protein phosphorylation systems in both cytotrophoblast and syncytiotrophoblast.

  1. A-kinase anchoring proteins: scaffolding proteins in the heart

    PubMed Central

    Dodge-Kafka, Kimberly L.; Li, Jinliang; Kapiloff, Michael S.

    2011-01-01

    The pleiotropic cyclic nucleotide cAMP is the primary second messenger responsible for autonomic regulation of cardiac inotropy, chronotropy, and lusitropy. Under conditions of prolonged catecholaminergic stimulation, cAMP also contributes to the induction of both cardiac myocyte hypertrophy and apoptosis. The formation of localized, multiprotein complexes that contain different combinations of cAMP effectors and regulatory enzymes provides the architectural infrastructure for the specialization of the cAMP signaling network. Scaffolds that bind protein kinase A are called “A-kinase anchoring proteins” (AKAPs). In this review, we discuss recent advances in our understanding of how PKA is compartmentalized within the cardiac myocyte by AKAPs and how AKAP complexes modulate cardiac function in both health and disease. PMID:21856912

  2. Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival

    PubMed Central

    Green, Benjamin D.; Barry, Emma F.; Ma, Yuefang; Woodcock, Joanna; Fitter, Stephen; Zannettino, Andrew C. W.; Pitson, Stuart M.; Hughes, Timothy P.; Lopez, Angel F.; Shepherd, Peter R.; Wei, Andrew H.; Ekert, Paul G.; Guthridge, Mark A.

    2013-01-01

    The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in

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

    PubMed

    Oh, Man-Ho; Wu, Xia; Kim, Hyoung Seok; Harper, Jeffrey F; Zielinski, Raymond E; Clouse, Steven D; Huber, Steven C

    2012-11-30

    Although calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases, autophosphorylation on tyrosine residues was observed for soybean CDPKβ and several Arabidopsis isoforms (AtCPK4 and AtCPK34). We identified Ser-8, Thr-17, Tyr-24 (in the kinase domain), Ser-304, and Ser-358 as autophosphorylation sites of His(6)-GmCDPKβ. Overall autophosphorylation increased kinase activity with synthetic peptides, but autophosphorylation of Tyr-24 appears to attenuate kinase activity based on studies with the Y24F directed mutant. While much remains to be done, it is clear that several CDPKs are dual-specificity kinases, which raises the possibility that phosphotyrosine signaling may play a role in Ca(2+)/CDPK-mediated processes. Published by Elsevier B.V.

  4. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    NASA Technical Reports Server (NTRS)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  5. Unveiling the Novel Dual Specificity Protein Kinases in Bacillus anthracis

    PubMed Central

    Arora, Gunjan; Sajid, Andaleeb; Arulanandh, Mary Diana; Singhal, Anshika; Mattoo, Abid R.; Pomerantsev, Andrei P.; Leppla, Stephen H.; Maiti, Souvik; Singh, Yogendra

    2012-01-01

    Dual specificity protein kinases (DSPKs) are unique enzymes that can execute multiple functions in the cell, which are otherwise performed exclusively by serine/threonine and tyrosine protein kinases. In this study, we have characterized the protein kinases Bas2152 (PrkD) and Bas2037 (PrkG) from Bacillus anthracis. Transcriptional analyses of these kinases showed that they are expressed in all phases of growth. In a serendipitous discovery, both kinases were found to be DSPKs. PrkD was found to be similar to the eukaryotic dual specificity Tyr phosphorylation-regulated kinase class of dual specificity kinases, which autophosphorylates on Ser, Thr, and Tyr residues and phosphorylates Ser and Thr residues on substrates. PrkG was found to be a bona fide dual specificity protein kinase that mediates autophosphorylation and substrate phosphorylation on Ser, Thr, and Tyr residues. The sites of phosphorylation in both of the kinases were identified through mass spectrometry. Phosphorylation on Tyr residues regulates the kinase activity of PrkD and PrkG. PrpC, the only known Ser/Thr protein phosphatase, was also found to possess dual specificity. Genistein, a known Tyr kinase inhibitor, was found to inhibit the activities of PrkD and PrkG and affect the growth of B. anthracis cells, indicating a possible role of these kinases in cell growth and development. In addition, the glycolytic enzyme pyruvate kinase was found to be phosphorylated by PrkD on Ser and Thr residues but not by PrkG. Thus, this study provides the first evidence of DSPKs in B. anthracis that belong to different classes and have different modes of regulation. PMID:22711536

  6. Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

    PubMed

    Nishikawa, K; Toker, A; Wong, K; Marignani, P A; Johannes, F J; Cantley, L C

    1998-09-04

    Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

  7. Crystal Structures of Two Aminoglycoside Kinases Bound with a Eukaryotic Protein Kinase Inhibitor

    PubMed Central

    Hwang, Jiyoung; Berghuis, Albert M.

    2011-01-01

    Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3′)-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3′)-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eurkaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides. PMID:21573013

  8. Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487

    PubMed Central

    Heathcote, Helen R.; Mancini, Sarah J.; Strembitska, Anastasiya; Jamal, Kunzah; Reihill, James A.; Palmer, Timothy M.; Gould, Gwyn W.; Salt, Ian P.

    2016-01-01

    The key metabolic regulator, AMP-activated protein kinase (AMPK), is reported to be down-regulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491, respectively, as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC-stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues. PMID:27784766

  9. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    USDA-ARS?s Scientific Manuscript database

    Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

  10. Dual activators of Protein Kinase R (PKR) and Protein Kinase R Like Kinase (PERK) Identify Common and Divergent Catalytic Targets

    PubMed Central

    Ming, Jie; Sun, Hong; Cao, Peng; Fusco, Dahlene N.; Chung, Raymond T.; Chorev, Michael; Jin, Qi; Aktas, Bertal H.

    2013-01-01

    Chemical genetics has evolved into a powerful tool for studying gene function in normal- and patho-biology. PKR and PERK, two eukaryotic translation initiation factor 2 alpha (eIF2α) kinases, play critical roles in maintenance of cellular hemostasis, metabolic stability, and anti-viral defenses. Both kinases interact with and phosphorylate additional substrates including tumor suppressor p53 and nuclear protein 90. Loss of function of both kinases has been studied by reverse genetics and recently identified inhibitors. In contrast, activating probes for studying the role of catalytic activity of these kinases are not available. We identified a 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5,7-dihydroxy-4H-chromen-4-one (DHBDC) as specific dual activator of PKR and PERK by screening a chemical library of 20,000 small molecules in a dual luciferase surrogate eIF2α phosphorylation assay. We present here extensive biological characterization and preliminary structure-activity relationship of DHBDC, which phosphorylate eIF2α by activating PKR and PERK but no other eIF2α kinases. These agents also activate downstream effectors of eIF2α phosphorylation; inducing CHOP and suppressing cyclin D1 expression and inhibiting cancer cell proliferation, all in a manner dependent on PKR and PERK. Consistent with the role of eIF2α phosphorylation in viral infection, DHBDC inhibits proliferation of human hepatitis C virus. Finally, DHBDC induces phosphorylation of Ikβα, and activates NF-κB pathway. Surprisingly, activation of NF-κB pathway is dependent on PERK but independent of PKR activity. These data indicate that DHBDC is an invaluable probe for elucidating the role of PKR and PERK in normal- and patho-biology. PMID:23784735

  11. Activation of cGMP-dependent protein kinase by protein kinase C.

    PubMed

    Hou, Yali; Lascola, Judith; Dulin, Nickolai O; Ye, Richard D; Browning, Darren D

    2003-05-09

    The cGMP-dependent protein kinases (PKG) are emerging as important components of mainstream signal transduction pathways. Nitric oxide-induced cGMP formation by stimulation of soluble guanylate cyclase is generally accepted as being the most widespread mechanism underlying PKG activation. In the present study, PKG was found to be a target for phorbol 12-myristate 13-acetate (PMA)-responsive protein kinase C (PKC). PKG1alpha became phosphorylated in HEK-293 cells stimulated with PMA and also in vitro using purified components. PKC-dependent phosphorylation was found to activate PKG as measured by phosphorylation of vasodilator-stimulated phosphoprotein, and by in vitro kinase assays. Although there are 11 potential PKC substrate recognition sites in PKG1alpha, threonine 58 was examined due to its proximity to the pseudosubstrate domain. Antibodies generated against the phosphorylated form of this region were used to demonstrate phosphorylation in response to PMA treatment of the cells with kinetics similar to vasodilator-stimulated phosphoprotein phosphorylation. A phospho-mimetic mutation at this site (T58E) generated a partially activated PKG that was more sensitive to cGMP levels. A phospho-null mutation (T58A) revealed that this residue is important but not sufficient for PKG activation by PKC. Taken together, these findings outline a novel signal transduction pathway that links PKC stimulation with cyclic nucleotide-independent activation of PKG.

  12. Regulation of tomato Prf by Pto-like protein kinases.

    PubMed

    Mucyn, Tatiana S; Wu, Ai-Jiuan; Balmuth, Alexi L; Arasteh, Julia Maryam; Rathjen, John P

    2009-04-01

    Tomato Prf encodes a nucleotide-binding domain shared by Apaf-1, certain R proteins, and CED-4 fused to C-terminal leucine-rich repeats (NBARC-LRR) protein that is required for bacterial immunity to Pseudomonas syringae and sensitivity to the organophosphate fenthion. The signaling pathways involve two highly related protein kinases. Pto kinase mediates direct recognition of the bacterial effector proteins AvrPto or AvrPtoB. Fen kinase is required for fenthion sensitivity and recognition of bacterial effectors related to AvrPtoB. The role of Pto and its association with Prf has been characterized but Fen is poorly described. We show that, similar to Pto, Fen requires N-myristoylation and kinase activity for signaling and interacts with the N-terminal domain of Prf. Thus, the mechanisms of activation of Prf by the respective protein kinases are similar. Prf-Fen interaction is underlined by coregulatory mechanisms in which Prf negatively regulates Fen, most likely by controlling kinase activity. We further characterized negative regulation of Prf by Pto, and show that regulation is mediated by the previously described negative regulatory patch. Remarkably, the effectors released negative regulation of Prf in a manner dependent on Pto kinase activity. The data suggest a model in which Prf associates generally with Pto-like kinases in tightly regulated complexes, which are activated by effector-mediated disruption of negative regulation. Release of negative regulation may be a general feature of activation of NBARC-LRR proteins by cognate effectors.

  13. Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK.

    PubMed Central

    Lefebvre, D L; Bai, Y; Shahmolky, N; Sharma, M; Poon, R; Drucker, D J; Rosen, C F

    2001-01-01

    Subtraction hybridization after the exposure of keratinocytes to ultraviolet radiation identified a differentially expressed cDNA that encodes a protein of 630 amino acid residues possessing significant similarity to the catalytic domain of the sucrose-non-fermenting protein kinase (SNF1)/AMP-activated protein kinase (AMPK) family of serine/threonine protein kinases. Northern blotting and reverse-transcriptase-mediated PCR demonstrated that mRNA transcripts for the SNF1/AMPK-related kinase (SNARK) were widely expressed in rodent tissues. The SNARK gene was localized to human chromosome 1q32 by fluorescent in situ hybridization. SNARK was translated in vitro to yield a single protein band of approx. 76 kDa; Western analysis of transfected baby hamster kidney (BHK) cells detected two SNARK-immunoreactive bands of approx. 76-80 kDa. SNARK was capable of autophosphorylation in vitro; immunoprecipitated SNARK exhibited phosphotransferase activity with the synthetic peptide substrate HMRSAMSGLHLVKRR (SAMS) as a kinase substrate. SNARK activity was significantly increased by AMP and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) in rat keratinocyte cells, implying that SNARK might be activated by an AMPK kinase-dependent pathway. Furthermore, glucose deprivation increased SNARK activity 3-fold in BHK fibroblasts. These findings identify SNARK as a glucose- and AICAriboside-regulated member of the AMPK-related gene family that represents a new candidate mediator of the cellular response to metabolic stress. PMID:11284715

  14. VZV ORF47 serine protein kinase and its viral substrates.

    PubMed

    Kenyon, Teri K; Grose, Charles

    2010-01-01

    ORF47, a serine protein kinase of varicella-zoster virus (VZV) and homolog of herpes simplex virus UL13, is an interesting modulator of VZV pathogenesis. This chapter summarizes research showing that ORF47 protein kinase activity, by virtue of phosphorylation of or binding to various viral substrates, regulates VZV proteins during all phases of viral infection and has a pronounced effect on the trafficking of gE, the predominant VZV glycoprotein, which in turn is critical for cell-to-cell spread of the virus. Casein kinase II, an ubiquitous cellular protein kinase, recognizes a similar but less stringent phosphorylation consensus sequence and can partially compensate for lack of ORF47 activity in VZV-infected cells. Differences between the phosphorylation consensus sites of the viral and cellular kinases are outlined in detail.

  15. Binding of exogenous brain protein kinase C to liver nuclei

    SciTech Connect

    Misra, U.K.; Wolf, M.; Besterman, J.; Cuatrecasas, P.; Sahyoun, N.

    1986-05-01

    Protein kinase C is found both in the cytosol and bound to membranes. Binding of the enzyme to plasma membranes is controlled by calcium whereas enzyme activators regulate both its membrane binding and enzyme catalysis. Activation of protein kinase C has been implicated in several regulatory processes including gene expression. Accordingly, the possibility of direct interaction of protein kinase C with the nucleus was examined utilizing /sup 3/H-PDBu binding to detect the enzyme. Purified protein kinase C from rat brain could bind to purified rat liver nuclei at 4/sup 0/C or at 21/sup 0/C, and the reaction was completed by 20 min. The binding was linearly dependent on protein kinase C concentration and required free Ca/sup 2 +/ with a K/sub m/sub app// of 0.5 ..mu..M. Chelation of Ca/sup 2 +/ with EGTA resulted in a rapid dissociation of protein kinase C from the nuclei. Differential extraction experiments suggested that about 50% of the enzyme was bound to chromatin and 25% was associated with the nuclear matrix. Moreover, protein kinase C bound to nuclei was able to phosphorylate several endogenous nuclear substrates, including chromatin proteins, in a Ca/sup 2 +/ phosphatidyl serine dependent reaction.

  16. Developing irreversible inhibitors of the protein kinase cysteinome

    PubMed Central

    Liu, Qingsong; Sabnis, Yogesh; Zhao, Zheng; Zhang, Tinghu; Buhrlage, Sara J.; Jones, Lyn H.; Gray, Nathanael S.

    2013-01-01

    Protein kinases are a large family of approximately 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues such as tyrosine, serine and threonine which serves as a ubiquitous mechanism for cellular signal transduction. The clinical success of a number of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large number of kinases may represent therapeutically relevant targets. To-date the majority of clinical and preclinical kinase inhibitors are ATP-competitive, non-covalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a number of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency and ability to validate pharmacological specificity through mutation of the reactive cysteine residue. Here we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biological activity profiles. In addition, we provided an informatics assessment of the potential ‘kinase-cysteinome’ and discuss strategies for the efficient development of new covalent inhibitors. PMID:23438744

  17. Signal-transduction pathways that regulate visceral smooth muscle function. III. Coupling of muscarinic receptors to signaling kinases and effector proteins in gastrointestinal smooth muscles.

    PubMed

    Gerthoffer, William T

    2005-05-01

    Stimulation of muscarinic M3 and M2 receptors on gastrointestinal smooth muscle elicits contraction via activation of G proteins that are coupled to a diverse set of downstream signaling pathways and effector proteins. Many studies suggest a canonical excitation-contraction coupling pathway that includes activation of phospholipases, production of inositol 1,4,5-trisphosphate and diacylglycerol, release of calcium from the sarcoplasmic reticulum, activation of L-type calcium channels, and activation of nonselective cation channels. These events lead to elevated intracellular calcium concentration, which activates myosin light chain kinase to phosphorylate and activate myosin II thus causing contraction. In addition, muscarinic receptors are coupled to signaling pathways that modulate the effect of activator calcium. The Rho/Rho kinase pathway inhibits myosin light chain phosphatase, one of the key steps in sensitization of the contractile proteins to calcium. Phosphatidylinositol 3-kinases and Src family tyrosine kinases are also activated by muscarinic agonists. Src family tyrosine kinases regulate L-type calcium and nonselective cation channels. Src activation also leads to activation of ERK and p38 MAPKs. ERK MAPKs phosphorylate caldesmon, an actin filament binding protein. P38 MAPKs activate phospholipases and MAPKAP kinase 2/3, which phosphorylate HSP27. HSP27 may regulate cross-bridge function, actin filament formation, and actin filament attachment to the cell membrane. In addition to the well-known role of M3 muscarinic receptors to regulate myoplasmic calcium levels, the integrated effect of muscarinic activation probably also includes signaling pathways that modulate phospholipases, cyclic nucleotides, contractile protein function, and cytoskeletal protein function.

  18. Resolution of thylakoid polyphenol oxidase and a protein kinase

    SciTech Connect

    Race, H.L.; Davenport, J.W.; Hind, G.

    1995-12-31

    The predominant protein kinase activity in octylglucoside (OG) extracts of spinach thylakoids has been attributed to a 64-kDa protein, tp64. Recent work calls into question the relation between tp64 and protein kinase activity, which were fractionated apart using fluid phase IEF and hydroxylapatite chromatography. Hind et al. sequenced tp64 from the cDNA and showed it to be a polyphenol oxidase (PPO) homolog. Its transit peptide indicates a location for the mature protein within the thylakoid lumen, where there is presumably no ATP and where it is remote from the presumed kinase substrates: the stromally exposed regions of integral PS-II membrane proteins. Here the authors suggest that the kinase is a 64-kDa protein distinct from tp64.

  19. Protein kinase C as a tumor suppressor.

    PubMed

    Newton, Alexandra C

    2017-05-02

    Protein kinase C (PKC) has historically been considered an oncoprotein. This stems in large part from the discovery in the early 1980s that PKC is directly activated by tumor-promoting phorbol esters. Yet three decades of clinical trials using PKC inhibitors in cancer therapies not only failed, but in some cases worsened patient outcome. Why has targeting PKC in cancer eluded successful therapies? Recent studies looking at the disease for insight provide an explanation: cancer-associated mutations in PKC are generally loss-of-function (LOF), supporting an unexpected function as tumor suppressors. And, contrasting with LOF mutations in cancer, germline mutations that enhance the activity of some PKC isozymes are associated with degenerative diseases such as Alzheimer's disease. This review provides a background on the diverse mechanisms that ensure PKC is only active when, where, and for the appropriate duration needed and summarizes recent findings converging on a paradigm reversal: PKC family members generally function by suppressing, rather than promoting, survival signaling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. The specificities of protein kinase inhibitors: an update.

    PubMed Central

    Bain, Jenny; McLauchlan, Hilary; Elliott, Matthew; Cohen, Philip

    2003-01-01

    We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent protein kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 protein kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated kinase (PRAK; IC(50)=1.0 microM). PMID:12534346

  1. Auto-phosphorylation Represses Protein Kinase R Activity.

    PubMed

    Wang, Die; de Weerd, Nicole A; Willard, Belinda; Polekhina, Galina; Williams, Bryan R G; Sadler, Anthony J

    2017-03-10

    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity.

  2. Auto-phosphorylation Represses Protein Kinase R Activity

    PubMed Central

    Wang, Die; de Weerd, Nicole A.; Willard, Belinda; Polekhina, Galina; Williams, Bryan R. G.; Sadler, Anthony J.

    2017-01-01

    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity. PMID:28281686

  3. Protein kinase C-associated kinase can activate NFkappaB in both a kinase-dependent and a kinase-independent manner.

    PubMed

    Moran, Stewart T; Haider, Khaleda; Ow, Yongkai; Milton, Peter; Chen, Luojing; Pillai, Shiv

    2003-06-13

    Protein kinase C-associated kinase (PKK, also known as RIP4/DIK) activates NFkappaB when overexpressed in cell lines and is required for keratinocyte differentiation in vivo. However, very little is understood about the factors upstream of PKK or how PKK activates NFkappaB. Here we show that certain catalytically inactive mutants of PKK can activate NFkappaB, although to a lesser degree than wild type PKK. The deletion of specific domains of wild type PKK diminishes the ability of this enzyme to activate NFkappaB; the same deletions made on a catalytically inactive PKK background completely ablate NFkappaB activation. PKK may be phosphorylated by two specific mitogen-activated protein kinase kinase kinases, MEKK2 and MEKK3, and this interaction may in part be mediated through a critical activation loop residue, Thr184. Catalytically inactive PKK mutants that block phorbol ester-induced NFkappaB activation do not interfere with, but unexpectedly enhance, the activation of NFkappaB by these two mitogen-activated protein kinase kinase kinases. Taken together, these data indicate that PKK may function in both a kinase-dependent as well as a kinase-independent manner to activate NFkappaB.

  4. Activation of fat cell adenylate cyclase by protein kinase C

    SciTech Connect

    Naghshineh, S.; Noguchi, M.; Huang, K.P.; Londos, C.

    1986-05-01

    Purified protein kinase C (C-kinase) from guinea pig pancreas and rat brain stimulated adenylate cyclase activity in purified rat adipocyte membranes. Cyclase stimulation occurred over 100 to 1000 mU/ml of C-kinase activity, required greater than 10 ..mu..M calcium, proceeded without a lag, was not readily reversible, and required no exogenous phospholipid. Moreover, C-kinase inhibitors, such as chlorpromazine and palmitoyl carnitine, inhibited selectively adenylate cyclase which was activated by C-kinase and calcium. Depending on assay conditions, 10 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) either enhanced or was required for kinase action on cyclase. Also, TPA plus calcium promoted the quantitative association of C-kinase with membranes. Adenylate cyclase activation by C-kinase was seen both in the presence and absence of exogenous GTP, indicating that the kinase effect does not result from an action on the GTP-binding, inhibitory regulatory component (N/sub i/) of the cyclase system. Moreover, the kinase effect was seen in the presence of non-phosphorylating ATP analogs, such as AppNHp and AppCH/sub 2/p, suggesting that the effects of C-kinase described herein may result from association with, rather than phosphorylation of, adenylate cyclase.

  5. The Link between Protein Kinase CK2 and Atypical Kinase Rio1

    PubMed Central

    Kubiński, Konrad; Masłyk, Maciej

    2017-01-01

    The atypical kinase Rio1 is widespread in many organisms, ranging from Archaebacteria to humans, and is an essential factor in ribosome biogenesis. Little is known about the protein substrates of the enzyme and small-molecule inhibitors of the kinase. Protein kinase CK2 was the first interaction partner of Rio1, identified in yeast cells. The enzyme from various sources undergoes CK2-mediated phosphorylation at several sites and this modification regulates the activity of Rio1. The aim of this review is to present studies of the relationship between the two different kinases, with respect to CK2-mediated phosphorylation of Rio1, regulation of Rio1 activity, and similar susceptibility of the kinases to benzimidazole inhibitors. PMID:28178206

  6. [The role of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis].

    PubMed

    Nerusheva, O O; Dorogova, N V; Gubanova, N V; Omel'ianchuk, L V

    2008-09-01

    The cellular function of the gilgamesh mutation (89B9-12) of casein kinase gene in Drosophila spermatogenesis was studied. It was demonstrated that the sterility resulting from this mutation is connected with the abnormalities in spermatid individualization. A phylogenetic study of the protein sequences of casein kinases 1 from various organisms was conducted. The Gilgamesh protein was shown to be phylogenetically closer to the cytoplasmic casein kinase family, represented by the YCK3, YCK2, and YCK1 proteins of Saccharomyces cerevisiae and animal gamma-casein kinases. It is known that these yeast casein kinases are involved in vesicular trafficking, which, in turn, is related in its genetic control to the cell membrane remodeling during spermatid individualization. Thus, the data of phylogenetic analysis fit well the results obtained by studying the mutation phenotype.

  7. A family of human cdc2-related protein kinases.

    PubMed Central

    Meyerson, M; Enders, G H; Wu, C L; Su, L K; Gorka, C; Nelson, C; Harlow, E; Tsai, L H

    1992-01-01

    The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins. Images PMID:1639063

  8. Autophosphorylating protein kinase activity in titin-like arthropod projectin.

    PubMed

    Maroto, M; Vinós, J; Marco, R; Cervera, M

    1992-03-20

    The function of the high molecular weight structural proteins from muscle, namely vertebrate titin, arthropod projectin and nematode twitchin, remains to be established. Using a simple method for the purification of projectin from crayfish and Drosophila melanogaster, a polyclonal antibody has been raised against crayfish projectin, and shown to immunocrossreact with Drosophila projectin but not with rat titin. In this study, evidence is presented that projectin and twitchin may share functional protein kinase domains, indicating a possible relationship between them. Projectin has a serine/threonine protein kinase activity. This supports the relationship with twitchin since, in sequence analysis of the latter, a protein-kinase-like domain has been found. Moreover, projectin is capable of autophosphorylation in vitro. These kinase activities imply regulatory functions for this group of proteins, extending its previously assumed structural role in the sarcomere. We also show here that projectin is phosphorylated in vivo at serine residues, as described for titin.

  9. The protein activator of protein kinase R, PACT/RAX, negatively regulates protein kinase R during mouse anterior pituitary development.

    PubMed

    Dickerman, Benjamin K; White, Christine L; Kessler, Patricia M; Sadler, Anthony J; Williams, Bryan R G; Sen, Ganes C

    2015-12-01

    The murine double-stranded RNA-binding protein termed protein kinase R (PKR)-associated protein X (RAX) and the human homolog, protein activator of PKR (PACT), were originally characterized as activators of PKR. Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR-deficient mice, the phenotype has been attributed to PKR-independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax(-/-) mice deficient in PKR, or carrying a kinase-inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eukaryotic translation initiation factor 2 α subunit (eIF2α) (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax(-/-) mice. Generating rax(-/-) mice with a kinase-inactive mutant of PKR resulted in similar rescue, confirming that the rax(-/-) defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax(-/-) mice that were heterozygous for an unphosphorylatable mutant eIF2α provides partial rescue of the rax(-/-) defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR-regulated cyclin-dependent kinase inhibitor p21(WAF1/CIP1). These results demonstrate that PKR kinase activity is required for onset of the rax(-/-) phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development.

  10. RAF protein-serine/threonine kinases: Structure and regulation

    SciTech Connect

    Roskoski, Robert

    2010-08-27

    Research highlights: {yields} The formation of unique side-to-side RAF dimers is required for full kinase activity. {yields} RAF kinase inhibitors block MEK activation in cells containing oncogenic B-RAF. {yields} RAF kinase inhibitors can lead to the paradoxical increase in RAF kinase activity. -- Abstract: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.

  11. Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

    NASA Technical Reports Server (NTRS)

    Li, H.; Roux, S. J.

    1992-01-01

    Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

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

    USDA-ARS?s Scientific Manuscript database

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

  13. Protein kinase C activators inhibit capillary endothelial cell growth

    SciTech Connect

    Doctrow, S.R.

    1986-05-01

    Phorbol 12,13-dibutyrate (PDBu) binds specifically to bovine capillary endothelial (BCE) cells (K/sub d/ = 8nM) and inhibits the proliferation (K/sub 50/ = 6 +/- 4 nM). Under similar conditions, PDBu does not inhibit the growth of bovine aortic endothelial or smooth muscle cells. PDBu markedly attenuates the response of BCE cells to purified human hepatoma-derived growth factor which, in the absence of PDBu, stimulates BCE cell growth by about 3-fold. Several observations suggest that the inhibition of BCE cell growth by PDBu is mediated by protein kinase C: (1) different phorbol compounds inhibit BCE cell growth according to the relative potencies as protein kinase C activators (12-tetradecanoylphorbol 13-acetate > PDBu >> phorbol 12,13-diacetate >>>..beta..-phorbol; ..cap alpha..-phorbol 12,13-didecanoate). (2) Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol (diC/sub 8/), a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. (3) A cytosolic extract from BCE cells contains a Ca/sup 2 +//phosphatidylserine-dependent kinase that is activated by diC/sub 8/ and PDBu, but not by ..beta..-phorbol. These results support a role for protein kinase C in suppressing capillary endothelial cell growth and may therefore have implications in the intracellular regulation of angiogenesis.

  14. Interaction of SNF1 Protein Kinase with Its Activating Kinase Sak1▿

    PubMed Central

    Liu, Yang; Xu, Xinjing; Carlson, Marian

    2011-01-01

    The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change. PMID:21216941

  15. How protein kinases co-ordinate mitosis in animal cells.

    PubMed

    Ma, Hoi Tang; Poon, Randy Y C

    2011-04-01

    Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule-kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.

  16. Regulatory Crosstalk by Protein Kinases on CFTR Trafficking and Activity

    PubMed Central

    Farinha, Carlos M.; Swiatecka-Urban, Agnieszka; Brautigan, David L.; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e., channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease. PMID:26835446

  17. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  18. Characterization of microtubule-associated protein 1-associated protein kinases from rat brain.

    PubMed

    Fujii, T; Watanabe, M; Nakamura, A

    1996-01-01

    The microtubule-associated protein (MAP) 1 preparation, MAP1A and 1B, obtained from rat brain microtubules was associated with protein kinases that were insensitive to cAMP, cGMP, calcium, calcium/calmodulin and calcium/phosphatidylserine. The fractionation of highly purified MAP1 by phosphocellulose chromatography revealed that protein kinase activity to phosphorylate phosvitin was separated into three major peaks (MAP1 kinases A, B and C). MAP1 was recovered in the MAP1 kinase A fraction and phosphorylated by the contained kinase. MAP1 kinase A is a novel protein kinase that is remarkably activated by poly-L-lysine and poly-L-arginine, but very insensitive to heparin among the kinases. Photoaffinity labeling using [alpha-32P]8-azido ATP indicated that the 65 kDa polypeptide is identified as an ATP-binding protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the highly purified MAP1 and MAP1 kinase A fractions. MAP1 kinases B and C may be identified as casein kinase I- and II-like kinases. The present results show that MAP1 is associated with at least three kinases and provide an insight for understanding thoroughly the MAP1-mediated microtubule functions.

  19. Regulated protein kinases and phosphatases in cell cycle decisions.

    PubMed

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2010-12-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle.

  20. Regulated protein kinases and phosphatases in cell cycle decisions

    PubMed Central

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2013-01-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle. PMID:20678910

  1. Fluorescent sensors of protein kinases: from basics to biomedical applications.

    PubMed

    Nhu Ngoc Van, Thi; Morris, May C

    2013-01-01

    Protein kinases constitute a major class of enzymes underlying essentially all biological processes. These enzymes present similar structural folds, yet their mechanism of action and of regulation vary largely, as well as their substrate specificity and their subcellular localization. Classical approaches to study the function/activity of protein kinases rely on radioactive endpoint assays, which do not allow for characterization of their dynamic activity in their native environment. The development of fluorescent biosensors has provided a whole new avenue for studying protein kinase behavior and regulation in living cells in real time with high spatial and temporal resolution. Two major classes of biosensors have been developed: genetically encoded single-chain fluorescence resonance energy transfer biosensors and peptide/protein biosensors coupled to small synthetic fluorophores which are sensitive to changes in their environment. In this review, we discuss the developments in fluorescent biosensor technology related to protein kinase sensing and the different strategies employed to monitor protein kinase activity, conformation, or relative abundance, as well as kinase regulation and subcellular dynamics in living cells. Moreover, we discuss their application in biomedical settings, for diagnostics and therapeutics, to image disease progression and monitor response to therapeutics, in drug discovery programs, for high-throughput screening assays, for postscreen characterization of drug candidates, and for clinical evaluation of novel drugs. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Molecular cloning of plant transcripts encoding protein kinase homologs.

    PubMed Central

    Lawton, M A; Yamamoto, R T; Hanks, S K; Lamb, C J

    1989-01-01

    Oligonucleotides, corresponding to conserved regions of animal protein-serine/threonine kinases, were used to isolate cDNAs encoding plant homologs in the dicot bean (Phaseolus vulgaris L.) and the monocot rice (Oryzae sativa L.). The C-terminal regions of the deduced polypeptides encoded by the bean (PVPK-1) and rice (G11A) cDNAs, prepared from mRNAs of suspension cultures and leaves, respectively, contain features characteristic of the catalytic domains of eukaryotic protein-serine/threonine kinases, indicating that these cDNAs encode plant protein kinases. The putative catalytic domains are most closely related to cyclic nucleotide-dependent protein kinases and the protein kinase C family, suggesting the plant homologs may likewise transduce extracellular signals. However, outside these domains, PVPK-1 and G11A exhibit no homology either to each other or to regulatory domains of other protein kinases, indicating the plant homologs are modulated by other signals. PVPK-1 corresponds to a 2.4-kb transcript in suspension cultured bean cells. Southern blots of genomic DNA indicate that PVPK-1 and G11A correspond to single copy genes that form part of a family of related plant sequences. Images PMID:2541432

  3. The protein activator of protein kinase R, PACT/RAX, negatively regulates protein kinase R during mouse anterior pituitary development

    PubMed Central

    Dickerman, Benjamin K.; White, Christine L.; Kessler, Patricia M.; Sadler, Anthony J.; Williams, Bryan R.G.; Sen, Ganes C.

    2015-01-01

    The murine double-stranded RNA-binding protein RAX and the human homolog PACT were originally characterized as activators of protein kinase R (PKR). Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR-deficient mice, the phenotype has been attributed to PKR-independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax−/− mice deficient in PKR, or carrying a kinase-inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eIF2α (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax−/− mice. Generating rax−/− mice with a kinase-inactive mutant of PKR resulted in similar rescue, confirming that the rax−/− defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax−/− mice that were heterozygous for an unphosphorylatable mutant eIF2α provides partial rescue of the rax−/− defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR-regulated cyclin-dependent kinase inhibitor p21WAF1/CIP1. These results demonstrate that PKR kinase activity is required for onset of the rax−/− phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development. PMID:26414443

  4. The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

    SciTech Connect

    Papazyan, Romeo; Rozengurt, Enrique; Rey, Osvaldo . E-mail: orey@mednet.ucla.edu

    2006-04-14

    We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.

  5. Protein Kinases and Parkinson’s Disease

    PubMed Central

    Mehdi, Syed Jafar; Rosas-Hernandez, Hector; Cuevas, Elvis; Lantz, Susan M.; Barger, Steven W.; Sarkar, Sumit; Paule, Merle G.; Ali, Syed F.; Imam, Syed Z.

    2016-01-01

    Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson’s disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved and are used clinically for other indications. Kinase inhibitors represent a family of popular molecules for the treatment and prevention of various cancers, and have emerged as strong candidates for such repurposing because numerous serine/threonine and tyrosine kinases have been implicated in the pathobiology of Parkinson’s disease. This review focuses on various kinase-dependent pathways associated with the expression of Parkinson’s disease pathology, and evaluates how inhibitors of these pathways might play a major role as effective therapeutic molecules. PMID:27657053

  6. Targeting protein kinases in central nervous system disorders

    PubMed Central

    Chico, Laura K.; Van Eldik, Linda J.; Watterson, D. Martin

    2010-01-01

    Protein kinases are a growing drug target class in disorders in peripheral tissues, but the development of kinase-targeted therapies for central nervous system (CNS) diseases remains a challenge, largely owing to issues associated specifically with CNS drug discovery. However, several candidate therapeutics that target CNS protein kinases are now in various stages of preclinical and clinical development. We review candidate compounds and discuss selected CNS protein kinases that are emerging as important therapeutic targets. In addition, we analyse trends in small-molecule properties that correlate with key challenges in CNS drug discovery, such as blood–brain barrier penetrance and cytochrome P450-mediated metabolism, and discuss the potential of future approaches that will integrate molecular-fragment expansion with pharmacoinformatics to address these challenges. PMID:19876042

  7. Cadmium activates a mitogen-activated protein kinase gene and MBP kinases in rice.

    PubMed

    Yeh, Chuan-Ming; Hsiao, Lin-June; Huang, Hao-Jen

    2004-09-01

    Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, it has been evidenced that MAPKs play a role in the signaling of biotic and abiotic stresses, plant hormones, and cell cycle cues. However, the effect of heavy metals on plant MAPKs has not been well examined. The Northern blot analysis of OsMAPK mRNA levels has shown that only OsMAPK2, but not OsMAPK3 and OsMAPK4, expressed in suspension-cultured cells in response to 100-400 microM Cd treatments. The OsMAPK2 transcripts increased within 12 h upon 400 microM Cd treatment. In addition, we found that 42- and 50-kDa MBP kinases were significantly activated by Cd treatment in rice suspension-cultured cells. And 40-, 42-, 50- and 64-kDa MBP kinases were activated in rice roots. Furthermore, GSH inhibits Cd-induced 40-kDa MBP kinase activation. By immunoblot analysis and immunoprecipitation followed by in-gel kinase assay, we confirmed that Cd-activated 42-kDa MBP kinase is a MAP kinase. Our results suggest that a MAP kinase cascade may function in the Cd-signalling pathway in rice.

  8. Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways*

    PubMed Central

    Dickinson, Robin J.; Delavaine, Laurent; Cejudo-Marín, Rocío; Stewart, Graeme; Staples, Christopher J.; Didmon, Mark P.; Trinidad, Antonio Garcia; Alonso, Andrés; Pulido, Rafael; Keyse, Stephen M.

    2011-01-01

    MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site 55RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo. PMID:21908610

  9. Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa.

    PubMed

    Talevich, Eric; Mirza, Amar; Kannan, Natarajan

    2011-11-02

    The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK), calcium-dependent protein kinase (CDPK) and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the αC helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and evolutionary implications of these lineage-specific variations and

  10. Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa

    PubMed Central

    2011-01-01

    Background The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. Results We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK), calcium-dependent protein kinase (CDPK) and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the αC helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. Conclusions We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and evolutionary implications of these

  11. G protein-coupled receptor kinases: more than just kinases and not only for GPCRs

    PubMed Central

    Gurevich, Eugenia V.; Tesmer, John J. G.; Mushegian, Arcady; Gurevich, Vsevolod V.

    2011-01-01

    G protein-coupled receptor (GPCR) kinases (GRKs) are best known for their role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors and promote high affinity binding of arrestins, which precludes G protein coupling. GRKs have a multidomain structure, with the kinase domain inserted into a loop of a regulator of G protein signaling homology domain. Unlike many other kinases, GRKs do not need to be phosphorylated in their activation loop to achieve an activated state. Instead, they are directly activated by docking with active GPCRs. In this manner they are able to selectively phosphorylate Ser/Thr residues on only the activated form of the receptor, unlike related kinases such as protein kinase A. GRKs also phosphorylate a variety of non-GPCR substrates and regulate several signaling pathways via direct interactions with other proteins in a phosphorylation-independent manner. Multiple GRK subtypes are present in virtually every animal cell, with the highest expression levels found in neurons, with their extensive and complex signal regulation. Insufficient or excessive GRK activity was implicated in a variety of human disorders, ranging from heart failure to depression to Parkinson’s disease. As key regulators of GPCR-dependent and -independent signaling pathways, GRKs are emerging drug targets and promising molecular tools for therapy. Targeted modulation of expression and/or of activity of several GRK isoforms for therapeutic purposes was recently validated in cardiac disorders and Parkinson’s disease. PMID:21903131

  12. A Novel Mode of Protein Kinase Inhibition Exploiting Hydrophobic Motifs of Autoinhibited Kinases

    PubMed Central

    Eathiraj, Sudharshan; Palma, Rocio; Hirschi, Marscha; Volckova, Erika; Nakuci, Enkeleda; Castro, Jennifer; Chen, Chang-Rung; Chan, Thomas C. K.; France, Dennis S.; Ashwell, Mark A.

    2011-01-01

    Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases. PMID:21454610

  13. Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon.

    PubMed Central

    Schaap, D; van der Wal, J; van Blitterswijk, W J; van der Bend, R L; Ploegh, H L

    1993-01-01

    In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor. Images Figure 2 Figure 3 Figure 4 PMID:7679574

  14. Coupling phosphoryl transfer and substrate interactions in protein kinases.

    PubMed

    Lieser, Scot A; Aubol, Brandon E; Wong, Lilly; Jennings, Patricia A; Adams, Joseph A

    2005-12-30

    Protein kinases control cell signaling events through the ATP-dependent phosphorylation of serine, threonine and tyrosine residues in protein targets. The recognition of these protein substrates by the kinases relies on two principal factors: proper subcellular co-localization and molecular interactions between the kinase and substrate. In this review, we will focus on the kinetic role of the latter in conveying favorable substrate recognition. Using rapid mixing technologies, we demonstrate that the intrinsic thermodynamic affinities of two protein substrates for their respective kinases (Csk with Src and Sky1p with Npl3) are weak compared to their apparent affinities measured in traditional steady-state kinetic assays (i.e.--Km < Kd). The source of the high apparent affinities rests in a very fast and highly favorable phosphoryl transfer step that serves as a clamp for substrate recognition. In this mechanism, both Csk and Sky1p utilize this step to draw the substrate toward product, thereby, converting a high Kd into a low Km. We propose that this one form of substrate recognition employed by protein kinases is advantageous since it simultaneously facilitates high apparent substrate affinity and fast protein turnover.

  15. Extracellular cAMP-dependent protein kinase (ECPKA) in melanoma.

    PubMed

    Kita, Tsunekazu; Goydos, James; Reitman, Elena; Ravatn, Roald; Lin, Yong; Shih, Wei-Chung; Kikuchi, Yoshihiro; Chin, Khew-Voon

    2004-05-28

    Melanoma is one of the fastest rising malignancies in the United States. When detected early, primary melanomas are curable through surgery. However, despite significant improvements in diagnosis and surgical, local and systemic therapy, mortality rate in metastatic melanoma remains high. Furthermore, genetic alterations associated with the development and stepwise progression of melanoma, are still unclear. Previous reports show that the catalytic kinase subunit of the cAMP-dependent protein kinase is secreted by tumor cells and can be detected in the serum of cancer patients. We examine in this report the clinical significance of this secreted C subunit kinase termed extracellular protein kinase (ECPKA) in melanoma patients. Our results showed the presence of ECPKA activity in the serum of melanoma patients and correlate with the appearance and size of the tumor. Most importantly, surgical removal of melanoma causes a precipitous decrease in ECPKA activity in the sera of patients, suggesting that ECPKA may be a novel predictive marker in melanoma.

  16. Protein kinase CK2 in development and differentiation

    PubMed Central

    Götz, Claudia; Montenarh, Mathias

    2017-01-01

    Among the human kinomes, protein kinase CK2 (formerly termed casein kinase II) is considered to be essential, as it is implicated in the regulation of various cellular processes. Experiments with pharmacological inhibitors of the kinase activity of CK2 provide evidence that CK2 is essential for development and differentiation. Therefore, the present review addresses the role of CK2 during embryogenesis, neuronal, adipogenic, osteogenic and myogenic differentiation in established model cell lines, and in embryonic, neural and mesenchymal stem cells. CK2 kinase activity appears to be essential in the early stages of differentiation, as CK2 inhibition at early time points generally prevents differentiation. In addition, the present review reports on target proteins of CK2 in embryogenesis and differentiation. PMID:28357063

  17. Allosteric activation of apicomplexan calcium-dependent protein kinases

    SciTech Connect

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; Mandelbaum, Joseph; Ramek, Alexander; Shan, Yibing; Shaw, David E.; Schwartz, Thomas U.; Ploegh, Hidde L.; Lourido, Sebastian

    2015-08-24

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.

  18. Allosteric activation of apicomplexan calcium-dependent protein kinases

    DOE PAGES

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; ...

    2015-08-24

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics,more » the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.« less

  19. Allosteric activation of apicomplexan calcium-dependent protein kinases

    PubMed Central

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; Mandelbaum, Joseph; Ramek, Alexander; Shan, Yibing; Shaw, David E.; Schwartz, Thomas U.; Ploegh, Hidde L.; Lourido, Sebastian

    2015-01-01

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes. PMID:26305940

  20. Homology modeling of yeast cyclin-dependent protein kinase

    NASA Astrophysics Data System (ADS)

    Selwyne, R. A.; Kholmurodov, Kh. T.; Koltovaya, N. A.

    2007-07-01

    The important functions that CDKs perform in cell division and cell cycle regulation made central protein kinase of Saccharomyces cerevisiae CDC28 a target model for structural and functional analysis. The 3D models of CDC28 protein kinase using molecular modeling techniques will enlarge our understanding of the phosphorylation mechanism and the structural changes of mutant kinases. The structural template for S. cerevisiae CDC28 was identified from PDB (Protein Databank) using BLASTP (basic local alignment search tool for proteins). Template-target alignments were generated for homology modeling and checked manually for errors. The models were then generated using MODELLER and validated using PROCHECK followed by energy minimization and molecular dynamics calculations in AMBER force field.

  1. Light-assisted small molecule screening against protein kinases

    PubMed Central

    Inglés-Prieto, Álvaro; Reichhart, Eva; Muellner, Markus K.; Nowak, Matthias; Nijman, Sebastian M.; Grusch, Michael; Janovjak, Harald

    2015-01-01

    High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that obviates the addition of chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small molecule screen against human protein kinases including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes. PMID:26457372

  2. Evaluation of the enzyme activity of protozoan protein kinases by using an in vitro kinase assay.

    PubMed

    Kato, Kentaro

    2016-10-01

    The life cycles of parasites are more complicated than those of other biological species. Protein kinases (PKs) encoded by parasites are the main triggers of life stage conversions. Phosphorylation by cellular PKs regulates important cellular processes, and the protozoan genome contains many PKs. Some PK inhibitors inhibit specific parasite life cycle event. In this report, I present a practical approach to expressing and purifying protozoan PKs by using a wheat germ cell-free protein synthesis system and I assess the phosphorylation activities of protozoan PKs by using an in vitro kinase assay. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors

    PubMed Central

    Ojo, Kayode K; Larson, Eric T; Keyloun, Katelyn R; Castaneda, Lisa J; DeRocher, Amy E; Inampudi, Krishna K; Kim, Jessica E; Arakaki, Tracy L; Murphy, Ryan C; Zhang, Li; Napuli, Alberto J; Maly, Dustin J; Verlinde, Christophe LMJ; Buckner, Frederick S; Parsons, Marilyn; Hol, Wim GJ; Merritt, Ethan A; Van Voorhis, Wesley C

    2010-01-01

    New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds designed to be inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site at residue 128. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii cells expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to this class of selective kinase inhibitors. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable. PMID:20436472

  4. Protein Kinases of the Hippo Pathway: Regulation and Substrates

    PubMed Central

    Avruch, Joseph; Zhou, Dawang; Fitamant, Julien; Bardeesy, Nabeel; Mou, Fan; Barrufet, Laura Regué

    2012-01-01

    The “Hippo” signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in S. cerevisiae e.g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologues Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin e.g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP

  5. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

    PubMed Central

    Frost, J A; Xu, S; Hutchison, M R; Marcus, S; Cobb, M H

    1996-01-01

    The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway. PMID:8668187

  6. Protein-protein interactions of tandem affinity purified protein kinases from rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2009-08-19

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.

  7. Protein-Protein Interactions of Tandem Affinity Purified Protein Kinases from Rice

    PubMed Central

    Rohila, Jai S.; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L.; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E.

    2009-01-01

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex. PMID:19690613

  8. Pea DNA Topoisomerase I Is Phosphorylated and Stimulated by Casein Kinase 2 and Protein Kinase C

    PubMed Central

    Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

    2003-01-01

    DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg2+-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants. PMID:12913165

  9. Regulation of protein kinase C by the cytoskeletal protein calponin.

    PubMed

    Leinweber, B; Parissenti, A M; Gallant, C; Gangopadhyay, S S; Kirwan-Rhude, A; Leavis, P C; Morgan, K G

    2000-12-22

    Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase Cepsilon (PKCepsilon) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKCepsilon and PKCalpha with possible involvement of C1B. The C2 region of PKCepsilon binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKCbetaII, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKCalpha, -epsilon, and -betaII severalfold compared with control conditions. Similarly, calponin was found to increase the amount of (32)P-labeled phosphate incorporated into PKC from [gamma-(32)P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.

  10. The molecular basis of targeting protein kinases in cancer therapeutics.

    PubMed

    Tsai, Chung-Jung; Nussinov, Ruth

    2013-08-01

    In this paper, we provide an overview of targeted anticancer therapies with small molecule kinase inhibitors. First, we discuss why a single constitutively active kinase emanating from a variety of aberrant genetic alterations is capable of transforming a normal cell, leading it to acquire the hallmarks of a cancer cell. To draw attention to the fact that kinase inhibition in targeted cancer therapeutics differs from conventional cytotoxic chemotherapy, we exploit a conceptual framework explaining why suppressed kinase activity will selectively kill only the so-called oncogene 'addicted' cancer cell, while sparing the healthy cell. Second, we introduce the protein kinase superfamily in light of its common active conformation with precisely positioned structural elements, and the diversified auto-inhibitory conformations among the kinase families. Understanding the detailed activation mechanism of individual kinases is essential to relate the observed oncogenic alterations to the elevated constitutively active state, to identify the mechanism of consequent drug resistance, and to guide the development of the next-generation inhibitors. To clarify the vital importance of structural guidelines in studies of oncogenesis, we explain how somatic mutations in EGFR result in kinase constitutive activation. Third, in addition to the common theme of secondary (acquired) mutations that prevent drug binding from blocking a signaling pathway which is hijacked by the aberrant activated kinase, we discuss scenarios of drug resistance and relapse by compensating lesions that bypass the inactivated pathway in a vertical or horizontal fashion. Collectively, these suggest that the future challenge of cancer therapy with small molecule kinase inhibitors will rely on the discovery of distinct combinations of optimized drugs to target individual subtypes of different cancers. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

    DOE PAGES

    Tan, Li; Nomanbhoy, Tyzoon; Gurbani, Deepak; ...

    2014-07-17

    Here, we developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16more » and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. Lastly, a 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.« less

  12. 5'-AMP-activated protein kinase signaling in Caenorhabditis elegans.

    PubMed

    Beale, Elmus G

    2008-01-01

    5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.

  13. Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

    PubMed Central

    Ulloa, R M; Mesri, E; Esteva, M; Torres, H N; Téllez-Iñón, M T

    1988-01-01

    A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band. Images Fig. 7. Fig. 8. PMID:2848508

  14. Purification and characterization of a thylakoid protein kinase

    SciTech Connect

    Coughlan, S.J.; Hind, G.

    1986-01-01

    Control of state transitions in the thylakoid by reversible phosphorylation of the light-harvesting chlorophyll a/b protein complex of photosystem II (LHC-II) is modulated by a kinase. The kinase catalyzing this phosphorylation is associated with the thylakoid membrane, and is regulated by the redox state of the plastoquinone pool. The isolation and partial purification from spinach thylakoids of two protein kinases (CPK1, CPK2) of apparent molecular masses 25 kDa and 38 kDa has been reported. Neither enzyme utilizes isolated LHC-II as a substrate. The partial purification of a third protein kinase (LHCK) which can utilize both lysine-rich histones (IIIs and Vs) and isolated LHC-II as substrate has now been purified to homogeneity and characterized by SDS-polyacrylamide gel electrophoresis as a 64 kDa peptide. From a comparison of the two isolation procedures we have concluded that CPK1 is indeed a protein kinase, but has a lower specific activity than that of LHCK. 8 refs., 4 figs.

  15. Solution structure of the cAMP-dependent protein kinase

    SciTech Connect

    Trewhella, J.; Olah, G.A.; Walsh, D.A.; Mitchell, R.D.

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project as Los Alamos National Laboratory (LANL). Protein phosphorylation is well established as one of the most important mechanisms of signal transduction and cellular regulation. Two of the key enzymes that catalyze these phosphorylation reactions are the cAMP- (PKA) and cGMP- (PKG) dependent protein kinases. PKA has served as the prototypic model of this class of enzymes that now comprises in excess of 300 phylogenetically related proteins. A large number of these protein kinases are critical for the regulation of cell function and a full analysis of their similarities and differences is essential to understand their diverse physiological roles. The cAMP-dependent protein kinase has the subunit structure R2C2, in which C and R refer to the catalytic and regulatory subunits, respectively. The cGMP-dependent protein kinase (PKG) is highly homologous to PKA but is distinguished from it by having the regulatory and catalytic domains on a contiguous polypeptide. The studies described here use small-angle scattering and Fourier Transform InfraRed (FTIR) spectroscopy to study domain movements and conformational changes in these enzymes in different functional states in order to elucidate the molecular bases for the regulation of their activities.

  16. The Roles of NDR Protein Kinases in Hippo Signalling

    PubMed Central

    Hergovich, Alexander

    2016-01-01

    The Hippo tumour suppressor pathway has emerged as a critical regulator of tissue growth through controlling cellular processes such as cell proliferation, death, differentiation and stemness. Traditionally, the core cassette of the Hippo pathway includes the MST1/2 protein kinases, the LATS1/2 protein kinases, and the MOB1 scaffold signal transducer, which together regulate the transcriptional co-activator functions of the proto-oncoproteins YAP and TAZ through LATS1/2-mediated phosphorylation of YAP/TAZ. Recent research has identified additional kinases, such as NDR1/2 (also known as STK38/STK38L) and MAP4Ks, which should be considered as novel members of the Hippo core cassette. While these efforts helped to expand our understanding of Hippo core signalling, they also began to provide insights into the complexity and redundancy of the Hippo signalling network. Here, we focus on summarising our current knowledge of the regulation and functions of mammalian NDR kinases, discussing parallels between the NDR pathways in Drosophila and mammals. Initially, we provide a general overview of the cellular functions of NDR kinases in cell cycle progression, centrosome biology, apoptosis, autophagy, DNA damage signalling, immunology and neurobiology. Finally, we put particular emphasis on discussing NDR1/2 as YAP kinases downstream of MST1/2 and MOB1 signalling in Hippo signalling. PMID:27213455

  17. Ethanol increases affinity of protein kinase C for phosphatidylserine

    SciTech Connect

    Chin, J.H.

    1986-03-01

    Protein kinase C is a calcium-dependent enzyme that requires phospholipid for its activation. It is present in relatively high concentration in the brain and may be involved in neuronal function. The present experiments test whether the membrane disorder induced by ethanol affects the activity of kinase C by changing its interaction with membrane lipid. Fractions rich in kinase C were purified from rat brain cytosol by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Enzyme activity was assayed by measuring the phosphorylation of histone H1. As expected, phosphatidylserine activated the enzyme, and the stimulation was further increased by the addition of calcium and/or diacylglycerol. At low concentration of free calcium (0.5-1..mu..M), ethanol (800 mM0 enhanced kinase C activity if the presence of phospholipid. similar results were observed in the absence of calcium. Double reciprocal plots of the data showed that ethanol increased the affinity of the enzyme for phosphatidylserine without affecting the V/sub max. The stimulation of kinase C activity by ethanol was not observed at high calcium concentrations. These experiments suggest that ethanol may activated protein kinase C at physiological levels of calcium by facilitating its transfer into the hydrophobic membrane environment.

  18. Atypical protein kinase Clambda binds and regulates p70 S6 kinase.

    PubMed Central

    Akimoto, K; Nakaya, M; Yamanaka, T; Tanaka, J; Matsuda, S; Weng, Q P; Avruch, J; Ohno, S

    1998-01-01

    p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKClambda, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKClambda associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKClambda results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKClambda, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpresion of the active form of PKClambda, however, fails to activate p70 S6K. These results suggest that PKClambda is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression. PMID:9761742

  19. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    PubMed Central

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent S.

    2016-01-01

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology. PMID:27185916

  20. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    SciTech Connect

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent S.

    2016-05-16

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

  1. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    DOE PAGES

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; ...

    2016-05-16

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis;more » however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.« less

  2. Phosphorylation of spore coat proteins by a family of atypical protein kinases

    SciTech Connect

    Nguyen, Kim B.; Sreelatha, Anju; Durrant, Eric S.; Lopez-Garrido, Javier; Muszewska, Anna; Dudkiewicz, Małgorzata; Grynberg, Marcin; Yee, Samantha; Pogliano, Kit; Tomchick, Diana R.; Pawłowski, Krzysztof; Dixon, Jack E.; Tagliabracci, Vincent S.

    2016-05-16

    The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. In this paper, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Finally and collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

  3. 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

  4. Protein kinase Calpha activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase.

    PubMed

    Andreozzi, Francesco; Melillo, Rosa Marina; Carlomagno, Francesca; Oriente, Francesco; Miele, Claudia; Fiory, Francesca; Santopietro, Stefania; Castellone, Maria Domenica; Beguinot, Francesco; Santoro, Massimo; Formisano, Pietro

    2003-05-15

    We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCalpha, PKCdelta and PKCzeta activity and that PKCalpha, not PKCdelta and PKCzeta, forms a ligand-dependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCalpha. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGF-induced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCalpha. We conclude that RET binds to and activates PKCalpha. PKCalpha, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity.

  5. The regulation of AMP-activated protein kinase by phosphorylation.

    PubMed Central

    Stein, S C; Woods, A; Jones, N A; Davison, M D; Carling, D

    2000-01-01

    The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr(172) on AMPK activity. Mutation of Thr(172) to an aspartic acid residue (T172D) in either alpha1 or alpha2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr(172) accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the alpha subunit and one site on the beta subunit. Furthermore, we provide evidence that phosphorylation of Thr(172) may be involved in the sensitivity of the AMPK complex to AMP. PMID:10642499

  6. Targeting protein kinase A in cancer therapy: an update

    PubMed Central

    Sapio, Luigi; Di Maiolo, Francesca; Illiano, Michela; Esposito, Antonietta; Chiosi, Emilio; Spina, Annamaria; Naviglio, Silvio

    2014-01-01

    Protein Kinase A (PKA) is a well known member of the serine-threonin protein kinase superfamily. PKA, also known as cAMP-dependent protein kinase, is a multi-unit protein kinase that mediates signal transduction of G-protein coupled receptors through its activation upon cAMP binding. The widespread expression of PKA subunit genes, and the myriad of mechanisms by which cAMP is regulated within a cell suggest that PKA signaling is one of extreme importance to cellular function. It is involved in the control of a wide variety of cellular processes from metabolism to ion channel activation, cell growth and differentiation, gene expression and apoptosis. Importantly, since it has been implicated in the initiation and progression of many tumors, PKA has been proposed as a novel biomarker for cancer detection, and as a potential molecular target for cancer therapy. Here, we highlight some features of cAMP/PKA signaling that are relevant to cancer biology and present an update on targeting PKA in cancer therapy. PMID:26417307

  7. The elusive activity of the Yersinia protein kinase A kinase domain is revealed.

    PubMed

    Laskowski-Arce, Michelle A; Orth, Kim

    2007-10-01

    Yersinia spp. pathogens use their type III secretion system to translocate effectors that manipulate host signaling pathways during infection. Although molecular targets for five of the six known Yersinia effectors are known, the target for the serine/threonine kinase domain of Yersinia protein kinase A (YpkA) has remained elusive. Recently, Navarro et al. (2007) demonstrated that YpkA phosphorylates Galphaq, and inhibits Galphaq-mediated signaling. Inhibition by YpkA could contribute to one of the most documented symptoms of Yersinia pestis infection, extensive bleeding.

  8. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    NASA Astrophysics Data System (ADS)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  9. Comparative analysis of human and bovine protein kinases reveals unique relationship and functional diversity.

    PubMed

    Kabir, Nuzhat N; Kazi, Julhash U

    2011-10-01

    Reversible protein phosphorylation by protein kinases and phosphatases is a common event in various cellular processes. The eukaryotic protein kinase superfamily, which is one of the largest superfamilies of eukaryotic proteins, plays several roles in cell signaling and diseases. We identified 482 eukaryotic protein kinases and 39 atypical protein kinases in the bovine genome, by searching publicly accessible genetic-sequence databases. Bovines have 512 putative protein kinases, each orthologous to a human kinase. Whereas orthologous kinase pairs are, on an average, 90.6% identical, orthologous kinase catalytic domain pairs are, on an average, 95.9% identical at the amino acid level. This bioinformatic study of bovine protein kinases provides a suitable framework for further characterization of their functional and structural properties.

  10. Identification of a fungi-specific lineage of protein kinases closely related to tyrosine kinases.

    PubMed

    Zhao, Zhongtao; Jin, Qiaojun; Xu, Jin-Rong; Liu, Huiquan

    2014-01-01

    Tyrosine kinases (TKs) specifically catalyze the phosphorylation of tyrosine residues in proteins and play essential roles in many cellular processes. Although TKs mainly exist in animals, recent studies revealed that some organisms outside the Opisthokont clade also contain TKs. The fungi, as the sister group to animals, are thought to lack TKs. To better understand the origin and evolution of TKs, it is important to investigate if fungi have TK or TK-related genes. We therefore systematically identified possible TKs across the fungal kingdom by using the profile hidden Markov Models searches and phylogenetic analyses. Our results confirmed that fungi lack the orthologs of animal TKs. We identified a fungi-specific lineage of protein kinases (FslK) that appears to be a sister group closely related to TKs. Sequence analysis revealed that members of the FslK clade contain all the conserved protein kinase sub-domains and thus are likely enzymatically active. However, they lack key amino acid residues that determine TK-specific activities, indicating that they are not true TKs. Phylogenetic analysis indicated that the last common ancestor of fungi may have possessed numerous members of FslK. The ancestral FslK genes were lost in Ascomycota and Ustilaginomycotina and Pucciniomycotina of Basidiomycota during evolution. Most of these ancestral genes, however, were retained and expanded in Agaricomycetes. The discovery of the fungi-specific lineage of protein kinases closely related to TKs helps shed light on the origin and evolution of TKs and also has potential implications for the importance of these kinases in mushroom fungi.

  11. Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.

    PubMed

    Wilkes, Edmund H; Casado, Pedro; Rajeeve, Vinothini; Cutillas, Pedro R

    2017-09-01

    Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed kinase activity ranking using phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output. Application of KARP to the analysis of eight hematological cell lines revealed that cyclin-dependent kinase (CDK) 1/2, casein kinase (CK) 2, extracellular signal-related kinase (ERK), and p21-activated kinase (PAK) were the most frequently highly ranked kinases in these cell models. The patterns of kinase activation were cell-line specific yet showed a significant association with cell viability as a function of kinase inhibitor treatment. Thus, our study exemplifies KARP as an untargeted approach to empirically and systematically identify regulatory kinases within signaling networks. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Mitogen-Activated Protein Kinases and Mitogen Kinase Phosphatase 1: A Critical Interplay in Macrophage Biology

    PubMed Central

    Lloberas, Jorge; Valverde-Estrella, Lorena; Tur, Juan; Vico, Tania; Celada, Antonio

    2016-01-01

    Macrophages are necessary in multiple processes during the immune response or inflammation. This review emphasizes the critical role of the mitogen-activated protein kinases (MAPKs) and mitogen kinase phosphatase-1 (MKP-1) in the functional activities of macrophages. While the phosphorylation of MAPKs is required for macrophage activation or proliferation, MKP-1 dephosphorylates these kinases, thus playing a balancing role in the control of macrophage behavior. MKP-1 is a nuclear-localized dual-specificity phosphatase whose expression is regulated at multiple levels, including at the transcriptional and post-transcriptional level. The regulatory role of MKP-1 in the interplay between MAPK phosphorylation/dephosphorylation makes this molecule a critical regulator of macrophage biology and inflammation. PMID:27446931

  13. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    ERIC Educational Resources Information Center

    Sossin, Wayne S.

    2007-01-01

    Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used "Aplysia" as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented…

  14. Targeting protein kinases to reverse multidrug resistance in sarcoma.

    PubMed

    Chen, Hua; Shen, Jacson; Choy, Edwin; Hornicek, Francis J; Duan, Zhenfeng

    2016-02-01

    Sarcomas are a group of cancers that arise from transformed cells of mesenchymal origin. They can be classified into over 50 subtypes, accounting for approximately 1% of adult and 15% of pediatric cancers. Wide surgical resection, radiotherapy, and chemotherapy are the most common treatments for the majority of sarcomas. Among these therapies, chemotherapy can palliate symptoms and prolong life for some sarcoma patients. However, sarcoma cells can have intrinsic or acquired resistance after treatment with chemotherapeutics drugs, leading to the development of multidrug resistance (MDR). MDR attenuates the efficacy of anticancer drugs and results in treatment failure for sarcomas. Therefore, overcoming MDR is an unmet need for sarcoma therapy. Certain protein kinases demonstrate aberrant expression and/or activity in sarcoma cells, which have been found to be involved in the regulation of sarcoma cell progression, such as cell cycle, apoptosis, and survival. Inhibiting these protein kinases may not only decrease the proliferation and growth of sarcoma cells, but also reverse their resistance to chemotherapeutic drugs to subsequently reduce the doses of anticancer drugs and decrease drug side-effects. The discovery of novel strategies targeting protein kinases opens a door to a new area of sarcoma research and provides insight into the mechanisms of MDR in chemotherapy. This review will focus on the recent studies in targeting protein kinase to reverse chemotherapeutic drug resistance in sarcoma. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Targeting Protein Kinases to Reverse Multidrug Resistance in Sarcoma

    PubMed Central

    Chen, Hua; Shen, Jacson; Choy, Edwin; Hornicek, Francis J.; Duan, Zhenfeng

    2015-01-01

    Sarcomas are a group of cancers that arise from transformed cells of mesenchymal origin. They can be classified into over 50 subtypes, accounting for approximately 1% of adult and 15% of pediatric cancers. Wide surgical resection, radiotherapy, and chemotherapy are the most common treatments for the majority of sarcomas. Among these therapies, chemotherapy can palliate symptoms and prolong life for some sarcoma patients. However, sarcoma cells can have intrinsic or acquired resistance after treatment with chemotherapeutics drugs, leading to the development of multidrug resistance (MDR). MDR attenuates the efficacy of anticancer drugs and results in treatment failure for sarcomas. Therefore, overcoming MDR is an unmet need for sarcoma therapy. Certain protein kinases demonstrate aberrant expression and/or activity in sarcoma cells, which have been found to be involved in the regulation of sarcoma cell progression, such as cell cycle, apoptosis, and survival. Inhibiting these protein kinases may not only decrease the proliferation and growth of sarcoma cells, but also reverse their resistance to chemotherapeutic drugs to subsequently reduce the doses of anticancer drugs and decrease drug side-effects. The discovery of novel strategies targeting protein kinases opens a door to a new area of sarcoma research and provides insight into the mechanisms of MDR in chemotherapy. This review will focus on the recent studies in targeting protein kinase to reverse chemotherapeutic drug resistance in sarcoma. PMID:26827688

  16. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    ERIC Educational Resources Information Center

    Sossin, Wayne S.

    2007-01-01

    Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used "Aplysia" as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented…

  17. The Two Faces of Receptor Interacting Protein Kinase-1

    PubMed Central

    Weinlich, Ricardo; Green, Douglas R.

    2014-01-01

    Receptor Interacting Protein Kinase-1 (RIPK1), a key player in inflammation and cell death, assumes opposite functions depending on the cellular context and its posttranslational modifications. Genetic evidence supported by biochemical and cellular biology approaches shed light on the circumstances in which RIPK1 promotes or inhibits these processes. PMID:25459879

  18. Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

    PubMed Central

    Jiang, Youwei; Cypess, Aaron M.; Muse, Evan D.; Wu, Cui-Rong; Unson, Cecilia G.; Merrifield, R. B.; Sakmar, Thomas P.

    2001-01-01

    We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation. PMID:11517300

  19. Protein kinase C-associated kinase regulates NF-κB activation through inducing IKK activation.

    PubMed

    Kim, Sang-Woo; Schifano, Matthew; Oleksyn, David; Jordan, Craig T; Ryan, Daniel; Insel, Richard; Zhao, Jiyong; Chen, Luojing

    2014-10-01

    Activation of the transcription factor NF-κB induced by extracellular stimuli requires IKKα and IKKβ kinase activity. How IKKα and IKKβ are activated by various upstream signaling molecules is not fully understood. We previously showed that protein kinase C-associated kinase (PKK, also known as DIK/RIP4), which belongs to the receptor-interacting protein (RIP) kinase family, mediates the B cell activating factor of the TNF family (BAFF)-induced NF-κB activation in diffuse large B cell lymphoma (DLBCL) cell lines. Here we have investigated the mechanism underlying NF-κB activation regulated by PKK. Our results suggest that PKK can activate both the classical and the alternative NF-κB activation pathways. PKK associates with IKKα and IKKβ in mammalian cells and induces activation of both IKKα and IKKβ via phosphorylation of their serine residues 176/180 and 177/181, respectively. Unlike other members of the RIP family that activate NF-κB through a kinase-independent pathway, PKK appears to activate IKK and NF-κB mainly in a kinase-dependent manner. Suppression of PKK expression by RNA interference inhibits phosphorylation of IKKα and IKKβ as well as activation of NF-κB in human cancer cell lines. Thus, PKK regulates NF-κB activation by modulating activation of IKKα and IKKβ in mammalian cells. We propose that PKK may provide a critical link between IKK activation and various upstream signaling cascades, and may represent a potential target for inhibiting abnormal NF-κB activation in human cancers.

  20. Phosphorylation of Yeast Phosphatidylserine Synthase by Protein Kinase A

    PubMed Central

    Choi, Hyeon-Son; Han, Gil-Soo; Carman, George M.

    2010-01-01

    The CHO1-encoded phosphatidylserine synthase from Saccharomyces cerevisiae is phosphorylated and inhibited by protein kinase A in vitro. CHO1 alleles bearing Ser46 → Ala and/or Ser47 → Ala mutations were constructed and expressed in a cho1Δ mutant lacking phosphatidylserine synthase. In vitro, the S46A/S47A mutation reduced the total amount of phosphorylation by 90% and abolished the inhibitory effect protein kinase A had on phosphatidylserine synthase activity. The enzyme phosphorylation by protein kinase A, which was time- and dose-dependent and dependent on the concentration of ATP, caused a electrophoretic mobility shift from a 27-kDa form to a 30-kDa form. The two electrophoretic forms of phosphatidylserine synthase were present in exponential phase cells, whereas only the 27-kDa form was present in stationary phase cells. In vivo labeling with 32Pi and immune complex analysis showed that the 30-kDa form was predominantly phosphorylated when compared with the 27-kDa form. However, the S46A/S47A mutations abolished the protein kinase A-mediated electrophoretic mobility shift. The S46A/S47A mutations also caused a 55% reduction in the total amount of phosphatidylserine synthase in exponential phase cells and a 66% reduction in the amount of enzyme in stationary phase cells. In phospholipid composition analysis, cells expressing the S46A/S47A mutant enzyme exhibited a 57% decrease in phosphatidylserine and a 40% increase in phosphatidylinositol. These results indicate that phosphatidylserine synthase is phosphorylated on Ser46 and Ser47 by protein kinase A, which results in a higher amount of enzyme for the net effect of stimulating the synthesis of phosphatidylserine. PMID:20145252

  1. Phosphoproteins and protein kinases of the Golgi apparatus membrane

    SciTech Connect

    Capasso, J.M.; Abeijon, C.; Hirschberg, C.B.

    1985-11-25

    Incubation of a highly purified fraction derived from rat liver Golgi apparatus with (gamma-TSP)ATP results in phosphorylation of several endogenous phosphoproteins. One phosphoprotein with an apparent Mr of 48,300 is radiolabeled to an apparent extent at least 5-fold higher than any other phosphoprotein as part of either the Golgi apparatus or highly purified rat liver fractions derived from the rough endoplasmic reticulum, mitochondria, plasma membrane, coated vesicles, cytosol, and total homogenate. Approximately 70% of the 48.3-kDa phosphoprotein appears to be a specific extrinsic Golgi membrane protein with the phosphorylated amino acid being threonine. The protein kinase which phosphorylates the 48.3-kDa protein is an intrinsic Golgi membrane protein and is dependent on MgS , independent of CaS , calmodulin, and cAMP, and is inhibited by N-ethylmaleimide. Preliminary evidence suggests that there are also intrinsic membrane protein kinases in the Golgi apparatus which are dependent on CaS and cAMP. The physiological role of the above phosphoproteins and protein kinases is not known.

  2. Ten things you should know about protein kinases: IUPHAR Review 14

    PubMed Central

    Fabbro, Doriano; Cowan-Jacob, Sandra W; Moebitz, Henrik

    2015-01-01

    Many human malignancies are associated with aberrant regulation of protein or lipid kinases due to mutations, chromosomal rearrangements and/or gene amplification. Protein and lipid kinases represent an important target class for treating human disorders. This review focus on ‘the 10 things you should know about protein kinases and their inhibitors', including a short introduction on the history of protein kinases and their inhibitors and ending with a perspective on kinase drug discovery. Although the ‘10 things’ have been, to a certain extent, chosen arbitrarily, they cover in a comprehensive way the past and present efforts in kinase drug discovery and summarize the status quo of the current kinase inhibitors as well as knowledge about kinase structure and binding modes. Besides describing the potentials of protein kinase inhibitors as drugs, this review also focus on their limitations, particularly on how to circumvent emerging resistance against kinase inhibitors in oncological indications. PMID:25630872

  3. Targeting mitogen-activated protein kinase kinase (MEK) in solid tumors.

    PubMed

    Duffy, Austin; Kummar, Shivaani

    2009-12-01

    The Raf-mitogen activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) protein kinase signaling cascade is an important intracellular pathway whose activation influences many fundamental cellular processes and whose aberrancy is associated with cancer cell growth. In addition to activation from within by, for example, Raf mutations, this pathway is frequently activated from above by mutated Ras or epidermal growth factor receptor (EGFR). Given the near ubiquity of derangements affecting at least part of this network in cancer, there is a strong and clear rationale for interrupting it. In recent times, in colorectal and lung cancer, Ras and EGFR mutant status have been shown to be critically important and mutually exclusive predictors of response to anti-EGFR therapies. These developments underline the importance of targeting downstream effectors, and MEK inhibition has been the subject of intense scientific and clinical research for some time now. This article reviews the current status of MEK inhibitors with regard to their clinical development.

  4. Specific inhibition of AGC protein kinases by antibodies against C-terminal epitopes.

    PubMed

    Traincard, François; Giacomoni, Véronique; Veron, Michel

    2004-08-13

    The sequences contributing to the catalytic site of protein kinases are not all comprised within the highly conserved catalytic core. Thus, in mammalian cAMP-dependent protein kinase (PKA), the C-terminal sequence participates in substrate binding. Using synthetic peptides mimicking the FxxF motif present at most C-termini of AGC kinases, we have raised highly specific antibodies which are potent and specific inhibitors of the catalytic activity of the cognate protein kinase. Taking into account the structure of PKA, these results point to the potential of the C-terminal region of protein kinases as a target for designing specific protein kinase inhibitors.

  5. Mitochondrial protein import under kinase surveillance

    PubMed Central

    Opalińska, Magdalena; Meisinger, Chris

    2014-01-01

    Despite the simplicity of the yeast Saccharomyces cerevisiae, its basic cellular machinery tremendously mirrors that of higher eukaryotic counterparts. Thus, this unicellular organism turned out to be an invaluable model system to study the countless mechanisms that govern life of the cell. Recently, it has also enabled the deciphering of signalling pathways that control flux of mitochondrial proteins to the organelle according to metabolic requirements. For decades mitochondria were considered autonomous organelles that are only partially incorporated into cellular signalling networks. Consequently, only little has been known about the role of reversible phosphorylation as a meaningful mechanism that orchestrates mitochondrial biology accordingly to cellular needs. Therefore, research in this direction has been vastly neglected. However, findings over the past few years have changed this view and new exciting fields in mitochondrial biology have emerged. Here, we summarize recent discoveries in the yeast model system that point towards a vital role of reversible phosphorylation in regulation of mitochondrial protein import. PMID:28357222

  6. Effects of butyltins on mitogen-activated-protein kinase kinase kinase and Ras activity in human natural killer cells.

    PubMed

    Celada, Lindsay J; Whalen, Margaret M

    2014-09-01

    Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT) diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 min of TBT exposure and the MAP3K, ASK1, after 1 h exposures to TBT. In addition, our results suggest that both TBT and DBT affect the regulation of c-Raf.

  7. Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase*

    PubMed Central

    Maiolica, Alessio; de Medina-Redondo, Maria; Schoof, Erwin M.; Chaikuad, Apirat; Villa, Fabrizio; Gatti, Marco; Jeganathan, Siva; Lou, Hua Jane; Novy, Karel; Hauri, Simon; Toprak, Umut H.; Herzog, Franz; Meraldi, Patrick; Penengo, Lorenza; Turk, Benjamin E.; Knapp, Stefan; Linding, Rune; Aebersold, Ruedi

    2014-01-01

    Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr3 of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg2 and Lys4 adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys4 of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser137 of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser137 resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser137 might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing. PMID:24732914

  8. [Regulation of G protein-coupled receptor kinase activity].

    PubMed

    Haga, T; Haga, K; Kameyama, K; Nakata, H

    1994-09-01

    Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

  9. Protein kinase A regulates the osteogenic activity of Osterix.

    PubMed

    He, Siyuan; Choi, You Hee; Choi, Joong-Kook; Yeo, Chang-Yeol; Chun, ChangJu; Lee, Kwang Youl

    2014-10-01

    Osterix belongs to the SP gene family and is a core transcription factor responsible for osteoblast differentiation and bone formation. Activation of protein kinase A (PKA), a serine/threonine kinase, is essential for controlling bone formation and BMP-induced osteoblast differentiation. However, the relationship between Osterix and PKA is still unclear. In this report, we investigated the precise role of the PKA pathway in regulating Osterix during osteoblast differentiation. We found that PKA increased the protein level of Osterix; PKA phosphorylated Osterix, increased protein stability, and enhanced the transcriptional activity of Osterix. These results suggest that Osterix is a novel target of PKA, and PKA modulates osteoblast differentiation partially through the regulation of Osterix.

  10. Isolation of Drosophila genes encoding G protein-coupled receptor kinases.

    PubMed Central

    Cassill, J A; Whitney, M; Joazeiro, C A; Becker, A; Zuker, C S

    1991-01-01

    G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades. Images PMID:1662381

  11. Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Perez, Adam M.; Finnigan, Gregory C.; Roelants, Françoise M.; Thorner, Jeremy

    2016-01-01

    Septins are a family of eukaryotic GTP-binding proteins that associate into linear rods, which, in turn, polymerize end-on-end into filaments, and further assemble into other, more elaborate super-structures at discrete subcellular locations. Hence, septin-based ensembles are considered elements of the cytoskeleton. One function of these structures that has been well-documented in studies conducted in budding yeast Saccharomyces cerevisiae is to serve as a scaffold that recruits regulatory proteins, which dictate the spatial and temporal control of certain aspects of the cell division cycle. In particular, septin-associated protein kinases couple cell cycle progression with cellular morphogenesis. Thus, septin-containing structures serve as signaling platforms that integrate a multitude of signals and coordinate key downstream networks required for cell cycle passage. This review summarizes what we currently understand about how the action of septin-associated protein kinases and their substrates control information flow to drive the cell cycle into and out of mitosis, to regulate bud growth, and especially to direct timely and efficient execution of cytokinesis and cell abscission. Thus, septin structures represent a regulatory node at the intersection of many signaling pathways. In addition, and importantly, the activities of certain septin-associated protein kinases also regulate the state of organization of the septins themselves, creating a complex feedback loop. PMID:27847804

  12. Protein kinase C mechanisms that contribute to cardiac remodelling

    PubMed Central

    Newton, Alexandra C.; Antal, Corina E.; Steinberg, Susan F.

    2016-01-01

    Protein phosphorylation is a highly-regulated and reversible process that is precisely controlled by the actions of protein kinases and protein phosphatases. Factors that tip the balance of protein phosphorylation lead to changes in a wide range of cellular responses, including cell proliferation, differentiation and survival. The protein kinase C (PKC) family of serine/threonine kinases sits at nodal points in many signal transduction pathways; PKC enzymes have been the focus of considerable attention since they contribute to both normal physiological responses as well as maladaptive pathological responses that drive a wide range of clinical disorders. This review provides a background on the mechanisms that regulate individual PKC isoenzymes followed by a discussion of recent insights into their role in the pathogenesis of diseases such as cancer. We then provide an overview on the role of individual PKC isoenzymes in the regulation of cardiac contractility and pathophysiological growth responses, with a focus on the PKC-dependent mechanisms that regulate pump function and/or contribute to the pathogenesis of heart failure. PMID:27433023

  13. Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

    PubMed

    Kole, H K; Abdel-Ghany, M; Racker, E

    1988-08-01

    Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein.

  14. Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

    PubMed Central

    Kole, H K; Abdel-Ghany, M; Racker, E

    1988-01-01

    Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein. Images PMID:2901092

  15. Modulation of Purinergic Neuromuscular Transmission by Phorbol Dibutyrate is Independent of Protein Kinase C in Murine Urinary Bladder

    PubMed Central

    Silinsky, E. M.

    2012-01-01

    Parasympathetic control of murine urinary bladder consists of contractile components mediated by both muscarinic and purinergic receptors. Using intracellular recording techniques, the purinergic component of transmission was measured as both evoked excitatory junctional potentials (EJPs) in response to electrical field stimulation and spontaneous events [spontaneous EJPs (sEJPs)]. EJPs, but not sEJPs, were abolished by the application of the Na+ channel blocker tetrodotoxin and the Ca2+ channel blocker Cd2+. Both EJPs and sEJPs were abolished by the application of the P2X1 antagonist 8,8′-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF279). Application of phorbol dibutyrate (PDBu) increased electrically evoked EJP amplitudes with no effect on mean sEJP amplitudes. Similar increases in EJP amplitudes were produced by PDBu in the presence of either the nonselective protein kinase inhibitor staurosporine or the specific protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X). These results suggest that PDBu increases the purinergic component of detrusor transmission through increasing neurogenic ATP release via a PKC-independent mechanism. PMID:22547572

  16. Phosphorylation of the mRNA cap binding protein and eIF-4A by different protein kinases

    SciTech Connect

    Hagedorn, C.H.

    1987-05-01

    These studies were done to determine the identity of a protein kinase that phosphorylates the mRNA cap binding protein (CBP). Two chromatographic steps (dye and ligand and ion exchange HPLC) produced a 500x purification of an enzyme activity in rabbit reticulocytes that phosphorylated CBP at serine residues. Isoelectric focusing analysis of kinase treated CBP demonstrated 5 isoelectric species of which the 2 most anodic species were phosphorylated (contained /sup 32/P). This kinase activity phosphorylated CBP when it was isolated or in the eIF-4F complex. Purified protein kinase C, cAMP or cGMP dependent protein kinase, casein kinase I or II, myosin light chain kinase or insulin receptor kinase did not significantly phosphorylate isolated CBP or CBP in the eIF-4F complex. However, cAMP and cGMP dependent protein kinases and casein kinase II phosphorylated eIF-4A but did not phosphorylate the 46 kDa component of eIF-4F. cAMP dependent protein kinase phosphorylated a approx. 220 kDa protein doublet in eIF-4F preparations. These studies indicate that CBP kinase activity probably represents a previously unidentified protein kinase. In addition, eIF-4A appears to be phosphorylated by several protein kinases whereas the 46 kDa component of the eIF-4F complex was not.

  17. A Fluorescence-Based Thermal Shift Assay Identifies Inhibitors of Mitogen Activated Protein Kinase Kinase 4

    PubMed Central

    Krishna, Sankar N.; Luan, Chi-Hao; Mishra, Rama K.; Xu, Li; Scheidt, Karl A.; Anderson, Wayne F.; Bergan, Raymond C.

    2013-01-01

    Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed an in vitro MAP2K4 kinase assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, to evaluate kinase inhibitory function. In this manner, we validated the performance of our initial FTS screen. We next applied this approach to a 2000 compound chemically diverse library, identified 7 hits, and confirmed them in the in vitro kinase assay. Finally, by coupling our structure-activity relationship data to MAP2K4's crystal structure, we constructed a model for ligand binding. It predicts binding of our identified inhibitory compounds to the ATP binding pocket. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors. PMID:24339940

  18. Monoclonal antibodies against type II rat brain protein kinase

    SciTech Connect

    Nakabayashi, C.H.; Huang, K.P.

    1987-05-01

    Three monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C (PKC) were used to carry out the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II PKC in a dose-dependent manner but did neither to type I nor type III isozyme. Purified type II PKC has a molecular weight of 82,000 and consists of heterogeneous isoelectric point species, all of which are cross reactive with these antibodies. Immunoblot analysis of the tryptic fragments from PKC revealed that all three antibodies recognized the 33-38-KDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-KDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained the kinase activity which was dependent on Ca/sup 2 +/ and phosphatidylserine (PS) and further activated by diacylglycerol. With antibody 8/1, the apparent Km values of the kinase for Ca/sup 2 +/ and PS were not influenced. The initial rate and final extent of autophosphorylation were reduced. The concentration of PS required for half-maximal (/sup 3/H)phorbol 12,13-dibutyrate (PDBu) binding was increased and the total PDBu binding was reduced. In the presence of optimum concentrations of Ca/sup 2 +/ and PS, the Kd of PDBu was unaffected by the antibody but the total binding was reduced. These results demonstrate that the PS/PDBu-binding domain contains the major epitope for the antibodies and the antibody mainly influences the PS/PDBu binding to the kinase.

  19. N-terminus of the protein kinase CLK1 induces SR protein hyperphosphorylation.

    PubMed

    Aubol, Brandon E; Plocinik, Ryan M; Keshwani, Malik M; McGlone, Maria L; Hagopian, Jonathan C; Ghosh, Gourisankar; Fu, Xiang-Dong; Adams, Joseph A

    2014-08-15

    SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine/serine-rich) domains by two major families of protein kinases. The SRPKs (SR-specific protein kinases) efficiently phosphorylate the arginine/serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs (Cdc2-like kinases) lack a docking groove and phosphorylate both arginine/serine and serine-proline dipeptides, modifications that generate a hyperphosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1 (SR protein splicing factor 1). This interaction not only is essential for facilitating hyperphosphorylation, but also induces co-operative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 (transformer 2β1) by 2-3-fold. These findings suggest that CLK1-dependent hyperphosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein.

  20. Protein interactome analysis of 12 mitogen-activated protein kinase kinase kinase in rice using a yeast two-hybrid system.

    PubMed

    Singh, Raksha; Lee, Jae-Eun; Dangol, Sarmina; Choi, Jihyun; Yoo, Ran Hee; Moon, Jae Sun; Shim, Jae-Kyung; Rakwal, Randeep; Agrawal, Ganesh Kumar; Jwa, Nam-Soo

    2014-01-01

    The mitogen-activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K-interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two-hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K-interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full-length cDNA in the rice KOME (http://cdna01.dna.affrc.go.jp/cDNA/) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead-associated domain-containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K-interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors.

  1. Protein Tyrosine Kinase Signaling During Oocyte Maturation and Fertilization

    PubMed Central

    McGinnis, Lynda K.; Carroll, David J.; Kinsey, William H.

    2011-01-01

    The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre-positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC-family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK-mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC-family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans. PMID:21681843

  2. Spatial distribution of protein kinase A activity during cell migration is mediated by A-kinase anchoring protein AKAP Lbc.

    PubMed

    Paulucci-Holthauzen, Adriana A; Vergara, Leoncio A; Bellot, Larry J; Canton, David; Scott, John D; O'Connor, Kathleen L

    2009-02-27

    Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility.

  3. Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

    PubMed Central

    Cai, H; Smola, U; Wixler, V; Eisenmann-Tappe, I; Diaz-Meco, M T; Moscat, J; Rapp, U; Cooper, G M

    1997-01-01

    The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo. PMID:9001227

  4. Spatial Distribution of Protein Kinase A Activity during Cell Migration Is Mediated by A-kinase Anchoring Protein AKAP Lbc*

    PubMed Central

    Paulucci-Holthauzen, Adriana A.; Vergara, Leoncio A.; Bellot, Larry J.; Canton, David; Scott, John D.; O'Connor, Kathleen L.

    2009-01-01

    Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility. PMID:19106088

  5. Regulation of polar auxin transport by protein and lipid kinases

    PubMed Central

    Jaillais, Yvon

    2016-01-01

    The directional transport of auxin, known as polar auxin transport, allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima and gradients that are instrumental in both organ initiation and shape determination. As such, polar auxin transport is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell-to-cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the ‘non-genomic’ regulation of auxin transport, putting an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some Receptor-Like Kinases (RLK) and two-component histidine kinase receptors in polar auxin transport, noticing that there are likely RLKs involved in coordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition as well as root gravitropism and shoot phototropism. PMID:27242371

  6. DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Ishizuka, T; Kajita, K; Miura, A; Ishizawa, M; Kanoh, Y; Itaya, S; Kimura, M; Muto, N; Mune, T; Morita, H; Yasuda, K

    1999-01-01

    We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 microM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-alpha, -beta, and -gamma. DHEA provoked the translocation of PKC-beta and -zeta from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.

  7. Protein kinase Cδ regulates vaccinia-related kinase 1 in DNA damage–induced apoptosis

    PubMed Central

    Park, Choon-Ho; Choi, Bo-Hwa; Jeong, Min-Woo; Kim, Sangjune; Kim, Wanil; Song, Yun Seon; Kim, Kyong-Tai

    2011-01-01

    Vaccinia-related kinase 1 (VRK1) is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase Cδ (PKCδ) in the regulation of murine VRK1. We show that PKCδ interacts with VRK1, phosphorylates the Ser-355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to a DNA-damaging agent. In addition, p53 played a critical role in the regulation of DNA damage–induced cell death accompanied by PKCδ-mediated modulation of VRK1. In p53-deficient cells, PKCδ-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCδ. Taken together, these results indicate that PKCδ regulates phosphorylation and down-regulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner. PMID:21346188

  8. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  9. Protein kinase C activation inhibits eosinophil degranulation through stimulation of intracellular cAMP production.

    PubMed

    Ezeamuzie, Charles I; Taslim, Najla

    2004-11-01

    The mechanism of inhibition of eosinophil degranulation by protein kinase C (PKC) was investigated in complement C5a (C5a)-stimulated degranulation of highly purified human eosinophils using the specific PKC activator - phorbol 12-myristate 13-acetate (PMA). C5a-induced release of eosinophil peroxidase and eosinophil cationic protein was potently inhibited in a concentration-dependent manner by PMA (IC(50): 3 and 5 nM, respectively). The inhibition by PMA, but not histamine, was significantly reversed by the specific, but isoform nonselective, PKC inhibitor Ro 31-8220 (1 microM). In the presence of phosphodiesterase inhibitor rolipram (5 microM), PMA stimulated a pronounced concentration-dependent increase in intracellular cAMP, with a potency 400 times that of histamine (EC(50): 55 nM vs 22.5 microM). The inactive PMA analogue, 4alpha-PMA, had no such effect. The cAMP production by PMA, but not histamine, was significantly reversed by Ro 31-8220 (1 microM) and the selective inhibitor of the novel PKCdelta, rottlerin (1-3 microM), but not the selective inhibitor of the classical PKC isoforms, Gö 6976 (0.01-0.1 microM). Western blot analysis revealed the presence of six PKC isoforms (alpha, betaI, betaII, delta, iota and zeta) in isolated eosinophils. Chelation of internal or external calcium had no effect on PMA-induced cAMP response, but abolished that induced by histamine. There was a good correlation between increase in intracellular cAMP and inhibition of degranulation. These results show, for the first time, that in human eosinophils, PMA, via activation of PKCdelta isoform, can stimulate cAMP production, and that this may be the basis for its potent anti-degranulatory effect.

  10. Protein kinase C activation inhibits eosinophil degranulation through stimulation of intracellular cAMP production

    PubMed Central

    Ezeamuzie, Charles I; Taslim, Najla

    2004-01-01

    The mechanism of inhibition of eosinophil degranulation by protein kinase C (PKC) was investigated in complement C5a (C5a)-stimulated degranulation of highly purified human eosinophils using the specific PKC activator – phorbol 12-myristate 13-acetate (PMA). C5a-induced release of eosinophil peroxidase and eosinophil cationic protein was potently inhibited in a concentration-dependent manner by PMA (IC50: 3 and 5 nM, respectively). The inhibition by PMA, but not histamine, was significantly reversed by the specific, but isoform nonselective, PKC inhibitor Ro 31-8220 (1 μM). In the presence of phosphodiesterase inhibitor rolipram (5 μM), PMA stimulated a pronounced concentration-dependent increase in intracellular cAMP, with a potency 400 times that of histamine (EC50: 55 nM vs 22.5 μM). The inactive PMA analogue, 4α-PMA, had no such effect. The cAMP production by PMA, but not histamine, was significantly reversed by Ro 31-8220 (1 μM) and the selective inhibitor of the novel PKCδ, rottlerin (1–3 μM), but not the selective inhibitor of the classical PKC isoforms, Gö 6976 (0.01–0.1 μM). Western blot analysis revealed the presence of six PKC isoforms (α, βI, βII, δ, ι and ζ) in isolated eosinophils. Chelation of internal or external calcium had no effect on PMA-induced cAMP response, but abolished that induced by histamine. There was a good correlation between increase in intracellular cAMP and inhibition of degranulation. These results show, for the first time, that in human eosinophils, PMA, via activation of PKCδ isoform, can stimulate cAMP production, and that this may be the basis for its potent anti-degranulatory effect. PMID:15504748

  11. 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

  12. Signals fly when kinases meet Rho-of-plants (ROP) small G-proteins.

    PubMed

    Fehér, Attila; Lajkó, Dézi Bianka

    2015-08-01

    Rho-type small GTP-binding plant proteins function as two-state molecular switches in cellular signalling. There is accumulating evidence that Rho-of-plants (ROP) signalling is positively controlled by plant receptor kinases, through the ROP guanine nucleotide exchange factor proteins. These signalling modules regulate cell polarity, cell shape, hormone responses, and pathogen defence, among other things. Other ROP-regulatory proteins might also be subjected to protein phosphorylation by cellular kinases (e.g., mitogen-activated protein kinases or calcium-dependent protein kinases), in order to integrate various cellular signalling pathways with ROP GTPase-dependent processes. In contrast to the role of kinases in upstream ROP regulation, much less is known about the potential link between ROP GTPases and downstream kinase signalling. In other eukaryotes, Rho-type G-protein-activated kinases are widespread and have a key role in many cellular processes. Recent data indicate the existence of structurally different ROP-activated kinases in plants, but their ROP-dependent biological functions still need to be validated. In addition to these direct interactions, ROPs may also indirectly control the activity of mitogen-activated protein kinases or calcium-dependent protein kinases. These kinases may therefore function as upstream as well as downstream kinases in ROP-mediated signalling pathways, such as the phosphatidylinositol monophosphate kinases involved in cell polarity establishment. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. AMP-activated protein kinase and metabolic control

    PubMed Central

    Viollet, Benoit; Andreelli, Fabrizio

    2011-01-01

    AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, is a major regulator of cellular and whole-body energy homeostasis that coordinates metabolic pathways in order to balance nutrient supply with energy demand. It is now recognized that pharmacological activation of AMPK improves blood glucose homeostasis, lipid profile and blood pressure in insulin-resistant rodents. Indeed, AMPK activation mimics the beneficial effects of physical activity or those of calorie restriction by acting on multiple cellular targets. In addition it is now demonstrated that AMPK is one of the probable (albeit indirect) targets of major antidiabetic drugs including, the biguanides (metformin) and thiazolidinediones, as well as of insulin sensitizing adipokines (e.g., adiponectin). Taken together, such findings highlight the logic underlying the concept of targeting the AMPK pathway for the treatment of metabolic syndrome and type 2 diabetes. PMID:21484577

  14. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  15. The Protein Kinase RSK Family - Roles in Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    of the prodrug, kaempferol 3-O-(2",3",4"-tri-O-acetyl-a-L-rhamnopyranoside)(3Ac- SL0101), a novel inhibitor of the Ser/Thr protein kinase, RSK...Y, Holman NJ, Hecht SM, Lannigan DA. Synthesis of the prodrug, kaempferol 3-O-(2", 3", 4"-tri-O-acetyl-a-L-rhamnopyranoside) (3Ac-SLO1 01), a novel

  16. Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

    PubMed Central

    Johnston, Jonathan D; Price, Sally A; Bristow, David R

    1998-01-01

    Acute flunitrazepam (1 μM) exposure for 1 h reduced GABAA receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABAA receptor protein expression, that appears to be dependent on protein kinase C activity. PMID:9723942

  17. Propranolol Targets Hemangioma Stem Cells via cAMP and Mitogen-Activated Protein Kinase Regulation

    PubMed Central

    Munabi, Naikhoba C.O.; England, Ryan W.; Edwards, Andrew K.; Kitajewski, Alison A.; Tan, Qian Kun; Weinstein, Andrew; Kung, Justin E.; Wilcox, Maya; Kitajewski, Jan K.; Shawber, Carrie J.

    2016-01-01

    Infantile hemangiomas (IHs) are the most common vascular tumor and arise from a hemangioma stem cell (HemSC). Propranolol has proved efficacious for problematic IHs. Propranolol is a nonselective β-adrenergic receptor (βAR) antagonist that can lower cAMP levels and activate the mitogen-activated protein kinase (MAPK) pathway downstream of βARs. We found that HemSCs express β1AR and β2AR in proliferating IHs and determined the role of these βARs and the downstream pathways in mediating propranolol’s effects. In isolated HemSCs, propranolol suppressed cAMP levels and activated extracellular signal-regulated kinase (ERK)1/2 in a dose-dependent fashion. Propranolol, used at doses of <10−4 M, reduced cAMP levels and decreased HemSC proliferation and viability. Propranolol at ≥10−5 M reduced cAMP levels and activated ERK1/2, and this correlated with HemSC apoptosis and cytotoxicity at ≥10−4 M. Stimulation with a βAR agonist, isoprenaline, promoted HemSC proliferation and rescued the antiproliferative effects of propranolol, suggesting that propranolol inhibits βAR signaling in HemSCs. Treatment with a cAMP analog or a MAPK inhibitor partially rescued the HemSC cell viability suppressed by propranolol. A selective β2AR antagonist mirrored propranolol’s effects on HemSCs in a dose-dependent fashion, and a selective β1AR antagonist had no effect, supporting a role for β2AR signaling in IH pathobiology. In a mouse model of IH, propranolol reduced the vessel caliber and blood flow assessed by ultrasound Doppler and increased activation of ERK1/2 in IH cells. We have thus demonstrated that propranolol acts on HemSCs in IH to suppress proliferation and promote apoptosis in a dose-dependent fashion via β2AR perturbation, resulting in reduced cAMP and MAPK activation. Significance The present study investigated the action of propranolol in infantile hemangiomas (IHs). IHs are the most common vascular tumor in children and have been proposed to arise from

  18. Propranolol Targets Hemangioma Stem Cells via cAMP and Mitogen-Activated Protein Kinase Regulation.

    PubMed

    Munabi, Naikhoba C O; England, Ryan W; Edwards, Andrew K; Kitajewski, Alison A; Tan, Qian Kun; Weinstein, Andrew; Kung, Justin E; Wilcox, Maya; Kitajewski, Jan K; Shawber, Carrie J; Wu, June K

    2016-01-01

    Infantile hemangiomas (IHs) are the most common vascular tumor and arise from a hemangioma stem cell (HemSC). Propranolol has proved efficacious for problematic IHs. Propranolol is a nonselective β-adrenergic receptor (βAR) antagonist that can lower cAMP levels and activate the mitogen-activated protein kinase (MAPK) pathway downstream of βARs. We found that HemSCs express β1AR and β2AR in proliferating IHs and determined the role of these βARs and the downstream pathways in mediating propranolol's effects. In isolated HemSCs, propranolol suppressed cAMP levels and activated extracellular signal-regulated kinase (ERK)1/2 in a dose-dependent fashion. Propranolol, used at doses of <10(-4) M, reduced cAMP levels and decreased HemSC proliferation and viability. Propranolol at ≥10(-5) M reduced cAMP levels and activated ERK1/2, and this correlated with HemSC apoptosis and cytotoxicity at ≥10(-4) M. Stimulation with a βAR agonist, isoprenaline, promoted HemSC proliferation and rescued the antiproliferative effects of propranolol, suggesting that propranolol inhibits βAR signaling in HemSCs. Treatment with a cAMP analog or a MAPK inhibitor partially rescued the HemSC cell viability suppressed by propranolol. A selective β2AR antagonist mirrored propranolol's effects on HemSCs in a dose-dependent fashion, and a selective β1AR antagonist had no effect, supporting a role for β2AR signaling in IH pathobiology. In a mouse model of IH, propranolol reduced the vessel caliber and blood flow assessed by ultrasound Doppler and increased activation of ERK1/2 in IH cells. We have thus demonstrated that propranolol acts on HemSCs in IH to suppress proliferation and promote apoptosis in a dose-dependent fashion via β2AR perturbation, resulting in reduced cAMP and MAPK activation. The present study investigated the action of propranolol in infantile hemangiomas (IHs). IHs are the most common vascular tumor in children and have been proposed to arise from a hemangioma

  19. Protein kinase d in the cardiovascular system: emerging roles in health and disease.

    PubMed

    Avkiran, Metin; Rowland, Alexandra J; Cuello, Friederike; Haworth, Robert S

    2008-02-01

    The protein kinase D (PKD) family is a recent addition to the calcium/calmodulin-dependent protein kinase group of serine/threonine kinases, within the protein kinase complement of the mammalian genome. Relative to their alphabetically superior cousins in the AGC group of kinases, namely the various isoforms of protein kinase A, protein kinase B/Akt, and protein kinase C, PKD family members have to date received limited attention from cardiovascular investigators. Nevertheless, increasing evidence now points toward important roles for PKD-mediated signaling pathways in the cardiovascular system, particularly in the regulation of myocardial contraction, hypertrophy and remodeling. This review provides a primer on PKD signaling, using information gained from studies in multiple cell types, and discusses recent data that suggest novel functions for PKD-mediated pathways in the heart and the circulation.

  20. Synthetic phosphorylation of p38α recapitulates protein kinase activity.

    PubMed

    Chooi, K Phin; Galan, Sébastien R G; Raj, Ritu; McCullagh, James; Mohammed, Shabaz; Jones, Lyn H; Davis, Benjamin G

    2014-02-05

    Through a "tag-and-modify" protein chemical modification strategy, we site-selectively phosphorylated the activation loop of protein kinase p38α. Phosphorylation at natural (180) and unnatural (172) sites created two pure phospho-forms. p38α bearing only a single phosphocysteine (pCys) as a mimic of pThr at 180 was sufficient to switch the kinase to an active state, capable of processing natural protein substrate ATF2; 172 site phosphorylation did not. In this way, we chemically recapitulated triggering of a relevant segment of the MAPK-signaling pathway in vitro. This allowed detailed kinetic analysis of global and stoichiometric phosphorylation events catalyzed by p38α and revealed that site 180 is a sufficient activator alone and engenders dominant mono-phosphorylation activity. Moreover, a survey of kinase inhibition using inhibitors with different (Type I/II) modes (including therapeutically relevant) revealed unambiguously that Type II inhibitors inhibit phosphorylated p38α and allowed discovery of a predictive kinetic analysis based on cooperativity to distinguish Type I vs II.

  1. Protein kinase CK2: a newcomer in the 'druggable kinome'.

    PubMed

    Pagano, M A; Cesaro, L; Meggio, F; Pinna, L A

    2006-12-01

    The acronym CK2 (derived from the misnomer 'casein kinase' 2) denotes one of the most pleiotropic members of the eukaryotic protein kinase superfamily, characterized by an acidic consensus sequence in which a carboxylic acid (or pre-phosphorylated) side chain at position n+3 relative to the target serine/threonine residue plays a crucial role. The latest repertoire of CK2 substrates includes approx. 300 proteins, but the analysis of available phosphopeptide databases from different sources suggests that CK2 alone may be responsible for the generation of a much larger proportion (10-20%) of the eukaryotic phosphoproteome. Although for the time being CK2 is not included among protein kinases whose inhibitors are in clinical practice or in advanced clinical trials, evidence is accumulating that elevated CK2 constitutive activity co-operates to induce a number of pathological conditions, including cancer, infectious diseases, neurodegeneration and cardiovascular pathologies. The development and usage of cell-permeant, selective inhibitors discloses a scenario whereby CK2 plays a global anti-apoptotic role, which under special circumstances may lead to untimely and pathogenic cell survival.

  2. Substrate thiophosphorylation by Arabidopsis mitogen-activated protein kinases.

    PubMed

    Leissing, Franz; Nomoto, Mika; Bocola, Marco; Schwaneberg, Ulrich; Tada, Yasuomi; Conrath, Uwe; Beckers, Gerold J M

    2016-02-24

    Mitogen-activated protein kinase (MPK) cascades are important to cellular signaling in eukaryotes. They regulate growth, development and the response to environmental challenges. MPK cascades function via reversible phosphorylation of cascade components, MEKK, MEK, and MPK, but also by MPK substrate phosphorylation. Using mass spectrometry, we previously identified many in vivo MPK3 and MPK6 substrates in Arabidopsis thaliana, and we disclosed their phosphorylation sites. We verified phosphorylation of several of our previously identified MPK3/6 substrates using a nonradioactive in vitro labeling assay. We engineered MPK3, MPK4, and MPK6 to accept bio-orthogonal ATPγS analogs for thiophosphorylating their appropriate substrate proteins. Subsequent alkylation of the thiophosphorylated amino acid residue(s) allows immunodetection using thiophosphate ester-specific antibodies. Site-directed mutagenesis of amino acids confirmed the protein substrates' site-specific phosphorylation by MPK3 and MPK6. A combined assay with MPK3, MPK6, and MPK4 revealed substrate specificity of the individual kinases. Our work demonstrates that the in vitro-labeling assay represents an effective, specific and highly sensitive test for determining kinase-substrate relationships.

  3. A cyclic nucleotide-independent protein kinase in Leishmania donovani.

    PubMed Central

    Das, S; Saha, A K; Mukhopadhyay, N K; Glew, R H

    1986-01-01

    Leishmania donovani promastigotes labelled for 2 h with 32Pi incorporated radioactivity into at least 21 different proteins, as determined by SDS/polyacrylamide-gel electrophoresis. Pulse-chase studies with 32Pi demonstrated that the labelled proteins were in a dynamic state: some radiolabelled proteins rapidly disappeared and others appeared after the chase. The possibility of an ectokinase on the parasite was examined; incubation of intact parasites for 10 min at 25 degrees C in an osmotically buffered medium containing [gamma-32P]ATP, but not [alpha-32P]ATP, resulted in the labelling of 10 different protozoal proteins, presumably localized to the surface of the organism's plasma membrane. Intact promastigotes also catalysed the transfer of 32P from [gamma-32P]ATP to histones. The histone-dependent kinase was solubilized by repeated freezing and thawing, and sonication, and purified 118-fold by chromatographing the high-speed (200,000 g, 1 h) supernatant fraction on QAE-Sephadex, Sephadex G-150 and hydroxyapatite columns. The kinase eluted as a single activity peak from all three columns. The partially purified histone-dependent kinase had the following properties: pH optimum, 7.0; optimum temperature, 37 degrees C; Km for mixed calf thymus histone, 0.15 mM; Km for ATP, 0.8 mM; preferred fractionated histone acceptors, H2b greater than H4 greater than H2a greater than H3 (H1 does not serve as an acceptor); optimum activity required 10-20 mM-Mg2+; inhibited 50-80% by 0.01 mM- and 1 mM-Ca2+; activity was not stimulated by calmodulin, cyclic AMP (1 mM) or cyclic GMP (1 mM) nor inhibited by a cyclic AMP-dependent protein kinase inhibitor (50 micrograms/assay); apparent Mr 75,000, as determined by Sephadex G-150 gel filtration chromatography; phosphorylated exclusively serine residues. Protein kinase activity was low in the early exponential phase of the growth curve and increased 6-fold upon entry into the stationary phase. PMID:3030283

  4. Protein kinases as targets for interventive biogerontology: overview and perspectives.

    PubMed

    Lai, Wing-Fu

    2012-04-01

    Protein kinases are enzymes that catalyze the transfer of γ phosphate from adenosine triphosphate to substrate proteins, and are important signal transduction mediators in a diversity of biological processes, ranging from apoptosis to energy metabolism. In this article, we will take this prominent class of proteins as an example to illustrate the involvement of proteins in modulation of aging and to highlight the prospects and challenges of protein-targeted interventions for anti-aging purposes. It is hoped that through this article, more empirical work on interventive gerontology will follow, and with collaborative endeavors among researchers, hurdles in anti-aging intervention development can be overcome in the near future. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation.

    PubMed

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-04-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kappaB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  6. Protein kinase D activity controls endothelial nitric oxide synthesis.

    PubMed

    Aicart-Ramos, Clara; Sánchez-Ruiloba, Lucía; Gómez-Parrizas, Mónica; Zaragoza, Carlos; Iglesias, Teresa; Rodríguez-Crespo, Ignacio

    2014-08-01

    Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone. © 2014. Published by The Company of Biologists Ltd.

  7. Implications of mitogen-activated protein kinase signaling in glioma.

    PubMed

    Pandey, Vimal; Bhaskara, Vasantha Kumar; Babu, Phanithi Prakash

    2016-02-01

    Gliomas are the most common primary central nervous system tumors. Gliomas originate from astrocytes, oligodendrocytes, and neural stem cells or their precursors. According to WHO classification, gliomas are classified into four different malignant grades ranging from grade I to grade IV based on histopathological features and related molecular aberrations. The induction and maintenance of these tumors can be attributed largely to aberrant signaling networks. In this regard, the mitogen-activated protein kinase (MAPK) network has been widely studied and is reported to be severely altered in glial tumors. Mutations in MAPK pathways most frequently affect RAS and B-RAF in the ERK, c-Jun N-terminal kinase (JNK), and p38 pathways leading to malignant transformation. Also, it is linked to both inherited and sequential accumulations of mutations that control receptor tyrosine kinase (RTK)-activated signal transduction pathways, cell cycle growth arrest pathways, and nonresponsive cell death pathways. Genetic alterations that modulate RTK signaling can also alter several downstream pathways, including RAS-mediated MAP kinases along with JNK pathways, which ultimately regulate cell proliferation and cell death. The present review focuses on recent literature regarding important deregulations in the RTK-activated MAPK pathway during gliomagenesis and progression.

  8. A catalytically inactive form of protein kinase C-associated kinase/receptor interacting protein 4, a protein kinase C beta-associated kinase that mediates NF-kappa B activation, interferes with early B cell development.

    PubMed

    Cariappa, Annaiah; Chen, Luojing; Haider, Khaleda; Tang, Mei; Nebelitskiy, Eugene; Moran, Stewart T; Pillai, Shiv

    2003-08-15

    Protein kinase C-associated kinase (PKK)/receptor interacting protein 4 (RIP4) is a protein kinase C (PKC) beta-associated kinase that links PKC to NF-kappaB activation. The kinase domain of PKK is similar to that of RIP, RIP2, and RIP3. We show in this study that PKK is expressed early during lymphocyte development and can be detected in common lymphoid progenitor cells. Targeting of a catalytically inactive version of PKK to lymphoid cells resulted in a marked impairment in pro-B cell generation in the bone marrow. Although peripheral B cell numbers were markedly reduced, differentiation into follicular and marginal zone B cells was not defective in these mice. B-1a and B-1b B cells could not be detected in these mice, but this might be a reflection of the overall defect in B cell production observed in these animals. In keeping with a possible link to PKCbeta, peripheral B cells in these mice exhibit a defect in anti-IgM-mediated proliferation. These studies suggest that PKK may be required early in B cell development and for BCR-mediated B cell proliferation.

  9. Activation of S6 kinase in human neutrophils by calcium pyrophosphate dihydrate crystals: protein kinase C-dependent and phosphatidylinositol-3-kinase-independent pathways.

    PubMed Central

    Tudan, C; Jackson, J K; Charlton, L; Pelech, S L; Sahl, B; Burt, H M

    1998-01-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown previously to be a central enzyme in crystal-induced neutrophil activation. Since activation of the 70 kDa S6 kinase (p70S6K) has been shown to be dependent on PI 3-kinase activation in mammalian cells, and since the former is a key enzyme in the transmission of signals to the cell nucleus, activation of p70(S6K) was investigated in crystal-stimulated neutrophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD)-crystal-activated neutrophils were separated by Mono Q chromatography and analysed for phosphotransferase activity using a range of substrates and probed by Western analysis using antibodies to p70(S6K) and mitogen-activated protein kinase (MAP kinase). CPPD crystals induced a robust, transient activation (peak activity at 2 min) of p70(S6K) that was fully inhibited by pretreatment with rapamycin. This is the first report of the activation of p70(S6K) in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p70(S6K) could also be inhibited by a protein kinase C (PKC) inhibitor (Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD crystals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive) and protein kinase B (wortmannin sensitive) in neutrophils. These data suggest that activation of p70(S6K) may proceed through a PI 3-kinase- and protein kinase B-independent but PKC-dependent pathway in crystal-activated neutrophils. PMID:9531494

  10. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

    PubMed Central

    Matsui, T; Amano, M; Yamamoto, T; Chihara, K; Nakafuku, M; Ito, M; Nakano, T; Okawa, K; Iwamatsu, A; Kaibuchi, K

    1996-01-01

    The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway. Images PMID:8641286

  11. Protein Kinase C: An Attractive Target for Cancer Therapy

    PubMed Central

    Marengo, Barbara; De Ciucis, Chiara; Ricciarelli, Roberta; Pronzato, Maria A.; Marinari, Umberto M.; Domenicotti, Cinzia

    2011-01-01

    Apoptosis plays an important role during all stages of carcinogenesis and the development of chemoresistance in tumor cells may be due to their selective defects in the intracellular signaling proteins, central to apoptotic pathways. Consequently, many studies have focused on rendering the chemotherapy more effective in order to prevent chemoresistance and pre-clinical and clinical data has suggested that protein kinase C (PKC) may represent an attractive target for cancer therapy. Therefore, a complete understanding of how PKC regulates apoptosis and chemoresistance may lead to obtaining a PKC-based therapy that is able to reduce drug dosages and to prevent the development of chemoresistance. PMID:24212628

  12. Protein Kinase A Opposes the Phosphorylation-dependent Recruitment of Glycogen Synthase Kinase 3β to A-kinase Anchoring Protein 220*

    PubMed Central

    Whiting, Jennifer L.; Nygren, Patrick J.; Tunquist, Brian J.; Langeberg, Lorene K.; Seternes, Ole-Morten; Scott, John D.

    2015-01-01

    The proximity of an enzyme to its substrate can influence rate and magnitude of catalysis. A-kinase anchoring protein 220 (AKAP220) is a multivalent anchoring protein that can sequester a variety of signal transduction enzymes. These include protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Using a combination of molecular and cellular approaches we show that GSK3β phosphorylation of Thr-1132 on AKAP220 initiates recruitment of this kinase into the enzyme scaffold. We also find that AKAP220 anchors GSK3β and its substrate β-catenin in membrane ruffles. Interestingly, GSK3β can be released from the multienzyme complex in response to PKA phosphorylation on serine 9, which suppresses GSK3β activity. The signaling scaffold may enhance this regulatory mechanism, as AKAP220 has the capacity to anchor two PKA holoenzymes. Site 1 on AKAP220 (residues 610–623) preferentially interacts with RII, whereas site 2 (residues 1633–1646) exhibits a dual specificity for RI and RII. In vitro affinity measurements revealed that site 2 on AKAP220 binds RII with ∼10-fold higher affinity than site 1. Occupancy of both R subunit binding sites on AKAP220 could provide a mechanism to amplify local cAMP responses and enable cross-talk between PKA and GSK3β. PMID:26088133

  13. Phospholipid metabolism and protein kinase C mediated protein phosphorylation in dietary protein deficiency in rat lung.

    PubMed

    Bansal, Surendra K; Kathayat, Rachna; Tyagi, Manoj; Taneja, Krishna K; Basir, Seemi F

    2005-07-01

    Nutritional deprivation of proteins decreases the protein kinase C (PKC) activity in rat lung. The activity of (PKC) is influenced by lipid metabolism. Changes in PKC activity may influence phosphorylation of its substrate proteins in the tissues. Therefore, alterations in phospholipid metabolism and PKC mediated protein phosphorylation in dietary protein deficiency in rat lung were envisaged. The study was conducted on rats fed on three different types of diet viz., casein (20% protein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threoning). Feeding of protein deficient diet caused reduction in incorporation of [3H] myo-inositol in the total phosphoinositides in lungs and an increase in total inositol phosphate pool. There was a significant reduction in the contents and turnover rate of phosphatidyl inositol and phosphatidyl inositol monophosphate. Supplementation of diet with L-lysine and DL-threonine had a reversing effect on total pool of phosphoinositides and, the metabolism of phosphatidyl inositol bisphosphate and phosphatidyl inositol. In phosphatidyl choline metabolism, the dietary protein deficiency led to a decrease in incorporation of [14C-methyl] choline-chloride in total phospholipids. In contrast, its incorporation increased in phosphatidyl choline pool. The contents of phosphatidyl choline and residue, incorporation of [14C-methyl] choline-chloride in them and their turnover rate also increased. Supplementation of diet had a reversal effect on most of these parameters. Phosphorylation of proteins of 84, 47, 35 and 16 kDa was identified to be mediated by PKC. In dietary protein deficiency, phosphorylation of all these proteins, except that of 47 kDa, increased. Supplementation of diet reversed the pattern except that of 84 kDa. The findings suggest that changes in phospholipid metabolism in dietary protein deficiency may effect the activity of PKC thereby influencing the

  14. The DNA-dependent protein kinase: A multifunctional protein kinase with roles in DNA double strand break repair and mitosis.

    PubMed

    Jette, Nicholas; Lees-Miller, Susan P

    2015-03-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes.

  15. Pivotal Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in Inflammatory Pulmonary Diseases

    PubMed Central

    Qian, Feng; Deng, Jing; Wang, Gang; Ye, Richard D.; Christman, John W.

    2016-01-01

    Mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2) is exclusively regulated by p38 MAPK in vivo. Upon activation of p38 MAPK, MK2 binds with p38 MAPK, leading to phosphorylation of TTP, Hsp27, Akt and Cdc25 that are involved in regulation of various essential cellular functions. In this review, we discuss current knowledge about molecular mechanisms of MK2 in regulation of TNF-α production, NADPH oxidase activation, neutrophil migration, and DNA-damage-induced cell cycle arrest which are involved in the molecular pathogenesis of acute lung injury, pulmonary fibrosis, and non-small-cell lung cancer. Collectively current and emerging new information indicate that developing MK2 inhibitors and blocking MK2-mediated signal pathways is a potential therapeutic strategy for treatment of inflammatory and fibrotic lung diseases and lung cancer. PMID:26119506

  16. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

    PubMed Central

    Jette, Nicholas; Lees-Miller, Susan P.

    2015-01-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes. PMID:25550082

  17. Protein kinase Cepsilon is important for migration of neuroblastoma cells

    PubMed Central

    Stensman, Helena; Larsson, Christer

    2008-01-01

    Background Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility. Methods PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot. Results Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS. Conclusion PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration. PMID:19077250

  18. A Quantitative Mass Spectrometry-based Approach for Identifying Protein Kinase-Clients and Quantifying Kinase Activity

    USDA-ARS?s Scientific Manuscript database

    The Homo sapiens and Arabidopsis thaliana genomes are believed to encode >500 and >1,000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Mass spectrometry (MS)-based approaches have been integral to the large-scale mapp...

  19. Discovery of cellular substrates for protein kinase A using a peptide array screening protocol.

    PubMed

    Smith, F Donelson; Samelson, Bret K; Scott, John D

    2011-08-15

    Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth. © The Authors Journal compilation © 2011 Biochemical Society

  20. A role for protein kinase casein kinase2 α-subunits in the Arabidopsis circadian clock.

    PubMed

    Lu, Sheen X; Liu, Hongtao; Knowles, Stephen M; Li, Jian; Ma, Ligeng; Tobin, Elaine M; Lin, Chentao

    2011-11-01

    Circadian rhythms are autoregulatory, endogenous rhythms with a period of approximately 24 h. A wide variety of physiological and molecular processes are regulated by the circadian clock in organisms ranging from bacteria to humans. Phosphorylation of clock proteins plays a critical role in generating proper circadian rhythms. Casein Kinase2 (CK2) is an evolutionarily conserved serine/threonine protein kinase composed of two catalytic α-subunits and two regulatory β-subunits. Although most of the molecular components responsible for circadian function are not conserved between kingdoms, CK2 is a well-conserved clock component modulating the stability and subcellular localization of essential clock proteins. Here, we examined the effects of a cka1a2a3 triple mutant on the Arabidopsis (Arabidopsis thaliana) circadian clock. Loss-of-function mutations in three nuclear-localized CK2α subunits result in period lengthening of various circadian output rhythms and central clock gene expression, demonstrating that the cka1a2a3 triple mutant affects the pace of the circadian clock. Additionally, the cka1a2a3 triple mutant has reduced levels of CK2 kinase activity and CIRCADIAN CLOCK ASSOCIATED1 phosphorylation in vitro. Finally, we found that the photoperiodic flowering response, which is regulated by circadian rhythms, was reduced in the cka1a2a3 triple mutant and that the plants flowered later under long-day conditions. These data demonstrate that CK2α subunits are important components of the Arabidopsis circadian system and their effects on rhythms are in part due to their phosphorylation of CIRCADIAN CLOCK ASSOCIATED1.

  1. Atypical protein kinase C controls sea urchin ciliogenesis

    PubMed Central

    Prulière, Gérard; Cosson, Jacky; Chevalier, Sandra; Sardet, Christian; Chenevert, Janet

    2011-01-01

    The atypical protein kinase C (aPKC) is part of the conserved aPKC/PAR6/PAR3 protein complex, which regulates many cell polarity events, including the formation of a primary cilium at the apical surface of epithelial cells. Cilia are highly organized, conserved, microtubule-based structures involved in motility, sensory processes, signaling, and cell polarity. We examined the distribution and function of aPKC in the sea urchin embryo, which forms a swimming blastula covered with motile cilia. We found that in the early embryo aPKC is uniformly cortical and becomes excluded from the vegetal pole during unequal cleavages at the 8- to 64-cell stages. During the blastula and gastrula stages the kinase localizes at the base of cilia, forming a ring at the transition zone between the basal body and the elongating axoneme. A dose-dependent and reversible inhibition of aPKC results in mislocalization of the kinase, defective ciliogenesis, and lack of swimming. Thus, as in the primary cilium of differentiated mammalian cells, aPKC controls the growth of motile cilia in invertebrate embryos. We suggest that aPKC might function to phosphorylate kinesin and so activate the transport of intraflagellar vesicles. PMID:21508313

  2. Calcium-Dependent Protein Kinase Genes in Corn Roots

    NASA Technical Reports Server (NTRS)

    Takezawa, D.; Patil, S.; Bhatia, A.; Poovaiah, B. W.

    1996-01-01

    Two cDNAs encoding Ca-2(+) - Dependent Protein Kinases (CDPKs), Corn Root Protein Kinase 1 and 2 (CRPK 1, CRPK 2) were isolated from the root tip library of corn (Zea mays L., cv. Merit) and their nucleotide sequences were determined. Deduced amino acid sequences of both the clones have features characteristic of plant CDPKS, including all 11 conserved serine/threonine kinase subdomains, a junction domain and a calmodulin-like domain with four Ca-2(+), -binding sites. Northern analysis revealed that CRPKI mRNA is preferentially expressed in roots, especially in the root tip; whereas, the expression of CRPK2 mRNA was very low in all the tissues tested. In situ hybridization experiments revealed that CRPKI mRNA is highly expressed in the root apex, as compared to other parts of the root. Partially purified CDPK from the root tip phosphorylates syntide-2, a common peptide substrate for plant CDPKs, and the phosphorylation was stimulated 7-fold by the addition of Ca-2(+). Our results show that two CDPK isoforms are expressed in corn roots and they may be involved in the Ca-2(+)-dependent signal transduction process.

  3. Protein kinase Cι: human oncogene, prognostic marker and therapeutic target

    PubMed Central

    Fields, Alan P.; Regala, Roderick P.

    2009-01-01

    The Protein kinase C (PKC) family of serine/threonine kinases has been the subject of intensive study in the field of cancer since their initial discovery as major cellular receptors for the tumor promoting phorbol esters nearly thirty years ago. However, despite these efforts, the search for a direct genetic link between members of the PKC family and human cancer has yielded only circumstantial evidence that any PKC isozyme is a true cancer gene. This situation changed in the past year with the discovery that atypical protein kinase C iota (PKCι) is a bonafide human oncogene. PKCι is required for the transformed growth of human cancer cells and the PKCι gene is the target of tumor-specific gene amplification in multiple forms of human cancer. PKCι participates in multiple aspects of the transformed phenotype of human cancer cells including transformed growth, invasion and survival. Herein, we review pertinent aspects of atypical PKC structure, function and regulation that relate to the role of these enzymes in oncogenesis. We discuss the evidence that PKCι is a human oncogene, review mechanisms controlling PKCι expression in human cancers, and describe the molecular details of PKCι-mediated oncogenic signaling. We conclude with a discussion of how oncogenic PKCι signaling has been successfully targeted to identify a novel, mechanism-based therapeutic drug currently entering clinical trials for treatment of human lung cancer. Throughout, we identify key unanswered questions and exciting future avenues of investigation regarding this important oncogenic molecule. PMID:17570678

  4. Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

    SciTech Connect

    Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

    2008-10-17

    We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK){zeta}, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGK{zeta} siRNA transfection decreased H{sub 2}O{sub 2}-induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGK{zeta} also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGK{zeta} rapidly translocated to the cytoplasm following H{sub 2}O{sub 2} treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGK{zeta}, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells.

  5. The New Role for an Old Kinase: Protein Kinase CK2 Regulates Metal Ion Transport

    PubMed Central

    Johnson, Adam J.; Wu, Ming J.

    2016-01-01

    The pleiotropic serine/threonine protein kinase CK2 was the first kinase discovered. It is renowned for its role in cell proliferation and anti-apoptosis. The complexity of this kinase is well reflected by the findings of past decades in terms of its heterotetrameric structure, subcellular location, constitutive activity and the extensive catalogue of substrates. With the advent of non-biased high-throughput functional genomics such as genome-wide deletion mutant screening, novel aspects of CK2 functionality have been revealed. Our recent discoveries using the model organism Saccharomyces cerevisiae and mammalian cells demonstrate that CK2 regulates metal toxicity. Extensive literature search reveals that there are few but elegant works on the role of CK2 in regulating the sodium and zinc channels. As both CK2 and metal ions are key players in cell biology and oncogenesis, understanding the details of CK2’s regulation of metal ion homeostasis has a direct bearing on cancer research. In this review, we aim to garner the recent data and gain insights into the role of CK2 in metal ion transport. PMID:28009816

  6. Genome-wide analysis and experimentation of plant serine/ threonine/tyrosine-specific protein kinases.

    PubMed

    Rudrabhatla, Parvathi; Reddy, Mamatha M; Rajasekharan, Ram

    2006-01-01

    Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogenesis. No classical protein tyrosine kinase has hitherto been cloned from plants. Does protein tyrosine kinase exist in plants? To address this, we have performed a genomic survey of protein tyrosine kinase motifs in plants using the delineated tyrosine phosphorylation motifs from the animal system. The Arabidopsis thaliana genome encodes 57 different protein kinases that have tyrosine kinase motifs. Animal non-receptor tyrosine kinases, SRC, ABL, LYN, FES, SEK, KIN and RAS have structural relationship with putative plant tyrosine kinases. In an extended analysis, animal receptor and non-receptor kinases, Raf and Ras kinases, mixed lineage kinases and plant serine/threonine/tyrosine (STY) protein kinases, form a well-supported group sharing a common origin within the superfamily of STY kinases. We report that plants lack bona fide tyrosine kinases, which raise an intriguing possibility that tyrosine phosphorylation is carried out by dual-specificity STY protein kinases in plants. The distribution pattern of STY protein kinase families on Arabidopsis chromosomes indicates that this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. Genome-wide analysis is supported by the functional expression and characterization of At2g24360 and phosphoproteomics of Arabidopsis. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, anti-phosphotyrosine immunoblotting, phosphoamino acid analysis and peptide mass fingerprinting. These results report the first comprehensive survey of genome-wide and tyrosine phosphoproteome analysis of plant STY protein kinases.

  7. Identification of protein kinase C activation as a novel mechanism for RGS2 protein upregulation through phenotypic screening of natural product extracts.

    PubMed

    Raveh, Avi; Schultz, Pamela J; Aschermann, Lauren; Carpenter, Colleen; Tamayo-Castillo, Giselle; Cao, Shugeng; Clardy, Jon; Neubig, Richard R; Sherman, David H; Sjögren, Benita

    2014-10-01

    Biochemical high-throughput screening is widely used in drug discovery, using a variety of small molecule libraries. However, broader screening strategies may be more beneficial to identify novel biologic mechanisms. In the current study we used a β-galactosidase complementation method to screen a selection of microbial-derived pre-fractionated natural product extracts for those that increase regulator of G protein signaling 2 (RGS2) protein levels. RGS2 is a member of a large family of proteins that all regulate signaling through G protein-coupled receptors (GPCRs) by accelerating GTPase activity on active Gα as well as through other mechanisms. RGS2(-/-) mice are hypertensive, show increased anxiety, and are prone to heart failure. RGS2 has a very short protein half-life due to rapid proteasomal degradation, and we propose that enhancement of RGS2 protein levels could be a beneficial therapeutic strategy. Bioassay-guided fractionation of one of the hit strains yielded a pure compound, Indolactam V, a known protein kinase C (PKC) activator, which selectively increased RGS2 protein levels in a time- and concentration-dependent manner. Similar results were obtained with phorbol 12-myristate 13-acetate as well as activation of the Gq-coupled muscarinic M3 receptor. The effect on RGS2 protein levels was blocked by the nonselective PKC inhibitor Gö6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), the PKCβ-selective inhibitor Ruboxastaurin, as well as small interfering RNA-mediated knockdown of PKCβ. Indolactam V-mediated increases in RGS2 protein levels also had functional effects on GPCR signaling. This study provides important proof-of-concept for our screening strategy and could define a negative feedback mechanism in Gq/Phospholipase C signaling through RGS2 protein upregulation.

  8. SARS-CoV nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity.

    PubMed

    Wei, Wei-Yen; Li, Hui-Chun; Chen, Chiung-Yao; Yang, Chee-Hing; Lee, Shen-Kao; Wang, Chia-Wen; Ma, Hsin-Chieh; Juang, Yue-Li; Lo, Shih-Yen

    2012-04-01

    The pathogenesis of SARS-CoV remains largely unknown. To study the function of the SARS-CoV nucleocapsid protein, we have conducted a yeast two-hybrid screening experiment to identify cellular proteins that may interact with the SARS-CoV nucleocapsid protein. Pyruvate kinase (liver) was found to interact with SARS-CoV nucleocapsid protein in this experiment. The binding domains of these two proteins were also determined using the yeast two-hybrid system. The physical interaction between the SARS-CoV nucleocapsid and cellular pyruvate kinase (liver) proteins was further confirmed by GST pull-down assay, co-immunoprecipitation assay and confocal microscopy. Cellular pyruvate kinase activity in hepatoma cells was repressed by SARS-CoV nucleocapsid protein in either transiently transfected or stably transfected cells. PK deficiency in red blood cells is known to result in human hereditary non-spherocytic hemolytic anemia. It is reasonable to assume that an inhibition of PKL activity due to interaction with SARS-CoV N protein is likely to cause the death of the hepatocytes, which results in the elevation of serum alanine aminotransferase and liver dysfunction noted in most SARS patients. Thus, our results suggest that SARS-CoV could reduce pyruvate kinase activity via its nucleocapsid protein, and this may in turn cause disease.

  9. Sensitization of TRPA1 by Protein Kinase A

    PubMed Central

    Meents, Jannis E.; Fischer, Michael J. M.; McNaughton, Peter A.

    2017-01-01

    The TRPA1 ion channel is expressed in nociceptive (pain-sensitive) somatosensory neurons and is activated by a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here, we investigate the enhancement of TRPA1 function caused by inflammatory mediators, which is thought to be important in lung conditions such as asthma and COPD. Protein kinase A is an important kinase acting downstream of inflammatory mediators to cause sensitization of TRPA1. By using site-directed mutagenesis, patch-clamp electrophysiology and calcium imaging we identify four amino acid residues, S86, S317, S428, and S972, as the principal targets of PKA-mediated phosphorylation and sensitization of TRPA1. PMID:28076424

  10. Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

    PubMed

    Altman, Amnon; Kong, Kok-Fai

    2016-05-20

    The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

  11. 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

  12. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase.

    PubMed

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

    2015-06-26

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

    PubMed Central

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun

    2016-01-01

    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  14. Altered protein kinase C regulation of pulmonary endothelial store- and receptor-operated Ca2+ entry after chronic hypoxia.

    PubMed

    Paffett, Michael L; Riddle, Melissa A; Kanagy, Nancy L; Resta, Thomas C; Walker, Benjimen R

    2010-09-01

    Chronic hypoxia (CH)-induced pulmonary hypertension is associated with decreased basal pulmonary artery endothelial cell (EC) Ca(2+), which correlates with reduced store-operated Ca(2+) (SOC) entry. Protein kinase C (PKC) attenuates SOC entry in ECs. Therefore, we hypothesized that PKC has a greater inhibitory effect on EC SOC and receptor-operated Ca(2+) entry after CH. To test this hypothesis, we assessed SOC in the presence or absence of the nonselective PKC inhibitor GF109203X [2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide] in freshly isolated, Fura-2-loaded ECs obtained from intrapulmonary arteries of control and CH rats (4 weeks at 0.5 atm). We found that SOC entry and 1-oleoyl-2-acetyl-sn-glycerol (OAG)- and ATP-induced Ca(2+) influx were attenuated in ECs from CH rats versus controls, and GF109203X restored SOC and OAG responses to the level of controls. In contrast, nonselective PKC inhibition with GF109203X or the selective PKC(epsilon) inhibitor myristoylated V1-2 attenuated ATP-induced Ca(2+) entry in ECs from control but not CH pulmonary arteries. ATP-induced Ca(2+) entry was also attenuated by the T-type voltage-gated Ca(2+) channel (VGCC) inhibitor mibefradil in control cells. Consistent with the presence of endothelial T-type VGCC, we observed depolarization-induced Ca(2+) influx in control cells that was inhibited by mibefradil. This response was largely absent in ECs from CH arteries. We conclude that CH enhances PKC-dependent inhibition of SOC- and OAG-induced Ca(2+) entry. Furthermore, these data suggest that CH may reduce the ATP-dependent Ca(2+) entry that is mediated, in part, by PKCepsilon and mibefradil-sensitive Ca(2+) channels in control cells.

  15. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    PubMed Central

    Villamor, Joji Grace; Kaschani, Farnusch; Colby, Tom; Oeljeklaus, Julian; Zhao, David; Kaiser, Markus; Patricelli, Matthew P.; van der Hoorn, Renier A. L.

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188. PMID:23722185

  16. Targeting of calcium/calmodulin-dependent protein kinase II.

    PubMed Central

    Colbran, Roger J

    2004-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) has diverse roles in virtually all cell types and it is regulated by a plethora of mechanisms. Local changes in Ca2+ concentration drive calmodulin binding and CaMKII activation. Activity is controlled further by autophosphorylation at multiple sites, which can generate an autonomously active form of the kinase (Thr286) or can block Ca2+/calmodulin binding (Thr305/306). The regulated actions of protein phosphatases at these sites also modulate downstream signalling from CaMKII. In addition, CaMKII targeting to specific subcellular microdomains appears to be necessary to account for the known signalling specificity, and targeting is regulated by Ca2+/calmodulin and autophosphorylation. The present review focuses on recent studies revealing the diversity of CaMKII interactions with proteins localized to neuronal dendrites. Interactions with various subunits of the NMDA (N-methyl-D-aspartate) subtype of glutamate receptor have attracted the most attention, but binding of CaMKII to cytoskeletal and several other regulatory proteins has also been reported. Recent reports describing the molecular basis of each interaction and their potential role in the normal regulation of synaptic transmission and in pathological situations are discussed. These studies have revealed fundamental regulatory mechanisms that are probably important for controlling CaMKII functions in many cell types. PMID:14653781

  17. Multiple functions of G protein-coupled receptor kinases.

    PubMed

    Watari, Kenji; Nakaya, Michio; Kurose, Hitoshi

    2014-03-06

    Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1-GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of β-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of β-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of β-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the β-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and β-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for β-arrestin-mediated signaling. Agonists that selectively activate GRK/β-arrestin-dependent signaling without affecting G protein signaling are known as β-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as

  18. The Predikin webserver: improved prediction of protein kinase peptide specificity using structural information

    PubMed Central

    Saunders, Neil F. W.

    2008-01-01

    The Predikin webserver allows users to predict substrates of protein kinases. The Predikin system is built from three components: a database of protein kinase substrates that links phosphorylation sites with specific protein kinase sequences; a perl module to analyse query protein kinases and a web interface through which users can submit protein kinases for analysis. The Predikin perl module provides methods to (i) locate protein kinase catalytic domains in a sequence, (ii) classify them by type or family, (iii) identify substrate-determining residues, (iv) generate weighted scoring matrices using three different methods, (v) extract putative phosphorylation sites in query substrate sequences and (vi) score phosphorylation sites for a given kinase, using optional filters. The web interface provides user-friendly access to each of these functions and allows users to obtain rapidly a set of predictions that they can export for further analysis. The server is available at http://predikin.biosci.uq.edu.au. PMID:18477637

  19. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27.

    PubMed

    Freshney, N W; Rawlinson, L; Guesdon, F; Jones, E; Cowley, S; Hsuan, J; Saklatvala, J

    1994-09-23

    An IL-1-stimulated protein kinase cascade resulting in phosphorylation of the small heat shock protein hsp27 has been identified in KB cells. It is distinct from the p42 MAP kinase cascade. An upstream activator kinase phosphorylated a 40 kDa kinase (p40) upon threonine and tyrosine residues, which in turn phosphorylated a 50 kDa kinase (p50) upon threonine (and some serine) residues. p50 phosphorylated hsp27 upon serine. p40 and p50 were purified to near homogeneity. All three components were inactivated by protein phosphatase 2A, and p40 was inactivated by protein tyrosine phosphatase 1B. The substrate specificity of p40 differed from that of p42 and p54 MAP kinases. The upstream activator was not a MAP kinase kinase. p50 resembled MAPKAPK-2 and may be identical.

  20. Prevention of neuronal apoptosis by phorbol ester-induced activation of protein kinase C: blockade of p38 mitogen-activated protein kinase.

    PubMed

    Behrens, M M; Strasser, U; Koh, J Y; Gwag, B J; Choi, D W

    1999-01-01

    Consistent with previous studies on cell lines and non-neuronal cells, specific inhibitors of protein kinase C induced mouse primary cultured neocortical neurons to undergo apoptosis. To examine the complementary hypothesis that activating protein kinase C would attenuate neuronal apoptosis, the cultures were exposed for 1 h to phorbol-12-myristate-13-acetate, which activated protein kinase C as evidenced by downstream enhancement of the mitogen-activated protein kinase pathway. Exposure to phorbol-12-myristate-13-acetate, or another active phorbol ester, phorbol-12,13-didecanoate, but not to the inactive ester, 4alpha-phorbol-12,13-didecanoate, markedly attenuated neuronal apoptosis induced by serum deprivation. Phorbol-12-myristate-13-acetate also attenuated neuronal apoptosis induced by exposure to beta-amyloid peptide 1-42, or oxygen-glucose deprivation in the presence of glutamate receptor antagonists. The neuroprotective effects of phorbol-12-myristate-13-acetate were blocked by brief (non-toxic) concurrent exposure to the specific protein kinase C inhibitors, but not by a specific mitogen-activated protein kinase 1 inhibitor. Phorbol-12-myristate-13-acetate blocked the induction of p38 mitogen-activated protein kinase activity and specific inhibition of this kinase by SB 203580 attenuated serum deprivation-induced apoptosis. c-Jun N-terminal kinase 1 activity was high at rest and not modified by phorbol-12-myristate-13-acetate treatment. These data strengthen the idea that protein kinase C is a key modulator of several forms of central neuronal apoptosis, in part acting through inhibition of p38 mitogen-activated protein kinase regulated pathways.

  1. Autophagy mediates nonselective RNA degradation in starving yeast

    PubMed Central

    Welter, Evelyn; Elazar, Zvulun

    2015-01-01

    During nitrogen starvation, a nonselective bulk degradation of cytosolic proteins and organelles including ribosomes, termed macro-autophagy (hereafter termed autophagy), is induced. The precise mechanism of RNA degradation by this pathway has not been yet elucidated. In this issue of the The EMBO Journal, Huang et al characterize an autophagy-dependent RNA catabolism in yeast and identify the enzymes responsible for the degradation process. PMID:25492883

  2. Protein kinases: mechanisms and downstream targets in inflammation-mediated obesity and insulin resistance.

    PubMed

    Nandipati, Kalyana C; Subramanian, Saravanan; Agrawal, Devendra K

    2017-02-01

    Obesity-induced low-grade inflammation (metaflammation) impairs insulin receptor signaling. This has been implicated in the development of insulin resistance. Insulin signaling in the target tissues is mediated by stress kinases such as p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, inhibitor of NF-kB kinase complex β (IKKβ), AMP-activated protein kinase, protein kinase C, Rho-associated coiled-coil containing protein kinase, and RNA-activated protein kinase. Most of these kinases phosphorylate several key regulators in glucose homeostasis. The phosphorylation of serine residues in the insulin receptor and IRS-1 molecule results in diminished enzymatic activity in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. This has been one of the key mechanisms observed in the tissues that are implicated in insulin resistance especially in type 2 diabetes mellitus (T2-DM). Identifying the specific protein kinases involved in obesity-induced chronic inflammation may help in developing the targeted drug therapies to minimize the insulin resistance. This review is focused on the protein kinases involved in the inflammatory cascade and molecular mechanisms and their downstream targets with special reference to obesity-induced T2-DM.

  3. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

    PubMed Central

    Prasad, K V; Janssen, O; Kapeller, R; Raab, M; Cantley, L C; Rudd, C E

    1993-01-01

    The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8394019

  4. Protein kinase C-zeta and protein kinase B regulate distinct steps of insulin endocytosis and intracellular sorting.

    PubMed

    Fiory, Francesca; Oriente, Francesco; Miele, Claudia; Romano, Chiara; Trencia, Alessandra; Alberobello, Anna Teresa; Esposito, Iolanda; Valentino, Rossella; Beguinot, Francesco; Formisano, Pietro

    2004-03-19

    We have investigated the molecular mechanisms regulating insulin internalization and intracellular sorting. Insulin internalization was decreased by 50% upon incubation of the cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. PI3K inhibition also reduced insulin degradation and intact insulin release by 50 and 75%, respectively. Insulin internalization was reduced by antisense inhibition of protein kinase C-zeta (PKCzeta) expression and by overexpression of a dominant negative PKCzeta mutant (DN-PKCzeta). Conversely, overexpression of PKCzeta increased insulin internalization as a function of the PKCzeta levels achieved in the cells. Expression of wild-type protein kinase B (PKB)-alpha or of a constitutively active form (myr-PKB) did not significantly alter insulin internalization and degradation but produced a 100% increase of intact insulin release. Inhibition of PKB by a dominant negative mutant (DN-PKB) or by the pharmacological inhibitor ML-9 reduced intact insulin release by 75% with no effect on internalization and degradation. In addition, overexpression of Rab5 completely rescued the effect of PKCzeta inhibition on insulin internalization but not that of PKB inhibition on intact insulin recycling. Indeed, PKCzeta bound to and activated Rab5. Thus, PI3K controls different steps within the insulin endocytic itinerary. PKCzeta appears to mediate the PI3K effect on insulin internalization in a Rab5-dependent manner, whereas PKB directs intracellular sorting toward intact insulin release.

  5. Characterization of serine 916 as an in vivo autophosphorylation site for protein kinase D/Protein kinase Cmu.

    PubMed

    Matthews, S A; Rozengurt, E; Cantrell, D

    1999-09-10

    Activation of the serine kinase protein kinase D (PKD)/PKCmicro is controlled by the phosphorylation of two serine residues within its activation loop via a PKC-dependent signaling cascade. In this study we have identified the C-terminal serine 916 residue as an in vivo phosphorylation site within active PKD/PKCmu. An antibody that recognized PKD/PKCmu proteins specifically phosphorylated on the serine 916 residue was generated and used to show that phosphorylation of Ser-916 is induced by phorbol ester treatment of cells. Thus, the pS916 antibody is a useful tool to study the regulation of PKD/PKCmu activity in vivo. Antigen receptor ligation of T and B lymphocytes also induced phosphorylation of the serine 916 residue of PKD/PKCmu. Furthermore the regulatory FcgammaRIIB receptor, which mediates vital negative feedback signals to the B cell antigen receptor complex, inhibited the antigen receptor-induced activation and serine 916 phosphorylation of PKD/PKCmu. The degree of serine 916 phosphorylation during lymphocyte activation and inhibition exactly correlated with the activation status of PKD/PKCmu. Moreover, using different mutants of PKD/PKCmu, we show that serine 916 is not trans-phosphorylated by an upstream kinase but is rather an autophosphorylation event that occurs following activation of PKD/PKCmu.

  6. Protein Kinase D–mediated Phosphorylation of Polycystin-2 (TRPP2) Is Essential for Its Effects on Cell Growth and Calcium Channel Activity

    PubMed Central

    Streets, Andrew J.; Needham, Andrew J.; Gill, Sharonjit K.

    2010-01-01

    PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease. The PKD2 protein, polycystin-2 or TRPP2, is a nonselective Ca2+-permeable cation channel that has been shown to function at several locations, including primary cilia, basolateral membrane, and at the endoplasmic reticulum (ER). Nevertheless, the factors that regulate the channel activity of polycystin-2 are not well understood. Polycystin-2 has been shown previously to be regulated by phosphorylation at two serine residues (Ser812 and Ser76) with distinct functional consequences. Here, we report the identification of a previously unrecognized phosphorylation site within the polycystin-2 C terminus (Ser801), and we demonstrate that it is phosphorylated by protein kinase D. Phosphorylation at this site was significantly increased in response to serum and epidermal growth factor stimulation. In nonciliated Madin-Darby canine kidney I cells, inducible expression of polycystin-2 inhibited cell proliferation compared with wild-type cells. Mutagenesis at Ser801 abolished these effects and reduced ATP-stimulated Ca2+ release from ER stores. Finally, we show that a pathogenic mutation (S804N) within the consensus kinase recognition sequence abolished Ser801 phosphorylation. These results suggest that growth factor-stimulated, protein kinase D-mediated phosphorylation of polycystin-2 is essential for its ER channel function and links extracellular stimuli to its effects on cell growth and intracellular calcium regulation. PMID:20881056

  7. Cross-talk phosphorylations by protein kinase C and Pho85p-Pho80p protein kinase regulate Pah1p phosphatidate phosphatase abundance in Saccharomyces cerevisiae.

    PubMed

    Su, Wen-Min; Han, Gil-Soo; Carman, George M

    2014-07-04

    Yeast Pah1p is the phosphatidate phosphatase that catalyzes the penultimate step in triacylglycerol synthesis and plays a role in the transcriptional regulation of phospholipid synthesis genes. The enzyme is multiply phosphorylated, some of which is mediated by Pho85p-Pho80p, Cdc28p-cyclin B, and protein kinase A. Here, we showed that Pah1p is a bona fide substrate of protein kinase C; the phosphorylation reaction was time- and dose-dependent and dependent on the concentrations of ATP (Km = 4.5 μm) and Pah1p (Km = 0.75 μm). The stoichiometry of the reaction was 0.8 mol of phosphate/mol of Pah1p. By combining mass spectrometry, truncation analysis, site-directed mutagenesis, and phosphopeptide mapping, we identified Ser-677, Ser-769, Ser-773, and Ser-788 as major sites of phosphorylation. Analysis of Pah1p phosphorylations by different protein kinases showed that prephosphorylation with protein kinase C reduces its subsequent phosphorylation with protein kinase A and vice versa. Prephosphorylation with Pho85p-Pho80p had an inhibitory effect on its subsequent phosphorylation with protein kinase C; however, prephosphorylation with protein kinase C had no effect on the subsequent phosphorylation with Pho85p-Pho80p. Unlike its phosphorylations by Pho85p-Pho80p and protein kinase A, which cause a significant reduction in phosphatidate phosphatase activity, the phosphorylation of Pah1p by protein kinase C had a small stimulatory effect on the enzyme activity. Analysis of phosphorylation-deficient forms of Pah1p indicated that protein kinase C does not have a major effect on its location or its function in triacylglycerol synthesis, but instead, the phosphorylation favors loss of Pah1p abundance when it is not phosphorylated with Pho85p-Pho80p. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Effects of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions.

    PubMed Central

    Ahmed, K; Wilson, M J; Goueli, S A; Williams-Ashman, H G

    1978-01-01

    Studies are presented on the influence of polyamines on prostatic chromatin- and non-histone-protein-associated protein kinase reactions involving both exogenous and endogenous substrates. The activities toward the model acidic protein substrate, dephosphophosvitin, were maximal at 160--200mM-NaCl (or -KCl or -NH4Cl). Under these conditions, spermidine and spermine added in concentrations up to 2mM were essentially without effect. However, without addition of NaCl to the medium, marked stimulation of these reactions was elicited by these polyamines at 1--2mM concentrations. The stimulatory effects were not due to non-specific changes in the ionic strength or to substitution of spermine for Mg2+, as maximal stimulation by 1 mM-spermine was observed only at optimal (2--4mM) Mg2+ concentrations. Qualitatively similar effects of polyamines were observed with enzyme preparations from the prostates of castrated rats, and with chromatin and non-histone-protein preparations from other tissues besides ventral prostate. When phosphorylation of endogenous non-histone proteins of the chromatin was measured, spermine stimulated both the initial rates and the final extent of transphosphorylation, even in the presence of optimal concentration of NaCl. By contrast, spermine or spermidine had no effect on the chromatin- and non-histone-protein-associated protein kinase reactions determined with lysine-rich histones as substrates. Chemically NN-dimethylated dephosphophosvitin was a less active substrate for the chromatin-associated protein kinase, but its phosphorylation was more markedly stimulated by spermine in comparison with unmodified dephosphophosvitin. These observations hint that the polyamine stimulations of the various protein kinase reactions may be due to effects on the conformations of the non-histone protein substrates rather than on the kinases themselves. PMID:747650

  9. Solubilized placental membrane protein inhibits insulin receptor tyrosine kinase activity

    SciTech Connect

    Strout, H.V. Jr.; Slater, E.E.

    1987-05-01

    Regulation of insulin receptor (IR) tyrosine kinase (TK) activity may be important in modulating insulin action. Utilizing an assay which measures IR phosphorylation of angiotensin II (AII), the authors investigated whether fractions of TX-100 solubilized human placental membranes inhibited IR dependent AII phosphorylation. Autophosphorylated IR was incubated with membrane fractions before the addition of AII, and kinase inhibition measured by the loss of TSP incorporated in AII. An inhibitory activity was detected which was dose, time, and temperature dependent. The inhibitor was purified 200-fold by sequential chromatography on wheat germ agglutinin, DEAE, and hydroxyapatite. This inhibitory activity was found to correlate with an 80 KD protein which was electroeluted from preparative slab gels and rabbit antiserum raised. Incubation of membrane fractions with antiserum before the IRTK assay immunoprecipitated the inhibitor. Protein immunoblots of crude or purified fractions revealed only the 80 KD protein. Since IR autophosphorylation is crucial to IRTK activity, the authors investigated the state of IR autophosphorylation after treatment with inhibitor; no change was detected by phosphoamino acid analysis.

  10. Protein kinase A signaling as an anti-aging target.

    PubMed

    Enns, Linda C; Ladiges, Warren

    2010-07-01

    Protein kinase A (PKA) is a multi-unit protein kinase that mediates signal transduction of G-protein-coupled receptors through its activation by adenyl cyclase (AC)-mediated cAMP. The vital importance of PKA signaling to cellular function is reflected in the widespread expression of PKA subunit genes. As one of its many functions, PKA plays a key role in the regulation of metabolism and triglyceride storage. The PKA pathway has become of great interest to the study of aging, since mutations that cause a reduction in PKA signaling have been shown to extend lifespan in yeast, and to both delay the incidence and severity of age-related disease, and to promote leanness and longevity, in mice. There is increasing interest in the potential for the inhibition or redistribution of adiposity to attenuate aging, since obesity is associated with impaired function of most organ systems, and is a strong risk factor for shortened life span. Its association with coronary heart disease, hypertension, type 2 diabetes, cancer, sleep apnea and osteoarthritis is leading to its accession as a major cause of global ill health. Therefore, gene signaling pathways such as PKA that promote adiposity are potential inhibitory targets for aging intervention. Since numerous plant compounds have been found that both prevent adipogenesis and inhibit PKA signaling, a focused investigation into their effects on biological systems and the corresponding molecular mechanisms would be of high relevance to the discovery of novel and non-toxic compounds that promote healthy aging.

  11. Disease Phenotypes in a Mouse Model of RNA Toxicity Are Independent of Protein Kinase Cα and Protein Kinase

    PubMed Central

    Kim, Yun K.; Yadava, Ramesh S.; Mandal, Mahua; Mahadevan, Karunasai; Yu, Qing; Leitges, Michael; Mahadevan, Mani S.

    2016-01-01

    Myotonic dystrophy type 1(DM1) is the prototype for diseases caused by RNA toxicity. RNAs from the mutant allele contain an expanded (CUG)n tract within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The toxic RNAs affect the function of RNA binding proteins leading to sequestration of muscleblind-like (MBNL) proteins and increased levels of CELF1 (CUGBP, Elav-like family member 1). The mechanism for increased CELF1 is not very clear. One favored proposition is hyper-phosphorylation of CELF1 by Protein Kinase C alpha (PKCα) leading to increased CELF1 stability. However, most of the evidence supporting a role for PKC-α relies on pharmacological inhibition of PKC. To further investigate the role of PKCs in the pathogenesis of RNA toxicity, we generated transgenic mice with RNA toxicity that lacked both the PKCα and PKCβ isoforms. We find that these mice show similar disease progression as mice wildtype for the PKC isoforms. Additionally, the expression of CELF1 is also not affected by deficiency of PKCα and PKCβ in these RNA toxicity mice. These data suggest that disease phenotypes of these RNA toxicity mice are independent of PKCα and PKCβ. PMID:27657532

  12. Rassf Proteins as Modulators of Mst1 Kinase Activity

    PubMed Central

    Bitra, Aruna; Sistla, Srinivas; Mariam, Jessy; Malvi, Harshada; Anand, Ruchi

    2017-01-01

    Rassf1A/5 tumor suppressors serve as adaptor proteins possessing a modular architecture with the C-terminal consisting of a coiled-coil SARAH (Salvador-Rassf-Hippo) domain and the central portion being composed of Ras associated (RA) domain. Here, we investigate the effect of Rassf effectors on Mst1 function by mapping the interaction of various domains of Rassf1A/5 and Mst1 kinase using surface plasmon resonance (SPR). The results revealed that apart from the C-terminal SARAH domain of Mst1 which interacts to form heterodimers with Rassf1A/5, the N-terminal kinase domain of Mst1 plays a crucial role in the stabilization of this complex. In addition, SPR experiments show that the RA domains play an important role in fine-tuning the Mst1-Rassf interaction, with Rassf5 being a preferred partner over a similar Rassf1A construct. It was also demonstrated that the activity profile of Mst1 in presence of Rassf adaptors completely switches. A Rassf-Mst1 complexed version of the kinase becomes apoptotic by positively regulating Mst1-H2B mediated serine 14 histone H2B phosphorylation, a hallmark of chromatin condensation. In contrast, the heterodimerization of Mst1 with Rassf1A/5 suppresses the phosphorylation of FoxO, thereby inhibiting the downstream Mst1-FoxO signalling pathway. PMID:28327630

  13. Rassf Proteins as Modulators of Mst1 Kinase Activity.

    PubMed

    Bitra, Aruna; Sistla, Srinivas; Mariam, Jessy; Malvi, Harshada; Anand, Ruchi

    2017-03-22

    Rassf1A/5 tumor suppressors serve as adaptor proteins possessing a modular architecture with the C-terminal consisting of a coiled-coil SARAH (Salvador-Rassf-Hippo) domain and the central portion being composed of Ras associated (RA) domain. Here, we investigate the effect of Rassf effectors on Mst1 function by mapping the interaction of various domains of Rassf1A/5 and Mst1 kinase using surface plasmon resonance (SPR). The results revealed that apart from the C-terminal SARAH domain of Mst1 which interacts to form heterodimers with Rassf1A/5, the N-terminal kinase domain of Mst1 plays a crucial role in the stabilization of this complex. In addition, SPR experiments show that the RA domains play an important role in fine-tuning the Mst1-Rassf interaction, with Rassf5 being a preferred partner over a similar Rassf1A construct. It was also demonstrated that the activity profile of Mst1 in presence of Rassf adaptors completely switches. A Rassf-Mst1 complexed version of the kinase becomes apoptotic by positively regulating Mst1-H2B mediated serine 14 histone H2B phosphorylation, a hallmark of chromatin condensation. In contrast, the heterodimerization of Mst1 with Rassf1A/5 suppresses the phosphorylation of FoxO, thereby inhibiting the downstream Mst1-FoxO signalling pathway.

  14. Secreted protein kinases regulate cyst burden during chronic toxoplasmosis.

    PubMed

    Jones, Nathaniel G; Wang, Qiuling; Sibley, L David

    2017-02-01

    Toxoplasma gondii is an apicomplexan parasite that secretes a large number of protein kinases and pseudokinases from its rhoptry organelles. Although some rhoptry kinases (ROPKs) act as virulence factors, many remain uncharacterized. In this study, predicted ROPKs were assessed for bradyzoite expression then prioritized for a reverse genetic analysis in the type II strain Pru that is amenable to targeted disruption. Using CRISPR/Cas9, we engineered C-terminally epitope tagged ROP21 and ROP27 and demonstrated their localization to the parasitophorous vacuole and cyst matrix. ROP21 and ROP27 were not secreted from microneme, rhoptry, or dense granule organelles, but rather were located in small vesicles consistent with a constitutive pathway. Using CRISPR/Cas9, the genes for ROP21, ROP27, ROP28, and ROP30 were deleted individually and in combination, and the mutant parasites were assessed for growth and their ability to form tissue cysts in mice. All knockouts lines were normal for in vitro growth and bradyzoite differentiation, but a combined ∆rop21/∆rop17 knockout led to a 50% reduction in cyst burden in vivo. Our findings question the existing annotation of ROPKs based solely on bioinformatic techniques and yet highlight the importance of secreted kinases in determining the severity of chronic toxoplasmosis.

  15. The pharmacogenomics of drug resistance to protein kinase inhibitors

    PubMed Central

    Gillis, Nancy K.; McLeod, Howard L.

    2016-01-01

    Dysregulation of growth factor cell signaling is a major driver of most human cancers. This has led to development of numerous drugs targeting protein kinases, with demonstrated efficacy in the treatment of a wide spectrum of cancers. Despite their high initial response rates and survival benefits, the majority of patients eventually develop resistance to these targeted therapies. This review article discusses examples of established mechanisms of drug resistance to anticancer therapies, including drug target mutations or gene amplifications, emergence of alternate signaling pathways, and pharmacokinetic variation. This reveals a role for pharmacogenomic analysis to identify and monitor for resistance, with possible therapeutic strategies to combat chemoresistance. PMID:27620953

  16. Protein kinase C epsilon in cell division: control of abscission.

    PubMed

    Saurin, Adrian T; Brownlow, Nicola; Parker, Peter J

    2009-02-15

    Cell division requires the separation and partitioning of sister chromatids to opposite ends of the cell before an actomyosin ring contracts the membrane in between during cytokinesis. The final irreversible step occurs during abscission when the ring breaks down and the membrane is sealed in its place. The physical mechanics of contraction depend on RhoA, which is stimulated by a centralspindlin complex around the cell equator. However exactly how these events are reversed to allow actomyosin breakdown and abscission were not well understood. Here we will discuss new findings that implicate Protein Kinase C epsilon (PKCepsilon) as a regulator of RhoA signalling required for abscission.

  17. The Dictyostelium Kinome—Analysis of the Protein Kinases from a Simple Model Organism

    PubMed Central

    Liu, Allen; Fey, Petra; Pilcher, Karen E; Xu, Yanji; Smith, Janet L

    2006-01-01

    Dictyostelium discoideum is a widely studied model organism with both unicellular and multicellular forms in its developmental cycle. The Dictyostelium genome encodes 285 predicted protein kinases, similar to the count of the much more advanced Drosophila. It contains members of most kinase classes shared by fungi and metazoans, as well as many previously thought to be metazoan specific, indicating that they have been secondarily lost from the fungal lineage. This includes the entire tyrosine kinase–like (TKL) group, which is expanded in Dictyostelium and includes several novel receptor kinases. Dictyostelium lacks tyrosine kinase group kinases, and most tyrosine phosphorylation appears to be mediated by TKL kinases. About half of Dictyostelium kinases occur in subfamilies not present in yeast or metazoa, suggesting that protein kinases have played key roles in the adaptation of Dictyostelium to its habitat. This study offers insights into kinase evolution and provides a focus for signaling analysis in this system. PMID:16596165

  18. Structure of the pseudokinase-kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition.

    PubMed

    Lupardus, Patrick J; Ultsch, Mark; Wallweber, Heidi; Bir Kohli, Pawan; Johnson, Adam R; Eigenbrot, Charles

    2014-06-03

    Janus kinases (JAKs) are receptor-associated multidomain tyrosine kinases that act downstream of many cytokines and interferons. JAK kinase activity is regulated by the adjacent pseudokinase domain via an unknown mechanism. Here, we report the 2.8-Å structure of the two-domain pseudokinase-kinase module from the JAK family member TYK2 in its autoinhibited form. We find that the pseudokinase and kinase interact near the kinase active site and that most reported mutations in cancer-associated JAK alleles cluster in or near this interface. Mutation of residues near the TYK2 interface that are analogous to those in cancer-associated JAK alleles, including the V617F and "exon 12" JAK2 mutations, results in increased kinase activity in vitro. These data indicate that JAK pseudokinases are autoinhibitory domains that hold the kinase domain inactive until receptor dimerization stimulates transition to an active state.

  19. Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex

    SciTech Connect

    Reenstra, W.W.; Bae, H.R.; Verkman, A.S. Univ. of California, San Francisco ); Sabolic, I. Harvard Medical School, Charlestown, MA )

    1992-01-14

    Regulation of Cl conductance by protein kinase A action, cell-free measurements of Cl transport and membrane protein phosphorylation were carried out in apical endocytic vesicles from rabbit kidney proximal tubule. Cl transport was measured by a stopped-flow quenching assay in endosomes labeled in vivo with the fluorescent Cl indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Phosphorylation was studied in a purified endosomal preparation by SDS-PAGE and autoradiography of membrane proteins labeled by ({gamma}-{sup 32}P)ATP. These results suggest that, in a cell-free system, protein kinase A increases Cl conductance in endosomes from kidney proximal tubule by a phosphorylation mechanism. The labeled protein has a size similar to that of the 64-kDa putative kidney Cl channel reported by Landry et al. but is much smaller than the {approximately}170-kDa cystic fibrosis transmembrane conductance regulatory protein.

  20. Identification of an autoinhibitory region in the activation loop of the Mos protein kinase.

    PubMed Central

    Robertson, S C; Donoghue, D J

    1996-01-01

    The Mos protein is a serine/threonine protein kinase which acts to regulate progression through meiosis in vertebrate oocytes. Although Mos function is dependent on its ability to act as a protein kinase, little is known about the factors which regulate Mos kinase activity. To understand the mechanism by which Mos kinase activity is regulated, we have used molecular modeling to construct a three-dimensional model of Mos based on the crystallographic coordinates of cyclic AMP-dependent kinase (PKA). This model identified a loop in Mos which is positioned near the active site and appears capable of blocking substrate access to the active site. Mutagenesis was used to construct altered forms of the Mos protein with deletions of parts or all of the loop. In vitro kinase assays showed that Mos proteins with the loop removed had up to a fourfold increase in kinase activity compared with the wild-type protein, indicating that the loop acts in an autoinhibitory manner for Mos kinase activity. Point mutations were also made on individual residues of the loop which were determined from the molecular model to be capable of reaching the active site. Determination of the kinase activities of these mutants showed that individual mutations in the loop region are capable of either increasing or decreasing kinase activity with regard to the wild-type protein. These data suggest that the loop identified in Mos acts as an autoinhibitor of kinase activity. PMID:8668163

  1. Isozymic forms of rat brain CA/sup 2 +/-activated and phospholipid-dependent protein kinase

    SciTech Connect

    Huang, K.P.; Huang, F.L.

    1986-05-01

    Three forms of Ca/sup 2 +/-activated and phospholipid-dependent protein kinase (protein kinase C) were purified from the cytosolic fraction of rat brain. These enzymes, designated as type I, II, and III protein kinase C, all have the similar molecular weight of 80 Kd, bind (/sup 3/H)-phorbol dibutyrate in the presence of Ca/sup 2 +/, and undergo autophosphorylation in the presence of Ca/sup 2 +/, phosphatidylserine, and diolein. Autophosphorylation of these kinases resulted in an incorporation of 1- 1.5 mol /sup 32/P/mol of enzyme. Analysis of the /sup 32/P-labeled tryptic peptides derived from the autophosphorylated protein kinase C by two-dimensional peptide mapping revealed that these kinases had different sites of autophosphorylation. Phosphoamino acid analysis revealed that the type I and type III protein kinase C mainly phosphorylated at Ser residue while the type II kinase phosphorylated at both Ser and Thr residues. In addition, polyclonal antibodies previously prepared against a mixed enzyme fraction preferentially inhibited the type I and type II enzymes but less effectively toward the type III enzyme. Monoclonal antibody specifically against the type II protein kinase C did not inhibit the type I or type III enzymes. These kinases also had different susceptibility to limited proteolysis by trypsin and upon proteolytic degradation they generate distinct fragments. These results demonstrate the presence of isozymic forms of protein kinase C in rat brain.

  2. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

    PubMed Central

    King, W G; Mattaliano, M D; Chan, T O; Tsichlis, P N; Brugge, J S

    1997-01-01

    Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases. PMID:9234699

  3. N-Terminus of the Protein Kinase CLK1 Induces SR Protein Hyper-Phosphorylation

    PubMed Central

    Aubol, Brandon E.; Plocinik, Ryan M.; Keshwani, Malik M.; McGlone, Maria L.; Hagopian, Jonathan C.; Ghosh, Gourisankar; Fu, Xiang-Dong; Adams, Joseph A.

    2016-01-01

    SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine-serine-rich) domains by two major families of protein kinases. The SRPKs efficiently phosphorylate the arginine-serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs lack a docking groove and phosphorylate both arginine-serine and serine-proline dipeptides, modifications that generate a hyper-phosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long, flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1. This interaction not only is essential for facilitating hyper-phosphorylation but also induces cooperative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 by 2–3-fold. These findings suggest that CLK1-dependent hyper-phosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein. PMID:24869919

  4. Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

    PubMed

    Nakamura, Shingo; Pourkheirandish, Mohammad; Morishige, Hiromi; Kubo, Yuta; Nakamura, Masako; Ichimura, Kazuya; Seo, Shigemi; Kanamori, Hiroyuki; Wu, Jianzhong; Ando, Tsuyu; Hensel, Goetz; Sameri, Mohammad; Stein, Nils; Sato, Kazuhiro; Matsumoto, Takashi; Yano, Masahiro; Komatsuda, Takao

    2016-03-21

    Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat.

  5. Homology modeling and ligand docking of Mitogen-activated protein kinase-activated protein kinase 5 (MK5)

    PubMed Central

    2013-01-01

    Background Mitogen-activated protein kinase-activated protein kinase 5 (MK5) is involved in one of the major signaling pathways in cells, the mitogen-activated protein kinase pathway. MK5 was discovered in 1998 by the groups of Houng Ni and Ligou New, and was found to be highly conserved throughout the vertebrates. Studies, both in vivo and in vitro, have shown that it is implicated in tumor suppression as well as tumor promotion, embryogenesis, anxiety, locomotion, cell motility and cell cycle regulation. Methods In order to obtain a molecular model of MK5 that can be used as a working tool for development of chemical probes, three MK5 models were constructed and refined based on three different known crystal structures of the closely related MKs; MK2 [PDB: 2OZA and PDB: 3M2W] and MK3 [PDB: 3FHR]. The main purpose of the present MK5 molecular modeling study was to identify the best suited template for making a MK5 model. The ability of the generated models to effectively discriminate between known inhibitors and decoys was analyzed using receiver operating characteristic (ROC) curves. Results According to the ROC curve analyzes, the refined model based on 3FHR was most effective in discrimination between known inhibitors and decoys. Conclusions The 3FHR-based MK5 model may serve as a working tool for development of chemical probes using computer aided drug design. The biological function of MK5 still remains elusive, but its role as a possible drug target may be elucidated in the near future. PMID:24034446

  6. Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

    SciTech Connect

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki

    2014-03-28

    Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

  7. Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

    NASA Technical Reports Server (NTRS)

    Li, H.; Roux, S. J.

    1992-01-01

    A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

  8. Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

    NASA Technical Reports Server (NTRS)

    Li, H.; Roux, S. J.

    1992-01-01

    A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

  9. Activation of Protein Kinase C Triggers Its Ubiquitination and Degradation†

    PubMed Central

    Lu, Zhimin; Liu, David; Hornia, Armand; Devonish, Wayne; Pagano, Michele; Foster, David A.

    1998-01-01

    Treatment of cells with tumor-promoting phorbol esters results in the activation but then depletion of phorbol ester-responsive protein kinase C (PKC) isoforms. The ubiquitin-proteasome pathway has been implicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. We report here that in 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms α, δ, and ɛ in response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Proteasome inhibitors also blocked the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which results from the depletion of PKC δ. Consistent with the involvement of the ubiquitin-proteasome pathway in the degradation of PKC isoforms, ubiquitinated PKC α, δ, and ɛ were detected within 30 min of TPA treatment. Diacylglycerol, the physiological activator of PKC, also stimulated ubiquitination and degradation of PKC, suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activation of PKC prevented both TPA- and diacylglycerol-induced PKC depletion and ubiquitination. Moreover, a kinase-dead ATP-binding mutant of PKC α could not be depleted by TPA treatment. These data are consistent with a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasome pathway. PMID:9447980

  10. Pyruvate kinase M2 is a phosphotyrosine-binding protein

    SciTech Connect

    Christofk, H.R.; Vander Heiden, M.G.; Wu, N.; Asara, J.M.; Cantley, L.C.

    2008-06-03

    Growth factors stimulate cells to take up excess nutrients and to use them for anabolic processes. The biochemical mechanism by which this is accomplished is not fully understood but it is initiated by phosphorylation of signalling proteins on tyrosine residues. Using a novel proteomic screen for phosphotyrosine-binding proteins, we have made the observation that an enzyme involved in glycolysis, the human M2 (fetal) isoform of pyruvate kinase (PKM2), binds directly and selectively to tyrosine-phosphorylated peptides. We show that binding of phosphotyrosine peptides to PKM2 results in release of the allosteric activator fructose-1,6-bisphosphate, leading to inhibition of PKM2 enzymatic activity. We also provide evidence that this regulation of PKM2 by phosphotyrosine signalling diverts glucose metabolites from energy production to anabolic processes when cells are stimulated by certain growth factors. Collectively, our results indicate that expression of this phosphotyrosine-binding form of pyruvate kinase is critical for rapid growth in cancer cells.

  11. Tumor promotion by depleting cells of protein kinase C delta.

    PubMed Central

    Lu, Z; Hornia, A; Jiang, Y W; Zang, Q; Ohno, S; Foster, D A

    1997-01-01

    Tumor-promoting phorbol esters activate, but then deplete cells of, protein kinase C (PKC) with prolonged treatment. It is not known whether phorbol ester-induced tumor promotion is due to activation or depletion of PKC. In rat fibroblasts overexpressing the c-Src proto-oncogene, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent growth and other transformation-related phenotypes. The appearance of transformed phenotypes induced by TPA in these cells correlated not with activation but rather with depletion of expressed PKC isoforms. Consistent with this observation, PKC inhibitors also induced transformed phenotypes in c-Src-overexpressing cells. Bryostatin 1, which inhibited the TPA-induced down-regulation of the PKCdelta isoform specifically, blocked the tumor-promoting effects of TPA, implicating PKCdelta as the target of the tumor-promoting phorbol esters. Consistent with this hypothesis, expression of a dominant negative PKCdelta mutant in cells expressing c-Src caused transformation of these cells, and rottlerin, a protein kinase inhibitor with specificity for PKCdelta, like TPA, caused transformation of c-Src-overexpressing cells. These data suggest that the tumor-promoting effect of phorbol esters is due to depletion of PKCdelta, which has an apparent tumor suppressor function. PMID:9154841

  12. Protein engineering of bacterial histidine kinase receptor systems.

    PubMed

    Xie, Wei; Blain, Katherine Y; Kuo, Mario Meng-Chiang; Choe, Senyon

    2010-07-01

    Two-component systems (TCS) involving the His-Asp phosphotransfer are commonly utilized for signal transduction in prokaryotes in which the two essential components are a sensor histidine kinase (HK) receptor and a response regulator (RR). Despite great efforts in structural and functional characterization of signal perception mechanisms, the exact signaling mechanisms remain elusive for many TCSs. Mimicking the natural TCS signaling pathways, chimeric receptor kinases and response regulators have been constructed through the process of swapping modular domains of related TCSs. To design chimeras with new signaling pathways, domains from different proteins that have little relationship at the primary structural level but carrying desirable functional properties can be conjoined to engineer novel TCSs. These chimeras maintain the ability to respond to environmental stimulants by regulating protein phosphorylation to produce downstream output signals. Depending on the nature of external signals, chimeric TCSs can serve as a novel tool not only to examine the natural signaling mechanisms in TCSs, but also for industrial and clinical applications.

  13. Protein kinase Cη is targeted to lipid droplets.

    PubMed

    Suzuki, Michitaka; Iio, Yuri; Saito, Naoaki; Fujimoto, Toyoshi

    2013-04-01

    Protein kinase C (PKC) is a family of kinases that regulate numerous cellular functions. They are classified into three subfamilies, i.e., conventional PKCs, novel PKCs, and atypical PKCs, that have different domain structures. Generally, PKCs exist as a soluble protein in the cytosol in resting cells and they are recruited to target membranes upon stimulation. In the present study, we found that PKCη tagged with EGFP distributed in lipid droplets (LD) and induced a significant reduction in LD size. Two other novel PKCs, PKCδ and PKCε, also showed some concentration around LDs, but it was less distinct and less frequent than that of PKCη. Conventional and atypical PKCs (α, βII, γ, and ζ) did not show any preferential distribution around LDs. 1,2-Diacylglycerol, which can activate novel PKCs without an increase of Ca(2+) concentration, is the immediate precursor of triacylglycerol and exists in LDs. The present results suggest that PKCη modifies lipid metabolism by phosphorylating unidentified targets in LDs.

  14. Protein Kinase CK2: Intricate Relationships within Regulatory Cellular Networks.

    PubMed

    Nuñez de Villavicencio-Diaz, Teresa; Rabalski, Adam J; Litchfield, David W

    2017-03-05

    Protein kinase CK2 is a small family of protein kinases that has been implicated in an expanding array of biological processes. While it is widely accepted that CK2 is a regulatory participant in a multitude of fundamental cellular processes, CK2 is often considered to be a constitutively active enzyme which raises questions about how it can be a regulatory participant in intricately controlled cellular processes. To resolve this apparent paradox, we have performed a systematic analysis of the published literature using text mining as well as mining of proteomic databases together with computational assembly of networks that involve CK2. These analyses reinforce the notion that CK2 is involved in a broad variety of biological processes and also reveal an extensive interplay between CK2 phosphorylation and other post-translational modifications. The interplay between CK2 and other post-translational modifications suggests that CK2 does have intricate roles in orchestrating cellular events. In this respect, phosphorylation of specific substrates by CK2 could be regulated by other post-translational modifications and CK2 could also have roles in modulating other post-translational modifications. Collectively, these observations suggest that the actions of CK2 are precisely coordinated with other constituents of regulatory cellular networks.

  15. Control of EGF receptor function by protein kinase C

    SciTech Connect

    Whiteley, B.J.

    1986-01-01

    Treatment of human epidermoid carcinoma A431 cells with nanomolar concentrations of the potent tumor promotor, phorbol 12-myristate 13-acetate (PMA), is shown to attentuate the ability of epidermal growth factor (EGF) or serum to activate Na/sup +//H/sup +/ exchange, which is measured as an amiloride-inhibitable pH/sub i/ increase or /sup 22/Na/sup +/ uptake. The ability of PMA to directly activate Na/sup +//H/sup +/ exchange is also reported, but PMA-induced pH/sub i/ increases are modest with respect to those of EGF or serum and require relatively high concentrations of PMA. The effects of PMA on mitogen receptor-stimulated Na/sup +//H/sup +/ exchange were examined in the mouse fibroblast NR6 cell line using platelet-derived growth factor (PDGF). The results were similar to those in A431 cells, except that PMA in NR6 cells causes pH/sub i/ increases at lower concentrations. Phorbol diester action is mediated by the activity of the enzyme protein kinase C. The results summarized above support the hypothesis that PMA-induced protein kinase C activity opposes mitogenic stimulation. The presumed endogenous PMA analog is diacylglycerol, which is generated by phosphoinositide hydrolysis and has been reported to be produced in response to the mitogens, EGF and PDGF.

  16. Functions of AMP-activated protein kinase in adipose tissue

    PubMed Central

    Daval, Marie; Foufelle, Fabienne; Ferré, Pascal

    2006-01-01

    AMP-activated protein kinase (AMPK) is involved in cellular energy homeostasis. Its functions have been extensively studied in muscles and liver. AMPK stimulates pathways which increase energy production (glucose transport, fatty acid oxidation) and switches off pathways which consume energy (lipogenesis, protein synthesis, gluconeogenesis). This has led to the concept that AMPK has an interesting pharmaceutical potential in situations of insulin resistance and it is indeed the target of existing drugs and hormones which improve insulin sensitivity. Adipose tissue is a key player in energy metabolism through the release of substrates and hormones involved in metabolism and insulin sensitivity. Activation of AMPK in adipose tissue can be achieved through situations such as fasting and exercise. Leptin and adiponectin as well as hypoglycaemic drugs are activators of adipose tissue AMPK. This activation probably involves changes in the AMP/ATP ratio and the upstream kinase LKB1. When activated, AMPK limits fatty acid efflux from adipocytes and favours local fatty acid oxidation. Since fatty acids have a key role in insulin resistance, especially in muscles, activating AMPK in adipose tissue might be found to be beneficial in insulin-resistant states, particularly as AMPK activation also reduces cytokine secretion in adipocytes. PMID:16709632

  17. Protein Kinase CK2: Intricate Relationships within Regulatory Cellular Networks

    PubMed Central

    Nuñez de Villavicencio-Diaz, Teresa; Rabalski, Adam J.; Litchfield, David W.

    2017-01-01

    Protein kinase CK2 is a small family of protein kinases that has been implicated in an expanding array of biological processes. While it is widely accepted that CK2 is a regulatory participant in a multitude of fundamental cellular processes, CK2 is often considered to be a constitutively active enzyme which raises questions about how it can be a regulatory participant in intricately controlled cellular processes. To resolve this apparent paradox, we have performed a systematic analysis of the published literature using text mining as well as mining of proteomic databases together with computational assembly of networks that involve CK2. These analyses reinforce the notion that CK2 is involved in a broad variety of biological processes and also reveal an extensive interplay between CK2 phosphorylation and other post-translational modifications. The interplay between CK2 and other post-translational modifications suggests that CK2 does have intricate roles in orchestrating cellular events. In this respect, phosphorylation of specific substrates by CK2 could be regulated by other post-translational modifications and CK2 could also have roles in modulating other post-translational modifications. Collectively, these observations suggest that the actions of CK2 are precisely coordinated with other constituents of regulatory cellular networks. PMID:28273877

  18. Molecular mechanisms regulating protein kinase Czeta turnover and cellular transformation.

    PubMed Central

    Le Good, J Ann; Brindley, David N

    2004-01-01

    The regulation of protein kinase C (PKC)zeta in relation to its turnover, cell growth and transformation was investigated in Rat2 fibroblasts by over-expressing wild-type or mutant forms of PKCzeta. Deletion of the pseudosubstrate site (PSS) produced the most active mutant (PKCzeta Delta PSS), but mutants designed to mimic phosphorylated PKCzeta had lower specific activities in an in vitro assay. The mutant lacking phosphorylation at the Thr-560 site (T560A) had similar specific activity to wild-type PKCzeta. The T560A mutant also protected PKCzeta against proteolysis, whereas phosphorylation at Thr-410 targeted it towards proteosomal degradation. Blocking proteosomal degradation with lactacystin caused the accumulation of full-length PKCzeta Delta PSS, T410E, PKCzeta Delta PSS T410/560E, PKCzeta and T560A. Expressed PKCzeta activity was paralleled by extracellular-regulated protein kinase activation, increased cell division, serum-independent growth and focus formation. These foci were seen for cells expressing higher PKCzeta activity (PKCzeta Delta PSS, PKCzeta Delta PSS T410/560E and T560A mutants), but these fibroblasts did not show significant anchorage-independent growth. This work provides novel information concerning the role of the PSS and phosphorylation sites in regulating the activity and turnover of an atypical PKC and shows how this activity can induce cell transformation with respect to focus formation. PMID:14580237

  19. Phosphorylation of the Goodpasture antigen by type A protein kinases.

    PubMed

    Revert, F; Penadés, J R; Plana, M; Bernal, D; Johansson, C; Itarte, E; Cervera, J; Wieslander, J; Quinones, S; Saus, J

    1995-06-02

    Collagen IV is the major component of basement membranes. The human alpha 3 chain of collagen IV contains an antigenic domain called the Goodpasture antigen that is the target for the circulating immunopathogenic antibodies present in patients with Goodpasture syndrome. Characteristically, the gene region encoding the Goodpasture antigen generates multiple alternative products that retain the antigen amino-terminal region with a five-residue motif (KRGDS). The serine therein appears to be the major in vitro cAMP-dependent protein kinase phosphorylation site in the isolated antigen and can be phosphorylated in vitro by two protein kinases of approximately 50 and 41 kDa associated with human kidney plasma membrane, suggesting that it can also be phosphorylated in vivo. Consistent with this, the Goodpasture antigen is isolated from human kidney in phosphorylated and non-phosphorylated forms and only the non-phosphorylated form is susceptible to phosphorylation in vitro. Since this motif is exclusive to the human alpha 3(IV) chain and includes the RGD cell adhesion motif, its phosphorylation might play a role in pathogenesis and influence cell attachment to basement membrane.

  20. Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization.

    PubMed

    Miele, Claudia; Paturzo, Flora; Teperino, Raffaele; Sakane, Fumio; Fiory, Francesca; Oriente, Francesco; Ungaro, Paola; Valentino, Rossella; Beguinot, Francesco; Formisano, Pietro

    2007-11-02

    Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.

  1. Properties of a microtubule-associated cofactor-independent protein kinase from pig brain.

    PubMed Central

    Scott, C W; Caputo, C B; Salama, A I

    1989-01-01

    A protein kinase activity was identified in pig brain that co-purified with microtubules through repeated cycles of temperature-dependent assembly and disassembly. The microtubule-associated protein kinase (MTAK) phosphorylated histone H1; this activity was not stimulated by cyclic nucleotides. Ca2+ plus calmodulin, phospholipids or polyamines. MTAK did not phosphorylate synthetic peptides which are substrates for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase. Ca2+/calmodulin-dependent protein kinase II, protein kinase C or casein kinase II. MTAK activity was inhibited by trifluoperazine [IC50 (median inhibitory concn.) = 600 microM] in a Ca2+-independent fashion. Ca2+ alone was inhibitory [IC50 = 4 mM). MTAK was not inhibited by heparin, a potent inhibitor of casein kinase II, nor a synthetic peptide inhibitor of cyclic AMP-dependent protein kinase. MTAK demonstrated a broad pH maximum (7.5-8.5) and an apparent Km for ATP of 45 microM. Mg2+ was required for enzyme activity and could not be replaced by Mn2+. MTAK phosphorylated serine and threonine residues on histone H1. MTAK is a unique cofactor-independent protein kinase that binds to microtubule structures. Images Fig. 1. Fig. 6. Fig. 7. PMID:2557823

  2. Functional modulation of AMP-activated protein kinase by cereblon.

    PubMed

    Lee, Kwang Min; Jo, Sooyeon; Kim, Hyunyoung; Lee, Jongwon; Park, Chul-Seung

    2011-03-01

    Mutations in cereblon (CRBN), a substrate binding component of the E3 ubiquitin ligase complex, cause a form of mental retardation in humans. However, the cellular proteins that interact with CRBN remain largely unknown. Here, we report that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK α1) and inhibits the activation of AMPK activation. The ectopic expression of CRBN reduces phosphorylation of AMPK α1 and, thus, inhibits the enzyme in a nutrient-independent manner. Moreover, AMPK α1 can be potently activated by suppressing endogenous CRBN using CRBN-specific small hairpin RNAs. Thus, CRBN may act as a negative modulator of the AMPK signaling pathway in vivo.

  3. GRK mythology: G-protein receptor kinases in cardiovascular disease.

    PubMed

    Dorn, Gerald W

    2009-05-01

    G-protein receptor kinases (GRKs) are indispensable for terminating signaling of G-protein coupled receptors (GPCR) through receptor desensitization and downregulation. Increased neurohormone levels in heart failure and the adverse consequences of constant neurohormonal stimulation suggest an important protective role for mechanisms that desensitize neurohormone receptor responses. For that reason, GRK2, the first GRK identified in the heart, has been extensively studied in heart failure, cardiac hypertrophy, and myocardial infarction. However, our understanding of the roles of GRKs in general, and the differential effects of cardiac receptor phosphorylation by individual cardiac-expressed GRKs, have evolved considerably in the last few years. Here, recent developments are reviewed, with an emphasis on novel GRK functions and signaling pathways.

  4. Pin1: Intimate involvement with the regulatory protein kinase networks in the global phosphorylation landscape.

    PubMed

    Litchfield, David W; Shilton, Brian H; Brandl, Christopher J; Gyenis, Laszlo

    2015-10-01

    Protein phosphorylation is a universal regulatory mechanism that involves an extensive network of protein kinases. The discovery of the phosphorylation-dependent peptidyl-prolyl isomerase Pin1 added an additional layer of complexity to these regulatory networks. We have evaluated interactions between Pin1 and the regulatory kinome and proline-dependent phosphoproteome taking into consideration findings from targeted studies as well as data that has emerged from systematic phosphoproteomic workflows and from curated protein interaction databases. The relationship between Pin1 and the regulatory protein kinase networks is not restricted simply to the recognition of proteins that are substrates for proline-directed kinases. In this respect, Pin1 itself is phosphorylated in cells by protein kinases that modulate its functional properties. Furthermore, the phosphorylation-dependent targets of Pin1 include a number of protein kinases as well as other enzymes such as phosphatases and regulatory subunits of kinases that modulate the actions of protein kinases. As a result of its interactions with numerous protein kinases and their substrates, as well as itself being a target for phosphorylation, Pin1 has an intricate relationship with the regulatory protein kinase and phosphoproteomic networks that orchestrate complex cellular processes and respond to environmental cues. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. 3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

    PubMed Central

    Sithanandam, G; Latif, F; Duh, F M; Bernal, R; Smola, U; Li, H; Kuzmin, I; Wixler, V; Geil, L; Shrestha, S

    1996-01-01

    NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity. PMID:8622688

  6. Regulation of the activity of protein kinases by endogenous heat stable protein inhibitors.

    PubMed

    Szmigielski, A

    1985-01-01

    Protein kinase activities are regulated by endogenous thermostable protein inhibitors. Type I inhibitor is a protein of MW 22,000-24,000 which inhibits specifically cyclic AMP-(cAMP) dependent protein kinase (APK) as a competitive inhibitor of catalytic subunits of the enzyme. Type I inhibitor activity changes inversely according to the activation of adenylate cyclase and the changes in cAMP content in tissues. It seems that type I inhibitor serves as a factor preventing spontaneous cAMP-dependent phosphorylation in unstimulated cell. The other thermostable protein which inhibits APK activity has been found in Sertoli cell-enriched testis (testis inhibitor). Physiological role of the testis inhibitor is unknown. Type II inhibitor is a protein of MW 15,000 which blocks phosphorylation mediated by cAMP and cyclic GMP (cGMP) dependent (APK and GPK) and cyclic nucleotide independent protein kinases as a competitive inhibitor of substrate proteins. Activity of this inhibitor specifically changes in reciprocal manner to the changes in cGMP content. It seems that type II inhibitor serves as a factor preventing the phosphorylation catalyzed by GPK when cGMP content is low. Stimulation of guanylate cyclase and activation of GPK is followed by a decrease of type II inhibitor activity. This change in relationship between activities of GPK and type II inhibitor allows for effective phosphorylation catalyzed by this enzyme when cGMP content is increased.

  7. Structural Basis of Protein Kinase C Isoform Function

    PubMed Central

    STEINBERG, SUSAN F.

    2010-01-01

    Protein kinase C (PKC) isoforms comprise a family of lipid-activated enzymes that have been implicated in a wide range of cellular functions. PKCs are modular enzymes comprised of a regulatory domain (that contains the membrane-targeting motifs that respond to lipid cofactors, and in the case of some PKCs calcium) and a relatively conserved catalytic domain that binds ATP and substrates. These enzymes are coexpressed and respond to similar stimulatory agonists in many cell types. However, there is growing evidence that individual PKC isoforms subserve unique (and in some cases opposing) functions in cells, at least in part as a result of isoform-specific subcellular compartmentalization patterns, protein-protein interactions, and posttranslational modifications that influence catalytic function. This review focuses on the structural basis for differences in lipid cofactor responsiveness for individual PKC isoforms, the regulatory phosphorylations that control the normal maturation, activation, signaling function, and downregulation of these enzymes, and the intra-/intermolecular interactions that control PKC isoform activation and subcellular targeting in cells. A detailed understanding of the unique molecular features that underlie isoform-specific posttranslational modification patterns, protein-protein interactions, and subcellular targeting (i.e., that impart functional specificity) should provide the basis for the design of novel PKC isoform-specific activator or inhibitor compounds that can achieve therapeutically useful changes in PKC signaling in cells. PMID:18923184

  8. Distribution of regulatory subunits of protein kinase A and A kinase anchor proteins (AKAP 95, 150) in rat pinealocytes.

    PubMed

    Koch, M; Korf, H-W

    2002-12-01

    The rat pineal organ is an established model to study signal transduction cascades that are activated by norepinephrine (NE) and cause increases in cAMP levels and stimulation of protein kinase A (PKA). PKA type II catalyzes the phosphorylation of the transcription factor cAMP-response-element-binding protein (CREB) which is essential for the transcriptional induction of the arylalkylamine- N-acetyltransferase (AANAT), the rate limiting enzyme of melatonin biosynthesis. Moreover, PKA may control protein levels and enzyme activity via two PKA-dependent phosphorylation sites in the AANAT molecule. Despite the functional importance of PKA very little is known about the distribution of its isoenzymes and of A-kinase anchor proteins (AKAPs) that target the PKA to specific membrane areas and organelles by binding to the regulatory (R) subunits of PKA. We have addressed this problem by demonstrating the R subunits alpha and beta of PKA type I and II and two AKAPs (150 and 95) in NE-stimulated and untreated rat pinealocytes by immunoblot and immunocytochemistry. The immunoreactions (IR) of all four R subunits were confined to granules evenly distributed in the pinealocyte cytoplasm. Immunoreactions of RIIalpha and RIIbeta were stronger than those of RIalpha and RIbeta. AKAP 150-IR was concentrated at the cell periphery; AKAP 95-IR was restricted to the nucleus. Amount and subcellular distribution of the immunoreactions of all proteins investigated did not change upon NE stimulation. A substantial colocalization was observed between RII-subunits and AKAP 150-IR, suggesting that, in rat pinealocytes, AKAP 150 primarily anchors the R subunits of PKA II.

  9. Differential effects of protein kinase C inhibitors on chemokine production in human synovial fibroblasts.

    PubMed Central

    Jordan, N. J.; Watson, M. L.; Yoshimura, T.; Westwick, J.

    1996-01-01

    1. Rheumatoid arthritis is associated with the accumulation and activation of selected populations of inflammatory cells within the arthritic joint. One putative signal for this process is the production, by resident cells, of a group of inflammatory mediators known as the chemokines. 2. The chemokines interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and RANTES (regulated on activation normal T-cell expressed and presumably secreted) are target-cell specific chemoattractants produced by synovial fibroblasts in response to stimulation with interleukin-1 alpha (IL-1 alpha) or tumour necrosis factor alpha (TNF alpha). The signalling pathways involved in their production are not well defined. We therefore used four different protein kinase C inhibitors to investigate the role of this kinase in the regulation of chemokine mRNA and protein expression in human cultured synovial fibroblasts. 3. The non-selective PKC inhibitor, staurosporine (1-300 nM) significantly increased the production of IL-1 alpha-induced IL-8 mRNA and protein. A specific PKC inhibitor, chelerythrine chloride (0.1-3 microM), also caused a small concentration-dependent increase in IL-8 mRNA and protein production. In contrast, 3-[1-[3-(amidinothio)propyl]-3-indoly]-4-(1-methyl-3-indolyl )- 1H-pyrrole-2,5-dione methanesulphonate (Ro 31-8220) and 2[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3- yl)-maleimide (GF 109203X), two selective PKC inhibitors of the substituted bisindolylmaleimide family had a concentration-dependent biphasic effect on IL-1 alpha or TNF alpha-induced chemokine expression. At low concentrations they caused a stimulation in chemokine production, which was especially evident at the mRNA level. At higher concentrations both inhibited IL-1 alpha or TNF alpha-induced chemokine mRNA and protein production. Ro 31-8220 was 10 fold more potent than GF 109203X, with an IC50 of 1.6 +/- 0.08 microM (mean +/- s.e.mean, n = 4) for IL-1 alpha induced IL-8 production. Ro 31

  10. Huntingtin-associated protein 1 (HAP1) is a cGMP-dependent kinase anchoring protein (GKAP) specific for the cGMP-dependent protein kinase Iβ isoform.

    PubMed

    Corradini, Eleonora; Burgers, Pepijn P; Plank, Michael; Heck, Albert J R; Scholten, Arjen

    2015-03-20

    Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ Isoform*

    PubMed Central

    Corradini, Eleonora; Burgers, Pepijn P.; Plank, Michael; Heck, Albert J. R.; Scholten, Arjen

    2015-01-01

    Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins. PMID:25653285

  12. Effects of obesity on protein kinase C, brain creatine kinase, transcription, and autophagy in cochlea.

    PubMed

    Hwang, Juen-Haur

    2017-06-01

    Diet-induced obesity (DIO) has been shown to exacerbate hearing degeneration via increased hypoxia, inflammatory responses, and cell loss via both caspase-dependent and caspase-independent apoptosis signaling pathways. This study aimed to investigate the effects of DIO on the mRNA expressions of protein kinase c-β (PKC-β), brain creatine kinase (CKB), transcription modification genes, and autophagy-related genes in the cochlea of CD/1 mice. Sixteen 4-week-old male CD/1 mice were randomly divided into 2 groups. For 16 weeks, the DIO group was fed a high fat diet (60% kcal fat) and the controls were fed a standard diet. Morphometry, biochemistry, auditory brainstem response thresholds, omental fat, and histopathology of the cochlea were compared. Results showed that body weight, body length, body-mass index, omental fat, plasma triglyceride, and auditory brainstem response thresholds were significantly elevated in the DIO group compared with those of the control group. The ratio of vessel wall thickness to radius in the stria vascularis was significantly higher in the DIO group. The cell densities in the spiral ganglion, but not in the spiral prominence, of the cochlea were significantly lower in the DIO group. The expression of histone deacetylation gene 1 (HDAC1) was significantly higher in the DIO group than the control group. However, the expressions of PKC-β, CKB, HDAC3, histone acetyltransferase gene (P300), lysosome-associated membrane protein 2 (Lamp2), and light chain 3 (Lc3) genes were not significantly different between two groups. These results suggest that DIO might exacerbate hearing degeneration possibly via increased HDAC1 gene expression in the cochlea of CD/1 mice.

  13. Thrombin-induced neuronal protection: role of the mitogen activated protein kinase/ribosomal protein S6 kinase pathway

    PubMed Central

    Hu, Haitao; Yamashita, Shiro; Hua, Ya; Keep, Richard F.; Liu, Wenquan; Xi, Guohua

    2010-01-01

    Our previous studies have found that intracerebral pretreatment with a low dose of thrombin (thrombin preconditioning, TPC) reduces infarct volume and attenuates brain edema after focal cerebral ischemia. In this study, we examined whether TPC protects against the neuronal death induced by oxygen glucose deprivation (OGD), and whether the protection is through thrombin receptors and the p44/42 mitogen activated protein kinases (MAPK)/ribosomal protein S6 kinases (p70 S6K) pathway. Expression of protease-activated receptors (PARs) mRNA was detected in cultured primary rat neurons and thrombin upregulated PAR-1 and PAR-4 mRNA expression. TPC reduced OGD-induced neuronal death (e.g. dead cells: 52.5±5.4% vs. 72.3±7.2% in the control group, n=6, p<0.01). Agonists of PAR-1 and PAR-4 mimicked the effects of thrombin and reduced OGD-induced neuronal death. Pretreatment with thrombin or PAR agonists induced the upregulation of activated p44/42 MAPK and p70S6K (Thr 421/Ser 424). PD98059, an inhibitor of p44/42 MAPK kinase, blocked thrombin-induced upregulation of activated p44/42 MAPK and p70S6K. It also reduced TPC-induced neuronal protection (e.g. dead cells: 68.2±5.2% vs. 56.9±4.6% in vehicle+TPC group, n=6, p<0.05). These results suggest that TPC-induced ischemic tolerance is through activation of thrombin receptors and the p44/42 MAPK/p70S6K pathway. PMID:20846511

  14. Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

    PubMed

    Santafé, M M; Garcia, N; Lanuza, M A; Tomàs, M; Tomàs, J

    2009-02-15

    We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release.

  15. Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca(2+)-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons.

    PubMed

    Cabell, L; Audesirk, G

    1993-06-01

    A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN62 (inhibitor of Ca(2+)-calmodulin-dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-acetate (an activator of protein kinase C) and 4 alpha-phorbol 12,13-didecanoate (an inactive phorbol ester) were also tested. At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4 alpha-phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron, and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every

  16. Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine.

    PubMed

    Stern, D F; Zheng, P; Beidler, D R; Zerillo, C

    1991-02-01

    A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.

  17. Rho kinase acts as a downstream molecule to participate in protein kinase Cε regulation of vascular reactivity after hemorrhagic shock in rats.

    PubMed

    Li, Tao; Zhu, Yu; Zang, Jia-tao; Peng, Xiao-yong; Lan, Dan; Yang, Guang-ming; Xu, Jing; Liu, Liang-ming

    2014-09-01

    Our previous study demonstrated that Rho kinase and protein kinase C (PKC) played important parts in the regulation of vascular reactivity after shock. Using superior mesenteric arteries (SMAs) from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs), relationship of PKCε regulation of vascular reactivity to Rho kinase, as well as the signal transduction after shock, was investigated. The results showed that inhibition of Rho kinase with the Rho kinase-specific inhibitor Y-27632 antagonized the PKCε-specific agonist carbachol and highly expressed PKCε-induced increase of vascular reactivity in SMAs and VSMCs, whereas inhibition of PKCε with its specific inhibitory peptide did not antagonize the Rho kinase agonist (U-46619)-induced increase of vascular reactivity in SMAs and VSMCs. Activation of PKCε or highly expressed PKCε upregulated the activity of Rho kinase and the phosphorylation of PKC-dependent phosphatase inhibitor 17 (CPI-17), zipper interacting protein kinase (ZIPK), and integrin-linked kinase (ILK), whereas activation of Rho kinase increased only CPI-17 phosphorylation. The specific neutralization antibodies of ZIPK and ILK antagonized PKCε-induced increases in the activity of Rho kinase, but CPI-17 neutralization antibody did not antagonize this effect. These results suggested that Rho kinase takes part in the regulation of PKCε on vascular reactivity after shock. Rho kinase is downstream of PKCε. Protein kinase Cε activates Rho kinase via ZIPK and ILK; CPI-17 is downstream of Rho kinase.

  18. Incorporating hidden Markov models for identifying protein kinase-specific phosphorylation sites.

    PubMed

    Huang, Hsien-Da; Lee, Tzong-Yi; Tzeng, Shih-Wei; Wu, Li-Cheng; Horng, Jorng-Tzong; Tsou, Ann-Ping; Huang, Kuan-Tsae

    2005-07-30

    Protein phosphorylation, which is an important mechanism in posttranslational modification, affects essential cellular processes such as metabolism, cell signaling, differentiation, and membrane transportation. Proteins are phosphorylated by a variety of protein kinases. In this investigation, we develop a novel tool to computationally predict catalytic kinase-specific phosphorylation sites. The known phosphorylation sites from public domain data sources are categorized by their annotated protein kinases. Based on the concepts of profile Hidden Markov Models (HMM), computational models are trained from the kinase-specific groups of phosphorylation sites. After evaluating the trained models, we select the model with highest accuracy in each kinase-specific group and provide a Web-based prediction tool for identifying protein phosphorylation sites. The main contribution here is that we have developed a kinase-specific phosphorylation site prediction tool with both high sensitivity and specificity.

  19. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    SciTech Connect

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  20. Protein Kinase D Regulates Cell Death Pathways in Experimental Pancreatitis

    PubMed Central

    Yuan, Jingzhen; Liu, Yannan; Tan, Tanya; Guha, Sushovan; Gukovsky, Ilya; Gukovskaya, Anna; Pandol, Stephen J.

    2012-01-01

    Inflammation and acinar cell necrosis are two major pathological responses of acute pancreatitis, a serious disorder with no current therapies directed to its molecular pathogenesis. Serine/threonine protein kinase D family, which includes PKD/PKD1, PKD2, and PKD3, has been increasingly implicated in the regulation of multiple physiological and pathophysiological effects. We recently reported that PKD/PKD1, the predominant PKD isoform expressed in rat pancreatic acinar cells, mediates early events of pancreatitis including NF-κB activation and inappropriate intracellular digestive enzyme activation. In current studies, we investigated the role and mechanisms of PKD/PKD1 in the regulation of necrosis in pancreatic acinar cells by using two novel small molecule PKD inhibitors CID755673 and CRT0066101 and molecular approaches in in vitro and in vivo experimental models of acute pancreatitis. Our results demonstrated that both CID755673 and CRT0066101 are PKD-specific inhibitors and that PKD/PKD1 inhibition by either the chemical inhibitors or specific PKD/PKD1 siRNAs attenuated necrosis while promoting apoptosis induced by pathological doses of cholecystokinin-octapeptide (CCK) in pancreatic acinar cells. Conversely, up-regulation of PKD expression in pancreatic acinar cells increased necrosis and decreased apoptosis. We further showed that PKD/PKD1 regulated several key cell death signals including inhibitors of apoptotic proteins, caspases, receptor-interacting protein kinase 1 to promote necrosis. PKD/PKD1 inhibition by CID755673 significantly ameliorated necrosis and severity of pancreatitis in an in vivo experimental model of acute pancreatitis. Thus, our studies indicate that PKD/PKD1 is a key mediator of necrosis in acute pancreatitis and that PKD/PKD1 may represent a potential therapeutic target in acute pancreatitis. PMID:22470346

  1. Phosphorylation of the mitochondrial protein Sab by stress-activated protein kinase 3.

    PubMed

    Court, Naomi W; Kuo, Ivana; Quigley, Oonagh; Bogoyevitch, Marie A

    2004-06-18

    Mitogen-activated protein kinases (MAPKs) transduce extracellular signals into responses such as growth, differentiation, and death through their phosphorylation of specific substrate proteins. Early studies showed the consensus sequence (Pro/X)-X-(Ser/Thr)-Pro to be phosphorylated by MAPKs. Docking domains such as the "kinase interaction motif" (KIM) also appear to be crucial for efficient substrate phosphorylation. Here, we show that stress-activated protein kinase-3 (SAPK3), a p38 MAPK subfamily member, localizes to the mitochondria. Activated SAPK3 phosphorylates the mitochondrial protein Sab, an in vitro substrate of c-Jun N-terminal kinase (JNK). Sab phosphorylation by SAPK3 was dependent on the most N-terminal KIM (KIM1) of Sab and occurred primarily on Ser321. This appeared to be dependent on the position of Ser321 within Sab and the sequence immediately surrounding it. Our results suggest that SAPK3 and JNK may share a common target at the mitochondria and provide new insights into the substrate recognition by SAPK3.

  2. Autophosphorylation Activity of a Soluble Hexameric Histidine Kinase Correlates with the Shift in Protein Conformational Equilibrium

    PubMed Central

    Wojnowska, Marta; Yan, Jun; Sivalingam, Ganesh N.; Cryar, Adam; Gor, Jayesh; Thalassinos, Konstantinos; Djordjevic, Snezana

    2013-01-01

    Summary In a commonly accepted model, in response to stimuli, bacterial histidine kinases undergo a conformational transition between an active and inactive form. Structural information on histidine kinases is limited. By using ion mobility-mass spectrometry (IM-MS), we demonstrate an exchange between two conformational populations of histidine kinase ExsG that are linked to different levels of kinase activity. ExsG is an atypical signaling protein that incorporates an uncommon histidine kinase catalytic core at the C terminus preceded by an N-terminal “receiver domain” that is normally associated with the response regulator proteins in two-component signal transduction systems. IM-MS analysis and enzymatic assays indicate that phosphorylation of the ExsG receiver domain stabilizes the “compact” form of the protein and inhibits kinase core activity; in contrast, nucleotide binding required for kinase activity is associated with the more open conformation of ExsG. PMID:24210218

  3. Using Bacteria to Determine Protein Kinase Specificity and Predict Target Substrates

    PubMed Central

    Lubner, Joshua M.; Church, George M.; Husson, Robert N.; Schwartz, Daniel

    2012-01-01

    The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A) and acidophilic (Casein Kinase II) kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence. PMID:23300758

  4. Protein kinase and phosphatase activities of thylakoid membranes

    SciTech Connect

    Michel, H.; Shaw, E.K.; Bennett, J.

    1987-01-01

    Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg/sup 2 +/ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg/sup 2 +/ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs.

  5. Spatio-temporal regulation of mitogen-activated protein kinase (MAPK) signalling by protein phosphatases.

    PubMed

    Karlsson, M; Mandl, M; Keyse, S M

    2006-11-01

    ERK (extracellular-signal-regulated kinase) is a MAPK (mitogen-activated protein kinase), which regulates diverse physiological functions including cell proliferation, differentiation, transformation and survival. It is now clear that in addition to the duration and magnitude of signalling through this MAPK pathway, the spatial restriction of MAPK activity plays a key role in determining the physiological outcome of signalling. Recent work has led to the discovery of MAPK-binding proteins, which contain either nuclear localization signals or nuclear export signals. These include MAPK activators and specific protein phosphatases, which may act to both regulate MAPK activity and the subcellular localization of their substrate. This represents a mechanism by which signalling in response to extracellular stimuli may be modulated in terms of both magnitude/duration and spatial restriction thus allowing differential access of the activated MAPK to target proteins and the interpretation of this information by cells to determine an appropriate physiological response.

  6. Recognition of a PP2C interaction motif in several plant protein kinases.

    PubMed

    Chakraborty, Niranjan; Ohta, Masaru; Zhu, Jian-Kang

    2007-01-01

    Protein phosphatase 2Cs (PP2Cs) constitute a major class of phosphatases in plants. PP2Cs play important roles in many signaling pathways by countering the action of specific protein kinases. In addition to their role in several environmental stress-related signal transduction pathways, they are also involved in plant metabolism. Protein phosphatases often physically associate with their protein kinase counterparts. One approach to understanding PP2C function is to identify their interacting protein kinases. We describe a yeast two-hybrid assay system used in our lab to determine the interaction between members of the PP2C family and protein kinases in the SOS2 family. This chapter and the cited articles describing related work might be of help in discovering interactions between other protein phosphatases and kinases.

  7. Activation of cyclin A-dependent protein kinases during apoptosis.

    PubMed Central

    Meikrantz, W; Gisselbrecht, S; Tam, S W; Schlegel, R

    1994-01-01

    Apoptosis was induced in S-phase-arrested HeLa cells by staurosporine, caffeine, 6-dimethylaminopurine, and okadaic acid, agents that activate M-phase-promoting factor and induce premature mitosis in similarly treated hamster cell lines. Addition of these agents to asynchronously growing HeLa cells or to cells arrested in early G1 phase with lovastatin had little or no effect. S-phase arrest also promoted tumor necrosis factor alpha-induced apoptosis, eliminating the normal requirement for simultaneous cycloheximide treatment. For all of the apoptosis-inducing agents tested, the appearance of condensed chromatin was accompanied by 2- to 7-fold increases in cyclin A-associated histone H1 kinase activity, levels approximating the mitotic value. Where examined, both Cdc2 and Cdk2, the catalytic subunits known to associate with cyclin A, were activated. Stable overexpression of bcl-2 suppressed the apoptosis-inducing activity of all agents tested and reduced the amount of Cdc2 and Cdk2 in the nucleus, suggesting a possible mechanism by which bcl-2 inhibits the chromatin condensation characteristic of apoptosis. These findings suggest that at least one of the biochemical steps required for mitosis, activation of cyclin A-dependent protein kinases, is also an important event during apoptosis. Images PMID:8170983

  8. Effects of AMP-activated protein kinase in cerebral ischemia.

    PubMed

    Li, Jun; McCullough, Louise D

    2010-03-01

    AMP-activated protein kinase (AMPK) is a serine threonine kinase that is highly conserved through evolution. AMPK is found in most mammalian tissues including the brain. As a key metabolic and stress sensor/effector, AMPK is activated under conditions of nutrient deprivation, vigorous exercise, or heat shock. However, it is becoming increasingly recognized that changes in AMPK activation not only signal unmet metabolic needs, but also are involved in sensing and responding to 'cell stress', including ischemia. The downstream effect of AMPK activation is dependent on many factors, including the severity of the stressor as well as the tissue examined. This review discusses recent in vitro and in vivo studies performed in the brain/neuronal cells and vasculature that have contributed to our understanding of AMPK in stroke. Recent data on the potential role of AMPK in angiogenesis and neurogenesis and the interaction of AMPK with 3-hydroxy-3-methy-glutaryl-CoA reductase inhibitors (statins) agents are highlighted. The interaction between AMPK and nitric oxide signaling is also discussed.

  9. Effects of AMP-activated protein kinase in cerebral ischemia

    PubMed Central

    Li, Jun; McCullough, Louise D

    2010-01-01

    AMP-activated protein kinase (AMPK) is a serine threonine kinase that is highly conserved through evolution. AMPK is found in most mammalian tissues including the brain. As a key metabolic and stress sensor/effector, AMPK is activated under conditions of nutrient deprivation, vigorous exercise, or heat shock. However, it is becoming increasingly recognized that changes in AMPK activation not only signal unmet metabolic needs, but also are involved in sensing and responding to ‘cell stress', including ischemia. The downstream effect of AMPK activation is dependent on many factors, including the severity of the stressor as well as the tissue examined. This review discusses recent in vitro and in vivo studies performed in the brain/neuronal cells and vasculature that have contributed to our understanding of AMPK in stroke. Recent data on the potential role of AMPK in angiogenesis and neurogenesis and the interaction of AMPK with 3-hydroxy-3-methy-glutaryl-CoA reductase inhibitors (statins) agents are highlighted. The interaction between AMPK and nitric oxide signaling is also discussed. PMID:20010958

  10. Regulation of Greatwall kinase by protein stabilization and nuclear localization

    PubMed Central

    Yamamoto, Tomomi M; Wang, Ling; Fisher, Laura A; Eckerdt, Frank D; Peng, Aimin

    2014-01-01

    Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. In this study we identified Hsp90, Cdc37 and members of the importin α and β families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. We found that Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. We identified KR456/457 as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, we showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry. PMID:25483093

  11. The WNKs: atypical protein kinases with pleiotropic actions

    PubMed Central

    McCormick, James A.; Ellison, David H.

    2011-01-01

    WNKs are serine/threonine kinases that comprise a unique branch of the kinome. They are so-named owing to the unusual placement of an essential catalytic lysine. WNKs have now been identified in diverse organisms. In humans and other mammals, four genes encoding WNKs. WNKs are widely expressed at the message level, although data on protein expression is more limited. Soon after the WNKs were identified, mutations in genes encoding WNK 1 and 4 were determined to cause the human disease, Familial Hyperkalemic Hypertension (also known as pseudohypoaldosteronism II, or Gordon’s Syndrome). For this reason, a major focus of investigation has been to dissect the role of WNK kinases in renal regulation of ion transport. More recently, a different mutation in WNK1 was identified as the cause of hereditary sensory and autonomic neuropathy type II (HSANII), an early-onset autosomal disease of peripheral sensory nerves. Thus, the WNKs represent an important family of potential targets for the treatment of human disease, and further elucidation of their physiological actions outside of the kidney and brain is necessary. In this review, we describe the gene structure and mechanisms regulating expression and activity of the WNKs. Subsequently, we outline substrates and targets of WNKs, and effects of WNKs on cellular physiology, both in the kidney and elsewhere. Next, consequences of these effects on integrated physiological function are outlined. Finally, we discuss the known and putative pathophysiological relevance of the WNKs. PMID:21248166

  12. Cell cycle regulation by the NEK family of protein kinases.

    PubMed

    Fry, Andrew M; O'Regan, Laura; Sabir, Sarah R; Bayliss, Richard

    2012-10-01

    Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer.

  13. Ischemia induced activation of heat shock protein 27 kinases and casein kinase 2 in the preconditioned rabbit heart.

    PubMed

    Kim, S O; Baines, C P; Critz, S D; Pelech, S L; Katz, S; Downey, J M; Cohen, M V

    1999-01-01

    Protein kinase C (PKC), p38 MAP kinase, and mitogen-activated protein kinase-activated kinases 2 and 3 (MAPKAPK2 and MAPKAPK3) have been implicated in ischemic preconditioning (PC) of the heart to reduce damage following a myocardial infarct. This study examined whether extracellular signal-regulated kinase (Erk) 1, p70 ribosomal S6 kinase (p70 S6K), casein kinase 2 (CK2), and other hsp27 kinases are also activated by PC, and if they are required for protection in rabbit hearts. CK2 and hsp27 kinase activities declined during global ischemia in control hearts, whereas PC with 5 min ischemia and 10 min reperfusion increased their activities during global ischemia. Resource Q chromatography resolved two distinct peaks of hsp27 phosphotransferase activities; the first peak (at 0.36 M NaCl) appeared to correspond to the 55-kDa MAPKAPK2. Erk1 activity was elevated in both control and PC hearts after post-ischemic reperfusion, but no change was observed in p70 S6K activity. Infarct size (measured by triphenyltetrazolium staining) in isolated rabbit hearts subjected to 30 min regional ischemia and 2 h reperfusion was 31.0+/-2.6% of the risk zone in controls and was 10.3+/-2.2% in PC hearts (p<0.001). Neither the CK2 inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) nor the Mek1/2 inhibitor PD98059 infused during ischemia blocked protection by PC. The activation of CK2 and Erk1 in ischemic preconditioned hearts appear to be epiphenomena and not required for the reduction of infarction from myocardial ischemia.

  14. Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway.

    PubMed

    Yang, Heping; Wu, Dapeng; Zhang, Xiaojie; Wang, Xiang; Peng, Yi; Hu, Zhiping

    2012-10-05

    Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU. In this study, we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process. Western blot analysis demonstrated that telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid, while they were expressed in PAJU cells transfected with a telencephalin expression plasmid. After treatment with 1.0 nM amyloid beta protein 42, expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished, while levels of phosphorylated ezrin/radixin/moesin increased. In addition, the high levels of telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

  15. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

    PubMed Central

    Massip, L; Garand, C; Labbé, A; Perreault, È; Turaga, RVN; Bohr, VA; Lebel, M

    2015-01-01

    Werner’s syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCδ and PKCβII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases. PMID:19966859

  16. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms.

    PubMed

    Massip, L; Garand, C; Labbé, A; Perreault, E; Turaga, R V N; Bohr, V A; Lebel, M

    2010-03-11

    Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.

  17. How Do Protein Kinases Take a Selfie (Autophosphorylate)?

    PubMed

    Beenstock, Jonah; Mooshayef, Navit; Engelberg, David

    2016-11-01

    Eukaryotic protein kinases (EPKs) control most biological processes and play central roles in many human diseases. To become catalytically active, EPKs undergo conversion from an inactive to an active conformation, an event that depends upon phosphorylation of their activation loop. Intriguingly, EPKs can use their own catalytic activity to achieve this critical phosphorylation. In other words, paradoxically, EPKs catalyze autophosphorylation when supposedly in their inactive state. This indicates the existence of another important conformation that specifically permits autophosphorylation at the activation loop, which in turn imposes adoption of the active conformation. This can be considered a prone-to-autophosphorylate conformation. Recent findings suggest that in prone-to-autophosphorylate conformations catalytic motifs are aligned allosterically, by dimerization or by regulators, and support autophosphorylation in cis or trans. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Protein kinases as targets for antiparasitic chemotherapy drugs.

    PubMed

    Canduri, Fernanda; Perez, Patrícia Cardoso; Caceres, Rafael A; de Azevedo, Walter F

    2007-03-01

    Parasitic protozoa infecting humans have a great impact on public health, especially in the developing countries. In many instances, the parasites have developed resistance against available chemotherapeutic agents, making the search for alternative drugs a priority. In line with the current interest in Protein Kinase (PK) inhibitors as potential drugs against a variety of diseases, the possibility that PKs may represent targets for novel anti-parasitic agents is being explored. Research into parasite PKs has benefited greatly from genome and EST sequencing projects, with the genomes from a few species fully sequenced (notably that from the malaria parasite Plasmodium falciparum) and several more under way, the structural features that are important to design specific inhibitors against these PKs will be reviewed in the present work.

  19. Protein kinase C and the antiviral effect of human interferon.

    PubMed

    Cernescu, C; Constantinescu, S N; Baltă, F; Popescu, L M; Cajal, N

    1989-01-01

    Protein kinase C (PKC) inhibitors: Hidaka's compounds H-7 (10 microM) and H-8 (20 microM), palmitoyl-carnitine (10 microM) and phloretin (50 microM), did not modify the antiviral effect of human natural or recombinant interferon alpha and of natural interferon beta. The tumor promoter 12-o-tetradecanoyl-phorbol-13-acetate (TPA) (200 nM), known as activator of PKC induced an antiviral state when tested on human embryo fibroblasts challenged with the vesicular stomatitis virus. The battery of PKC inhibitors used inhibited the antiviral effect induced by TPA. Palmitoyl-carnitine (10 microM) exerted a toxic effect that was reversed by interferon treatment (2,000 IU/ml interferon alpha). These results suggest that PKC, possibly activated by interferon-receptor interaction, is not essential for inducing the antiviral effect of interferon, but, probably, mediates the antiviral effect of TPA.

  20. Fhit is a physiological target of the protein kinase Src.

    PubMed

    Pekarsky, Yuri; Garrison, Preston N; Palamarchuk, Alexey; Zanesi, Nicola; Aqeilan, Rami I; Huebner, Kay; Barnes, Larry D; Croce, Carlo M

    2004-03-16

    The FHIT gene is a tumor suppressor that is frequently inactivated by genomic alterations at chromosomal region 3p14.2. In the last few years, a considerable amount of data describing inactivation of FHIT in a variety of human malignancies and demonstrating the tumor suppressor potential of Fhit have been reported. Despite the demonstration that FHIT functions as a tumor suppressor, the pathway through which Fhit induces apoptosis and inhibits growth of cancer cells is not known. Our data demonstrate that Fhit is a target of tyrosine phosphorylation by the Src protein kinase. We show that Src phosphorylates Y114 of Fhit in vitro and in vivo, providing insight into a biochemical pathway involved in Fhit signaling.

  1. The unique protein kinase Cη: implications for breast cancer (review).

    PubMed

    Pal, Deepanwita; Basu, Alakananda

    2014-08-01

    Deregulation of key signal transduction pathways that govern important cellular processes leads to cancer. The development of effective therapeutics for cancer warrants a comprehensive understanding of the signaling pathways that are deregulated in cancer. The protein kinase C (PKC) family has served as an attractive target for cancer therapy for decades owing to its crucial roles in several cellular processes. PKCη is a novel member of the PKC family that plays critical roles in various cellular processes such as growth, proliferation, differentiation and cell death. The regulation of PKCη appears to be unique compared to other PKC isozymes, and there are conflicting reports regarding its role in cancer. This review focuses on the unique aspects of PKCη in terms of its structure, regulation and subcellular distribution and speculates on how these features could account for its distinct functions. We have also discussed the functional implications of PKCη in cancer with particular emphasis on breast cancer.

  2. Protein kinase C coordinates histone H3 phosphorylation and acetylation

    PubMed Central

    Darieva, Zoulfia; Webber, Aaron; Warwood, Stacey; Sharrocks, Andrew D

    2015-01-01

    The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly. DOI: http://dx.doi.org/10.7554/eLife.09886.001 PMID:26468616

  3. Mitogen-activated protein kinases in male reproductive function

    PubMed Central

    Li, Michelle W.M.; Mruk, Dolores D.; Cheng, C. Yan

    2009-01-01

    Recent studies have shown that male reproductive function is modulated via the mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade is involved in numerous male reproductive processes, including spermatogenesis, sperm maturation and activation, capacitation and acrosome reaction, before fertilization of the oocyte. In this review, we discuss the latest findings in this rapidly developing field regarding the role of MAPK in male reproduction in animal models and in human spermatozoa in vitro. This research will facilitate the design of future studies in humans, although much work is needed before this information can be used to manage male infertility and environmental toxicant-induced testicular injury in men, such as blood–testis-barrier disruption. PMID:19303360

  4. Chromatographic resolution of altered forms of protein kinase C

    SciTech Connect

    Ashendel, C.L.; Minor, P.L.; Baudoin, P.A.; Carlos, M.

    1987-05-01

    Rapid chromatographic resolution of protein kinase C (PKC) in extracts of rat brain on DEAE-cellulose yielded two major peaks of activity. These fractions bound phorbol esters with identical affinity and specificity and had similar ratios of PKC to phorbol ester-binding activities. Chicken egg yolk antibodies raised to PKC in the first fraction reacted with 74 to 76 kilodalton peptides in the second fraction. Chromatography of each fraction on hydroxylapatite yielded similar distributions of three PKC isozymes. Rechromatography of the DEAE-cellulose fractions on DEAE-cellulose confirmed that these forms of PKC were not rapidly interconvertible. Results of experiments in which extracts or fractions were incubated with MgATP and phosphatase inhibitors were consistent with elution of dephospho-PKC in the first fraction while the second fraction contained phospho-PKC. If confirmed, this suggests that a substantial fraction of PKC in rat and mouse tissues exists in the phosphorylated form.

  5. Tyrosine kinase activity of a Ca{sup 2+}/calmodulin-dependent protein kinase II catalytic fragment

    SciTech Connect

    Sugiyama, Yasunori; Ishida, Atsuhiko; Sueyoshi, Noriyuki; Kameshita, Isamu

    2008-12-12

    A 30-kDa fragment of Ca{sup 2+}/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX{sub 40}-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca{sup 2+}/calmodulin-dependent protein kinase I (CX{sub 40}) were fused to the N-terminal end of 30K-CaMKII. Although CX{sub 40}-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX{sub 40} region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.

  6. Conformational selection of protein kinase A revealed by flexible-ligand flexible-protein docking.

    PubMed

    Huang, Zunnan; Wong, Chung F

    2009-03-01

    Protein kinases have high structural plasticity: their structure can change significantly, depending on what ligands are bound to them. Rigid-protein docking methods are not capable of describing such effects. Here, we present a new flexible-ligand flexible-protein docking model in which the protein can adopt conformations between two extremes observed experimentally. The model utilized a molecular dynamics-based simulated annealing cycling protocol and a distance-dependent dielectric model to perform docking. By testing this model on docking four diverse ligands to protein kinase A, we found that the ligands were able to dock successfully to the protein with the proper conformations of the protein induced. By imposing relatively soft conformational restraints to the protein during docking, this model reduced computational costs yet permitted essential conformational changes that were essential for these inhibitors to dock properly to the protein. For example, without adequate movement of the glycine-rich loop, it was difficult for the ligands to move from the surface of the protein to the binding site. In addition, these simulations called for better ways to compare simulation results with experiment other than using the popular root-mean-square deviation between the structure of a ligand in a docking pose and that in experiment because the structure of the protein also changed. In this work, we also calculated the correlation coefficient between protein-ligand/protein-protein distances in the docking structure and those in the crystal structure to check how well a ligand docked into the binding site of the protein and whether the proper conformation of the protein was induced.

  7. OncoPPi-informed discovery of mitogen-activated protein kinase kinase 3 as a novel binding partner of c-Myc | Office of Cancer Genomics

    Cancer.gov

    Mitogen-activated protein kinase kinase 3 (MKK3) is a dual threonine/tyrosine protein kinase that regulates inflammation, proliferation and apoptosis through specific phosphorylation and activation of the p38 mitogen-activated protein kinase. However, the role of MKK3 beyond p38-signaling remains elusive. Recently, we reported a protein-protein interaction (PPI) network of cancer-associated genes, termed OncoPPi, as a resource for the scientific community to generate new biological models. Analysis of the OncoPPi connectivity identified MKK3 as one of the major hub proteins in the network.

  8. Role of Protein Kinases and Their Inhibitors in Radiation Response of Tumor Cells.

    PubMed

    Kim, Byeong Mo; Yoon, Wonsuck; Shim, Jung-Hyun; Jung, Haiyoung; Lim, Ji Hong; Choi, Hyun-Jung; Seo, MiRan; Lee, Tae Ho; Min, Sang-Hyun

    2017-06-07

    Phosphorylation, the addition of a phosphate group to a molecule, is an effective way of regulating the biological properties of that molecule. Protein phosphorylation is a post translational modification of proteins and affects cellular signaling transduction. Protein kinases induce phosphorylation by catalyzing the transfer of phosphate groups to serine, threonine, and tyrosine residues on protein substrates. Consistent with their roles in cancer, protein kinases have emerged as one of the most clinically useful target molecules in pharmacological cancer therapy. Intrinsic or acquired resistance of cancers against anti-cancer therapeutics, such as ionizing radiation, is a major obstacle for the effective treatment of many cancers. In this review, we describe key aspects of various kinases acting on proteins. We also discuss the roles of protein kinases in the pathophysiology and treatment of cancer. Because protein kinases correlate with radiation resistance in various types of cancer, we focus on several kinases responsible for radiation resistance and/or sensitivity and their therapeutic implications. Finally, we suggest some ongoing radiation-sensitization strategies using genetic loss and/or kinase inhibitors that can counteract radiation resistance-related protein kinases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. Specific interactions among transmembrane 4 superfamily (TM4SF) proteins and phosphoinositide 4-kinase.

    PubMed Central

    Yauch, R L; Hemler, M E

    2000-01-01

    In earlier work we established that phosphoinositide 4-kinase (PI 4-kinase) may associate with transmembrane 4 superfamily (TM4SF, tetraspanin) proteins, but critical specificity issues were not addressed. Here we demonstrate that at least five different TM4SF proteins (CD9, CD63, CD81, CD151 and A15/TALLA1) can associate with a similar or identical 55 kDa type II PI 4-kinase. These associations were specific, since we found no evidence for other phosphoinositide kinases (e.g. phosphoinositide 3-kinase and phosphoinositide-4-phosphate 5-kinase) associating with TM4SF proteins, and many other TM4SF proteins (including CD82 and CD53) did not associate with PI 4-kinase. CD63-PI 4-kinase complexes were almost entirely intracellular, and thus are distinct from other TM4SF-PI 4-kinase complexes (e.g. involving CD9), which are largely located in the plasma membrane. These results suggest that a specific subset of TM4SF proteins may recruit PI 4-kinase to specific membrane locations, and thereby influence phosphoinositide-dependent signalling. PMID:11042117

  10. Characterization of Protein Kinase CK2 from Trypanosoma brucei

    PubMed Central

    Jensen, Bryan C; Kifer, Charles T; Brekken, Deirdre L; Randall, Amber C; Wang, Qin; Drees, Becky L.; Parsons, Marilyn

    2007-01-01

    CK2 is a ubiquitous but enigmatic kinase. The difficulty in assigning a role to CK2 centers on the fact that, to date, no biologically relevant modulator of its function has been identified. One common theme revolves around a constellation of known substrates involved in growth control, compatible with its concentration in the nucleus and nucleolus. We had previously described the identification of two catalytic subunits of CK2 in Trypanosoma brucei and characterized one of them. Here we report the characterization of the second catalytic subunit, CK2α’, and the identification and characterization of the regulatory subunit CK2β. All three subunits are primarily localized to the nucleolus in T. brucei. We also show that CK2β interacts with the nucleolar protein NOG1, adding to the interaction map which previously linked CK2α to the nucleolar protein NOPP44/46, which in turn associates with the rRNA binding protein p37. CK2 activity has four distinctive features: near equal affinity for GTP and ATP, heparin sensitivity, and stimulation by polyamines and polybasic peptides. Sequence comparison shows that the parasite orthologues have mutations in residues previously mapped as important in specifying affinity for GTP and stimulation by both polyamines and polybasic peptides. Studies of the enzymatic activity of the T. brucei CK2s show that both the affinity for GTP and stimulation by polyamines have been lost and only the features of heparin inhibition and stimulation by polybasic peptides are conserved. PMID:17097160

  11. Non-Selective Evolution of Growing Populations

    PubMed Central

    Jung, Heinrich; Frey, Erwin

    2015-01-01

    Non-selective effects, like genetic drift, are an important factor in modern conceptions of evolution, and have been extensively studied for constant population sizes (Kimura, 1955; Otto and Whitlock, 1997). Here, we consider non-selective evolution in the case of growing populations that are of small size and have varying trait compositions (e.g. after a population bottleneck). We find that, in these conditions, populations never fixate to a trait, but tend to a random limit composition, and that the distribution of compositions “freezes” to a steady state. This final state is crucially influenced by the initial conditions. We obtain these findings from a combined theoretical and experimental approach, using multiple mixed subpopulations of two Pseudomonas putida strains in non-selective growth conditions (Matthijs et al, 2009) as model system. The experimental results for the population dynamics match the theoretical predictions based on the Pólya urn model (Eggenberger and Pólya, 1923) for all analyzed parameter regimes. In summary, we show that exponential growth stops genetic drift. This result contrasts with previous theoretical analyses of non-selective evolution (e.g. genetic drift), which investigated how traits spread and eventually take over populations (fixate) (Kimura, 1955; Otto and Whitlock, 1997). Moreover, our work highlights how deeply growth influences non-selective evolution, and how it plays a key role in maintaining genetic variability. Consequently, it is of particular importance in life-cycles models (Melbinger et al, 2010; Cremer et al, 2011; Cremer et al, 2012) of periodically shrinking and expanding populations. PMID:26274606

  12. Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito.

    PubMed

    Dewald, D N; Steinmetz, E L; Walldorf, U

    2014-12-01

    The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. In the present study, we show that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and we map several phosphorylation sites of Nito. Furthermore, we were able to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase.

  13. Dataset of integrin-linked kinase protein: Protein interactions in cardiomyocytes identified by mass spectrometry.

    PubMed

    Traister, Alexandra; Lu, Mingliang; Coles, John G; Maynes, Jason T

    2016-06-01

    Using hearts from mice overexpressing integrin linked kinase (ILK) behind the cardiac specific promoter αMHC, we have performed immunoprecipitation and mass spectrometry to identify novel ILK protein:protein interactions that regulate cardiomyocyte activity and calcium flux. Integrin linked kinase complexes were captured from mouse heart lysates using a commercial antibody, with subsequent liquid chromatography tandem mass spectral analysis. Interacting partners were identified using the MASCOT server, and important interactions verified using reverse immunoprecipitation and mass spectrometry. All ILK interacting proteins were identified in a non-biased manner, and are stored in the ProteomeXchange Consortium via the PRIDE partner repository (reference ID PRIDE: PXD001053). The functional role of identified ILK interactions in cardiomyocyte function and arrhythmia were subsequently confirmed in human iPSC-cardiomyocytes.

  14. Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases

    PubMed Central

    Swatek, Kirby N.; Wilson, Rashaun S.; Ahsan, Nagib; Tritz, Rebecca L.; Thelen, Jay J.

    2014-01-01

    Plant 14-3-3 proteins are phosphorylated at multiple sites in vivo; however, the protein kinase(s) responsible are unknown. Of the 34 CPK (calcium-dependent protein kinase) paralogues in Arabidopsis thaliana, three (CPK1, CPK24 and CPK28) contain a canonical 14-3-3-binding motif. These three, in addition to CPK3, CPK6 and CPK8, were tested for activity against recombinant 14-3-3 proteins χ and ε. Using an MS-based quantitative assay we demonstrate phosphorylation of 14-3-3 χ and ε at a total of seven sites, one of which is an in vivo site discovered in Arabidopsis. CPK autophosphorylation was also comprehensively monitored by MS and revealed a total of 45 sites among the six CPKs analysed, most of which were located within the N-terminal variable and catalytic domains. Among these CPK autophosphorylation sites was Tyr463 within the calcium-binding EF-hand domain of CPK28. Of all CPKs assayed, CPK28, which contained an autophosphorylation site (Ser43) within a canonical 14-3-3-binding motif, showed the highest activity against 14-3-3 proteins. Phosphomimetic mutagenesis of Ser72 to aspartate on 14-3-3χ, which is adjacent to the 14-3-3-binding cleft and conserved among all 14-3-3 isoforms, prevented 14-3-3-mediated inhibition of phosphorylated nitrate reductase. PMID:24438037

  15. Targeting protein–protein interactions in complexes organized by A kinase anchoring proteins

    PubMed Central

    Calejo, Ana I.; Taskén, Kjetil

    2015-01-01

    Cyclic AMP is a ubiquitous intracellular second messenger involved in the regulation of a wide variety of cellular processes, a majority of which act through the cAMP – protein kinase A (PKA) signaling pathway and involve PKA phosphorylation of specific substrates. PKA phosphorylation events are typically spatially restricted and temporally well controlled. A-kinase anchoring proteins (AKAPs) directly bind PKA and recruit it to specific subcellular loci targeting the kinase activity toward particular substrates, and thereby provide discrete spatiotemporal control of downstream phosphorylation events. AKAPs also scaffold other signaling molecules into multi-protein complexes that function as crossroads between different signaling pathways. Targeting AKAP coordinated protein complexes with high-affinity peptidomimetics or small molecules to tease apart distinct protein–protein interactions (PPIs) therefore offers important means to disrupt binding of specific components of the complex to better understand the molecular mechanisms involved in the function of individual signalosomes and their pathophysiological role. Furthermore, development of novel classes of small molecules involved in displacement of AKAP-bound signal molecules is now emerging. Here, we will focus on mechanisms for targeting PPI, disruptors that modulate downstream cAMP signaling and their role, especially in the heart. PMID:26441649

  16. Steroidogenic Acute Regulatory Protein Overexpression Correlates with Protein Kinase A Activation in Adrenocortical Adenoma

    PubMed Central

    Xie, Jing; Su, Tingwei; Jiang, Lei; Jiang, Yiran; Cao, Yanan; Liu, Jianmin; Ning, Guang; Wang, Weiqing

    2016-01-01

    The association of pathological features of cortisol-producing adrenocortical adenomas (ACAs) with somatic driver mutations and their molecular classification remain unclear. In this study, we explored the association between steroidogenic acute regulatory protein (StAR) expression and the driver mutations activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling to identify the pathological markers of ACAs. Immunohistochemical staining for StAR and mutations in the protein kinase cAMP-activated catalytic subunit alpha (PRKACA), protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A) and guanine nucleotide binding protein, alpha stimulating (GNAS) genes were examined in 97 ACAs. The association of StAR expression with the clinical and mutational features of the ACAs was analyzed. ACAs with mutations in PRKACA, GNAS, and PRKAR1A showed strong immunopositive staining for StAR. The concordance between high StAR expression and mutations activating cAMP/PKA signaling in the ACAs was 99.0%. ACAs with high expression of StAR had significantly smaller tumor volume (P < 0.001) and higher urinary cortisol per tumor volume (P = 0.032) than those with low expression of StAR. Our findings suggest that immunohistochemical staining for StAR is a reliable pathological approach for the diagnosis and classification of ACAs with cAMP/PKA signaling-activating mutations. PMID:27606678

  17. Structural assembly of the signaling competent ERK2-RSK1 heterodimeric protein kinase complex.

    PubMed

    Alexa, Anita; Gógl, Gergő; Glatz, Gábor; Garai, Ágnes; Zeke, András; Varga, János; Dudás, Erika; Jeszenői, Norbert; Bodor, Andrea; Hetényi, Csaba; Reményi, Attila

    2015-03-03

    Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state.

  18. The DAP kinase family of pro-apoptotic proteins: novel players in the apoptotic game.

    PubMed

    Kögel, D; Prehn, J H; Scheidtmann, K H

    2001-04-01

    The DAP (Death Associated Protein) kinase family is a novel subfamily of pro-apoptotic serine/threonine kinases. All five DAP kinase family members identified to date are ubiquitously expressed in various tissues and are capable of inducing apoptosis. The sequence homology of the five kinases is largely restricted to the N-terminal kinase domain. In contrast, the adjacent C-terminal regions are very diverse and link individual family members to specific signal transduction pathways. There is increasing evidence that DAP kinase family members are involved in both extrinsic and intrinsic pathways of apoptosis and may play a role in tumor progression. This review will focus on structural composition and subcellular localization of DAP kinase family members and on signal transduction pathways leading to their activation. Potential mechanisms of DAP kinase family-mediated apoptosis will be discussed. BioEssays 23:352-358, 2001. Copyright 2001 John Wiley & Sons, Inc.

  19. Development of Certain Protein Kinase Inhibitors with the Components from Traditional Chinese Medicine

    PubMed Central

    Liu, Minghua; Zhao, Ge; Cao, Shousong; Zhang, Yangyang; Li, Xiaofang; Lin, Xiukun

    2017-01-01

    Traditional Chinese medicines (TCMs) have been used in China for more than two thousand years, and some of them have been confirmed to be effective in cancer treatment. Protein kinases play critical roles in control of cell growth, proliferation, migration, survival, and angiogenesis and mediate their biological effects through their catalytic activity. In recent years, numerous protein kinase inhibitors have been developed and are being used clinically. Anticancer TCMs represent a large class of bioactive substances, and some of them display anticancer activity via inhibiting protein kinases to affect the phosphoinositide 3-kinase, serine/threonine-specific protein kinases, pechanistic target of rapamycin (PI3K/AKT/mTOR), P38, mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) pathways. In the present article, we comprehensively reviewed several components isolated from anticancer TCMs that exhibited significantly inhibitory activity toward a range of protein kinases. These components, which belong to diverse structural classes, are reviewed herein, based upon the kinases that they inhibit. The prospects and problems in development of the anticancer TCMs are also discussed. PMID:28119606

  20. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila.

    PubMed

    Spindler, Stephen R; Li, Rui; Dhahbi, Joseph M; Yamakawa, Amy; Sauer, Frank

    2012-01-01

    Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

  1. Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein.

    PubMed

    Seo, Eun Joo; Liu, Furong; Kawagishi-Kobayashi, Makiko; Ung, Tekly L; Cao, Chune; Dar, Arvin C; Sicheri, Frank; Dever, Thomas E

    2008-11-04

    As part of the mammalian cell innate immune response, the double-stranded RNA activated protein kinase PKR phosphorylates the translation initiation factor eIF2alpha to inhibit protein synthesis and thus block viral replication. Poxviruses including vaccinia and smallpox viruses express PKR inhibitors such as the vaccinia virus K3L protein that resembles the N-terminal substrate-targeting domain of eIF2alpha. Whereas high-level expression of human PKR was toxic in yeast, this growth inhibition was suppressed by coexpression of the K3L protein. We used this yeast assay to screen for PKR mutants that are resistant to K3L inhibition, and we identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain. The PKR mutations specifically conferred resistance to the K3L protein both in yeast and in vitro. Consistently, the PKR-D486V mutation led to nearly a 15-fold decrease in K3L binding affinity yet did not impair eIF2alpha phosphorylation. Our results support the identification of the eIF2alpha-binding site on an extensive face of the C-terminal lobe of the kinase domain, and they indicate that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate vs. substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2alpha around the Ser-51 phosphorylation site.

  2. Phosphorylation and inhibition of. gamma. -glutamyl transferase activity by cAMP-dependent protein kinase

    SciTech Connect

    Kolesnichenko, L.S.; Chernov, N.N.

    1986-10-20

    It was shown that preparations of bovine kidney ..gamma..-glutamyl transferase of differing degrees of purity are phosphorylated by cAMP-dependent protein kinase. This is accompanied by a decrease in both the transferase and hydrolase activities of the enzyme. Consequently, ..gamma..-glutamyl transferase may serve as the substrate and target of the regulation of cAMP-dependent protein kinase.

  3. Studies on the Orientation of Cyclic AMP-Dependent Protein Kinase in Human Erythrocyte Membranes

    PubMed Central

    Rubin, Charles S.; Rosenfeld, Robert D.; Rosen, Ora M.

    1973-01-01

    The topographic location of cyclic AMP-dependent protein kinase has been studied in preparations of permeable and sealed membranes derived from human erythrocytes. Both the catalytic and cyclic AMP-binding components of protein kinase are localized on the inner, cytoplasmic, surface of the plasma membrane. PMID:4359486

  4. The cAMP Signaling Pathway and Direct Protein Kinase A Phosphorylation Regulate Polycystin-2 (TRPP2) Channel Function*

    PubMed Central

    Cantero, María del Rocío; Velázquez, Irina F.; Streets, Andrew J.; Ong, Albert C. M.; Cantiello, Horacio F.

    2015-01-01

    Polycystin-2 (PC2) is a TRP-type, Ca2+-permeable non-selective cation channel that plays an important role in Ca2+ signaling in renal and non-renal cells. The effect(s) of the cAMP pathway and kinase mediated phosphorylation of PC2 seem to be relevant to PC2 trafficking and its interaction with polycystin-1. However, the role of PC2 phosphorylation in channel function is still poorly defined. Here we reconstituted apical membranes of term human syncytiotrophoblast (hST), containing endogenous PC2 (PC2hst), and in vitro translated channel protein (PC2iv). Addition of the catalytic subunit of PKA increased by 566% the spontaneous PC2hst channel activity in the presence of ATP. Interestingly, 8-Br-cAMP also stimulated spontaneous PC2hst channel activity in the absence of the exogenous kinase. Either stimulation was inhibited by addition of alkaline phosphatase, which in turn, was reversed by the phosphatase inhibitor vanadate. Neither maneuver modified the single channel conductance but instead increased channel mean open time. PKA directly phosphorylated PC2, which increased the mean open time but not the single channel conductance of the channel. PKA phosphorylation did not modify either R742X truncated or S829A-mutant PC2iv channel function. The data indicate that the cAMP pathway regulates PC2-mediated cation transport in the hST. The relevant PKA site for PC2 channel regulation centers on a single residue serine 829, in the carboxyl terminus. PMID:26269590

  5. Nicotine stimulates adhesion molecular expression via calcium influx and mitogen-activated protein kinases in human endothelial cells.

    PubMed

    Wang, Yajing; Wang, Zhaoxia; Zhou, Ying; Liu, Liming; Zhao, Yangxing; Yao, Chenjiang; Wang, Lianyun; Qiao, Zhongdong

    2006-02-01

    To evaluate the effect of nicotine on endothelium dysfunction and development of vascular diseases, we investigated the influence on adhesion molecular expression mediated by nicotine and the mechanism of this effect in human umbilical vein endothelial cells (HUVECs). The result showed that nicotine could induce surface/soluble vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin) expression in a time-response decline manner and the peak appeared at 15 min. This action could be mediated by mitogen-activated protein kinase/extracellular signal regulated kinase 1/2 (MAPK/ERK1/2) and MAPK/p38 because their activation could be distinctly blocked by MAPK inhibitors, PD098059 or SB203580. Mecamylamine (non-selective nicotinic receptor inhibitor), alpha-bungarotoxin (alpha7 nicotinic receptor inhibitor) could block Ca2+ accumulation, and then, prevented the phosphorylation on ERK1/2 and p38. They also inhibited the surface/soluble VCAM-1, E-selectin production of HUVECs modulated by nicotine. Therefore, we concluded that: (i) nicotine obviously up-regulates VCAM-1 and E-selectin expression at 15 min in HUVECs, (ii) nicotine activates HUVECs triggered by the ERK1/2 and p38 phosphorylation with an involvement of intracellular calcium mobilization chiefly mediated by alpha7 nicotinic receptor, (iii) intracellular Ca2+ activates a sequential pathway from alpha7 nicotinic receptor to the phosphorylation of ERK1/2, p38. These elucidate that nicotine activates HUVECs through fast signal transduction pathway and arguments their capacity of adhesion molecular production. Further more nicotine may contribute its influence to the progression of vascular disease such as atherosclerotic lesion.

  6. Pharmacological modulation of protein kinases as a new approach to treat addiction to cocaine and opiates.

    PubMed

    García-Pardo, María Pilar; Roger-Sanchez, Concepción; Rodríguez-Arias, Marta; Miñarro, Jose; Aguilar, María Asunción

    2016-06-15

    Drug addiction shares brain mechanisms and molecular substrates with learning and memory processes, such as the stimulation of glutamate receptors and their downstream signalling pathways. In the present work we provide an up-to-date review of studies that have demonstrated the implication of the main memory-related calcium-dependent protein kinases in opiate and cocaine addiction. The effects of these drugs of abuse in different animal models of drug reward, dependence and addiction are altered by manipulation of the mitogen-activated protein kinase (MAPK) family, particularly extracellular signal regulated kinase (ERK), calcium/calmodulin-dependent kinase II (CaMKII), the protein kinase C (PKC) family (including PKMζ), cAMP-dependent protein kinase A (PKA), cGMP-dependent protein kinase G (PKG), the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream target mammalian target of Rapamycin (mTOR), cyclin-dependent kinase 5 (Cdk5), heat-shock proteins (Hsp) and other enzymes and proteins. Research suggests that drugs of abuse induce dependence and addiction by modifying the signalling pathways that involve these memory-related protein kinases, and supports the idea that drug addiction is an excessive aberrant learning disorder in which the maladaptive memory of drug-associated cues maintains compulsive drug use and contributes to relapse. Moreover, the studies we review offer new pharmacological strategies to treat opiate and cocaine dependence based on the manipulation of these protein kinases. In particular, disruption of reconsolidation of drug-related memories may have a high therapeutic value in the treatment of drug addiction.

  7. Haemophilus ducreyi LspA proteins are tyrosine phosphorylated by macrophage-encoded protein tyrosine kinases.

    PubMed

    Deng, Kaiping; Mock, Jason R; Greenberg, Steven; van Oers, Nicolai S C; Hansen, Eric J

    2008-10-01

    The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.

  8. PROTEIN KINASE B/AKT IS A NOVEL CYSTEINE STRING PROTEIN KINASE THAT REGULATES EXOCYTOSIS RELEASE KINETICS AND QUANTAL SIZE

    PubMed Central

    Evans, Gareth J. O.; Barclay, Jeff W.; Prescott, Gerald R.; Jo, Sung-Ro; Burgoyne, Robert D.; Birnbaum, Morris J.; Morgan, Alan

    2008-01-01

    Protein kinase B/Akt has been implicated in the insulin-dependent exocytosis of GLUT4-containing vesicles, and, more recently, insulin secretion. To determine if Akt also regulates insulin-independent exocytosis, we used adrenal chromaffin cells, a popular neuronal model. Akt1 was the predominant isoform expressed in chromaffin cells, although lower levels of Akt2 and Akt3 were also found. Secretory stimuli in both intact and permeabilized cells induced Akt phosphorylation on serine-473, and the time course of Ca2+-induced Akt phosphorylation was similar to that of exocytosis in permeabilized cells. To determine if Akt modulated exocytosis, we transfected chromaffin cells with Akt constructs and monitored catecholamine release by amperometry. Wild-type Akt had no effect on the overall number of exocytotic events, but slowed the kinetics of catecholamine release from individual vesicles, resulting in an increased quantal size. This effect was due to phosphorylation by Akt, as it was not seen in cells transfected with kinase-dead mutant Akt. As overexpression of cysteine string protein (CSP) results in a similar alteration in release kinetics and quantal size, we determined if CSP was an Akt substrate. In vitro 32P-phosphorylation studies revealed that Akt phosphorylates CSP on serine-10. Using phospho-serine10-specific antisera, we found that both transfected and endogenous cellular CSP is phosphorylated by Akt on this residue. Taken together, these findings reveal a novel role for Akt phosphorylation in regulating the late stages of exocytosis and suggest that this is achieved via the phosphorylation of CSP on serine-10. PMID:16243840

  9. A simple in vitro method to measure autophosphorylation of protein kinases

    PubMed Central

    2013-01-01

    Receptor-like protein kinases (RLKs) are a large and important group of plant proteins involved in numerous aspects of development and stress response. Within this family, homo-oligermization of receptors followed by autophosphorylation of the intracellular protein kinase domain appears to be a widespread mechanism to regulate protein kinase activity. In vitro studies of several RLKs have identified autophosphorylation sites involved in regulation of catalytic activity and signaling in vivo. Recent work has established that multiple RLKs are biochemically active when expressed in E. coli and readily autophosphorylate prior to purification or subsequent manipulation. This observation has led us to develop a simplified method for assaying RLK phosphorylation status as an indirect measure of intrinsic autophosphorylation activity. The method involves expressing a recombinant RLK protein kinase domain in E. coli, followed by SDS-PAGE of boiled cell lysate, and sequential staining with the phosphoprotein stain Pro-Q Diamond and a colloidal Coomassie total protein stain. We show this method can be used to measure and quantify in vitro autophosphorylation levels of recombinant wildtype and mutant versions of the Arabidopsis RLK HAESA, as well as to detect transphosphorylation activity of recombinant HAESA against a protein kinase inactive version of itself. Our method has several advantages over traditional protein kinase assays. It does not require protein purification, transfer, blotting, or radioactive reagents. It allows for rapid and quantitative assessment of autophosphorylation levels and should have general utility in the study of any autophosphorylating protein kinase expressed in E. coli. PMID:23803530

  10. Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C.

    PubMed Central

    Borowski, P; Heiland, M; Kornetzky, L; Medem, S; Laufs, R

    1998-01-01

    The catalytic domain of p72(syk) kinase (CDp72(syk)) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72(syk) preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5'-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the 'linker' region of p72(syk). By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72(syk). The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72(syk) autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72(syk) was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-mediated phosphorylation had a strong activating effect on the tyrosine kinase activity of CDp72(syk). Studies extended to conventional PKC isoforms revealed an isoform-dependent manner (alpha > betaI = betaII > gamma) of CDp72(syk) phosphorylation. The different phosphorylation efficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity. PMID:9531509

  11. Crystal Structure of the Ca2+/Calmodulin-dependent Protein Kinase Kinase in Complex with the Inhibitor STO-609*

    PubMed Central

    Kukimoto-Niino, Mutsuko; Yoshikawa, Seiko; Takagi, Tetsuo; Ohsawa, Noboru; Tomabechi, Yuri; Terada, Takaho; Shirouzu, Mikako; Suzuki, Atsushi; Lee, Suni; Yamauchi, Toshimasa; Okada-Iwabu, Miki; Iwabu, Masato; Kadowaki, Takashi; Minokoshi, Yasuhiko; Yokoyama, Shigeyuki

    2011-01-01

    Ca2+/calmodulin (CaM)-dependent protein kinase (CaMK) kinase (CaMKK) is a member of the CaMK cascade that mediates the response to intracellular Ca2+ elevation. CaMKK phosphorylates and activates CaMKI and CaMKIV, which directly activate transcription factors. In this study, we determined the 2.4 Å crystal structure of the catalytic kinase domain of the human CaMKKβ isoform complexed with its selective inhibitor, STO-609. The structure revealed that CaMKKβ lacks the αD helix and that the equivalent region displays a hydrophobic molecular surface, which may reflect its unique substrate recognition and autoinhibition. Although CaMKKβ lacks the activation loop phosphorylation site, the activation loop is folded in an active-state conformation, which is stabilized by a number of interactions between amino acid residues conserved among the CaMKK isoforms. An in vitro analysis of the kinase activity confirmed the intrinsic activity of the CaMKKβ kinase domain. Structure and sequence analyses of the STO-609-binding site revealed amino acid replacements that may affect the inhibitor binding. Indeed, mutagenesis demonstrated that the CaMKKβ residue Pro274, which replaces the conserved acidic residue of other protein kinases, is an important determinant for the selective inhibition by STO-609. Therefore, the present structure provides a molecular basis for clarifying the known biochemical properties of CaMKKβ and for designing novel inhibitors targeting CaMKKβ and the related protein kinases. PMID:21504895

  12. Requirement of protein kinase C zeta for stimulation of protein synthesis by insulin.

    PubMed Central

    Mendez, R; Kollmorgen, G; White, M F; Rhoads, R E

    1997-01-01

    The ability of insulin to stimulate protein synthesis and cellular growth is mediated through the insulin receptor (IR), which phosphorylates Tyr residues in the insulin receptor substrate-signaling proteins (IRS-1 and IRS-2), Gab-1, and Shc. These phosphorylated substrates directly bind and activate enzymes such as phosphatidylinositol 3'-kinase (PI3K) and the guanine nucleotide exchange factor for p21Ras (GRB-2/SOS), which are in turn required for insulin-stimulated protein synthesis, cell cycle progression, and prevention of apoptosis. We have now shown that one or more members of the atypical protein kinase C group, as exemplified by the zeta isoform (PKC zeta), are downstream of IRS-1 and P13K and mediate the effect of insulin on general protein synthesis. Ectopic expression of constitutively activated PKC zeta eliminates the requirement of IRS-1 for general protein synthesis but not for insulin-stimulated activation of 70-kDa S6 kinase (p70S6K), synthesis of growth-regulated proteins (e.g., c-Myc), or mitogenesis. The fact that PKC zeta stimulates general protein synthesis but not activation of p70S6K indicates that PKC zeta activation does not involve the proto-oncogene Akt, which is also activated by PI3K. Yet insulin is still required for the stimulation of general protein synthesis in the presence of constitutively active PKC zeta and in the absence of IRS-1, suggesting a requirement for the convergence of the IRS-1/PI3K/PKC zeta pathway with one or more additional pathways emanating from the IR, e.g., Shc/SOS/p21Ras/mitogen-activated protein kinase. Thus, PI3K appears to represent a bifurcation in the insulin signaling pathway, one branch leading through PKC zeta to general protein synthesis and one, through Akt and the target of rapamycin (mTOR), to growth-regulated protein synthesis and cell cycle progression. PMID:9271396

  13. Simple fluorescence-based detection of protein kinase A activity using a molecular beacon probe.

    PubMed

    Ma, Changbei; Lv, Xiaoyuan; Wang, Kemin; Jin, Shunxin; Liu, Haisheng; Wu, Kefeng; Zeng, Weimin

    2017-06-08

    Protein kinase A was detected by quantifying the amount of ATP used after a protein kinase reaction. The ATP assay was performed using the T4 DNA ligase and a molecular beacon (MB). In the presence of ATP, DNA ligase catalyzed the ligation of short DNA. The ligation product then hybridized to MB, resulting in a fluorescence enhancement of the MB. This assay was capable of determining protein kinase A in the range of 12.5∼150 nM, with a detection limit of 1.25 nM. Furthermore, this assay could also be used to investigate the effect of genistein on protein kinase A. It was a universal, non-radioisotopic, and homogeneous method for assaying protein kinase A.

  14. Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries.

    PubMed

    Miller, Chad J; Turk, Benjamin E

    2016-01-01

    Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors.

  15. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions

    PubMed Central

    Nguyen, Thi Mong Diep; Combarnous, Yves; Praud, Christophe; Duittoz, Anne; Blesbois, Elisabeth

    2016-01-01

    Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca2+, or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca2+. Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca2+ entry in sperm through the Ca2+/CaM/CaMKKs/CaMKI pathway. The Ca2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca2+ entry in the cells

  16. PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum.

    PubMed Central

    Bracchi-Ricard, V; Barik, S; Delvecchio, C; Doerig, C; Chakrabarti, R; Chakrabarti, D

    2000-01-01

    We have isolated a novel protein kinase cDNA, PfPK6, by differential display RT-PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic stages of Plasmodium falciparum, which shows sequence similarity to both cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) family members. The 915 bp open reading frame (ORF) is interrupted by seven introns and encodes a 305-residue polypeptide with a predicted molecular mass of 35848 Da. Several cDNA clones with some of the intron sequences were isolated, indicating alternate or defective splicing of PfPK6 transcripts because the gene seems to be a single copy located on chromosome 13. The similarity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3% and 49.6%, respectively. The signature PSTAIRE (single-letter amino acid codes) CDK motif is changed to SKCILRE in PfPK6. The TXY residues that are phosphorylated in MAPKs for their activation are T(173)PT in PfPK6. Three size classes of PfPK6 transcripts of 6.5, 2.0 and 1.1 kb are up-regulated during the transition of P. falciparum from ring to trophozoite. Western blot analysis suggested the expression of a 35 kDa polypeptide in trophozoites and schizonts. Immunofluorescence studies indicated both nuclear and cytoplasmic localization of PfPK6 in trophozoite, schizont and segmenter stages. In vitro, recombinant PfPK6 phosphorylated itself and also exogenous substrates, histone and the small subunit of the malarial ribonucleotide reductase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors such as olomoucine and roscovitine. PfPK6 showed a preference for Mn(2+) over Mg(2+) ions as a cofactor. The Lys(38)-->Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in catalytic rate for R2 phosphorylation. PMID:10727426

  17. [Effects of phosphatidylinositol-3 kinase/protein kinase b/bone morphogenetic protein-15 pathway on the follicular development in the mammalian ovary].

    PubMed

    Wu, Yan-qing; Chen, Li-yun; Zhang, Zheng-hong; wang, Zheng-chao

    2013-04-01

    In mammals, ovarian follicle is made of an oocyte with its surrounding granulosa cells and theca cells. Follicular growth and development is a highly coordinated programmable process, which guarantees the normal oocyte maturation and makes it having the fertilizing capacity. The paracrine and autocrine between oocytes and granulosa cells are essential for the follicular development to provide a suitable microenvironment. Phosphatidylinositol-3 kinase /protein kinase B is one of these important regulatory signaling pathways during this developmental process, and bone morphogenetic protein-15 an oocyte-specific secreted signal molecule, which regulates the follicular development by paracrine in the mammalian ovary. The present article overviewed the role of phosphatidylinositol-3 kinase / protein kinase B signaling during the follicular development based on our previous investigation about protein kinase B /forkhead transcription factor forkhead family of transcription factors -3a, and then focused on the regulatory effects of bone morphogenetic protein-15, as a downstream signal molecule of phosphatidylinositol-3 kinase / forkhead family of transcription factors -3a pathway, on ovarian follicular development, which helped to further understand the molecular mechanism regulating the follicular development and to treat ovarian diseases like infertility.

  18. Anxiety phenotype in mice that overexpress protein kinase A.

    PubMed

    Keil, Margaret F; Briassoulis, George; Gokarn, Nirmal; Nesterova, Maria; Wu, T John; Stratakis, Constantine A

    2012-06-01

    The role of cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling in the molecular pathways involved in fear and memory is well established. Prior studies in our lab reported that transgenic mice with an inactivating mutation in Prkar1a gene (codes for the 1-alpha regulatory subunit (R1α) of PKA) exhibited behavioral abnormalities including anxiety and depression. In the present study, we examined the role of altered PKA signaling on anxiety-like behaviors in Prkar1a(+/-) mice compared to wild-type (WT) littermates. The elevated plus maze (EPM) and marble bury (MB) tests were used to assess anxiety-like behavior. The hotplate test was performed to evaluate analgesia. We further examined the impact of the Prkar1a inactivating mutation on PKA activity in specific nuclei of the brain associated with anxiety-like behavior. Results for the MB test showed a genotype effect, with increased anxiety-like behavior in Prkar1a(+/-) mice, compared to WT littermates (p<0.05). MANOVA analysis showed a significant genotype difference in anxiety-like behavior in the EPM between WT and Prkar1a(+/-) mice on combined dependent variables (open arm time and open to total time ratio; p<0.05). Results of hotplate testing showed no genotype effect however; the expected sex difference was noted. Analysis of PKA activity showed the loss of one Prkar1a allele led to an increase in basal and cAMP-stimulated kinase activity in both the basolateral and central amygdala. These results suggest that the alteration in PKA signaling in Prkar1a(+/-) mice is not a ubiquitous effect; and supports the importance of cAMP/PKA pathway in neurobiological processes involved in anxiety and fear sensitization. Published by Elsevier Ltd.

  19. Fluorescent biosensors for high throughput screening of protein kinase inhibitors.

    PubMed

    Prével, Camille; Pellerano, Morgan; Van, Thi Nhu Ngoc; Morris, May C

    2014-02-01

    High throughput screening assays aim to identify small molecules that interfere with protein function, activity, or conformation, which can serve as effective tools for chemical biology studies of targets involved in physiological processes or pathways of interest or disease models, as well as templates for development of therapeutics in medicinal chemistry. Fluorescent biosensors constitute attractive and powerful tools for drug discovery programs, from high throughput screening assays, to postscreen characterization of hits, optimization of lead compounds, and preclinical evaluation of candidate drugs. They provide a means of screening for inhibitors that selectively target enzymatic activity, conformation, and/or function in vitro. Moreover, fluorescent biosensors constitute useful tools for cell- and image-based, multiplex and multiparametric, high-content screening. Application of fluorescence-based sensors to screen large and complex libraries of compounds in vitro, in cell-based formats or whole organisms requires several levels of optimization to establish robust and reproducible assays. In this review, we describe the different fluorescent biosensor technologies which have been applied to high throughput screens, and discuss the prerequisite criteria underlying their successful application. Special emphasis is placed on protein kinase biosensors, since these enzymes constitute one of the most important classes of therapeutic targets in drug discovery.

  20. Choristoneura fumiferana Granulovirus pk-1: a baculoviral protein kinase.

    PubMed

    Giannopoulos, Paresa N; Nassoury, Nasha; Lamontagne, Lucie; Guertin, Claude; Rashidan, Kianoush Khajeh

    2005-07-31

    Open reading frame (ORF) 3 on the Choristoneura fumiferana granulovirus (ChfuGV), located in the 11 kb fragment of the BamHI genomic bank encodes a predicted 32-kDa putative kinase protein. Bioinformatics analysis on the predicted amino acid sequence of ChfuGV PK-1 revealed the existence of 11 catalytic subdomains. Sequence analysis within the 5'-untranslated region (5'-UTR) of ChfuGV pk- 1 indicates the presence of both putative early and late promoter motifs, indicating that pk-1 may be expressed throughout the infection cycle. Promoter sequence analysis reveals that pk-1 is deprived of a TATA box and appears instead to be regulated by other cis-acting transcriptional regulatory elements. Temporal transcription analysis by RT-PCR confirms the appearance of transcripts detected from 2 h p.i. until 72 h p.i. Northern blot hybridization characterizes pk-1 transcription as a 1.2 kb transcript. Homology comparisons reveal that ChfuGV PK-1 protein is most closely related to Phthorimaea operculalla granulovirus (PoGV) with 80 % amino acid identity.

  1. Anti-Inflammatory Effects of Protein Kinase Inhibitor Pyrrol Derivate

    PubMed Central

    Yena, Maryna S.; Kotlyar, Iryna P.; Ogloblya, Olexandr V.; Rybalchenko, Volodymyr K.

    2016-01-01

    In our previous studies we showed antitumor and anti-inflammatory activities of protein kinases inhibitor pyrrol derivate 1-(4-Cl-benzyl)-3-Cl-4-(CF3-fenylamino)-1H-pyrrol-2,5-dione (MI-1) on rat colon cancer model. Therefore anti-inflammatory effect of MI-1 on rat acetic acid induced ulcerative colitis (UC) model was aimed to be discovered. The anti-inflammatory effects of MI-1 (2.7 mg/kg daily) compared to reference drug Prednisolone (0.7 mg/kg daily) after 14-day usage were evaluated on macro- and light microscopy levels and expressed in 21-grade scale. Redox status of bowel mucosa was also estimated. It was shown that in UC group the grade of total injury (GTI) was equal to 9.6 (GTIcontrol = 0). Increase of malonic dialdehyde (MDA) by 89% and protein carbonyl groups (PCG) by 60% and decrease of superoxide dismutase (SOD) by 40% were also observed. Prednisolone decreased GTI to 3 and leveled SOD activity, but MDA and PCG remained higher than control ones by 52% and 42%, respectively. MI-1 restored colon mucosa integrity and decreased mucosa inflammation down to GTI = 0.5 and leveled PCG and SOD. Thus, MI-1 possessed anti-inflammatory properties, which were more expressed that Prednisolone ones, as well as normalized mucosa redox balance, and so has a prospect for correction of inflammatory processes. PMID:28101521

  2. Anti-Inflammatory Effects of Protein Kinase Inhibitor Pyrrol Derivate.

    PubMed

    Kuznietsova, Halyna M; Yena, Maryna S; Kotlyar, Iryna P; Ogloblya, Olexandr V; Rybalchenko, Volodymyr K

    2016-01-01

    In our previous studies we showed antitumor and anti-inflammatory activities of protein kinases inhibitor pyrrol derivate 1-(4-Cl-benzyl)-3-Cl-4-(CF3-fenylamino)-1H-pyrrol-2,5-dione (MI-1) on rat colon cancer model. Therefore anti-inflammatory effect of MI-1 on rat acetic acid induced ulcerative colitis (UC) model was aimed to be discovered. The anti-inflammatory effects of MI-1 (2.7 mg/kg daily) compared to reference drug Prednisolone (0.7 mg/kg daily) after 14-day usage were evaluated on macro- and light microscopy levels and expressed in 21-grade scale. Redox status of bowel mucosa was also estimated. It was shown that in UC group the grade of total injury (GTI) was equal to 9.6 (GTIcontrol = 0). Increase of malonic dialdehyde (MDA) by 89% and protein carbonyl groups (PCG) by 60% and decrease of superoxide dismutase (SOD) by 40% were also observed. Prednisolone decreased GTI to 3 and leveled SOD activity, but MDA and PCG remained higher than control ones by 52% and 42%, respectively. MI-1 restored colon mucosa integrity and decreased mucosa inflammation down to GTI = 0.5 and leveled PCG and SOD. Thus, MI-1 possessed anti-inflammatory properties, which were more expressed that Prednisolone ones, as well as normalized mucosa redox balance, and so has a prospect for correction of inflammatory processes.

  3. ATM kinase: Much more than a DNA damage responsive protein.

    PubMed

    Guleria, Ayushi; Chandna, Sudhir

    2016-03-01

    ATM, mutation of which causes Ataxia telangiectasia, has emerged as a cardinal multifunctional protein kinase during past two decades as evidenced by various studies from around the globe. Further to its well established and predominant role in DNA damage response, ATM has also been understood to help in maintaining overall functional integrity of cells; since its mutation, inactivation or deficiency results in a variety of pathological manifestations besides DNA damage. These include oxidative stress, metabolic syndrome, mitochondrial dysfunction as well as neurodegeneration. Recently, high throughput screening using proteomics, metabolomics and transcriptomic studies revealed several proteins which might be acting as substrates of ATM. Studies that can help in identifying effective regulatory controls within the ATM-mediated pathways/mechanisms can help in developing better therapeutics. In fact, more in-depth understanding of ATM-dependent cellular signals could also help in the treatment of variety of other disease conditions since these pathways seem to control many critical cellular functions. In this review, we have attempted to put together a detailed yet lucid picture of the present-day understanding of ATM's role in various pathophysiological conditions involving DNA damage and beyond. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Huntingtin-Interacting Protein 1 Phosphorylation by Receptor Tyrosine Kinases

    PubMed Central

    Ames, Heather M.; Wang, Anmin A.; Coughran, Alanna; Evaul, Kristen; Huang, Sha; Graves, Chiron W.; Soyombo, Abigail A.

    2013-01-01

    Huntingtin-interacting protein 1 (HIP1) binds inositol lipids, clathrin, actin, and receptor tyrosine kinases (RTKs). HIP1 is elevated in many tumors, and its expression is prognostic in prostate cancer. HIP1 overexpression increases levels of the RTK epidermal growth factor receptor (EGFR) and transforms fibroblasts. Here we report that HIP1 is tyrosine phosphorylated in the presence of EGFR and platelet-derived growth factor β receptor (PDGFβR) as well as the oncogenic derivatives EGFRvIII, HIP1/PDGFβR (H/P), and TEL/PDGFβR (T/P). We identified a four-tyrosine “HIP1 phosphorylation motif” (HPM) in the N-terminal region of HIP1 that is required for phosphorylation mediated by both EGFR and PDGFβR but not by the oncoproteins H/P and T/P. We also identified a tyrosine residue (Y152) within the HPM motif of HIP1 that inhibits HIP1 tyrosine phosphorylation. The HPM tyrosines are conserved in HIP1's only known mammalian relative, HIP1-related protein (HIP1r), and are also required for HIP1r phosphorylation. Tyrosine-to-phenylalanine point mutations in the HPM of HIP1 result in proapoptotic activity, indicating that an intact HPM may be necessary for HIP1's role in cellular survival. These data suggest that phosphorylation of HIP1 by RTKs in an N-terminal region contributes to the promotion of cellular survival. PMID:23836884

  5. A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2.

    PubMed

    Wang, Hong; Brautigan, David L

    2002-12-20

    Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.

  6. The CDC2-related kinase PITALRE is the catalytic subunit of active multimeric protein complexes.

    PubMed Central

    Garriga, J; Mayol, X; Graña, X

    1996-01-01

    PITALRE is a human protein kinase identified by means of its partial sequence identity to the cell division cycle regulatory kinase CDC2. Immunopurified PITALRE protein complexes exhibit an in vitro kinase activity that phosphorylates the retinoblastoma protein, suggesting that PITALRE catalyses this phosphorylation reaction. However, the presence of other kinases in the immunopurified complex could not be ruled out. In the present work, an inactive mutant of the PITALRE kinase has been used to demonstrate that PITALRE is the catalytic subunit responsible for the PITALRE-complex-associated kinase activity, Ectopic overexpression of PITALRE did not increase the total PITALRE kinase activity in the cell, suggesting that PITALRE is regulated by limiting cellular factor(s). Characterization of the PITALRE-containing protein complexes indicated that most of the cellular PITALRE protein exists as a subunit in at least two different active multimeric complexes. Although monomeric PITALRE is also active in vitro, PITALRE present in multimeric complexes exhibits several-fold higher activity than monomeric PITALRE. In addition, overexpression of PITALRE demonstrated the existence of two new associated proteins of approx. 48 and 98 kDa. Altogether these results suggest that, in contrast to the situation with cyclin-dependent kinases, monomeric PITALRE is active, and that association with other proteins modulates its activity and/or its ability to recognize substrates in vivo. PMID:8870681

  7. Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.

    PubMed Central

    Holt, K H; Kasson, B G; Pessin, J E

    1996-01-01

    The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase. PMID:8552085

  8. ProNormz--an integrated approach for human proteins and protein kinases normalization.

    PubMed

    Subramani, Suresh; Raja, Kalpana; Natarajan, Jeyakumar

    2014-02-01

    The task of recognizing and normalizing protein name mentions in biomedical literature is a challenging task and important for text mining applications such as protein-protein interactions, pathway reconstruction and many more. In this paper, we present ProNormz, an integrated approach for human proteins (HPs) tagging and normalization. In Homo sapiens, a greater number of biological processes are regulated by a large human gene family called protein kinases by post translational phosphorylation. Recognition and normalization of human protein kinases (HPKs) is considered to be important for the extraction of the underlying information on its regulatory mechanism from biomedical literature. ProNormz distinguishes HPKs from other HPs besides tagging and normalization. To our knowledge, ProNormz is the first normalization system available to distinguish HPKs from other HPs in addition to gene normalization task. ProNormz incorporates a specialized synonyms dictionary for human proteins and protein kinases, a set of 15 string matching rules and a disambiguation module to achieve the normalization. Experimental results on benchmark BioCreative II training and test datasets show that our integrated approach achieve a fairly good performance and outperforms more sophisticated semantic similarity and disambiguation systems presented in BioCreative II GN task. As a freely available web tool, ProNormz is useful to developers as extensible gene normalization implementation, to researchers as a standard for comparing their innovative techniques, and to biologists for normalization and categorization of HPs and HPKs mentions in biomedical literature. URL: http://www.biominingbu.org/pronormz. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Sti1 and Cdc37 can stabilize Hsp90 in chaperone complexes with a protein kinase.

    PubMed

    Lee, Paul; Shabbir, Arsalan; Cardozo, Christopher; Caplan, Avrom J

    2004-04-01

    Hsp90 functions in association with several cochaperones for folding of protein kinases and transcription factors, although the relative contribution of each to the overall reaction is unknown. We assayed the role of nine different cochaperones in the activation of Ste11, a Saccharomyces cerevisiae mitogen-activated protein kinase kinase kinase. Studies on signaling via this protein kinase pathway was measured by alpha-factor-stimulated induction of FIG1 or lacZ, and repression of HHF1. Several cochaperone mutants tested had reduced FIG1 induction or HHF1 repression, although to differing extents. The greatest defects were in cpr7Delta, sse1Delta, and ydj1Delta mutants. Assays of Ste11 kinase activity revealed a pattern of defects in the cochaperone mutant strains that were similar to the gene expression studies. Overexpression of CDC37, a chaperone required for protein kinase folding, suppressed defects the sti1Delta mutant back to wild-type levels. CDC37 overexpression also restored stable Hsp90 binding to the Ste11 protein kinase domain in the sti1Delta mutant strain. These data suggest that Cdc37 and Sti1 have functional overlap in stabilizing Hsp90:client complexes. Finally, we show that Cns1 functions in MAP kinase signaling in association with Cpr7.

  10. SOcK, MiSTs, MASK and STicKs: the GCKIII (germinal centre kinase III) kinases and their heterologous protein-protein interactions.

    PubMed

    Sugden, Peter H; McGuffin, Liam J; Clerk, Angela

    2013-08-15

    The GCKIII (germinal centre kinase III) subfamily of the mammalian Ste20 (sterile 20)-like group of serine/threonine protein kinases comprises SOK1 (Ste20-like/oxidant-stress-response kinase 1), MST3 (mammalian Ste20-like kinase 3) and MST4. Initially, GCKIIIs were considered in the contexts of the regulation of mitogen-activated protein kinase cascades and apoptosis. More recently, their participation in multiprotein heterocomplexes has become apparent. In the present review, we discuss the structure and phosphorylation of GCKIIIs and then focus on their interactions with other proteins. GCKIIIs possess a highly-conserved, structured catalytic domain at the N-terminus and a less-well conserved C-terminal regulatory domain. GCKIIIs are activated by tonic autophosphorylation of a T-loop threonine residue and their phosphorylation is regulated primarily through protein serine/threonine phosphatases [especially PP2A (protein phosphatase 2A)]. The GCKIII regulatory domains are highly disorganized, but can interact with more structured proteins, particularly the CCM3 (cerebral cavernous malformation 3)/PDCD10 (programmed cell death 10) protein. We explore the role(s) of GCKIIIs (and CCM3/PDCD10) in STRIPAK (striatin-interacting phosphatase and kinase) complexes and their association with the cis-Golgi protein GOLGA2 (golgin A2; GM130). Recently, an interaction of GCKIIIs with MO25 has been identified. This exhibits similarities to the STRADα (STE20-related kinase adaptor α)-MO25 interaction (as in the LKB1-STRADα-MO25 heterotrimer) and, at least for MST3, the interaction may be enhanced by cis-autophosphorylation of its regulatory domain. In these various heterocomplexes, GCKIIIs associate with the Golgi apparatus, the centrosome and the nucleus, as well as with focal adhesions and cell junctions, and are probably involved in cell migration, polarity and proliferation. Finally, we consider the association of GCKIIIs with a number of human diseases, particularly

  11. Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system.

    PubMed

    Naik, U P; Kornecki, E; Ehrlich, Y H

    1991-04-17

    We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.

  12. Analysis of mitogen-activated protein kinase pathways used by interleukin 1 in tissues in vivo: activation of hepatic c-Jun N-terminal kinases 1 and 2, and mitogen-activated protein kinase kinases 4 and 7.

    PubMed Central

    Finch, A; Davis, W; Carter, W G; Saklatvala, J

    2001-01-01

    The effects of interleukin 1 (IL-1) are mediated by the activation of protein kinase signalling pathways, which have been well characterized in cultured cells. We have investigated the activation of these pathways in rabbit liver and other tissues after the systemic administration of IL-1alpha. In liver there was 30-40-fold activation of c-Jun N-terminal kinase (JNK) and 5-fold activation of both JNK kinases, mitogen-activated protein kinase (MAPK) kinase (MKK)4 and MKK7. IL-1alpha also caused 2-3-fold activation of p38 MAPK and degradation of the inhibitor of nuclear factor kappaB ('IkappaB'), although no activation of extracellular signal-regulated protein kinase (ERK) (p42/44 MAPK) was observed. The use of antibodies against specific JNK isoforms showed that, in liver, short (p46) JNK1 and long (p54) JNK2 are the predominant forms activated, with smaller amounts of long JNK1 and short JNK2. No active JNK3 was detected. A similar pattern of JNK activation was seen in lung, spleen, skeletal muscle and kidney. Significant JNK3 activity was detectable only in the brain, although little activation of the JNK pathway in response to IL-1alpha was observed in this tissue. This distribution of active JNK isoforms probably results from a different expression of JNKs within the tissues, rather than from a selective activation of isoforms. We conclude that IL-1alpha might activate a more restricted set of signalling pathways in tissues in vivo than it does in cultured cells, where ERK and JNK3 activation are often observed. Cultured cells might represent a 'repair' phenotype that undergoes a broader set of responses to the cytokine. PMID:11139391

  13. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    NASA Astrophysics Data System (ADS)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-11-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  14. Rim15 and Sch9 kinases are involved in induction of autophagic degradation of ribosomes in budding yeast.

    PubMed

    Waliullah, Talukdar Muhammad; Yeasmin, Akter Mst; Kaneko, Atsuki; Koike, Naoki; Terasawa, Mashu; Totsuka, Takaya; Ushimaru, Takashi

    2017-02-01

    Autophagic degradation of ribosomes is promoted by nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1). Here we show that selective autophagic degradation of ribosomes (called ribophagy) after TORC1 inactivation requires the specific autophagy receptor Atg11. Rim15 protein kinase upregulated ribophagy, while it downregulated non-selective degradation of ribosomes.

  15. Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

    PubMed Central

    Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

    2014-01-01

    Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

  16. Inhibitory activity for the interferon-induced protein kinase is associated with the reovirus serotype 1 sigma 3 protein.

    PubMed Central

    Imani, F; Jacobs, B L

    1988-01-01

    In this report we demonstrate that reovirus serotype 1-infected cells contain an inhibitor of the interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase. We provide evidence that suggests that the virus-encoded sigma 3 protein is likely responsible for this kinase inhibitory activity. We could not detect activation of the dsRNA-dependent protein kinase in extracts prepared from either interferon-treated or untreated reovirus serotype 1-infected mouse L cells under conditions that led to activation of the kinase in extracts prepared from either interferon-treated or untreated, uninfected cells. Extracts from reovirus-infected cells blocked activation of kinase in extracts from interferon-treated cells when the two were mixed prior to assay. The kinase inhibitory activity in extracts of reovirus-infected cells could be overcome by adding approximately 100-fold excess of dsRNA over the amount required to activate kinase in extracts of uninfected cells. Kinase inhibitory activity in extracts of interferon-treated, virus-infected cells could be overcome with somewhat less dsRNA (approximately 10-fold excess). Most of the inhibitory activity in the extracts could be removed by adsorption with immobilized anti-reovirus sigma 3 serum or immobilized dsRNA, suggesting that the dsRNA-binding sigma 3 protein is necessary for kinase inhibitory activity. Purified sigma 3 protein, when added to reaction mixtures containing partially purified kinase, inhibited enzyme activation. Control of activation of this kinase, which can modify eukaryotic protein synthesis initiation factor 2, may be relevant to the sensitivity of reovirus replication to treatment of cells with interferon and to the shutoff of host protein synthesis in reovirus-infected cells. Images PMID:2460857

  17. Structural assembly of the signaling competent ERK2–RSK1 heterodimeric protein kinase complex

    PubMed Central

    Alexa, Anita; Gógl, Gergő; Glatz, Gábor; Garai, Ágnes; Zeke, András; Varga, János; Dudás, Erika; Jeszenői, Norbert; Bodor, Andrea; Hetényi, Csaba; Reményi, Attila

    2015-01-01

    Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase–kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 “docking” groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they “readjust,” whereas generic kinase domain surface contacts bring them into a catalytically competent state. PMID:25730857

  18. Deciphering Protein Kinase Specificity through Large-Scale Analysis of Yeast Phosphorylation Site Motifs

    PubMed Central

    Mok, Janine; Kim, Philip M.; Lam, Hugo Y. K.; Piccirillo, Stacy; Zhou, Xiuqiong; Jeschke, Grace R.; Sheridan, Douglas L.; Parker, Sirlester A.; Desai, Ved; Jwa, Miri; Cameroni, Elisabetta; Niu, Hengyao; Good, Matthew; Remenyi, Attila; Ma, Jia-Lin Nianhan; Sheu, Yi-Jun; Sassi, Holly E.; Sopko, Richelle; Chan, Clarence S. M.; De Virgilio, Claudio; Hollingsworth, Nancy M.; Lim, Wendell A.; Stern, David F.; Stillman, Bruce; Andrews, Brenda J.; Gerstein, Mark B.; Snyder, Michael; Turk, Benjamin E.

    2010-01-01

    Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases are known to target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisae. Correlation of these motifs with kinase primary sequence has uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P−3 Arg specificity among members of the CMGC group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo, and we identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of new kinase substrates across eukaryotes. PMID:20159853

  19. The identification of novel cyclic AMP-dependent protein kinase anchoring proteins using bioinformatic filters and peptide arrays

    PubMed Central

    McLaughlin, William A.; Hou, Tingjun; Taylor, Susan S.; Wang, Wei

    2011-01-01

    A-kinase anchoring proteins (AKAPs) localize cyclic AMP-dependent protein kinase (PKA) to specific regions in the cell and place PKA in proximity to its phosphorylation targets. A computational model was created to identify AKAPs that bind to the docking/dimerization domain of the RII alpha isoform of the regulatory subunit of PKA. The model was used to search the entire human proteome, and the top candidates were tested for an interaction using peptide array experiments. Verified interactions include sphingosine kinase interacting protein and retinoic acid-induced protein 16. These interactions highlight new signaling pathways mediated by PKA. PMID:21115539

  20. Increased protein glycation in fructosamine 3-kinase-deficient mice

    PubMed Central

    da-Cunha, Maria VEIGA; Jacquemin, Patrick; Delpierre, Ghislain; Godfraind, Catherine; Théate, Ivan; Vertommen, Didier; Clotman, Frédéric; Lemaigre, Frédéric; Devuyst, Olivier; Van Schaftingen, Emile

    2006-01-01

    Amines, including those present on proteins, spontaneously react with glucose to form fructosamines in a reaction known as glycation. In the present paper, we have explored, through a targeted gene inactivation approach, the role of FN3K (fructosamine 3-kinase), an intracellular enzyme that phosphorylates free and protein-bound fructose-ϵ-lysines and which is potentially involved in protein repair. Fn3k−/− mice looked healthy and had normal blood glucose and serum fructosamine levels. However, their level of haemoglobin-bound fructosamines was approx. 2.5-fold higher than that of control (Fn3k+/+) or Fn3k+/− mice. Other intracellular proteins were also significantly more glycated in Fn3k−/− mice in erythrocytes (1.8–2.2-fold) and in brain, kidney, liver and skeletal muscle (1.2–1.8-fold), indicating that FN3K removes fructosamines from intracellular proteins in vivo. The urinary excretion of free fructose-ϵ-lysine was 10–20-fold higher in fed mice compared with mice starved for 36 h, and did not differ between fed Fn3k+/+ and Fn3k−/− mice, indicating that food is the main source of urinary fructose-ϵ-lysine in these mice and that FN3K does not participate in the metabolism of food-derived fructose-ϵ-lysine. However, in starved animals, the urinary excretion of fructose-ϵ-lysine was 2.5-fold higher in Fn3k−/− mice compared with Fn3k+/+ or Fn3k+/− mice. Furthermore, a marked increase (5–13-fold) was observed in the concentration of free fructose-ϵ-lysine in tissues of fed Fn3k−/− mice compared with control mice, indicating that FN3K participates in the metabolism of endogenously produced fructose-ϵ-lysine. Taken together, these data indicate that FN3K serves as a protein repair enzyme and also in the metabolism of endogenously produced free fructose-ϵ-lysine. PMID:16819943

  1. Small molecule modulators of eukaryotic initiation factor 2α kinases, the key regulators of protein synthesis.

    PubMed

    Joshi, Manali; Kulkarni, Abhijeet; Pal, Jayanta K

    2013-11-01

    Eukaryotic initiation factor 2 alpha kinases (eIF-2α kinases) are key mediators of stress response in cells. In mammalian cells, there are four eIF-2α kinases, namely HRI (Heme-Regulated Inhibitor), PKR (RNA-dependent Protein Kinase), PERK (PKR-like ER Kinase) and GCN2 (General Control Non-derepressible 2). These kinases get activated during diverse cytoplasmic stress conditions and phosphorylate the alpha-subunit of eIF2, leading to global protein synthesis inhibition. Therefore, eIF-2α kinases play a vital role in various cellular processes such as proliferation, differentiation, apoptosis and cell signaling. Deregulation of eIF-2α kinases and protein synthesis has been linked to numerous pathological conditions such as certain cancers, anemia and neurodegenerative disorders. Thus, modulation of these kinases by small molecules holds a great therapeutic promise. In this review we have compiled the available information on inhibitors and activators of these four eIF-2α kinases. The review concludes with a note on the selectivity issue of currently available modulators and future perspectives for the design of specific small molecule probes.

  2. Transmembrane neuregulins interact with LIM kinase 1, a cytoplasmic protein kinase implicated in development of visuospatial cognition.

    PubMed

    Wang, J Y; Frenzel, K E; Wen, D; Falls, D L

    1998-08-07

    The neuregulins are receptor tyrosine kinase ligands that play a critical role in the development of the heart, nervous system, and breast. Unlike many extracellular signaling molecules, such as the neurotrophins, most neuregulins are synthesized as transmembrane proteins. To determine the functions of the highly conserved neuregulin cytoplasmic tail, a yeast two-hybrid screen was performed to identify proteins that interact with the 157-amino acid sequence common to the cytoplasmic tails of all transmembrane neuregulin isoforms. This screen revealed that the neuregulin cytoplasmic tail interacts with the LIM domain region of the nonreceptor protein kinase LIM kinase 1 (LIMK1). Interaction between the neuregulin cytoplasmic tail and full-length LIMK1 was demonstrated by in vitro binding and co-immunoprecipitation assays. Transmembrane neuregulins with each of the three known neuregulin cytoplasmic tail isoforms interacted with LIMK1. In contrast, the cytoplasmic tail of TGF-alpha did not interact with LIMK1. In vivo, neuregulin and LIMK1 are co-localized at the neuromuscular synapse, suggesting that LIMK1, like neuregulin, may play a role in synapse formation and maintenance. To our knowledge, LIMK1 is the first identified protein shown to interact with the cytoplasmic tail of a receptor tyrosine kinase ligand.

  3. Protein Kinase Activity Decreases with Higher Braak Stages of Alzheimer’s Disease Pathology

    PubMed Central

    Rosenberger, Andrea F.N.; Hilhorst, Riet; Coart, Elisabeth; García Barrado, Leandro; Naji, Faris; Rozemuller, Annemieke J.M.; van der Flier, Wiesje M.; Scheltens, Philip; Hoozemans, Jeroen J.M.; van der Vies, Saskia M.

    2015-01-01

    Alzheimer’s disease (AD) is characterized by a long pre-clinical phase (20–30 years), during which significant brain pathology manifests itself. Disease mechanisms associated with pathological hallmarks remain elusive. Most processes associated with AD pathogenesis, such as inflammation, synaptic dysfunction, and hyper-phosphorylation of tau are dependent on protein kinase activity. The objective of this study was to determine the involvement of protein kinases in AD pathogenesis. Protein kinase activity was determined in postmortem hippocampal brain tissue of 60 patients at various stages of AD and 40 non-demented controls (Braak stages 0-VI) using a peptide-based microarray platform. We observed an overall decrease of protein kinase activity that correlated with disease progression. The phosphorylation of 96.7% of the serine/threonine peptides and 37.5% of the tyrosine peptides on the microarray decreased significantly with increased Braak stage (p-value <0.01). Decreased activity was evident at pre-clinical stages of AD pathology (Braak I-II). Increased phosphorylation was not observed for any peptide. STRING analysis in combination with pathway analysis and identification of kinases responsible for peptide phosphorylation showed the interactions between well-known proteins in AD pathology, including the Ephrin-receptor A1 (EphA1), a risk gene for AD, and sarcoma tyrosine kinase (Src), which is involved in memory formation. Additionally, kinases that have not previously been associated with AD were identified, e.g., protein tyrosine kinase 6 (PTK6/BRK), feline sarcoma oncogene kinase (FES), and fyn-associated tyrosine kinase (FRK). The identified protein kinases are new biomarkers and potential drug targets for early (pre-clinical) intervention. PMID:26519433

  4. Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester.

    PubMed

    Muto, Akihiro; Ruland, Jürgen; McAllister-Lucas, Linda M; Lucas, Peter C; Yamaoka, Shoji; Chen, Felicia F; Lin, Amy; Mak, Tak W; Núñez, Gabriel; Inohara, Naohiro

    2002-08-30

    Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.

  5. Mutational Analysis of Glycogen Synthase KinaseProtein Kinase Together with Kinome-Wide Binding and Stability Studies Suggests Context-Dependent Recognition of Kinases by the Chaperone Heat Shock Protein 90

    PubMed Central

    Pasculescu, Adrian; Dai, Anna Yue; Williton, Kelly; Taylor, Lorne; Savitski, Mikhail M.; Bantscheff, Marcus; Woodgett, James R.; Pawson, Tony; Colwill, Karen

    2016-01-01

    The heat shock protein 90 (HSP90) and cell division cycle 37 (CDC37) chaperones are key regulators of protein kinase folding and maturation. Recent evidence suggests that thermodynamic properties of kinases, rather than primary sequences, are recognized by the chaperones. In concordance, we observed a striking difference in HSP90 binding between wild-type (WT) and kinase-dead (KD) glycogen synthase kinase 3β (GSK3β) forms. Using model cell lines stably expressing these two GSK3β forms, we observed no interaction between WT GSK3β and HSP90, in stark contrast to KD GSK3β forming a stable complex with HSP90 at a 1:1 ratio. In a survey of 91 ectopically expressed kinases in DLD-1 cells, we compared two parameters to measure HSP90 dependency: static binding and kinase stability following HSP90 inhibition. We observed no correlation between HSP90 binding and reduced stability of a kinase after pharmacological inhibition of HSP90. We expanded our stability study to >50 endogenous kinases across four cell lines and demonstrated that HSP90 dependency is context dependent. These observations suggest that HSP90 binds to its kinase client in a particular conformation that we hypothesize to be associated with the nucleotide-processing cycle. Lastly, we performed proteomics profiling of kinases and phosphopeptides in DLD-1 cells to globally define the impact of HSP90 inhibition on the kinome. PMID:26755559

  6. Protein kinase C is essential for viability of the rice blast fungus M agnaporthe oryzae

    PubMed Central

    Penn, Tina J.; Wood, Mark E.; Soanes, Darren M.; Csukai, Michael; Corran, Andrew John

    2015-01-01

    Summary Protein kinase C constitutes a family of serine–threonine kinases found in all eukaryotes and implicated in a wide range of cellular functions, including regulation of cell growth, cellular differentiation and immunity. Here, we present three independent lines of evidence which indicate that protein kinase C is essential for viability of M agnaporthe oryzae. First, all attempts to generate a target deletion of PKC 1, the single copy protein kinase C‐encoding gene, proved unsuccessful. Secondly, conditional gene silencing of PKC 1 by RNA interference led to severely reduced growth of the fungus, which was reversed by targeted deletion of the Dicer2‐encoding gene, MDL 2. Finally, selective kinase inhibition of protein kinase C by targeted allelic replacement with an analogue‐sensitive PKC 1AS allele led to specific loss of fungal viability in the presence of the PP1 inhibitor. Global transcriptional profiling following selective PKC inhibition identified significant changes in gene expression associated with cell wall re‐modelling, autophagy, signal transduction and secondary metabolism. When considered together, these results suggest protein kinase C is essential for growth and development of M . oryzae with extensive downstream targets in addition to the cell integrity pathway. Targeting protein kinase C signalling may therefore prove an effective means of controlling rice blast disease. PMID:26192090

  7. Protein kinase regulation of a cloned epithelial Na+ channel

    PubMed Central

    1996-01-01

    We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC- expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of - 100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel's open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be

  8. Transient Receptor Potential Channel 1 Deficiency Impairs Host Defense and Proinflammatory Responses to Bacterial Infection by Regulating Protein Kinase Cα Signaling

    PubMed Central

    Zhou, Xikun; Ye, Yan; Sun, Yuyang; Li, Xuefeng; Wang, Wenxue; Privratsky, Breanna; Tan, Shirui; Zhou, Zongguang; Huang, Canhua; Wei, Yu-Quan; Birnbaumer, Lutz

    2015-01-01

    Transient receptor potential channel 1 (TRPC1) is a nonselective cation channel that is required for Ca2+ homeostasis necessary for cellular functions. However, whether TRPC1 is involved in infectious disease remains unknown. Here, we report a novel function for TRPC1 in host defense against Gram-negative bacteria. TRPC1−/− mice exhibited decreased survival, severe lung injury, and systemic bacterial dissemination upon infection. Furthermore, silencing of TRPC1 showed decreased Ca2+ entry, reduced proinflammatory cytokines, and lowered bacterial clearance. Importantly, TRPC1 functioned as an endogenous Ca2+ entry channel critical for proinflammatory cytokine production in both alveolar macrophages and epithelial cells. We further identified that bacterium-mediated activation of TRPC1 was dependent on Toll-like receptor 4 (TLR4), which induced endoplasmic reticulum (ER) store depletion. After activation of phospholipase Cγ (PLC-γ), TRPC1 mediated Ca2+ entry and triggered protein kinase Cα (PKCα) activity to facilitate nuclear translocation of NF-κB/Jun N-terminal protein kinase (JNK) and augment the proinflammatory response, leading to tissue damage and eventually mortality. These findings reveal that TRPC1 is required for host defense against bacterial infections through the TLR4-TRPC1-PKCα signaling circuit. PMID:26031335

  9. Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

    PubMed Central

    Cargnello, Marie; Roux, Philippe P.

    2011-01-01

    Summary: The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries. PMID:21372320

  10. Prediction of Protein Kinase-Ligand Interactions through 2.5D Kinochemometrics.

    PubMed

    Bosc, Nicolas; Wroblowski, Berthold; Meyer, Christophe; Bonnet, Pascal

    2017-01-23

    So far, 518 protein kinases have been identified in the human genome. They share a common mechanism of protein phosphorylation and are involved in many critical biological processes of eukaryotic cells. Deregulation of the kinase phosphorylation function induces severe illnesses such as cancer, diabetes, or inflammatory diseases. Many actors in the pharmaceutical domain have made significant efforts to design potent and selective protein kinase inhibitors as new potential drugs. Because the ATP binding site is highly conserved in the protein kinase family, the design of selective inhibitors remains a challenge and has negatively impacted the progression of drug candidates to late-stage clinical development. The work presented here adopts a 2.5D kinochemometrics (KCM) approach, derived from proteochemometrics (PCM), in which protein kinases are depicted by a novel 3D descriptor and the ligands by 2D fingerprints. We demonstrate in two examples that the protein descriptor successfully classified protein kinases based on their group membership and their Asp-Phe-Gly (DFG) conformation. We also compared the performance of our models with those obtained from a full 2D KCM model and QSAR models. In both cases, the internal validation of the models demonstrated good capabilities to distinguish "active" from "inactive" protein kinase-ligand pairs. However, the external validation performed on two independent data sets showed that the two statistical models tended to overestimate the number of "inactive" pairs.

  11. The Role of Specific Mitogen-Activated Protein Kinase Signaling Cascades in the Regulation of Steroidogenesis

    PubMed Central

    Manna, Pulak R.; Stocco, Douglas M.

    2011-01-01

    Mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine kinases that are activated by a large variety of extracellular stimuli and play integral roles in controlling many cellular processes, from the cell surface to the nucleus. The MAPK family includes four distinct MAPK cascades, that is, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, c-Jun N-terminal kinase or stress-activated protein kinase, and ERK5. These MAPKs are essentially operated through three-tiered consecutive phosphorylation events catalyzed by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs lie in protein kinase cascades. The MAPK signaling pathways have been demonstrated to be associated with events regulating the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenesis in steroidogenic tissues. However, it has become clear that the regulation of MAPK-dependent StAR expression and steroid synthesis is a complex process and is context dependent. This paper summarizes the current level of understanding concerning the roles of the MAPK signaling cascades in the regulation of StAR expression and steroidogenesis in different steroidogenic cell models. PMID:21637381

  12. Characterisation of a plant 3-phosphoinositide-dependent protein kinase-1 homologue which contains a pleckstrin homology domain.

    PubMed

    Deak, M; Casamayor, A; Currie, R A; Downes, C P; Alessi, D R

    1999-05-28

    A plant homologue of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been identified in Arabidopsis and rice which displays 40% overall identity with human 3-phosphoinositide-dependent protein kinase-1. Like the mammalian 3-phosphoinositide-dependent protein kinase-1, Arabidopsis 3-phosphoinositide-dependent protein kinase-1 and rice 3-phosphoinositide-dependent protein kinase-1 possess a kinase domain at N-termini and a pleckstrin homology domain at their C-termini. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 can rescue lethality in Saccharomyces cerevisiae caused by disruption of the genes encoding yeast 3-phosphoinositide-dependent protein kinase-1 homologues. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 interacts via its pleckstrin homology domain with phosphatidic acid, PtdIns3P, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and to a lesser extent with PtdIns(4,5)P2 and PtdIns4P. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is able to activate human protein kinase B alpha (PKB/AKT) in the presence of PtdIns(3,4,5)P3. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is only the second plant protein reported to possess a pleckstrin homology domain and the first plant protein shown to bind 3-phosphoinositides.

  13. Mixed - Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states.

    PubMed

    Craige, Siobhan M; Reif, Michaella M; Kant, Shashi

    2016-09-01

    Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs).

    PubMed

    Barrows, Douglas; He, John Z; Parsons, Ramon

    2016-09-16

    Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.

  15. Calcium-dependent but calmodulin-independent protein kinase from soybean. [Glycine max

    SciTech Connect

    Harmon, A.C.; Putnam-Evans, C.; Cormier, M.J.

    1987-04-01

    A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (greater than or equal to 2 micromolar). The protein kinase activity was stimulated 100-fold by greater than or equal to 10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (less than or equal to 2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound /sup 45/Ca/sup 2 +/ in the presence of KCl and MgCl/sub 2/, which indicated that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity.

  16. Defining mitogen-activated protein kinase pathways with mass spectrometry-based approaches.

    PubMed

    Powell, David W; Pierce, William M; McLeish, Kenneth R

    2005-01-01

    Mitogen-activated protein kinases are a group of ubiquitously expressed kinase pathways that have been conserved from yeast through humans. They control a large number of critical cell functions. Identification of targets of those kinases is necessary to define signal transduction pathways that lead to cell responses. The application of a number of mass spectrometry-based techniques to the identification of phosphoproteins is reviewed. A new proteomic approach is described for the identification of the downstream targets of specific kinases that combines phosphorylation of cell lysates in in vitro kinase reactions by active recombinant kinase with protein separation by two-dimensional (2D) gel electrophoresis or SDS-PAGE and phosphoprotein identification by MALDI-TOF mass spectrometry or by phosphopeptide enrichment and tandem mass spectrometry. The results suggested that a combination of multiple approaches will be required to fully identify phosphoproteomes. (c) 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:847-864, 2005.

  17. Molecular Physiology of SPAK and OSR1: Two Ste20-Related Protein Kinases Regulating Ion Transport

    PubMed Central

    Gagnon, Kenneth B.; Delpire, Eric

    2015-01-01

    SPAK (Ste20-related proline alanine rich kinase) and OSR1 (oxidative stress responsive kinase) are members of the germinal center kinase VI sub-family of the mammalian Ste20 (Sterile20)-related protein kinase family. Although there are 30 enzymes in this protein kinase family, their conservation across the fungi, plant and animal kingdom confirms their evolutionary importance. Already, a large volume of work has accumulated on the tissue distribution, binding partners, signaling cascades, and physiological roles of mammalian SPAK and OSR1 in multiple organ systems. After reviewing this basic information, we will examine newer studies that demonstrate the pathophysiological consequences to SPAK and/or OSR1 disruption, discuss the development and analysis of genetically-engineered mouse models, and address the possible role these serine/threonine kinases might have in cancer proliferation and migration. PMID:23073627

  18. Cancer-associated mutations are preferentially distributed in protein kinase functional sites.

    PubMed

    Izarzugaza, Jose M G; Redfern, Oliver C; Orengo, Christine A; Valencia, Alfonso

    2009-12-01

    Protein kinases are a superfamily involved in many crucial cellular processes, including signal transmission and regulation of cell cycle. As a consequence of this role, kinases have been reported to be associated with many types of cancer and are considered as potential therapeutic targets. We analyzed the distribution of pathogenic somatic point mutations (drivers) in the protein kinase superfamily with respect to their location in the protein, such as in structural, evolutionary, and functionally relevant regions. We find these driver mutations are more clearly associated with key protein features than other somatic mutations (passengers) that have not been directly linked to tumor progression. This observation fits well with the expected implication of the alterations in protein kinase function in cancer pathogenicity. To explain the relevance of the detected association of cancer driver mutations at the molecular level in the human kinome, we compare these with genetically inherited mutations (SNPs). We find that the subset of nonsynonymous SNPs that are associated to disease, but sufficiently mild to the point of being widespread in the population, tend to avoid those key protein regions, where they could be more detrimental for protein function. This tendency contrasts with the one detected for cancer associated-driver-mutations, which seems to be more directly implicated in the alteration of protein function. The detailed analysis of protein kinase groups and a number of relevant examples, confirm the relation between cancer associated-driver-mutations and key regions for protein kinase structure and function.

  19. Transient Receptor Potential Canonical 7 (TRPC7): A Diacylglycerol-Activated Non-Selective Cation Channel

    PubMed Central

    Zhang, Xuexin

    2016-01-01

    Transient receptor potential canonical 7 (TRPC7) channel is the seventh member of the mammalian TRPC channel family. TRPC7 mRNA, protein and channel activity have been detected in many tissues and organs from mouse, rat and human. TRPC7 has high sequence homology with TRPC3 and TRPC6 and all three channels are activated by membrane receptors that couple to isoforms of phospholipase C (PLC) and mediate non-selective cation currents. TRPC7, along with TRPC3 and TRPC6 can be activated by direct exogenous application of diacylglycerol (DAG) analogs and by pharmacological maneuvers that increase endogenous DAG in cells. TRPC7 shows distinct properties of activation, such as constitutive activity, susceptibility to negative regulation by extracellular Ca2+ and by protein kinase C. TRPC7 can form heteromultimers with TRPC3 and TRPC6. Although TRPC7 remains one of the least studied TRPC channel, its role in various cell types and physiological and pathophysiological conditions is begining to emerge. PMID:24756707

  20. Archaeal protein kinases and protein phosphatases: insights from genomics and biochemistry.

    PubMed Central

    Kennelly, Peter J

    2003-01-01

    Protein phosphorylation/dephosphorylation has long been considered a recent addition to Nature's regulatory arsenal. Early studies indicated that this molecular regulatory mechanism existed only in higher eukaryotes, suggesting that protein phosphorylation/dephosphorylation had emerged to meet the particular signal-transduction requirements of multicellular organisms. Although it has since become apparent that simple eukaryotes and even bacteria are sites of protein phosphorylation/dephosphorylation, the perception widely persists that this molecular regulatory mechanism emerged late in evolution, i.e. after the divergence of the contemporary phylogenetic domains. Only highly developed cells, it was reasoned, could afford the high 'overhead' costs inherent in the acquisition of dedicated protein kinases and protein phosphatases. The advent of genome sequencing has provided an opportunity to exploit Nature's phylogenetic diversity as a vehicle for critically examining this hypothesis. In tracing the origins and evolution of protein phosphorylation/dephosphorylation, the members of the Archaea, the so-called 'third domain of life', will play a critical role. Whereas several studies have demonstrated that archaeal proteins are subject to modification by covalent phosphorylation, relatively little is known concerning the identities of the proteins affected, the impact on their functional properties, or the enzymes that catalyse these events. However, examination of several archaeal genomes has revealed the widespread presence of several ostensibly 'eukaryotic' and 'bacterial' protein kinase and protein phosphatase paradigms. Similar findings of 'phylogenetic trespass' in members of the Eucarya (eukaryotes) and the Bacteria suggest that this versatile molecular regulatory mechanism emerged at an unexpectedly early point in development of 'life as we know it'. PMID:12444920

  1. A mitogen-activated protein kinase kinase inhibitor induced compound skin toxicity with oedema in metastatic malignant melanoma.

    PubMed

    Thomas, C L; Mortimer, P S; Larkin, J M; Basu, T N; Gore, M E; Fearfield, L

    2016-04-01

    We report three cases of skin toxicity associated with oral mitogen-activated protein kinase kinase (MEK) inhibitor treatment for metastatic malignant melanoma (MM). All three patients developed oedema, and a single patient experienced eyelash trichomegaly. This is the first known report of eyelash trichomegaly secondary to MEK inhibitor use. We also discuss possible mechanisms for MEK inhibitor-associated oedema development. This series supports the role of the dermatologist in the screening and management of patients in the rapidly developing oncology setting, as new targeted agents can give rise to marked skin toxicity.

  2. Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis.

    PubMed

    Takiar, Vinita; Nishio, Saori; Seo-Mayer, Patricia; King, J Darwin; Li, Hui; Zhang, Li; Karihaloo, Anil; Hallows, Kenneth R; Somlo, Stefan; Caplan, Michael J

    2011-02-08

    Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD.

  3. Protein Kinase CK2 Content in GL261 Mouse Glioblastoma.

    PubMed

    Ferrer-Font, Laura; Alcaraz, Estefania; Plana, Maria; Candiota, Ana Paula; Itarte, Emilio; Arús, Carles

    2016-07-01

    Glioblastoma (GBM) is the most prevalent and aggressive human glial tumour with a median survival of 14-15 months. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment. Unfortunately, chemoresistence always ensues with concomitant tumour regrowth. Protein kinase CK2 (CK2) contributes to tumour development, proliferation, and suppression of apoptosis in cancer and it is overexpressed in human GBM. Targeting CK2 in GBM treatment may benefit patients. With this translational perspective in mind, we have studied the CK2 expression level by Western blot analysis in a preclinical model of GBM: GL261 cells growing orthotopically in C57BL/6 mice. The expression level of the CK2 catalytic subunit (CK2α) was higher in tumour (about 4-fold) and in contralateral brain parenchyma (more than 2-fold) than in normal brain parenchyma (p < 0.05). In contrast, no significant changes were found in CK2 regulatory subunit (CK2β) expression, suggesting an increased unbalance of CK2α/CK2β in GL261 tumours with respect to normal brain parenchyma, in agreement with a differential role of these two subunits in tumours.

  4. Protein kinase GCN2 mediates responses to glyphosate in Arabidopsis.

    PubMed

    Faus, Isabel; Zabalza, Ana; Santiago, Julia; Nebauer, Sergio G; Royuela, Mercedes; Serrano, Ramon; Gadea, Jose

    2015-01-21

    The increased selection pressure of the herbicide glyphosate has played a role in the evolution of glyphosate-resistance in weedy species, an issue that is becoming a threat to global agriculture. The molecular components involved in the cellular toxicity response to this herbicide at the expression level are still unidentified. In this study, we identify the protein kinase GCN2 as a cellular component that fosters the action of glyphosate in the model plant Arabidopsis thaliana. Comparative studies using wild-type and gcn2 knock-out mutant seedlings show that the molecular programme that the plant deploys after the treatment with the herbicide, is compromised in gcn2. Moreover, gcn2 adult plants show a lower inhibition of photosynthesis, and both seedlings and adult gcn2 plants accumulate less shikimic acid than wild-type after treatment with glyphosate. These results points to an unknown GCN2-dependent factor involved in the cascade of events triggered by glyphosate in plants. Data suggest either that the herbicide does not equally reach the target-enzyme in a gcn2 background, or that a decreased flux in the shikimate pathway in a gcn2 plants minimize the impact of enzyme inhibition.

  5. Death Associated Protein Kinases: Molecular Structure and Brain Injury

    PubMed Central

    Nair, Syam; Hagberg, Henrik; Krishnamurthy, Rajanikant; Thornton, Claire; Mallard, Carina

    2013-01-01

    Perinatal brain damage underlies an important share of motor and neurodevelopmental disabilities, such as cerebral palsy, cognitive impairment, visual dysfunction and epilepsy. Clinical, epidemiological, and experimental studies have revealed that factors such as inflammation, excitotoxicity and oxidative stress contribute considerably to both white and grey matter injury in the immature brain. A member of the death associated protein kinase (DAPk) family, DAPk1, has been implicated in cerebral ischemic damage, whereby DAPk1 potentiates NMDA receptor-mediated excitotoxicity through interaction with the NR2BR subunit. DAPk1 also mediate a range of activities from autophagy, membrane blebbing and DNA fragmentation ultimately leading to cell death. DAPk mRNA levels are particularly highly expressed in the developing brain and thus, we hypothesize that DAPk1 may play a role in perinatal brain injury. In addition to reviewing current knowledge, we present new aspects of the molecular structure of DAPk domains, and relate these findings to interacting partners of DAPk1, DAPk-regulation in NMDA-induced cerebral injury and novel approaches to blocking the injurious effects of DAPk1. PMID:23880846

  6. Death associated protein kinases: molecular structure and brain injury.

    PubMed

    Nair, Syam; Hagberg, Henrik; Krishnamurthy, Rajanikant; Thornton, Claire; Mallard, Carina

    2013-07-04

    Perinatal brain damage underlies an important share of motor and neurodevelopmental disabilities, such as cerebral palsy, cognitive impairment, visual dysfunction and epilepsy. Clinical, epidemiological, and experimental studies have revealed that factors such as inflammation, excitotoxicity and oxidative stress contribute considerably to both white and grey matter injury in the immature brain. A member of the death associated protein kinase (DAPk) family, DAPk1, has been implicated in cerebral ischemic damage, whereby DAPk1 potentiates NMDA receptor-mediated excitotoxicity through interaction with the NR2BR subunit. DAPk1 also mediate a range of activities from autophagy, membrane blebbing and DNA fragmentation ultimately leading to cell death. DAPk mRNA levels are particularly highly expressed in the developing brain and thus, we hypothesize that DAPk1 may play a role in perinatal brain injury. In addition to reviewing current knowledge, we present new aspects of the molecular structure of DAPk domains, and relate these findings to interacting partners of DAPk1, DAPk-regulation in NMDA-induced cerebral injury and novel approaches to blocking the injurious effects of DAPk1.

  7. Properties of the protein kinase C-phorbol ester interaction

    SciTech Connect

    Bazzi, M.D.; Nelsestuen, G.L. )

    1989-04-18

    The properties of the protein kinase C (PKC)-phorbol ester interaction were highly dependent on assay methods and conditions. Binding to cation-exchange materials or adsorption to gel matrices resulted in PKC that was capable of binding phorbol 12,13-dibutyrate (PDBu). The extraneous interactions were eliminated by measuring phorbol ester binding with a gel filtration chromatography assay in the presence of bovine serum albumin (BSA). In the absence of calcium, free PKC did not bind PDBu or phospholipids. Calcium caused structural changes in PKC which enhanced its interaction with surfaces such as the gel chromatography matrix. While BSA prevented this interaction, it did not interfere with PKC association with acidic phospholipids. Interaction of PKC with phospholipid resulted in two forms of membrane-associated PKC. Once PKC was inserted into a phospholipid bilayer, it bound PDBu in the presence and in the absence of Ca{sup 2+}. Calcium enhanced the affinity of PKC-PDBu interaction and decreased the dissociation rate. These results showed that dramatic changes occurred in the in vitro properties of PKC upon the formation of the irreversible PKC-membrane complex. These properties may be related to cellular events that induce formation of the chelator-resistant form of membrane-bound PKC.

  8. Positive feedback of protein kinase C proteolytic activation during apoptosis.

    PubMed Central

    Leverrier, Sabrina; Vallentin, Alice; Joubert, Dominique

    2002-01-01

    In contrast with protein kinase Calpha (PKCalpha) and PKCepsilon, which are better known for promoting cell survival, PKCdelta is known for its pro-apoptotic function, which is exerted mainly through a caspase-3-dependent proteolytic activation pathway. In the present study, we used the rat GH3B6 pituitary adenoma cell line to show that PKCalpha and PKCepsilon are activated and relocalized together with PKCdelta when apoptosis is induced by a genotoxic stress. Proteolytic activation is a crucial step used by the three isoforms since: (1) the catalytic domains of the PKCalpha, PKCepsilon or PKCdelta isoforms (CDalpha, CDepsilon and CDdelta respectively) accumulated, and this accumulation was dependent on the activity of both calpain and caspase; and (2) transient expression of CDalpha, CDepsilon or CDdelta sufficed to induce apoptosis. However, following this initial step of proteolytic activation, the pathways diverge; cytochrome c release and caspase-3 activation are induced by CDepsilon and CDdelta, but not by CDalpha. Another interesting finding of the present study is the proteolysis of PKCdelta induced by CDepsilon expression that revealed the existence of a cross-talk between PKC isoforms during apoptosis. Hence the PKC family may participate in the apoptotic process of pituitary adenoma cells at two levels: downstream of caspase and calpain, and via retro-activation of caspase-3, resulting in the amplification of its own proteolytic activation. PMID:12238950

  9. A Rice Kinase-Protein Interaction Map1[W][OA

    PubMed Central

    Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A.; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E.; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G.; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E.; Ronald, Pamela C.; Song, Wen-Yuan

    2009-01-01

    Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed. PMID:19109415

  10. Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

    PubMed Central

    Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

    1993-01-01

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

  11. Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols.

    PubMed Central

    Ganong, B R; Loomis, C R; Hannun, Y A; Bell, R M

    1986-01-01

    The specificity of protein kinase C activation by sn-1,2-diacylglycerols and analogues was investigated by using a Triton X-100 mixed micellar assay [Hannun, Y. A., Loomis, C. R. & Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043]. Analogues containing acyl or alkyl chains eight carbons in length were synthesized because sn-1,2-dioctanoylglycerol is an effective cell-permeant activator of protein kinase C. These analogues were tested as activators and antagonists of rat brain protein kinase C to determine the exact structural features important for activity. The analogues established that activation of protein kinase C by diacylglycerols is highly specific. Several analogues established that both carbonyl moieties of the oxygen esters are required for maximal activity and that the 3-hydroxyl moiety is also required. None of the analogues were antagonists. These data, combined with previous investigations, permitted formulation of a model of protein kinase C activation. A three-point attachment of sn-1,2-diacylglycerol to the surface-bound protein kinase C-phosphatidylserine-Ca2+ complex is envisioned to cause activation. Direct ligation of diacylglycerol to Ca2+ is proposed to be an essential step in the mechanism of activation of protein kinase C. Images PMID:3456578

  12. Interactions between hematopoietic progenitor kinase 1 and its adaptor proteins (Review).

    PubMed

    Zhang, Qing; Ding, Shu; Zhang, Huilin

    2017-09-13

    Hematopoietic progenitor kinase 1 (HPK1), also known as mitogen‑activated protein kinase kinase kinase kinase 1 is a serine/threonine protein kinase. It is involved in various cellular events, including mitogen‑activated protein kinase signaling, nuclear factor‑κB signaling, cytokine signaling, cellular proliferation and apoptosis, T cell receptor/B cell receptor signaling and T/B/dendritic cell‑mediated immune responses. Therefore, HPK1 has variety of roles in immunity, and is associated with the pathogenesis of autoimmune diseases, cancer, and the inflammatory response. In these cellular and immune events, HPK1 interacts with several adaptor proteins, including caspase recruitment domain family, member 11, hematopoietic cell‑specific protein 1, HPK1‑interacting protein of 55 kDa, the growth factor receptor‑bound protein 2 family, linker for activated T‑cells, the SH2 domain‑containing leukocyte protein of 76 kDa family, the v‑crk avian sarcoma virus CT10 oncogene homolog family, B‑cell adaptor molecule of 32 kDa and non‑catalytic region of tyrosine kinase adaptor protein. These adaptor proteins can couple HPK1 with various effector molecules, leading to the transmission of upstream signals to downstream targets. They are crucial in regulating the relocation, phosphorylation, activation and functions of HPK1. HPK1 can also phosphorylate certain proteins, consequently modulating their functions. This review aims to describe the adaptor proteins, which interact with HPK1, particularly focusing on their modes of interaction with HPK1, and the effects that these interactions cause.

  13. Further characterization of protein kinase C in mouse mast cells

    SciTech Connect

    White, J.R.; Ishizaka, T.

    1986-03-01

    Bridging of cell-bound IgE antibody molecules on colony stimulating factor dependent mouse mast cell line (PT-18) cells by multivalent antigen induces the mobilization and uptake of Ca/sup 2 +/ monitored by Quin-2 and the production of diacylglycerol. Exposure of the sensitized cells to antigen also induces a substantial increase in protein kinase C (PKC) activity in the plasma membrane (340 units to 1375 units: 1 unit = 1 pmol of /sup 32/P incorporated into Histone H-1/min/10/sup 7/ cells), within 30 seconds. There is also an increase in /sup 3/H phorbol-12, 13-dibutyrate (/sup 3/H-PDB) binding which parallels the increase in PKC activity both in kinetics and antigen dose dependency. Determination of K/sub m/ and V/sub max/ for PKC revealed no difference between the cytosolic and membranous forms of PKC. Partial purification of PKC from the membrane of sensitized mast cells which had been labeled with /sup 32/P and stimulated with DNP-HSA revealed a protein of 80-84,000 molecular weight, which migrated on polyacrylamide gel electrophoresis just above an authentic standard of PKC purified from rat brain. Treatment of the PKC from mouse mast cell membrane with alkaline phosphatase resulted in a reduction of phosphorylating activity and bindability of /sup 3/H-PDB. In conclusion, the authors speculate that activation of mouse mast cells by cross-linking IgE results in the phosphorylation of a silent-pool of PKC converting it from an inactive state to an activated form.

  14. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    SciTech Connect

    Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

    2010-10-22

    Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  15. Neurochondrin is an atypical RIIα-specific A-kinase anchoring protein