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Sample records for ii protein kinase

  1. Angiotensin II stimulates melanogenesis via the protein kinase C pathway

    PubMed Central

    LIU, LI-HONG; FAN, XIN; XIA, ZHI-KUAN; AN, XU-XI; YANG, RONG-YA

    2015-01-01

    Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which serve a crucial function in hyperpigmentation. The aim of the present study was to determine the effects of angiotensin II (Ang II) on melanogenesis and to elucidate the molecular events of Ang II-induced melanogenesis. Experiments were performed on human melanocytes to elucidate the pigmenting effect of Ang II and the underlying mechanisms. The elements involved in melanogenesis, including melanin content, tyrosinase (TYR) activity, and microphthalmia-associated transcription factor (MITF) and TYR expression at the mRNA and protein levels were evaluated. Melanin content and TYR activity increased in response to Ang II treatment in a concentration-dependent manner. MITF and TYR mRNA and protein expression levels were increased significantly in response to Ang II in a concentration-dependent manner. The Ang II-induced increase in melanin synthesis was reduced significantly in response to co-treatment with Ro-32-0432, a protein kinase C (PKC) inhibitor, whereas co-treatment with H-89, a PKA inhibitor, did not attenuate the Ang II-induced increase in melanin levels. These results suggest that PKC is required for Ang II-induced pigmentation in human melanocytes and that the mechanism involves the PKC pathway and MITF upregulation. PMID:26622519

  2. IMMUNOCYTOCHEMICAL LOCALIZATION OF CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE II IN RAT BRAIN

    EPA Science Inventory

    Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is a prominent enzyme in mammalian brain capable of phosphorylating a variety of substrate proteins. In the present investigation, the subcellular and regional distribution of CaM kinase II has been studied by light a...

  3. 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

  4. AKAP79 Selectively Enhances Protein Kinase C Regulation of GluR1 at a Ca2+-Calmodulin-dependent Protein Kinase II/Protein Kinase C Site*

    PubMed Central

    Tavalin, Steven J.

    2008-01-01

    Enhancement of AMPA receptor activity in response to synaptic plasticity inducing stimuli may arise, in part, through phosphorylation of the GluR1 AMPA receptor subunit at Ser-831. This site is a substrate for both Ca2+-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). However, neuronal protein levels of CaMKII may exceed those of PKC by an order of magnitude. Thus, it is unclear how PKC could effectively regulate this common target site. The multivalent neuronal scaffold A-kinase-anchoring protein 79 (AKAP79) is known to bind PKC and is linked to GluR1 by synapse-associated protein 97 (SAP97). Here, biochemical studies demonstrate that AKAP79 localizes PKC activity near the receptor, thus accelerating Ser-831 phosphorylation. Complementary electrophysiological studies indicate that AKAP79 selectively shifts the dose-dependence for PKC modulation of GluR1 receptor currents ∼20-fold, such that low concentrations of PKC are as effective as much higher CaMKII concentrations. By boosting PKC activity near a target substrate, AKAP79 provides a mechanism to overcome limitations in kinase abundance thereby ensuring faithful signal propagation and efficient modification of AMPA receptor-mediated responses. PMID:18305116

  5. 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.

  6. Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity

    PubMed Central

    Lim, Butaek; Park, Ji-In; Lee, Kyung Jin; Lee, Jin-Won; Kim, Tae-Wuk; Kim, Young-Pil

    2015-01-01

    We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity. PMID:26213934

  7. 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

  8. Protein kinase that phosphorylates light-harvesting complex is autophosphorylated and is associated with photosystem II

    SciTech Connect

    Coughlan, S.J.; Hind, G.

    1987-10-06

    Thylakoid membranes were phosphorylated with (..gamma..-/sup 32/P)ATP and extracted with octyl glucoside and cholate. Among the radiolabeled phosphoproteins in the extract was a previously characterized protein kinase of 64-kDa apparent mass. The ability of this enzyme to undergo autophosphorylation in situ was used to monitor its distribution in the membrane. Fractionation studies showed that the kinase is confined to granal regions of the thylakoid, where it appears to be associated with the light-harvesting chlorophyll-protein complex of photosystem II. The kinetics of kinase autophosphorylation were investigated both in situ and in extracted, purified enzyme. In the membrane, autophosphorylation saturated within 20-30 min and was reversed with a half-time of 7-8 min upon removal of ATP or oxidative inactivation of the kinase; the accompanying dephosphorylation of light-harvesting complex was slower and kinetically complex. Fluoride (10 mM) inhibited these dephosphorylations. Autophosphorylation of the isolated kinase was independent of enzyme concentration, indicative of an intramolecular mechanism. A maximum of one serine residue per mole of kinase was esterified. Autophosphorylation was more rapid in the presence of histone IIIs, an exogenous substrate. Dephosphorylation of the isolated enzyme was not observed.

  9. A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

    PubMed Central

    Guilfoyle, T J

    1989-01-01

    A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit. PMID:2535525

  10. Protein kinase C beta II suppresses colorectal cancer by regulating IGF-1 mediated cell survival.

    PubMed

    Dowling, Catríona M; Phelan, James; Callender, Julia A; Cathcart, Mary Clare; Mehigan, Brian; McCormick, Paul; Dalton, Tara; Coffey, John C; Newton, Alexandra C; O'Sullivan, Jacintha; Kiely, Patrick A

    2016-04-12

    Despite extensive efforts, cancer therapies directed at the Protein Kinase C (PKC) family of serine/threonine kinases have failed in clinical trials. These therapies have been directed at inhibiting PKC and have, in some cases, worsened disease outcome. Here we examine colon cancer patients and show not only that PKC Beta II is a tumour suppressor, but patients with low levels of this isozyme have significantly decreased disease free survival. Specifically, analysis of gene expression levels of all PKC genes in matched normal and cancer tissue samples from colon cancer patients revealed a striking down-regulation of the gene coding PKC Beta in the cancer tissue (n = 21). Tissue microarray analysis revealed a dramatic down-regulation of PKC Beta II protein levels in both the epithelial and stromal diseased tissue (n = 166). Of clinical significance, low levels of the protein in the normal tissue of patients is associated with a low (10%) 10 year survival compared with a much higher (60%) survival in patients with relatively high levels of the protein. Consistent with PKC Beta II levels protecting against colon cancer, overexpression of PKC Beta II in colon cancer cell lines reveals that PKC Beta II reverses transformation in cell based assays. Further to this, activation of PKC Beta II results in a dramatic downregulation of IGF-I-induced AKT, indicating a role for PKCs in regulating IGF-1 mediated cell survival. Thus, PKC Beta II is a tumour suppressor in colon cancer and low levels serve as a predictor for poor survival outcome. PMID:26989024

  11. Protein kinase C beta II suppresses colorectal cancer by regulating IGF-1 mediated cell survival

    PubMed Central

    Dowling, Catríona M.; Phelan, James; Callender, Julia A.; Cathcart, Mary Clare; Mehigan, Brian; McCormick, Paul; Dalton, Tara; Coffey, John C.; Newton, Alexandra C.; O'sullivan, Jacintha; Kiely, Patrick A.

    2016-01-01

    Despite extensive efforts, cancer therapies directed at the Protein Kinase C (PKC) family of serine/threonine kinases have failed in clinical trials. These therapies have been directed at inhibiting PKC and have, in some cases, worsened disease outcome. Here we examine colon cancer patients and show not only that PKC Beta II is a tumour suppressor, but patients with low levels of this isozyme have significantly decreased disease free survival. Specifically, analysis of gene expression levels of all PKC genes in matched normal and cancer tissue samples from colon cancer patients revealed a striking down-regulation of the gene coding PKC Beta in the cancer tissue (n = 21). Tissue microarray analysis revealed a dramatic down-regulation of PKC Beta II protein levels in both the epithelial and stromal diseased tissue (n = 166). Of clinical significance, low levels of the protein in the normal tissue of patients is associated with a low (10%) 10 year survival compared with a much higher (60%) survival in patients with relatively high levels of the protein. Consistent with PKC Beta II levels protecting against colon cancer, overexpression of PKC Beta II in colon cancer cell lines reveals that PKC Beta II reverses transformation in cell based assays. Further to this, activation of PKC Beta II results in a dramatic downregulation of IGF-I-induced AKT, indicating a role for PKCs in regulating IGF-1 mediated cell survival. Thus, PKC Beta II is a tumour suppressor in colon cancer and low levels serve as a predictor for poor survival outcome. PMID:26989024

  12. Muscarinic activation of Ca2+/calmodulin-dependent protein kinase II in pancreatic islets. Temporal dissociation of kinase activation and insulin secretion.

    PubMed Central

    Babb, E L; Tarpley, J; Landt, M; Easom, R A

    1996-01-01

    We have demonstrated previously that glucose activates the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in isolated rat pancreatic islets in a manner consistent with a role of this enzyme in the regulation of insulin secretion [Wenham, Landt and Easom (1994) J. Biol. Chem. 269, 4947-4952]. In the current study, the muscarinic agonist, carbachol, has been shown to induce the conversion of CaM kinase II into a Ca(2+)-independent, autonomous form indicative of its activation. Maximal activation (2-fold) was achieved by 15 s, followed by a rapid return to basal levels by 1 min. This response was primarily the result of the mobilization of Ca2+ from intracellular stores since it was not affected by a concentration (20 microM) of verapamil that completely prevented the activation of CaM kinase II by glucose. Surprisingly, carbachol added prior to, or simultaneously with, glucose attenuated nutrient activation of CaM kinase II. This effect was mimicked by cholecystokinin-8 (CCK-8) and thapsigargin, suggesting its mediation by phospholipase C and the mobilization of intracellular Ca2+. In contrast, carbachol, CCK-8 and thapsigargin markedly potentiated glucose (12 mM)-induced insulin secretion. These results suggest that CaM kinase II activation can be temporally dissociated from insulin secretion but do not exclude the potential dependence of insulin exocytosis on CaM kinase II-mediated protein phosphorylation. PMID:8694759

  13. Human biliverdin reductase, a previously unknown activator of protein kinase C betaII.

    PubMed

    Maines, Mahin D; Miralem, Tihomir; Lerner-Marmarosh, Nicole; Shen, Jenny; Gibbs, Peter E M

    2007-03-16

    Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) betaII, activated by insulin and free radicals (Miralem, T., Hu, Z., Torno, M. D., Lelli, K. M., and Maines, M. D. (2005) J. Biol. Chem. 280, 17084-17092; Lerner-Marmarosh, N., Shen, J., Torno, M. D., Kravets, A., Hu, Z., and Maines, M. D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7109-7114). Here, by using 293A cells co-transfected with pcDNA3-hBVR and PKC betaII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC betaII, but not the reductase and PKC zeta, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC betaII K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC betaII for myelin basic protein; activation was independent of phospholipids and extended to the phosphorylation of S2, a PKC-specific substrate. The increase in substrate phosphorylation was blocked by specific inhibitors of conventional PKCs and attenuated by sihBVR. The effect of the latter could be rescued by subsequent overexpression of hBVR. To a large extent, the activation was a function of the hBVR N-terminal chain of valines and intact ATP-binding site and the cysteine-rich C-terminal segment. The cobalt protoporphyrin-activated hBVR phosphorylated a threonine in a peptide corresponding to the Thr500 in the human PKC betaII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC betaII nor PKC zeta activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR.PKC betaII immunocomplex. The potential biological relevance of the hBVR activation of PKC betaII was suggested by the finding that in cells transfected with the PKC betaII, h

  14. Phosphorylation of Alzheimer disease amyloid precursor peptide by protein kinase C and Ca sup 2+ /calmodulin-dependent protein kinase II

    SciTech Connect

    Gandy, S.; Czernik, A.J.; Greengard, P. )

    1988-08-01

    The amino acid sequence of the Alzheimer disease amyloid precursor (ADAP) has been deduced from the corresponding cDNA, and hydropathy analysis of the sequence suggest a receptor-like structure with a single transmembrane domain. The putative cytoplasmic domain of ADAP contains potential sites for serine and threonine phosphorylation. In the present study, synthetic peptides derived from this domain were used as model substrates for various purified protein kinases. Protein kinase C rapidly catalyzed the phosphorylation of a peptide corresponding to amino acid residues 645-661 of ADAP. Ca{sup 2+}/calmodulin-dependent protein kinase II phosphorylated ADAP peptide (645-661) on Thr-654 and Ser-655. Using rat cerebral cortex synaptosomes prelabeled with {sup 32}P{sub i}, a {sup 32}P-labeled phosphoprotein of {approx}135 kDa was immunoprecipitated by using antisera prepared against ADAP peptide(597-624), consistent with the possibility that the holoform of ADAP in rat brain is a phosphoprotein. Based on analogy with the effect of phosphorylation by protein kinase C of juxtamembrane residues in the cytoplasmic domain of the epidermal growth factor receptor and the interleukin 2 receptor, phosphorylation of ADAP may target it for internalization.

  15. Role of cyclooxygenase 2 in protein kinase C beta II-mediated colon carcinogenesis.

    PubMed

    Yu, Wangsheng; Murray, Nicole R; Weems, Capella; Chen, Lu; Guo, Huiping; Ethridge, Richard; Ceci, Jeffrey D; Evers, B Mark; Thompson, E Aubrey; Fields, Alan P

    2003-03-28

    Elevated expression of protein kinase C beta II (PKC beta II) is an early promotive event in colon carcinogenesis (Gokmen-Polar, Y., Murray, N. R., Velasco, M. A., Gatalica, Z., and Fields, A. P. (2001) Cancer Res. 61, 1375-1381). Expression of PKC beta II in the colon of transgenic mice leads to hyperproliferation and increased susceptibility to colon carcinogenesis due, at least in part, to repression of transforming growth factor beta type II receptor (TGF-beta RII) expression (Murray, N. R., Davidson, L. A., Chapkin, R. S., Gustafson, W. C., Schattenberg, D. G., and Fields, A. P. (1999) J. Cell Biol., 145, 699-711). Here we report that PKC beta II induces the expression of cyclooxygenase type 2 (Cox-2) in rat intestinal epithelial (RIE) cells in vitro and in transgenic PKC beta II mice in vivo. Cox-2 mRNA increases more than 10-fold with corresponding increases in Cox-2 protein and PGE2 production in RIE/PKC beta II cells. PKC beta II activates the Cox-2 promoter by 2- to 3-fold and stabilizes Cox-2 mRNA by at least 4-fold. The selective Cox-2 inhibitor Celecoxib restores expression of TGF-beta RII both in vitro and in vivo and restores TGF beta-mediated transcription in RIE/PKC beta II cells. Likewise, the omega-3 fatty acid eicosapentaenoic acid (EPA), which inhibits PKC beta II activity and colon carcinogenesis, causes inhibition of Cox-2 protein expression, re-expression of TGF-beta RII, and restoration of TGF-beta1-mediated transcription in RIE/PKC beta II cells. Our data demonstrate that PKC beta II promotes colon cancer, at least in part, through induction of Cox-2, suppression of TGF-beta signaling, and establishment of a TGF-beta-resistant, hyperproliferative state in the colonic epithelium. Our data define a procarcinogenic PKC beta II --> Cox-2 --> TGF-beta signaling axis within the colonic epithelium, and provide a molecular mechanism by which dietary omega-3 fatty acids and nonsteroidal antiinflammatory agents such as Celecoxib suppress colon

  16. Mitogen-activated protein kinase ERK1/2 regulates the class II transactivator.

    PubMed

    Voong, Lilien N; Slater, Allison R; Kratovac, Sebila; Cressman, Drew E

    2008-04-01

    The expression of major histocompatibility class II genes is necessary for proper antigen presentation and induction of an immune response. This expression is initiated by the class II transactivator, CIITA. The establishment of the active form of CIITA is controlled by a series of post-translational events, including GTP binding, ubiquitination, and dimerization. However, the role of phosphorylation is less clearly defined as are the consequences of phosphorylation on CIITA activity and the identity of the kinases involved. In this study we show that the extracellular signal-regulated kinases 1 and 2 (ERK1/2) interact directly with CIITA, targeting serine residues in the amino terminus of the protein, including serine 288. Inhibition of this phosphorylation by dominant-negative forms of ERK or by treatment of cells with the ERK inhibitor PD98059 resulted in the increase in CIITA-mediated gene expression from a class II promoter, enhanced the nuclear concentration of CIITA, and impaired its ability to bind to the nuclear export factor, CRM1. In contrast, inhibition of ERK1/2 activity had little effect on serine-to-alanine mutant forms of CIITA. These data suggest a model whereby ERK1/2-mediated phosphorylation of CIITA down-regulates CIITA activity by priming it for nuclear export, thus providing a means for cells to tightly regulate the extent of antigen presentation. PMID:18245089

  17. Catalytic and molecular properties of highly purified phosvitin/casein kinase type II from human epithelial cells in culture (HeLa) and relation to ecto protein kinase.

    PubMed

    Pyerin, W; Burow, E; Michaely, K; Kübler, D; Kinzel, V

    1987-03-01

    Phosvitin/casein type II kinase was purified from HeLa cell extracts to homogeneity and characterized. The kinase prefers phosvitin over casein (Vmax phosvitin greater than Vmax casein; apparent Km 0.5 microM phosvitin and 3.3 microM casein) and utilizes as cosubstrate ATP (apparent Km 3-4 microM), GTP (apparent Km 4-5 microM) and other purine nucleoside triphosphates, including dATP and dGTP but not pyrimidine nucleoside triphosphates. Enzyme reaction is optimal at pH 6-8 and at 10-25 mM Mg2+.Mg2+ cannot be replaced by, but is antagonized by other divalent metal ions. The kinase is stimulated by polycations (spermine) and monovalent cations (Na+,K+), and is inhibited by fluoride, 2,3-diphosphoglycerate, and low levels of heparin (50% inhibition at 0.1 microgram/ml). The HeLa enzyme is composed of three subunits with Mr of approximately 43,000 (alpha), 38,000 (alpha'), and 28,000 (beta) forming alpha alpha'beta 2 and alpha'2 beta 2 structures with obvious sequence homology of alpha with alpha' but not with beta. Photoaffinity labeling with [alpha-32P]- and [gamma-32P]8-azido-ATP revealed high affinity binding sites on subunits alpha and alpha' but not on subunit beta. The kinase autophosphorylates subunit beta and, much weaker, subunits alpha and alpha'. Ecto protein kinase, detectable only by its enzyme activity but not yet as a protein (J. Biol. Chem. 257, 322-329), was characterized in cell-bound form and in released form, and the released form both with and without prior separation from phosvitin which was employed to induce the kinase release from intact HeLa cells (Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025). Ratios of phosvitin/casein phosphorylation (greater than 2) and of ATP/GTP utilization (1.5-2.1), inhibition by heparin (50% inhibition at 0.1 microgram/ml), and amino-acid side chains phosphorylated in phosvitin and casein (serine, threonine) are comparable for cell-bound and released form. These properties resemble those of type II kinase as does Mr

  18. Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II.

    PubMed Central

    Hudmon, Andy; Schulman, Howard

    2002-01-01

    Ca2+/calmodulin (CaM)-dependent protein kinase (CaMKII) is a ubiquitous mediator of Ca2+-linked signalling that phosphorylates a wide range of substrates to co-ordinate and regulate Ca2+-mediated alterations in cellular function. The transmission of information by the kinase from extracellular stimuli and the intracellular Ca2+ rise is not passive. Rather, its multimeric structure and autoregulation enable this enzyme to participate actively in the sensitivity, timing and location of its action. CaMKII can: (i) be activated in a Ca2+-spike frequency-dependent manner; (ii) become independent of its initial Ca2+/CaM activators; and (iii) undergo a 'molecular switch-like' behaviour, which is crucial for certain forms of learning and memory. CaMKII is derived from a family of four homologous but distinct genes, with over 30 alternatively spliced isoforms described at present. These isoforms possess diverse developmental and anatomical expression patterns, as well as subcellular localization. Six independent catalytic/autoregulatory domains are connected by a narrow stalk-like appendage to each hexameric ring within the dodecameric structure. Ca2+/CaM binding activates the enzyme by disinhibiting the autoregulatory domain; this process initiates an intra-holoenzyme autophosphorylation reaction that induces complex changes in the enzyme's sensitivity to Ca2+/CaM, including the generation of Ca2+/CaM-independent (autonomous) activity and marked increase in affinity for CaM. The role of CaMKII in Ca2+ signal transduction is shaped by its autoregulation, isoenzymic type and subcellular localization. The molecular determinants and mechanisms producing these processes are discussed as they relate to the structure-function of this multifunctional protein kinase. PMID:11931644

  19. Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II.

    PubMed

    Hudmon, Andy; Schulman, Howard

    2002-06-15

    Ca2+/calmodulin (CaM)-dependent protein kinase (CaMKII) is a ubiquitous mediator of Ca2+-linked signalling that phosphorylates a wide range of substrates to co-ordinate and regulate Ca2+-mediated alterations in cellular function. The transmission of information by the kinase from extracellular stimuli and the intracellular Ca2+ rise is not passive. Rather, its multimeric structure and autoregulation enable this enzyme to participate actively in the sensitivity, timing and location of its action. CaMKII can: (i) be activated in a Ca2+-spike frequency-dependent manner; (ii) become independent of its initial Ca2+/CaM activators; and (iii) undergo a 'molecular switch-like' behaviour, which is crucial for certain forms of learning and memory. CaMKII is derived from a family of four homologous but distinct genes, with over 30 alternatively spliced isoforms described at present. These isoforms possess diverse developmental and anatomical expression patterns, as well as subcellular localization. Six independent catalytic/autoregulatory domains are connected by a narrow stalk-like appendage to each hexameric ring within the dodecameric structure. Ca2+/CaM binding activates the enzyme by disinhibiting the autoregulatory domain; this process initiates an intra-holoenzyme autophosphorylation reaction that induces complex changes in the enzyme's sensitivity to Ca2+/CaM, including the generation of Ca2+/CaM-independent (autonomous) activity and marked increase in affinity for CaM. The role of CaMKII in Ca2+ signal transduction is shaped by its autoregulation, isoenzymic type and subcellular localization. The molecular determinants and mechanisms producing these processes are discussed as they relate to the structure-function of this multifunctional protein kinase. PMID:11931644

  20. Kinetics of the inhibition of calcium/calmodulin-dependent protein kinase II by pea protein-derived peptides.

    PubMed

    Li, Huan; Aluko, Rotimi E

    2005-11-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) catalyzes the phosphorylation of various cellular proteins and excessive activities have been implicated in the pathogenesis of various chronic diseases. We hypothesized that positively charged peptides can be produced through enzymatic hydrolysis of pea proteins; such peptides could then bind to negatively charged calmodulin (CaM) at a physiological pH level and inhibit CaMKII activity. Pea protein isolate was hydrolyzed with an alkaline protease (alcalase) and filtered through a 1000-mol wt cutoff membrane. The permeate, which contained low-molecular weight peptides, was used to isolate cationic peptides on an SP-Sepharose column by ion exchange chromatography. Separation of the permeate on the SP-Sepharose column yielded two fractions with net positive charges that were subsequently used for enzyme inhibition studies. Fraction I eluted earlier from the column and contained lower contents of lysine and arginine than Fraction II, which eluted later. Results show that both peptide fractions inhibited CaMKII activity mostly in a competitive manner, although kinetic data suggested that inhibition by Fraction II may be of the mixed type. Kinetic analysis (K(m) and K(i)) showed that affinity of peptides in Fraction II for CaM was more than that in Fraction I, which was directly correlated with the higher inhibitory properties of Fraction II against CaMKII. The results suggest that it may be possible to use pea protein-derived cationic peptides to modulate CaMKII activities. PMID:16111873

  1. 2,5-hexanedione (HD) treatment alters calmodulin, Ca{sup 2+}/calmodulin-dependent protein kinase II, and protein kinase C in rats' nerve tissues

    SciTech Connect

    Wang Qingshan Hou Liyan; Zhang Cuili; Zhao Xiulan; Yu Sufang; Xie, Ke-Qin

    2008-10-01

    Calcium-dependent mechanisms, particularly those mediated by Ca{sup 2+}/calmodulin (CaM)-dependent protein kinase II (CaMKII), have been implicated in neurotoxicant-induced neuropathy. However, it is unknown whether similar mechanisms exist in 2,5-hexanedione (HD)-induced neuropathy. For that, we investigated the changes of CaM, CaMKII, protein kinase C (PKC) and polymerization ratios (PRs) of NF-L, NF-M and NF-H in cerebral cortex (CC, including total cortex and some gray), spinal cord (SC) and sciatic nerve (SN) of rats treated with HD at a dosage of 1.75 or 3.50 mmol/kg for 8 weeks (five times per week). The results showed that CaM contents in CC, SC and SN were significantly increased, which indicated elevation of Ca{sup 2+} concentrations in nerve tissues. CaMKII contents and activities were also increased in CC and were positively correlated with gait abnormality, but it could not be found in SC and SN. The increases of PKC contents and activities were also observed in SN and were positively correlated with gait abnormality. Except for that of NF-M in CC, the PRs of NF-L, NF-M and NF-H were also elevated in nerve tissues, which was consistent with the activation of protein kinases. The results suggested that CaMKII might be partly (in CC but not in SC and SN) involved in HD-induced neuropathy. CaMKII and PKC might mediate the HD neurotoxicity by altering the NF phosphorylation status and PRs.

  2. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

    PubMed Central

    Nichols, Grant S.; DeBello, William M.

    2015-01-01

    Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB) provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII) in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX), the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is “off” in adults. PMID:25789177

  3. A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II.

    PubMed Central

    Lee, J M; Greenleaf, A L

    1989-01-01

    The unique C-terminal repeat domain (CTD) of the largest subunit (IIa) of eukaryotic RNA polymerase II consists of multiple repeats of the heptapeptide consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats ranges from 26 in yeast to 42 in Drosophila to 52 in mouse. The CTD is essential in vivo, but its structure and function are not yet understood. The CTD can be phosphorylated at multiple serine and threonine residues, generating a form of the largest subunit (II0) with markedly reduced mobility in NaDodSO4/polyacrylamide gels. To investigate this extensive phosphorylation, which presumably modulates functional properties of RNA polymerase II, we began efforts to purify a specific CTD kinase. Using CTD-containing fusion proteins as substrates, we have purified a CTD kinase from the yeast Saccharomyces cerevisiae. The enzyme extensively phosphorylates the CTD portion of both the fusion proteins and intact subunit IIa, producing products with reduced electrophoretic mobilities. The properties of the CTD kinase suggest that it is distinct from previously described protein kinases. Analogous activities were also detected in Drosophila and HeLa cell extracts. Images PMID:2657724

  4. Live imaging of endogenous Ca²⁺/calmodulin-dependent protein kinase II in neurons reveals that ischemia-related aggregation does not require kinase activity.

    PubMed

    Barcomb, Kelsey; Goodell, Dayton J; Arnold, Don B; Bayer, K Ulrich

    2015-11-01

    The Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) forms 12meric holoenzymes. These holoenzymes cluster into larger aggregates within neurons under ischemic conditions and in vitro when ischemic conditions are mimicked. This aggregation is thought to be mediated by interaction between the regulatory domain of one kinase subunit with the T-site of another kinase subunit in a different holoenzyme, an interaction that requires stimulation by Ca(2+) /CaM and nucleotide for its induction. This model makes several predictions that were verified here: Aggregation in vitro was reduced by the CaMKII inhibitors tatCN21 and tatCN19o (which block the T-site) as well as by KN93 (which is CaM-competitive). Notably, these and previously tested manipulations that block CaMKII activation all reduced aggregation, suggesting an alternative mechanism that instead requires kinase activity. However, experiments with the nucleotide-competitive broad-spectrum kinase inhibitors staurosporin and H7 showed that this is not the case. In vitro, staurosporine and H7 enabled CaMKII aggregation even in the absence of nucleotide. Within rat hippocampal neurons, an intra-body enabled live monitoring of endogenous CaMKII aggregation. This aggregation was blocked by tatCN21, but not by staurosporine, even though both effectively inhibit CaMKII activity. These results support the mechanistic model for CaMKII aggregation and show that kinase activity is not required. CaMKII aggregation is prevented by inhibiting kinase activity with mutations (red italics; shown previously) or inhibitors (red bold; shown here), indicating requirement of kinase activity. However, we show here that nucleotide-competitive inhibitors (green) allow CaMKII aggregation (including endogenous CaMKII within neurons), demonstrating that kinase activity is not required and supporting the current mechanistic model for CaMKII aggregation. PMID:26212614

  5. Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

    SciTech Connect

    Stein, J.C.

    1985-01-01

    Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several /sup 37/P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the /sup 32/P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex.

  6. Calm down when the heart is stressed: Inhibiting calmodulin-dependent protein kinase II for antiarrhythmias

    PubMed Central

    Duan, Dayue Darrel

    2015-01-01

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a pivotal role in many regulatory processes of cellular functions ranging from membrane potentials and electric–contraction (E-C) coupling to mitochondrial integrity and survival of cardiomyocytes. The review article by Hund and Mohler in this issue of Trends in Cardiovascular Medicine highlights the importance of the elevated CaMKII signaling pathways under stressed conditions such as myocardial hypertrophy and ischemia in the detrimental remodeling of ion channels and in the genesis of cardiac arrhythmias. Down-regulation of the elevated CaMKII is now emerging as a powerful therapeutic strategy for the treatment of cardiac arrhythmias and other forms of heart disease such as hypertrophic and ischemic heart failure. The development of new specific and effective CaMKII inhibitors as therapeutic agents for cardiac arrhythmias is challenged by the tremendous complexity of CaMKII expression and distribution of multi isoforms, as well as the multitude of downstream targets in the CaMKII signaling pathways and regulatory processes. A systematic understanding of the structure and regulation of the CaMKII signaling and functional network under the scope of genome and phenome may improve and extend our knowledge about the role of CaMKII in cardiac health and disease and accelerate the discovery of new CaMKII inhibitors that target not only the ATP-binding site but also the regulation sites in the CaMKII signaling and functional network. PMID:25910598

  7. Calm down when the heart is stressed: Inhibiting calmodulin-dependent protein kinase II for antiarrhythmias.

    PubMed

    Duan, Dayue Darrel

    2015-07-01

    Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a pivotal role in many regulatory processes of cellular functions ranging from membrane potentials and electric-contraction (E-C) coupling to mitochondrial integrity and survival of cardiomyocytes. The review article by Hund and Mohler in this issue of Trends in Cardiovascular Medicine highlights the importance of the elevated CaMKII signaling pathways under stressed conditions such as myocardial hypertrophy and ischemia in the detrimental remodeling of ion channels and in the genesis of cardiac arrhythmias. Down-regulation of the elevated CaMKII is now emerging as a powerful therapeutic strategy for the treatment of cardiac arrhythmias and other forms of heart disease such as hypertrophic and ischemic heart failure. The development of new specific and effective CaMKII inhibitors as therapeutic agents for cardiac arrhythmias is challenged by the tremendous complexity of CaMKII expression and distribution of multi isoforms, as well as the multitude of downstream targets in the CaMKII signaling pathways and regulatory processes. A systematic understanding of the structure and regulation of the CaMKII signaling and functional network under the scope of genome and phenome may improve and extend our knowledge about the role of CaMKII in cardiac health and disease and accelerate the discovery of new CaMKII inhibitors that target not only the ATP-binding site but also the regulation sites in the CaMKII signaling and functional network. PMID:25910598

  8. Regulation of type II transforming-growth-factor-beta receptors by protein kinase C iota.

    PubMed Central

    Chuang, Lea-Yea; Guh, Jinn-Yuh; Liu, Shu-Fen; Hung, Min-Yuan; Liao, Tung-Nan; Chiang, Tai-An; Huang, Jau-Shyang; Huang, Yu-Lun; Lin, Chi-Fong; Yang, Yu-Lin

    2003-01-01

    TGF-beta (transforming growth factor-beta) is implicated in the pathogenesis of diabetic nephropathy. We previously demonstrated that up-regulation of type II TGF-beta receptor (TbetaRII) induced by high glucose might contribute to distal tubular hypertrophy [Yang, Guh, Yang, Lai, Tsai, Hung, Chang and Chuang (1998) J. Am. Soc. Nephrol. 9, 182-193]. We have elucidated the mechanism by using cultured Madin-Darby canine kidney cells. Enhancer assay and electrophoretic-mobility-shift assay were used to estimate the involvement of transcription factors. Western blotting and an in vitro kinase assay were used to evaluate the level and activity of protein kinase. We showed that glucose (100-900 mg/dl) induced an increase in mRNA level and promoter activity of TbetaRII (note: 'mg/dl' are the units commonly used in diabetes studies). The promoter region -209 to -177 appeared to contribute to positive transactivation of TbetaRII promoter by comparing five TbetaRII-promoter-CAT (chloramphenicol acetyl-transferase) plasmids. Moreover, the transcription factor AP-1 (activator protein 1) was significantly activated and specifically binds to TbetaRII promoter (-209 to -177). More importantly, we found that atypical PKC iota might be pivotal for high glucose-induced increase in both AP-1 binding and TbetaRII promoter activity. First, high glucose induced cytosolic translocation, activation and autophosphorylation of PKC iota. Secondly, antisense PKC iota expression plasmids attenuated high-glucose-induced increase in AP-1 binding and TbetaRII promoter activity; moreover, sense PKC iota expression plasmids enhanced these instead. Finally, we showed that antisense PKC iota expression plasmids might partly attenuate a high-glucose/TGF-beta1-induced increase in fibronectin. We conclude that PKC iota might mediate high-glucose-induced increase in TbetaRII promoter activity. In addition, antisense PKC iota expression plasmid effectively suppressed up-regulation of TbetaRII and

  9. Concentrated expression of Ca2+/ calmodulin-dependent protein kinase II and protein kinase C in the mushroom bodies of the brain of the honeybee Apis mellifera L.

    PubMed

    Kamikouchi, A; Takeuchi, H; Sawata, M; Natori, S; Kubo, T

    2000-02-21

    We have previously used the differential display method to identify a gene that is expressed preferentially in the mushroom bodies of worker honeybees and to show that it encodes a putative inositol 1,4,5-trisphosphate receptor (IP3R) homologue (Kamikouchi et al. [1998] Biochem. Biophys. Res. Commun. 242:181-186). In the present study, we examined whether the expression of some of the genes for proteins involved in the intracellular Ca2+ signal transduction is also concentrated in the mushroom bodies of the honeybee by isolating cDNA fragments that encode the Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) homologues of the honeybee. In situ hybridization analysis revealed that the expression of these genes was also concentrated in the mushroom bodies of the honeybee brain: The CaMKII gene was expressed preferentially in the large-type Kenyon cells of the mushroom bodies, whereas that for PKC was expressed in both the large and small types of Kenyon cells. The expression of the genes for IP3R and CaMKII was concentrated in the mushroom bodies of the queen and drone as well as in those of the worker bee. Furthermore, the enzymatic activities of CaMKII and PKC were found to be higher in the mushroom bodies/central bodies than in the optic and antennal lobes of the worker bee brain. These results suggest that the function of the intracellular Ca2+ signal transduction is enhanced in Kenyon cells in comparison to other neuronal cell types in the honeybee brain. PMID:10701869

  10. Human type II receptor for bone morphogenic proteins (BMPs): extension of the two-kinase receptor model to the BMPs.

    PubMed Central

    Liu, F; Ventura, F; Doody, J; Massagué, J

    1995-01-01

    Bone morphogenic proteins (BMPs) are universal regulators of animal development. We report the identification and cloning of the BMP type II receptor (BMPR-II), a missing component of this receptor system in vertebrates. BMPR-II is a transmembrane serine/threonine kinase that binds BMP-2 and BMP-7 in association with multiple type I receptors, including BMPR-IA/Brk1, BMPR-IB, and ActR-I, which is also an activin type I receptor. Cloning of BMPR-II resulted from a strong interaction of its cytoplasmic domain with diverse transforming growth factor beta family type I receptor cytoplasmic domains in a yeast two-hybrid system. In mammalian cells, however, the interaction of BMPR-II is restricted to BMP type I receptors and is ligand dependent. BMPR-II binds BMP-2 and -7 on its own, but binding is enhanced by coexpression of type I BMP receptors. BMP-2 and BMP-7 can induce a transcriptional response when added to cells coexpressing ActR-I and BMPR-II but not to cells expressing either receptor alone. The kinase activity of both receptors is essential for signaling. Thus, despite their ability to bind to type I and II receptors receptors separately, BMPs appear to require the cooperation of these two receptors for optimal binding and for signal transduction. The combinatorial nature of these receptors and their capacity to crosstalk with the activin receptor system may underlie the multifunctional nature of their ligands. PMID:7791754

  11. Noradrenaline synthesis after sympathetic nerve activation in rat atria and its dependence on calcium but not CAM kinase II and protein kinases A or C.

    PubMed Central

    Kotsonis, P.; Binko, J.; Majewski, H.

    1996-01-01

    1. The biosynthesis of noradrenaline following sympathetic nerve activation was investigated in rat atria. In particular the time course of noradrenaline synthesis changes, the relationship of changes in synthesis to transmitter release and the possible roles of second messengers and protein kinases were examined. 2. Rat atria incubated with the precursor [3H]-tyrosine synthesized [3H]-noradrenaline. Synthesis was enhanced following pulsatile electrical field stimulation (3 Hz for 5 min) with the bulk of the increase occurring in the first 45 min after the commencement of electrical stimulation. In separate experiments rat atria were pre-incubated with [3H]-noradrenaline and the radioactive outflow in response to electrical field stimulation (3 Hz for 5 min) was taken as an index of noradrenaline release. 3. Stimulation-induced (S-I) noradrenaline synthesis was significantly correlated to S-I noradrenaline release for a variety of procedures which modulate noradrenaline release by mechanisms altering Ca2+ entry into the neurone (r2 = 0.99): those which decreased release: tetrodotoxin (0.3 microM), Ca(2+)-free medium, lowering the frequency of nerve activation to 1 Hz, and those which increased release, tetraethylammonium (0.3 mM), phentolamine (1 microM) and the combination of phentolamine (1 microM) and adenosine (10 microM). On the strength of this relationship we suggest that Ca2+ entry is a determining factor in S-I synthesis changes rather than the amount of noradrenaline released. Indeed the reduction in noradrenaline release with the calmodulin-dependent protein (CAM) kinase II inhibitor KN-62 (10 microM) which acts subsequent to Ca2+ entry, did not affect S-I synthesis. 4. The cell permeable cyclic AMP analogue, 8-bromoadenosine 3',5'-monophosphate (BrcAMP, 90 and 270 microM), dose-dependently increased basal [3H]-noradrenaline synthesis in unstimulated rat atria. This effect was antagonized by the selective protein kinase A (PKA) antagonist, Rp-8

  12. Ca2+/calmodulin-dependent protein kinase II and protein kinase C activities mediate extracellular glucose-regulated hippocampal synaptic efficacy.

    PubMed

    Moriguchi, Shigeki; Oomura, Yutaka; Shioda, Norifumi; Han, Feng; Hori, Nobuaki; Aou, Shuji; Fukunaga, Kohji

    2011-01-01

    To define how extracellular glucose levels affect synaptic efficacy and long-term potentiation (LTP), we evaluated electrophysiological and neurochemical properties in hippocampal CA1 regions following alterations in glucose levels in the ACSF. In rat hippocampal slices prepared in ACSF with 3.5mM glucose, fEPSPs generated by Schaffer collateral/commissural stimulation markedly increased when ACSF glucose levels were increased from 3.5 to 7.0mM. The paired-pulse facilitation reflecting presynaptic transmitter release efficacy was significantly suppressed by elevation to 7.0mM glucose because of potentiation of the input-output relationship (I/O relationship) of fEPSPs by single pulse stimulation. Prolonged potentiation of fEPSPs by elevation to 7.0mM glucose coincided with increased autophosphorylation both of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and protein kinase Cα (PKCα). The increased I/O relationship of fEPSPs was also associated with markedly increased synapsin I phosphorylation by CaMKII. Transmitter-evoked postsynaptic currents were also measured in CA1 neurons by electrophoretical application of NMDA and AMPA to the apical dendrites of pyramidal neurons. NMDA- and AMPA-evoked currents were significantly augmented by elevation to 7.0mM. Notably, high frequency stimulation of the Schaffer collateral/commissural pathway failed to induce LTP in the CA1 region at 3.5mM glucose but LTP was restored dose-dependently by increasing glucose levels to 7.0 and 10.0mM. LTP induction in the presence of 7.0mM glucose was closely associated with further increases in CaMKII autophosphorylation without changes in PKCα autophosphorylation. Taken together, CaMKII and PKC activation likely mediate potentiation of fEPSPs by elevated glucose levels, and CaMKII activity is also associated with LTP induction in the hippocampal CA1 region. PMID:20807573

  13. Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey

    SciTech Connect

    Benson, D.L.; Isackson, P.J.; Hendry, S.H.; Jones, E.G. )

    1991-06-01

    In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin-dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non-GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate.

  14. The Octopamine Receptor OAMB Mediates Ovulation via Ca2+/Calmodulin-Dependent Protein Kinase II in the Drosophila Oviduct Epithelium

    PubMed Central

    Lee, Hyun-Gwan; Rohila, Suman; Han, Kyung-An

    2009-01-01

    Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. We have previously shown that OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine), is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3) with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. Here, we investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, we explored protein kinases functionally interacting with OAMB by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg transfer from

  15. Type II cGMP-dependent protein kinase directly inhibits HER2 activation of gastric cancer cells.

    PubMed

    Zhu, Miaolin; Yao, Xiaoyuan; Wu, Min; Qian, Hai; Wu, Yan; Chen, Yongchang

    2016-02-01

    Our previous study demonstrated that type II cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF)-induced phosphorylation/activation of epidermal growth factor receptor (EGFR). Since human epidermal growth factor receptor 2 (HER2) has a similar molecular structure to EGFR, the present study was designed to investigate whether PKG II also inhibits HER2 activation. The human gastric cancer cell line HGC‑27 was infected with an adenoviral construct encoding cDNA of PKG II (Ad‑PKG II) to increase the expression of PKG II and treated with 8‑(4‑chlorophenylthio)guanosine‑3',5'‑cyclic monophosphate (8‑pCPT‑cGMP) to activate the kinase. Western blotting was performed to detect the tyrosine and serine/threonine phosphorylation of HER2. Co‑immunoprecipitation was performed in order to determine the binding between PKG II and HER2. In addition, a QuikChange Lightning Site‑Directed Mutagenesis kit was used to mutate threonine 686 of HER2 to glutamic acid or alanine. The results demonstrated that EGF treatment increased the tyrosine phosphorylation (activation) of HER2. Increasing the PKG II activity of HGC‑27 cells through infection with Ad‑PKG II and stimulation with 8‑pCPT‑cGMP inhibited the EGF‑induced tyrosine phosphorylation/activation of HER2. PKG II bound directly with HER2 and caused phosphorylation of threonine 686. When threonine 686 of HER2 was mutated to alanine, which could not be phosphorylated by PKG II, the inhibitory effect of PKG II on the activation of HER2 was eradicated. When threonine 686 of HER2 was mutated to glutamic acid, which mimicked the phosphorylation of this site, treatment with EGF had no stimulating effect on tyrosine phosphorylation/activation of the mutant HER2. The results suggested that PKG II inhibits EGF‑induced activation of HER2 through binding with and causing threonine 686 phosphorylation of this oncogenic protein. PMID:26676300

  16. Identification of peptides in wheat germ hydrolysate that demonstrate calmodulin-dependent protein kinase II inhibitory activity.

    PubMed

    Kumrungsee, Thanutchaporn; Akiyama, Sayaka; Guo, Jian; Tanaka, Mitsuru; Matsui, Toshiro

    2016-12-15

    Hydrolysis of wheat germ by proteases resulted in bioactive peptides that demonstrated an inhibitory effect against the vasoconstrictive Ca(2+)-calmodulin (CaM)-dependent protein kinase II (CaMK II). The hydrolysate by thermolysin (1.0wt%, 5h) showed a particularly potent CaMK II inhibition. As a result of mixed mode high-performance liquid chromatography of thermolysin hydrolysate with pH elution gradient ranging between 4.8 and 8.9, the fraction eluted at pH 8.9 was the most potent CaMK II inhibitor. From this fraction, Trp-Val and Trp-Ile were identified as CaMK II inhibitors. In Sprague-Dawley rats, an enhanced aortic CaMK II activity by 1μM phenylephrine was significantly (p<0.05) suppressed by 15-min incubation with 300μM Trp-Val or Trp-Ile. On the basis of Ca(2+)-chelating fluorescence and CaMK II activity assays, it was concluded that Trp-Val and Trp-Ile competed with Ca(2+)-CaM complex to bind to CaMK II with Ki values of 5.4 and 3.6μM, respectively. PMID:27451188

  17. Ethanol alters angiotensin II stimulated mitogen activated protein kinase in hepatocytes: agonist selectivity and ethanol metabolic independence.

    PubMed

    Weng, Y; Shukla, S D

    2000-06-23

    Angiotensin II activated mitogen-activated protein kinase (MAPK) (p42 and p44) in rat hepatocytes exposed to ethanol and the relevance of ethanol metabolism on this activation was investigated. Hepatocytes, isolated from rat liver, were treated with or without ethanol for 24 h. Angiotensin II, vasopressin, insulin, serum and epinephrine significantly increased hepatocyte MAPK activity. Platelet activating factor (PAF), tumor necrosis factor-alpha (TNF-alpha), and insulin-like growth factor-1 (IGF-1) had little effect on MAPK activation. Interestingly, among the above agonists, which activated hepatocyte MAPK, ethanol exposure potentiated only angiotensin II and epinephrine-stimulated MAPK. Thus, potentiation of MAPK by ethanol exhibited agonist selectivity. In contrast to several other cells, there was prevalence of p42 over p44 MAPK band in hepatocytes. Angiotensin II treatment caused a rapid activation (peak 5 min) of MAPK followed by a decrease to basal levels in 30 min. Exposure with 100 mM ethanol potentiated the angiotensin II stimulated MAPK activity. This potentiation was partially blocked by pertussis toxin suggesting it to be a G-protein-dependent event. Treatment of the hepatocytes with pyrazole (an inhibitor of ethanol metabolism) or acetaldehyde (an ethanol metabolite) had no effect on potentiation. Thus, ethanol potentiation of hepatocyte MAPK is agonist-selective and independent of ethanol metabolism. PMID:10862821

  18. Protein kinase C activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of actomyosin contractility

    PubMed Central

    Ivanov, Andrei I; Samarin, Stanislav N; Bachar, Moshe; Parkos, Charles A; Nusrat, Asma

    2009-01-01

    Background Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled in vitro by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium. Results Exposure of HPAF-II human pancreatic adenocarcinoma cell monolayers to either OI-V or 12-O-tetradecanoylphorbol-13-acetate caused rapid disruption and internalization of AJs and TJs. Activity of classical PKC isoenzymes was responsible for the loss of cell-cell contacts which was accompanied by cell rounding, phosphorylation and relocalization of the F-actin motor nonmuscle myosin (NM) II. The OI-V-induced disruption of AJs and TJs was prevented by either pharmacological inhibition of NM II with blebbistatin or by siRNA-mediated downregulation of NM IIA. Furthermore, AJ/TJ disassembly was attenuated by inhibition of Rho-associated kinase (ROCK) II, but was insensitive to blockage of MLCK, calmodulin, ERK1/2, caspases and RhoA GTPase. Conclusion Our data suggest that stimulation of PKC disrupts epithelial apical junctions via ROCK-II dependent activation of NM II, which increases contractility of perijunctional actin filaments. This mechanism is likely to be important for cancer cell dissociation and tumor metastasis. PMID:19422706

  19. Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes.

    PubMed

    Fan, Heng-Yu; Huo, Li-Jun; Meng, Xiao-Qian; Zhong, Zhi-Sheng; Hou, Yi; Chen, Da-Yuan; Sun, Qing-Yuan

    2003-11-01

    Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins

  20. Alpha calcium/calmodulin dependent protein kinase II in learning-dependent plasticity of mouse somatosensory cortex.

    PubMed

    Skibinska-Kijek, A; Radwanska, A; Kossut, M

    2008-02-01

    Calcium/calmodulin dependent protein kinase II (CaMKII), and more specifically its alpha subunit, is widely believed to be fundamental for hippocampal synaptic plasticity. In the cerebral cortex, deprivation-evoked plasticity was shown to depend on alphaCaMKII autophosphorylation abilities. Here we analyzed how learning-induced functional reorganization of cortical representations affected alphaCaMKII in adult Swiss mice. Mice were subjected to short-lasting sensory training in which stimulation of whiskers was paired with tail shock. The pairing results in enlargement of functional representation of vibrissae activated during the training. alphaCaMKII protein and its autophosphorylation level were determined by Western-blotting in somatosensory cortex crude synaptosomal fraction (P2) and postsynaptic protein-enriched, Triton X-100 insoluble fraction (TIF). The first training session resulted in an increase in alphaCaMKII autophosphorylation at autonomy site observed in TIF. A similar increase was also observed after the first session of just whiskers stimulation, which alone does not induce rearrangement of cortical representations. These data indicate that increased autophosphorylation of postsynaptic alphaCaMKII is not a correlate of induction phase of plasticity related reorganization of cortical representation of vibrissae. The increase observed in both experimental groups was transient and did not persist in the maintenance phase of the plastic change. Furthermore, we found that the training caused a delayed upregulation of alphaCaMKII protein level in crude synaptosomal fraction, but not in TIF, and the upregulation was not accompanied by an increase in autophosphorylation level of the kinase. The result indicates alphaCaMKII involvement in the late phase of plastic change and suggests the participation of a presynaptic pool of kinase rather than postsynaptic at this point. PMID:18164137

  1. Cross-Linking Proteins To Show Complex Formation: A Laboratory That Visually Demonstrates Calmodulin Binding to Calmodulin Kinase II.

    ERIC Educational Resources Information Center

    Porta, Angela R.

    2003-01-01

    Presents a laboratory experiment demonstrating the binding of calcium/calmodulin to calmodulin kinase II, which is important in the metabolic and physiological activities of the cell. Uses SDS polyacrylamide gel electrophoresis (PAGE). (YDS)

  2. Protein kinase C betaII peptide inhibitor exerts cardioprotective effects in rat cardiac ischemia/reperfusion injury.

    PubMed

    Omiyi, Didi; Brue, Richard J; Taormina, Philip; Harvey, Margaret; Atkinson, Norrell; Young, Lindon H

    2005-08-01

    Ischemia followed by reperfusion (I/R) in the presence of polymorphonuclear leukocytes (PMNs) results in a marked cardiac contractile dysfunction. A cell-permeable protein kinase C (PKC) betaII peptide inhibitor was used to test the hypothesis that PKC betaII inhibition could attenuate PMN-induced cardiac dysfunction by suppression of superoxide production from PMNs and increase NO release from vascular endothelium. The effects of the PKC betaII peptide inhibitor were examined in isolated ischemic (20 min) and reperfused (45 min) rat hearts with PMNs. The PKC betaII inhibitor (10 microM; n = 7) significantly attenuated PMN-induced cardiac dysfunction compared with I/R hearts (n = 9) receiving PMNs alone in left ventricular developed pressure (LVDP) and the maximal rate of LVDP (+dP/dt(max)) cardiac function indices (p < 0.01). The PKC betaII inhibitor at 10 microM significantly increased endothelial NO release from a basal value of 1.85 +/- 0.18 pmol NO/mg tissue to 3.49 +/- 0.62 pmol NO/mg tissue from rat aorta. It also significantly inhibited superoxide release (i.e., absorbance) from N-formyl-L-methionyl-L-leucyl-L-phenylalanine-stimulated rat PMNs from 0.13 +/- 0.01 to 0.02 +/- 0.004 (p < 0.01) at 10 microM. Histological analysis of the left ventricle of representative rat hearts from each group showed that the PKC betaII peptide inhibitor-treated hearts experienced a marked reduction in PMN vascular adherence and infiltration into the postreperfused cardiac tissue compared with I/R + PMN hearts (p < 0.01). These results suggest that the PKC betaII peptide inhibitor attenuates PMN-induced post-I/R cardiac contractile dysfunction by increasing endothelial NO release and by inhibiting superoxide release from PMNs. PMID:15878997

  3. Calcium/calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways

    PubMed Central

    Timmins, Jenelle M.; Ozcan, Lale; Seimon, Tracie A.; Li, Gang; Malagelada, Cristina; Backs, Johannes; Backs, Thea; Bassel-Duby, Rhonda; Olson, Eric N.; Anderson, Mark E.; Tabas, Ira

    2009-01-01

    ER stress–induced apoptosis is implicated in various pathological conditions, but the mechanisms linking ER stress–mediated signaling to downstream apoptotic pathways remain unclear. Using human and mouse cell culture and in vivo mouse models of ER stress–induced apoptosis, we have shown that cytosolic calcium resulting from ER stress induces expression of the Fas death receptor through a pathway involving calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ) and JNK. Remarkably, CaMKIIγ was also responsible for processes involved in mitochondrial-dependent apoptosis, including release of mitochondrial cytochrome c and loss of mitochondrial membrane potential. CaMKII-dependent apoptosis was also observed in a number of cultured human and mouse cells relevant to ER stress–induced pathology, including cultured macrophages, endothelial cells, and neuronal cells subjected to proapoptotic ER stress. Moreover, WT mice subjected to systemic ER stress showed evidence of macrophage mitochondrial dysfunction and apoptosis, renal epithelial cell apoptosis, and renal dysfunction, and these effects were markedly reduced in CaMKIIγ-deficient mice. These data support an integrated model in which CaMKII serves as a unifying link between ER stress and the Fas and mitochondrial apoptotic pathways. Our study also revealed what we believe to be a novel proapoptotic function for CaMKII, namely, promotion of mitochondrial calcium uptake. These findings raise the possibility that CaMKII inhibitors could be useful in preventing apoptosis in pathological settings involving ER stress–induced apoptosis. PMID:19741297

  4. Ca2+/calmodulin-dependent protein kinase II regulates cardiac Na+ channels

    PubMed Central

    Wagner, Stefan; Dybkova, Nataliya; Rasenack, Eva C.L.; Jacobshagen, Claudius; Fabritz, Larissa; Kirchhof, Paulus; Maier, Sebastian K.G.; Zhang, Tong; Hasenfuss, Gerd; Brown, Joan Heller; Bers, Donald M.; Maier, Lars S.

    2006-01-01

    In heart failure (HF), Ca2+/calmodulin kinase II (CaMKII) expression is increased. Altered Na+ channel gating is linked to and may promote ventricular tachyarrhythmias (VTs) in HF. Calmodulin regulates Na+ channel gating, in part perhaps via CaMKII. We investigated effects of adenovirus-mediated (acute) and Tg (chronic) overexpression of cytosolic CaMKIIδC on Na+ current (INa) in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch clamp). Both acute and chronic CaMKIIδC overexpression shifted voltage dependence of Na+ channel availability by –6 mV (P < 0.05), and the shift was Ca2+ dependent. CaMKII also enhanced intermediate inactivation and slowed recovery from inactivation (prevented by CaMKII inhibitors autocamtide 2–related inhibitory peptide [AIP] or KN93). CaMKIIδC markedly increased persistent (late) inward INa and intracellular Na+ concentration (as measured by the Na+ indicator sodium-binding benzofuran isophthalate [SBFI]), which was prevented by CaMKII inhibition in the case of acute CaMKIIδC overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na+ channels. In vivo, transgenic CaMKIIδC overexpression prolonged QRS duration and repolarization (QT intervals), decreased effective refractory periods, and increased the propensity to develop VT. We conclude that CaMKII associates with and phosphorylates cardiac Na+ channels. This alters INa gating to reduce availability at high heart rate, while enhancing late INa (which could prolong action potential duration). In mice, enhanced CaMKIIδC activity predisposed to VT. Thus, CaMKII-dependent regulation of Na+ channel function may contribute to arrhythmogenesis in HF. PMID:17124532

  5. Endogenous expression of type II cGMP-dependent protein kinase mRNA and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator.

    PubMed Central

    Markert, T; Vaandrager, A B; Gambaryan, S; Pöhler, D; Häusler, C; Walter, U; De Jonge, H R; Jarchau, T; Lohmann, S M

    1995-01-01

    Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia. Images PMID:7543493

  6. Involvement of Ca2+/calmodulin-dependent protein kinase II in the activation of carnitine palmitoyltransferase I by okadaic acid in rat hepatocytes.

    PubMed Central

    Velasco, G; Guzmán, M; Zammit, V A; Geelen, M J

    1997-01-01

    The present work was undertaken to study the mechanism by which okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, stimulates carnitine palmitoyltransferase I (CPT-I) in isolated rat hepatocytes [Guzmán, Kolodziej, Caldwell, Costorphine and Zammit (1994) Biochem. J. 300, 693-699]. The OA-induced stimulation of CPT-I was abolished by the general protein kinase inhibitor K-252a as well as by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CM-PKII). However, neither the protein kinase C-specific inhibitor bisindolylmaleimide nor the protein kinase A/protein kinase C inhibitor H-7 was able to prevent the OA-induced stimulation of CPT-I. Hepatocyte-shrinkage-induced stimulation of CPT-I as well as OA-induced hepatocyte shrinkage was prevented by KN-62. KN-62 also antagonized the OA-enhanced release of lactate dehydrogenase from digitonin-permeabilized hepatocytes. Exposure of 32P-labelled hepatocytes to OA increased the degree of phosphorylation of Ca2+/CM-PKII, as immunoprecipitated by a monoclonal antibody raised against the alpha-subunit of rat brain kinase. This effect of OA was also antagonized by KN-62. The results thus indicate that the OA-dependent stimulation of CPT-I may be mediated (at least in part) by increased phosphorylation and subsequent activation of Ca2+/CM-PKII. PMID:9003421

  7. Ca2+/Calmodulin-Dependent Protein Kinase II Is a Modulator of CARMA1-Mediated NF-κB Activation†

    PubMed Central

    Ishiguro, Kazuhiro; Green, Todd; Rapley, Joseph; Wachtel, Heather; Giallourakis, Cosmas; Landry, Aimee; Cao, Zhifang; Lu, Naifang; Takafumi, Ando; Goto, Hidemi; Daly, Mark J.; Xavier, Ramnik J.

    2006-01-01

    CARMA1 is a central regulator of NF-κB activation in lymphocytes. CARMA1 and Bcl10 functionally interact and control NF-κB signaling downstream of the T-cell receptor (TCR). Computational analysis of expression neighborhoods of CARMA1-Bcl10MALT 1 for enrichment in kinases identified calmodulin-dependent protein kinase II (CaMKII) as an important component of this pathway. Here we report that Ca2+/CaMKII is redistributed to the immune synapse following T-cell activation and that CaMKII is critical for NF-κB activation induced by TCR stimulation. Furthermore, CaMKII enhances CARMA1-induced NF-κB activation. Moreover, we have shown that CaMKII phosphorylates CARMA1 on Ser109 and that the phosphorylation facilitates the interaction between CARMA1 and Bcl10. These results provide a novel function for CaMKII in TCR signaling and CARMA1-induced NF-κB activation. PMID:16809782

  8. Molecular mechanism of activation-triggered subunit exchange in Ca2+/calmodulin-dependent protein kinase II

    PubMed Central

    Bhattacharyya, Moitrayee; Stratton, Margaret M; Going, Catherine C; McSpadden, Ethan D; Huang, Yongjian; Susa, Anna C; Elleman, Anna; Cao, Yumeng Melody; Pappireddi, Nishant; Burkhardt, Pawel; Gee, Christine L; Barros, Tiago; Schulman, Howard; Williams, Evan R; Kuriyan, John

    2016-01-01

    Activation triggers the exchange of subunits in Ca2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones. DOI: http://dx.doi.org/10.7554/eLife.13405.001 PMID:26949248

  9. Multiplexed Dendritic Targeting of α Calcium Calmodulin-dependent Protein Kinase II, Neurogranin, and Activity-regulated Cytoskeleton-associated Protein RNAs by the A2 Pathway

    PubMed Central

    Gao, Yuanzheng; Tatavarty, Vedakumar; Korza, George; Levin, Mikhail K.

    2008-01-01

    In neurons, many different RNAs are targeted to dendrites where local expression of the encoded proteins mediates synaptic plasticity during learning and memory. It is not known whether each RNA follows a separate trafficking pathway or whether multiple RNAs are targeted to dendrites by the same pathway. Here, we show that RNAs encoding α calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein are coassembled into the same RNA granules and targeted to dendrites by the same cis/trans-determinants (heterogeneous nuclear ribonucleoprotein [hnRNP] A2 response element and hnRNP A2) that mediate dendritic targeting of myelin basic protein RNA by the A2 pathway in oligodendrocytes. Multiplexed dendritic targeting of different RNAs by the same pathway represents a new organizing principle for coordinating gene expression at the synapse. PMID:18305102

  10. Myotonic dystrophy protein kinase (DMPK) prevents ROS-induced cell death by assembling a hexokinase II-Src complex on the mitochondrial surface

    PubMed Central

    Pantic, B; Trevisan, E; Citta, A; Rigobello, M P; Marin, O; Bernardi, P; Salvatori, S; Rasola, A

    2013-01-01

    The biological functions of myotonic dystrophy protein kinase (DMPK), a serine/threonine kinase whose gene mutations cause myotonic dystrophy type 1 (DM1), remain poorly understood. Several DMPK isoforms exist, and the long ones (DMPK-A/B/C/D) are associated with the mitochondria, where they exert unknown activities. We have studied the isoform A of DMPK, which we have found to be prevalently associated to the outer mitochondrial membrane. The kinase activity of mitochondrial DMPK protects cells from oxidative stress and from the ensuing opening of the mitochondrial permeability transition pore (PTP), which would otherwise irreversibly commit cells to death. We observe that DMPK (i) increases the mitochondrial localization of hexokinase II (HK II), (ii) forms a multimeric complex with HK II and with the active form of the tyrosine kinase Src, binding its SH3 domain and (iii) it is tyrosine-phosphorylated by Src. Both interaction among these proteins and tyrosine phosphorylation of DMPK are increased under oxidative stress, and Src inhibition selectively enhances death in DMPK-expressing cells after HK II detachment from the mitochondria. Down-modulation of DMPK abolishes the appearance of muscle markers in in vitro myogenesis, which is rescued by oxidant scavenging. Our data indicate that, together with HK II and Src, mitochondrial DMPK is part of a multimolecular complex endowed with antioxidant and pro-survival properties that could be relevant during the function and differentiation of muscle fibers. PMID:24136222

  11. R-subunit Isoform Specificity in Protein Kinase A: Distinct Features of Protein Interfaces in PKA Types I and II by Amide H/2H exchange Mass Spectrometry

    PubMed Central

    Anand, Ganesh S.; Hotchko, Matthew; Brown, Simon H.J.; Ten Eyck, Lynn F.; Komives, Elizabeth A.; Taylor, Susan S.

    2009-01-01

    The two isoforms (RI and RII) of the regulatory (R) subunit of cAMP-dependent protein kinase or protein kinase A (PKA) are similar in sequence yet have different biochemical properties and physiological functions. To further understand the molecular basis for R-isoform-specificity, the interactions of the RIIβ isoform with the PKA catalytic (C) subunit were analyzed by amide H/2H exchange mass spectrometry to compare solvent accessibility of RIIβ and the C subunit in their free and complexed states. Direct mapping of the RIIβ-C interface revealed important differences between the intersubunit interfaces in the type I and type II holoenzyme complexes. These differences are seen in both the R-subunits as well as the C-subunit. Unlike the type I isoform, the type II isoform complexes require both cAMP-binding domains, and ATP is not obligatory for high affinity interactions with the C-subunit. Surprisingly, the C-subunit mediates distinct, overlapping surfaces of interaction with the two R-isoforms despite a strong homology in sequence and similarity in domain organization. Identification of a remote allosteric site on the C-subunit that is essential for interactions with RII, but not RI subunits, further highlights the considerable diversity in interfaces found in higher order protein complexes mediated by the C-subunit of PKA. PMID:17942118

  12. Curcumin Attenuates Opioid Tolerance and Dependence by Inhibiting Ca2+/Calmodulin-Dependent Protein Kinase II α Activity

    PubMed Central

    Hu, Xiaoyu; Huang, Fang; Szymusiak, Magdalena

    2015-01-01

    Chronic use of opioid analgesics has been hindered by the development of opioid addiction and tolerance. We have reported that curcumin, a natural flavonoid from the rhizome of Curcuma longa, attenuated opioid tolerance, although the underlying mechanism remains unclear. In this study, we tested the hypothesis that curcumin may inhibit Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα), a protein kinase that has been previously proposed to be critical for opioid tolerance and dependence. In this study, we used state-of-the-art polymeric formulation technology to produce poly(lactic-co-glycolic acid) (PLGA)-curcumin nanoparticles (nanocurcumin) to overcome the drug’s poor solubility and bioavailability, which has made it extremely difficult for studying in vivo pharmacological actions of curcumin. We found that PLGA-curcumin nanoparticles reduced the dose requirement by 11- to 33-fold. Pretreatment with PLGA-curcumin (by mouth) prevented the development of opioid tolerance and dependence in a dose-dependent manner, with ED50 values of 3.9 and 3.2 mg/kg, respectively. PLGA-curcumin dose-dependently attenuated already-established opioid tolerance (ED50 = 12.6 mg/kg p.o.) and dependence (ED50 = 3.1 mg/kg p.o.). Curcumin or PLGA-curcumin did not produce antinociception by itself or affect morphine (1–10 mg/kg) antinociception. Moreover, we found that the behavioral effects of curcumin on opioid tolerance and dependence correlated with its inhibition of morphine-induced CaMKIIα activation in the brain. These results suggest that curcumin may attenuate opioid tolerance and dependence by suppressing CaMKIIα activity. PMID:25515789

  13. Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

    SciTech Connect

    Muchir, Antoine; Wu, Wei; Sera, Fusako; Homma, Shunichi; Worman, Howard J.

    2014-10-03

    Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna{sup H222P/H222P} mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna{sup H222P/H222P} mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna{sup H222P/H222P} mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna{sup H222P/H222P} mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left

  14. Urotensin-II Receptor Stimulation of Cardiac L-type Ca2+ Channels Requires the βγ Subunits of Gi/o-protein and Phosphatidylinositol 3-Kinase-dependent Protein Kinase C β1 Isoform*

    PubMed Central

    Zhang, Yuan; Ying, Jiaoqian; Jiang, Dongsheng; Chang, Zhigang; Li, Hua; Zhang, Guoqiang; Gong, Shan; Jiang, Xinghong; Tao, Jin

    2015-01-01

    Recent studies have demonstrated that urotensin-II (U-II) plays important roles in cardiovascular actions including cardiac positive inotropic effects and increasing cardiac output. However, the mechanisms underlying these effects of U-II in cardiomyocytes still remain unknown. We show by electrophysiological studies that U-II dose-dependently potentiates L-type Ca2+ currents (ICa,L) in adult rat ventricular myocytes. This effect was U-II receptor (U-IIR)-dependent and was associated with a depolarizing shift in the voltage dependence of inactivation. Intracellular application of guanosine-5′-O-(2-thiodiphosphate) and pertussis toxin pretreatment both abolished the stimulatory effects of U-II. Dialysis of cells with the QEHA peptide, but not scrambled peptide SKEE, blocked the U-II-induced response. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin as well as the class I PI3K antagonist CH132799 blocked the U-II-induced ICa,L response. Protein kinase C antagonists calphostin C and chelerythrine chloride as well as dialysis of cells with 1,2bis(2aminophenoxy)ethaneN,N,N′,N′-tetraacetic acid abolished the U-II-induced responses, whereas PKCα inhibition or PKA blockade had no effect. Exposure of ventricular myocytes to U-II markedly increased membrane PKCβ1 expression, whereas inhibition of PKCβ1 pharmacologically or by shRNA targeting abolished the U-II-induced ICa,L response. Functionally, we observed a significant increase in the amplitude of sarcomere shortening induced by U-II; blockade of U-IIR as well as PKCβ inhibition abolished this effect, whereas Bay K8644 mimicked the U-II response. Taken together, our results indicate that U-II potentiates ICa,L through the βγ subunits of Gi/o-protein and downstream activation of the class I PI3K-dependent PKCβ1 isoform. This occurred via the activation of U-IIR and contributes to the positive inotropic effect on cardiomyocytes. PMID:25678708

  15. Adult cardiac fibroblast proliferation is modulated by calcium/calmodulin-dependent protein kinase II in normal and hypertrophied hearts.

    PubMed

    Martin, Tamara P; Lawan, Ahmed; Robinson, Emma; Grieve, David J; Plevin, Robin; Paul, Andrew; Currie, Susan

    2014-02-01

    Increased adult cardiac fibroblast proliferation results in an increased collagen deposition responsible for the fibrosis accompanying pathological remodelling of the heart. The mechanisms regulating cardiac fibroblast proliferation remain poorly understood. Using a minimally invasive transverse aortic banding (MTAB) mouse model of cardiac hypertrophy, we have assessed fibrosis and cardiac fibroblast proliferation. We have investigated whether calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) regulates proliferation in fibroblasts isolated from normal and hypertrophied hearts. It is known that CaMKIIδ plays a central role in cardiac myocyte contractility, but nothing is known of its role in adult cardiac fibroblast function. The MTAB model used here produces extensive hypertrophy and fibrosis. CaMKIIδ protein expression and activity is upregulated in MTAB hearts and, specifically, in cardiac fibroblasts isolated from hypertrophied hearts. In response to angiotensin II, cardiac fibroblasts isolated from MTAB hearts show increased proliferation rates. Inhibition of CaMKII with autocamtide inhibitory peptide inhibits proliferation in cells isolated from both sham and MTAB hearts, with a significantly greater effect evident in MTAB cells. These results are the first to show selective upregulation of CaMKIIδ in adult cardiac fibroblasts following cardiac hypertrophy and to assign a previously unrecognised role to CaMKII in regulating adult cardiac fibroblast function in normal and diseased hearts. PMID:23881186

  16. Structural Properties of Human CaMKII Ca2+ /Calmodulin-Dependent Protein Kinase II using X-ray Crystallography

    NASA Astrophysics Data System (ADS)

    Cao, Yumeng Melody; McSpadden, Ethan; Kuriyan, John; Department of Molecular; Cell Biology; Department of Chemistry Team

    To this day, human memory storage remains a mystery as we can at most describe the process vaguely on a cellular level. Switch-like properties of Calcium/Calmodulin-Dependent Protein Kinase II make it a leading candidate in understanding the molecular basis of human memory. The protein crystal was placed in the beam of a synchrotron source and the x-ray crystallography data was collected as reflections on a diffraction pattern that undergo Fourier transform to obtain the electron density. We observed two drastic differences from our solved structure at 2.75Å to a similar construct of the mouse CaMKII association domain. Firstly, our structure is a 6-fold symmetric dodecamer, whereas the previously published construct was a 7-fold symmetric tetradecamer. This suggests the association domain of human CaMKII is a dynamic structure that is triggered subunit exchange process. Secondly, in our structure the N-terminal tag is docked as an additional beta-strand on an uncapped beta-sheet present in each association domain protomer. This is concrete evidence of the involvement of the polypeptide docking site in the molecular mechanism underlining subunit exchange. In the future, we would like to selectively inhibit the exchange process while not disrupting the other functionalities of CaMKII.

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

    PubMed Central

    Sadoshima, J; Izumo, S

    1996-01-01

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

  18. Involvement of Ca2+/calmodulin-dependent protein kinase II in the modulation of indolamines in diabetic and hyperglycemic rats.

    PubMed

    Ramakrishnan, R; Prabhakaran, K; Jayakumar, A R; Gunasekaran, P; Sheeladevi, R; Suthanthirarajan, N

    2005-05-15

    Hyperglycemia and acidosis are the key factors in diabetic complications. It has been shown that acute or chronic diabetes alters serotonin levels in brain. However, the mechanism of hyperglycemia- or acidosis-induced changes in serotonin levels remains poorly understood. Because Ca2+-dependent protein kinases play a major role in the regulation of serotonin synthesis and release, we investigated the effect of diabetes, hyperglycemia, and acidosis on the level of indolamines [5-hydroxytryptamine (5-HT) and/or 5-hydroxyindoleacetic acid (5-HIAA)] and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) enzyme activity or protein expression in different brain regions. Alloxan-induced (45 mg/kg bw) diabetic rats (30 days) showed increased level of 5-HT in striatum (ST; 183%), midbrain (MB; 199%), pons medulla (PM; 151%), cerebellum (CB; 214%), and cerebral cortex (CCX; 162%) compared with control (P < 0.05), and these changes were reversed after insulin administration. Rats treated with glucose (500 mg/kg bw) for 30 days showed a 146%, 183%, 208%, and 177% (P < 0.05) increase in 5-HT levels in ST, PM, CB, and CCX, respectively. 5-HIAA level increased in hippocampus (HC; 172%) and in MB (145%; P < 0.05). In addition, rats treated with sodium acetoacetate (NaAcAc) for 30 days (60 mg/kg bw) showed significant increases (P < 0.05) of 5-HT level in ST (152%) and MB (174%). However, the levels of 5-HIAA increased only in MB (151%, P < 0.05). Rats treated with NH4Cl, which induced acidosis (150 mg/kg bw), showed an increased level of 5-HT only in HC (165%, P < 0.05). The increased activity and protein expression of CaMKII in ST, MB, PM, CB, and CCX under diabetic conditions were correlated with the levels of indolamines changes during diabetic, hyperglycemic, or acidotic conditions. These results suggest that CaMKII may be involved in the regulation of indolamines in diabetic animals. PMID:15846780

  19. A nonsense mutation in cGMP-dependent type II protein kinase (PRKG2) causes dwarfism in American Angus cattle

    PubMed Central

    Koltes, James E.; Mishra, Bishnu P.; Kumar, Dinesh; Kataria, Ranjit S.; Totir, Liviu R.; Fernando, Rohan L.; Cobbold, Rowland; Steffen, David; Coppieters, Wouter; Georges, Michel; Reecy, James M.

    2009-01-01

    Historically, dwarfism was the major genetic defect in U.S. beef cattle. Aggressive culling and sire testing were used to minimize its prevalence; however, neither of these practices can eliminate a recessive genetic defect. We assembled a 4-generation pedigree to identify the mutation underlying dwarfism in American Angus cattle. An adaptation of the Elston-Steward algorithm was used to overcome small pedigree size and missing genotypes. The dwarfism locus was fine-mapped to BTA6 between markers AFR227 and BM4311. Four candidate genes were sequenced, revealing a nonsense mutation in exon 15 of cGMP-dependant type II protein kinase (PRKG2). This C/T transition introduced a stop codon (R678X) that truncated 85 C-terminal amino acids, including a large portion of the kinase domain. Of the 75 mutations discovered in this region, only this mutation was 100% concordant with the recessive pattern of inheritance in affected and carrier individuals (log of odds score = 6.63). Previous research has shown that PRKG2 regulates SRY (sex-determining region Y) box 9 (SOX9)-mediated transcription of collagen 2 (COL2). We evaluated the ability of wild-type (WT) or R678X PRKG2 to regulate COL2 expression in cell culture. Real-time PCR results confirmed that COL2 is overexpressed in cells that overexpressed R678X PRKG2 as compared with WT PRKG2. Furthermore, COL2 and COL10 mRNA expression was increased in dwarf cattle compared with unaffected cattle. These experiments indicate that the R678X mutation is functional, resulting in a loss of PRKG2 regulation of COL2 and COL10 mRNA expression. Therefore, we present PRKG2 R678X as a causative mutation for dwarfism cattle. PMID:19887637

  20. Identification of the Interactome of a Palmitoylated Membrane Protein, Phosphatidylinositol 4-Kinase Type II Alpha.

    PubMed

    Gokhale, Avanti; Ryder, Pearl V; Zlatic, Stephanie A; Faundez, Victor

    2016-01-01

    Phosphatidylinositol 4-kinases (PI4K) are enzymes responsible for the production of phosphatidylinositol 4-phosphates, important intermediates in several cell signaling pathways. PI4KIIα is the most abundant membrane-associated kinase in mammalian cells and is involved in a variety of essential cellular functions. However, the precise role(s) of PI4KIIα in the cell is not yet completely deciphered. Here we present an experimental protocol that uses a chemical cross-linker, DSP, combined with immunoprecipitation and immunoaffinity purification to identify novel PI4KIIα interactors. As predicted, PI4KIIα participates in transient, low-affinity interactions that are stabilized by the use of DSP. Using this optimized protocol we have successfully identified actin cytoskeleton regulators-the WASH complex and RhoGEF1, as major novel interactors of PI4KIIα. While this chapter focuses on the PI4KIIα interactome, this protocol can and has been used to generate other membrane interactome networks. PMID:26552673

  1. Intrathecal inhibition of calcium/calmodulin-dependent protein kinase II in diabetic neuropathy adversely affects pain-related behavior.

    PubMed

    Jelicic Kadic, Antonia; Boric, Matija; Ferhatovic, Lejla; Banozic, Adriana; Sapunar, Damir; Puljak, Livia

    2013-10-25

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is considered an important enzyme contributing to the pathogenesis of persistent pain. The aim of this study was to test whether intrathecal injection of CaMKII inhibitors may reduce pain-related behavior in diabetic rats. Male Sprague-Dawley rats were used. Diabetes was induced with intraperitoneal injection of 55mg/kg streptozotocin. Two weeks after diabetes induction, CaMKII inhibitor myristoil-AIP or KN-93 was injected intrathecally. Behavioral testing with mechanical and thermal stimuli was performed before induction of diabetes, the day preceding the injection, as well as 2h and 24h after the intrathecal injection. The expression of total CaMKII and its alpha isoform in dorsal horn was quantified using immunohistochemistry. Intrathecal injection of mAIP and KN-93 resulted in significant decrease in expression of total CaMKII and CaMKII alpha isoform activity. Also, mAIP and KN93 injection significantly increased sensitivity to a mechanical stimulus 24h after i.t. injection. Intrathecal inhibition of CaMKII reduced the expression of total CaMKII and its CaMKII alpha isoform activity in diabetic dorsal horn, which was accompanied with an increase in pain-related behavior. Further studies about the intrathecal inhibition of CaMKII should elucidate its role in nociceptive processes of diabetic neuropathy. PMID:24035897

  2. The Effect of Calcium on the Binding of Calmodulin to Calcium/Calmodulin Protein Kinase II.

    ERIC Educational Resources Information Center

    Porta, Angela R.

    2000-01-01

    Introduces a follow-up laboratory experiment demonstrating the formation change when calcium binds to calmodulin. This conformation change allows this complex to bind to a target protein. Presents the necessary information to conduct the experiment and discusses the results. (YDS)

  3. Regulation of Cardiac ATP-sensitive Potassium Channel Surface Expression by Calcium/Calmodulin-dependent Protein Kinase II*

    PubMed Central

    Sierra, Ana; Zhu, Zhiyong; Sapay, Nicolas; Sharotri, Vikas; Kline, Crystal F.; Luczak, Elizabeth D.; Subbotina, Ekaterina; Sivaprasadarao, Asipu; Snyder, Peter M.; Mohler, Peter J.; Anderson, Mark E.; Vivaudou, Michel; Zingman, Leonid V.; Hodgson-Zingman, Denice M.

    2013-01-01

    Cardiac ATP-sensitive potassium (KATP) channels are key sensors and effectors of the metabolic status of cardiomyocytes. Alteration in their expression impacts their effectiveness in maintaining cellular energy homeostasis and resistance to injury. We sought to determine how activation of calcium/calmodulin-dependent protein kinase II (CaMKII), a central regulator of calcium signaling, translates into reduced membrane expression and current capacity of cardiac KATP channels. We used real-time monitoring of KATP channel current density, immunohistochemistry, and biotinylation studies in isolated hearts and cardiomyocytes from wild-type and transgenic mice as well as HEK cells expressing wild-type and mutant KATP channel subunits to track the dynamics of KATP channel surface expression. Results showed that activation of CaMKII triggered dynamin-dependent internalization of KATP channels. This process required phosphorylation of threonine at 180 and 224 and an intact 330YSKF333 endocytosis motif of the KATP channel Kir6.2 pore-forming subunit. A molecular model of the μ2 subunit of the endocytosis adaptor protein, AP2, complexed with Kir6.2 predicted that μ2 docks by interaction with 330YSKF333 and Thr-180 on one and Thr-224 on the adjacent Kir6.2 subunit. Phosphorylation of Thr-180 and Thr-224 would favor interactions with the corresponding arginine- and lysine-rich loops on μ2. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac KATP channel subunits. This mechanism couples the surface expression of cardiac KATP channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance. PMID:23223335

  4. Role of AMP-activated protein kinase α1 in angiotensin-II-induced renal Tgfß-activated kinase 1 activation.

    PubMed

    Mia, Sobuj; Castor, Tatsiana; Musculus, Katharina; Voelkl, Jakob; Alesutan, Ioana; Lang, Florian

    2016-08-01

    Angiotensin-II is a key factor in renal fibrosis. Obstructive nephropathy induces an isoform shift from catalytic Ampkα2 towards Ampkα1 which contributes to signaling involved in renal tissue injury. The present study explored whether the Ampkα1 isoform contributes to the renal effects of angiotensin-II. To this end, angiotensin-II was infused by subcutaneous implantation of osmotic minipumps in gene-targeted mice lacking functional Ampkα1 (Ampkα1(-/-)) and corresponding wild-type mice (Ampkα1(+/+)). Western blotting and qRT-PCR were employed to determine protein abundance and mRNA levels, respectively, in renal tissue. In Ampkα1(+/+) mice, angiotensin-II increased renal Ampkα1 protein expression without significantly modifying renal Ampkα2 protein expression. The renal phosphorylated Ampkα (Thr(172)) protein abundance was not affected by angiotensin-II in neither genotypes, but was significantly lower in Ampkα1(-/-) mice than Ampkα1(+/+) mice. Angiotensin-II increased the phosphorylation of Tak1 (Ser(412)) in renal tissue of Ampkα1(+/+) mice, an effect virtually absent in the Ampkα1(-/-) mice. Furthermore, angiotensin-II treatment significantly increased renal protein and mRNA expression of α-smooth muscle actin (αSma) as well as Tak1-target gene expression: Cox2, Il6 and Pai1 in Ampkα1(+/+) mice, all effects significantly less pronounced in Ampkα1(-/-) mice. In conclusion, angiotensin-II up-regulates the Ampkα1 isoform in renal tissue. Ampkα1 participates in renal Tak1 activation and Tak1-dependent signaling induced by angiotensin-II. PMID:27230958

  5. Casein Kinase II Induced Polymerization of Soluble TDP-43 into Filaments Is Inhibited by Heat Shock Proteins

    PubMed Central

    Davis, Mary; Lin, Wen-Lang; Cook, Casey; Dunmore, Judy; Tay, William; Menkosky, Kyle; Cao, Xiangkun; Petrucelli, Leonard; DeTure, Michael

    2014-01-01

    Background Trans-activation Response DNA-binding Protein-43 (TDP-43) lesions are observed in Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP) and 25–50% of Alzheimer's Disease (AD) cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The filamentous TDP-43 accumulations typically contain clean 10–12 nm filaments though wider 18–20 nm coated filaments may be observed. The TDP-43 present within these lesions is phosphorylated, truncated and ubiquitinated, and these modifications appear to be abnormal as they are linked to both a cellular heat shock response and microglial activation. The mechanisms associated with this abnormal TDP-43 accumulation are believed to result in a loss of TDP-43 function, perhaps due to the post-translational modifications or resulting from physical sequestration of the TDP-43. The formation of TDP-43 inclusions involves cellular translocation and conversion of TDP-43 into fibrillogenic forms, but the ability of these accumulations to sequester normal TDP-43 and propagate this behavior between neurons pathologically is mostly inferred. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades. Results The protocols described here generate soluble, full-length and untagged TDP-43 allowing for a direct assessment of the impact of various posttranslational modifications on TDP-43 function. We demonstrate that Casein Kinase II (CKII) promotes the polymerization of this soluble TDP-43 into 10 nm diameter filaments that resemble the most common TDP-43 structures observed in disease. Furthermore, these filaments are recognized as abnormal by Heat Shock Proteins (HSPs) which can inhibit TDP-43 polymerization or directly promote TDP-43 filament depolymerization. Conclusion These

  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. Angiotensin II regulates ACE and ACE2 in neurons through p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 signaling

    PubMed Central

    Xiao, Liang; Haack, Karla K. V.

    2013-01-01

    Brain ANG II plays an important role in modulating sympathetic function and homeostasis. The generation and degradation of ANG II are carried out, to a large extent, through the angiotensin-converting enzyme (ACE) and ACE2, respectively. In disease states, such as hypertension and chronic heart failure, central expression of ACE is upregulated and ACE2 is decreased in central sympathoregulatory neurons. In this study, we determined the expression of ACE and ACE2 in response to ANG II in a neuronal cell culture and the subsequent signaling mechanism(s) involved. A mouse catecholaminergic neuronal cell line (CATH.a) was treated with ANG II (30, 100, and 300 nM) for 24 h, and protein expression was determined by Western blot analysis. ANG II induced a significant dose-dependent increase in ACE and decrease in ACE2 mRNA and protein expression in CATH.a neurons. This effect was abolished by pretreatment of the cells with the p38 MAPK inhibitor SB-203580 (10 μM) 30 min before administration of ANG II or the ERK1/2 inhibitor U-0126 (10 μM). These data suggest that ANG II increases ACE and attenuates ACE2 expression in neurons via the ANG II type 1 receptor, p38 MAPK, and ERK1/2 signaling pathways. PMID:23535237

  8. Molecular basis for the modulation of native T-type Ca2+ channels in vivo by Ca2+ /calmodulin-dependent protein kinase II

    PubMed Central

    Yao, Junlan; Davies, Lucinda A.; Howard, Jason D.; Adney, Scott K.; Welsby, Philip J.; Howell, Nancy; Carey, Robert M.; Colbran, Roger J.; Barrett, Paula Q.

    2006-01-01

    Ang II receptor activation increases cytosolic Ca2+ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II–elicited signaling cascade that serves as a key intracellular Ca2+ sensor to feedback-regulate Ca2+ entry through voltage-gated Ca2+ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca2+ entry remain unresolved. We show here that CaMKII forms a signaling complex with α1H T-type Ca2+ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca2+ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression. PMID:16917542

  9. 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

  10. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    SciTech Connect

    Grose, C.; Jackson, W. ); Traugh, J.A. )

    1989-09-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.

  11. Protein Crystals of Raf Kinase

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This image shows crystals of the protein raf kinase grown on Earth (photo a) and on USML-2 (photo b). The space-grown crystals are an order of magnitude larger. Principal Investigator: Dan Carter of New Century Pharmaceuticals

  12. Type III Transforming Growth Factor-β Receptor Drives Cardiac Hypertrophy Through β-Arrestin2-Dependent Activation of Calmodulin-Dependent Protein Kinase II.

    PubMed

    Lou, Jie; Zhao, Dan; Zhang, Ling-Ling; Song, Shu-Ying; Li, Yan-Chao; Sun, Fei; Ding, Xiao-Qing; Yu, Chang-Jiang; Li, Yuan-Yuan; Liu, Mei-Tong; Dong, Chang-Jiang; Ji, Yong; Li, Hongliang; Chu, Wenfeng; Zhang, Zhi-Ren

    2016-09-01

    The role of type III transforming growth factor-β receptor (TβRIII) in the pathogenesis of heart diseases remains largely unclear. Here, we investigated the functional role and molecular mechanisms of TβRIII in the development of myocardial hypertrophy. Western blot and quantitative real time-polymerase chain reaction analyses revealed that the expression of TβRIII was significantly elevated in human cardiac hypertrophic samples. Consistently, TβRIII expression was substantially increased in transverse aortic constriction (TAC)- and isoproterenol-induced mouse cardiac hypertrophy in vivo and in isoproterenol-induced cardiomyocyte hypertrophy in vitro. Overexpression of TβRIII resulted in cardiomyocyte hypertrophy, whereas isoproterenol-induced cardiomyocyte hypertrophy was greatly attenuated by knockdown of TβRIII in vitro. Cardiac-specific transgenic expression of TβRIII independently led to cardiac hypertrophy in mice, which was further aggravated by isoproterenol and TAC treatment. Cardiac contractile function of the mice was not altered in TβRIII transgenic mice; however, TAC led to significantly decreased cardiac contractile function in TβRIII transgenic mice compared with control mice. Conversely, isoproterenol- and TAC-induced cardiac hypertrophy and TAC-induced cardiac contractile function impairment were partially reversed by suppression of TβRIII in vivo. Our data suggest that TβRIII mediates stress-induced cardiac hypertrophy through activation of Ca(2+)/calmodulin-dependent protein kinase II, which requires a physical interaction of β-arrestin2 with both TβRIII and calmodulin-dependent protein kinase II. Our findings indicate that stress-induced increase in TβRIII expression results in cardiac hypertrophy through β-arrestin2-dependent activation of calmodulin-dependent protein kinase II and that transforming growth factor-β and β-adrenergic receptor signaling are not involved in spontaneous cardiac hypertrophy in cardiac

  13. Characterization and differential expression of protein kinase C isoforms in PC12 cells. Differentiation parallels an increase in PKC beta II.

    PubMed

    Wooten, M W; Seibenhener, M L; Soh, Y; Ewald, S J; White, K R; Lloyd, E D; Olivier, A; Parker, P J

    1992-02-17

    Nerve growth factor (NGF) treatment of PC12 cells induced a 2.8-fold increase in protein kinase C activity concomitant with differentiation and acquisition of neuritis. PKC protein isoforms were separated by sequential chromatography on DEAE-Sephacel/hydroxylapatite. A broad peak of PKC activity eluted which corresponded to the alpha PKC isoform. In control cells, message for all six PKC isoforms was detected and expressed as epsilon greater than zeta = gamma greater than delta greater than beta greater than alpha. Western blot of whole cell lysates revealed a large increase in the beta II, while slight changes were observed for the other five PKC isoforms during treatment (1-14 days) with NGF (50 ng/ml). In parallel, coordinate changes in the expression of the individual transcripts for the six isoforms occurred during NGF treatment. Induction and accumulation of PKC beta II may play a role in maintenance of neuronal morphology. PMID:1544425

  14. Post-synaptic density-95 promotes calcium/calmodulin-dependent protein kinase II-mediated Ser847 phosphorylation of neuronal nitric oxide synthase.

    PubMed Central

    Watanabe, Yasuo; Song, Tao; Sugimoto, Katsuyoshi; Horii, Mariko; Araki, Nobukazu; Tokumitsu, Hiroshi; Tezuka, Tohru; Yamamoto, Tadashi; Tokuda, Masaaki

    2003-01-01

    Post-synaptic density-95 (PSD-95) is a neuronal scaffolding protein that associates with N -methyl-D-aspartate (NMDA) receptors and links them to intracellular signalling molecules. In neurons, neuronal nitric oxide synthase (nNOS) binds selectively to the second PDZ domain (PDZ2) of PSD-95, thereby exhibiting physiological activation triggered via NMDA receptors. We have demonstrated previously that Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaM-K IIalpha) directly phosphorylates nNOS at residue Ser(847), and can attenuate the catalytic activity of the enzyme in neuronal cells [Komeima, Hayashi, Naito and Watanabe (2000) J. Biol. Chem. 275, 28139-28143]. In the present study, we examined how CaM-K II participates in the phosphorylation by analysing the functional interaction between nNOS and PSD-95 in cells. The results showed that PSD-95 directly promotes the nNOS phosphorylation at Ser(847) induced by endogenous CaM-K II. In transfected cells, this effect of PSD-95 required its dual palmitoylation and the PDZ2 domain, but did not rely on its guanylate kinase domain. CaM-K Ialpha and CaM-K IV failed to phosphorylate nNOS at Ser(847) in transfected cells. Thus PSD-95 mediates cellular trafficking of nNOS, and may be required for the efficient phosphorylation of nNOS at Ser(847) by CaM-K II in neuronal cells. PMID:12630910

  15. 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.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2010-01-01

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

  18. Atlas of transgenic Tet-Off Ca2+/calmodulin-dependent protein kinase II and prion protein promoter activity in the mouse brain.

    PubMed

    Odeh, Francis; Leergaard, Trygve B; Boy, Jana; Schmidt, Thorsten; Riess, Olaf; Bjaalie, Jan G

    2011-02-14

    Conditional transgenic mouse models are important tools for investigations of neurodegenerative diseases and evaluation of potential therapeutic interventions. A popular conditional transgenic system is the binary tetracycline-responsive gene (Tet-Off) system, in which the expression of the gene of interest depends on a tetracycline-regulatable transactivator (tTA) under the control of a specific promoter construct. The most frequently used Tet-Off promoter mouse lines are the Ca(2+)/calmodulin-dependent protein kinase II (CamKII) and prion protein (PrP) promoter lines, respectively. To target the regulated gene of interest to relevant brain regions, a priori knowledge about the spatial distribution of the regulated gene expression in the brain is important. Such distribution patterns can be investigated using double transgenic mice in which the promoter construct regulates a LacZ reporter gene encoding the marker β-galactosidase which can be histologically detected using its substrate X-gal. We have previously published an atlas showing the brain-wide expression mediated by the Tet-Off PrP promoter mouse line, but the distribution of activity in the Tet-Off CamKII promoter mouse line is less well known. To compare promoter activity distributions in these two Tet-Off mouse lines, we have developed an online digital atlas tailored for side-by-side comparison of histological section images. The atlas provides a comprehensive list of brain regions containing X-gal labeling and an interactive dual image viewer tool for panning and zooming of corresponding section images. Comparison of spatial expression patterns between the two lines show considerable regional and cellular differences, relevant in context of generation and analysis of inducible models based on these two tetracycline responsive promoter mouse lines. PMID:21093594

  19. 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.

  20. 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.

  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 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome†

    PubMed Central

    Phatnani, Hemali P.; Jones, Janice C.; Greenleaf, Arno L.

    2009-01-01

    CTD kinase I (CTDK-I) of Saccharomyces cerevisiae is required for normal phosphorylation of the C-terminal repeat domain (CTD) on elongating RNA polymerase II. To elucidate cellular roles played by this kinase and the hyperphosphorylated CTD (phosphoCTD) it generates, we systematically searched yeast extracts for proteins that bound to the phosphoCTD made by CTDK-I in vitro. Initially, using a combination of far-western blotting and phosphoCTD affinity chromatography, we discovered a set of novel phosphoCTD-associating proteins (PCAPs) implicated in a variety of nuclear functions. We identified the phosphoCTD-interacting domains of a number of these PCAPs, and in several test cases (namely, Set2, Ssd1, and Hrr25) adduced evidence that phosphoCTD binding is functionally important in vivo. Employing surface plasmon resonance (BIACORE) analysis, we found that recombinant versions of these and other PCAPs bind preferentially to CTD repeat peptides carrying SerPO4 residues at positions 2 and 5 of each seven amino acid repeat, consistent with the positional specificity of CTDK-I in vitro [Jones, J. C., et al. (2004) J. Biol. Chem. 279, 24957–24964]. Subsequently, we used a synthetic CTD peptide with three doubly phosphorylated repeats (2,5P) as an affinity matrix, greatly expanding our search for PCAPs. This resulted in identification of approximately 100 PCAPs and associated proteins representing a wide range of functions (e.g., transcription, RNA processing, chromatin structure, DNA metabolism, protein synthesis and turnover, RNA degradation, snRNA modification, and snoRNP biogenesis). The varied nature of these PCAPs and associated proteins points to an unexpectedly diverse set of connections between Pol II elongation and other processes, conceptually expanding the role played by CTD phosphorylation in functional organization of the nucleus. PMID:15595826

  10. CDKF;1 and CDKD protein kinases regulate phosphorylation of serine residues in the C-terminal domain of Arabidopsis RNA polymerase II.

    PubMed

    Hajheidari, Mohsen; Farrona, Sara; Huettel, Bruno; Koncz, Zsuzsa; Koncz, Csaba

    2012-04-01

    Phosphorylation of conserved Y₁S₂P₃T₄S₅P₆S₇ repeats in the C-terminal domain of largest subunit of RNA polymerase II (RNAPII CTD) plays a central role in the regulation of transcription and cotranscriptional RNA processing. Here, we show that Ser phosphorylation of Arabidopsis thaliana RNAPII CTD is governed by CYCLIN-DEPENDENT KINASE F;1 (CDKF;1), a unique plant-specific CTD S₇-kinase. CDKF;1 is required for in vivo activation of functionally redundant CYCLIN-DEPENDENT KINASE Ds (CDKDs), which are major CTD S₅-kinases that also phosphorylate in vitro the S₂ and S₇ CTD residues. Inactivation of CDKF;1 causes extreme dwarfism and sterility. Inhibition of CTD S₇-phosphorylation in germinating cdkf;1 seedlings is accompanied by 3'-polyadenylation defects of pre-microRNAs and transcripts encoding key regulators of small RNA biogenesis pathways. The cdkf;1 mutation also decreases the levels of both precursor and mature small RNAs without causing global downregulation of the protein-coding transcriptome and enhances the removal of introns that carry pre-microRNA stem-loops. A triple cdkd knockout mutant is not viable, but a combination of null and weak cdkd;3 alleles in a triple cdkd123* mutant permits semidwarf growth. Germinating cdkd123* seedlings show reduced CTD S₅-phosphorylation, accumulation of uncapped precursor microRNAs, and a parallel decrease in mature microRNA. During later development of cdkd123* seedlings, however, S₇-phosphorylation and unprocessed small RNA levels decline similarly as in the cdkf;1 mutant. Taken together, cotranscriptional processing and stability of a set of small RNAs and transcripts involved in their biogenesis are sensitive to changes in the phosphorylation of RNAPII CTD by CDKF;1 and CDKDs. PMID:22547781

  11. Intra-nucleus accumbens administration of the calcium/calmodulin-dependent protein kinase II inhibitor AIP induced antinociception in rats with mononeuropathy.

    PubMed

    Bian, Hui; Yu, Long-Chuan

    2015-07-10

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine- dependent protein kinase, which has been implicated in pain modulation at different levels of the central nervous system. The present study was performed in rats with mononeuropathy induced by left common sciatic nerve ligation. Unilateral sciatic nerve loose ligation produced decreases in the hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation. Intra-nucleus accumbens (NAc) injection of 3 μg, 6 μg and 12 μg of myristoylated autocamtide-2-inhibitory peptide (AIP), the CaMKII inhibitor, dose-dependently increased the HWL to noxious thermal and mechanical stimulation in rats with mononeuropathy. Furthermore, intra-NAc administration of morphine, the HWL to noxious thermal and mechanical stimulation increased markedly, and there were no significant differences between morphine group and AIP group. Taken together, the results showed that intra-NAc injection of AIP induced significant antinociceptive effects in rats with mononeuropathy, indicating that CaMKII may play an important role in the transmission and/or modulation of nociceptive information in the NAc in rats with mononeuropathy. PMID:26022629

  12. Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates.

    PubMed

    Praseeda, Mullasseril; Pradeep, Kurup K; Krupa, Ananth; Krishna, S Sri; Leena, Suseela; Kumar, R Rajeev; Cheriyan, John; Mayadevi, Madhavan; Srinivasan, Narayanaswamy; Omkumar, Ramakrishnapillai V

    2004-03-01

    CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B ( N -methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60-->Gly mutation. PMID:14558884

  13. The specificity of the protein kinase C alpha, betaII and gamma isoforms as assessed by an unnatural alcohol-appended peptide library.

    PubMed Central

    Yan, X; Curley, K; Lawrence, D S

    2000-01-01

    Previous studies using conventional peptide-based libraries have demonstrated that homologous protein-processing enzymes [e.g. the alpha, betaII and gamma isoforms of protein kinase (PKC)] typically display identical amino acid consensus sequences. These observations have hampered the acquisition of selective synthetic substrates for the individual members of these enzyme families. We describe here a parallel synthesis strategy, readily adaptable to the preparation of large libraries, that has led to the emergence of the first examples of selective substrates for the conventional PKC isoforms. In addition, we have found that a wide variety of structurally diverse N-appended alcohol-containing residues, including tyrosine, serve as substrates for the PKC alpha, betaII and gamma isoforms. This broad active-site substrate specificity with respect to both natural and unnatural residues may prove to be especially applicable to the construction of transition-state analogues and suicide substrates, species that often require the presence of structurally elaborate functionality. PMID:10903131

  14. Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells.

    PubMed

    Chi, Mengna; Evans, Hamish; Gilchrist, Jackson; Mayhew, Jack; Hoffman, Alexander; Pearsall, Elizabeth Ann; Jankowski, Helen; Brzozowski, Joshua Stephen; Skelding, Kathryn Anne

    2016-01-01

    Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis. PMID:27605043

  15. Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells

    PubMed Central

    Chi, Mengna; Evans, Hamish; Gilchrist, Jackson; Mayhew, Jack; Hoffman, Alexander; Pearsall, Elizabeth Ann; Jankowski, Helen; Brzozowski, Joshua Stephen; Skelding, Kathryn Anne

    2016-01-01

    Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis. PMID:27605043

  16. cAMP signaling in neurons: patterns of neuronal expression and intracellular localization for a novel protein, AKAP 150, that anchors the regulatory subunit of cAMP-dependent protein kinase II beta.

    PubMed Central

    Glantz, S B; Amat, J A; Rubin, C S

    1992-01-01

    In mammalian brain, physiological signals carried by cyclic AMP (cAMP) seem to be targeted to effector sites via the tethering of cAMP-dependent protein kinase II beta (PKAII beta) to intracellular structures. Recently characterized A kinase anchor proteins (AKAPs) are probable mediators of the sequestration of PKAII beta because they contain a high-affinity binding site for the regulatory subunit (RII beta) of the kinase and a distinct intracellular targeting domain. To establish a cellular basis for this targeting mechanism, we have employed immunocytochemistry to 1) identify the types of neurons that are enriched in AKAPs, 2) determine the primary intracellular location of the anchor protein, and 3) demonstrate that an AKAP and RII beta are coenriched and colocalized in neurons that utilize the adenylate cyclase-cyclic AMP-dependent protein kinase (PKA) signaling pathway. Antibodies directed against rat brain AKAP 150 were used to elucidate the regional, cellular and intracellular distribution of a prototypic anchor protein in the CNS. AKAP 150 is abundant in Purkinje cells and in neurons of the olfactory bulb, basal ganglia, cerebral cortex, and other forebrain regions. In contrast, little AKAP 150 is detected in neurons of the thalamus, hypothalamus, midbrain, and hindbrain. A high proportion of total AKAP 150 is concentrated in primary branches of dendrites, where it is associated with microtubules. We also discovered that the patterns of accumulation and localization of RII beta (and PKAII beta) in brain are similar to those of AKAP 150. The results suggest that bifunctional AKAP 150 tethers PKAII beta to the dendritic cytoskeleton, thereby creating a discrete target site for the reception and propagation of signals carried by cAMP. Images PMID:1333841

  17. Calcium/Calmodulin-dependent Protein Kinase II is a Ubiquitous Molecule in Human Long-term Memory Synaptic Plasticity: A Systematic Review

    PubMed Central

    Ataei, Negar; Sabzghabaee, Ali Mohammad; Movahedian, Ahmad

    2015-01-01

    Background: Long-term memory is based on synaptic plasticity, a series of biochemical mechanisms include changes in structure and proteins of brain's neurons. In this article, we systematically reviewed the studies that indicate calcium/calmodulin kinase II (CaMKII) is a ubiquitous molecule among different enzymes involved in human long-term memory and the main downstream signaling pathway of long-term memory. Methods: All of the observational, case–control and review studies were considered and evaluated by the search engines PubMed, Cochrane Central Register of Controlled Trials and ScienceDirect Scopus between 1990 and February 2015. We did not carry out meta-analysis. Results: At the first search, it was fined 1015 articles which included “synaptic plasticity” OR “neuronal plasticity” OR “synaptic density” AND memory AND “molecular mechanism” AND “calcium/calmodulin-dependent protein kinase II” OR CaMKII as the keywords. A total of 335 articles were duplicates in the databases and eliminated. A total of 680 title articles were evaluated. Finally, 40 articles were selected as reference. Conclusions: The studies have shown the most important intracellular signal of long-term memory is calcium-dependent signals. Calcium linked calmodulin can activate CaMKII. After receiving information for learning and memory, CaMKII is activated by Glutamate, the most important neurotransmitter for memory-related plasticity. Glutamate activates CaMKII and it plays some important roles in synaptic plasticity modification and long-term memory. PMID:26445635

  18. Haploinsufficiency for either one of the type-II regulatory subunits of protein kinase A improves the bone phenotype of Prkar1a+/- mice.

    PubMed

    Liu, Sisi; Saloustros, Emmanouil; Mertz, Edward L; Tsang, Kitman; Starost, Matthew F; Salpea, Paraskevi; Faucz, Fabio R; Szarek, Eva; Nesterova, Maria; Leikin, Sergey; Stratakis, Constantine A

    2015-11-01

    Carney Complex (CNC), a human genetic syndrome predisposing to multiple neoplasias, is associated with bone lesions such as osteochondromyxomas (OMX). The most frequent cause for CNC is PRKAR1A deficiency; PRKAR1A codes for type-I regulatory subunit of protein kinase A (PKA). Prkar1a(+/-) mice developed OMX, fibrous dysplasia-like lesions (FDL) and other tumors. Tumor tissues in these animals had increased PKA activity due to an unregulated PKA catalytic subunit and increased PKA type II (PKA-II) activity mediated by the PRKAR2A and PRKAR2B subunits. To better understand the effect of altered PKA activity on bone, we studied Prkar2a and Prkar2b knock out (KO) and heterozygous mice; none of these mice developed bone lesions. When Prkar2a(+/-) and Prkar2b(+/-) mice were used to generate Prkar1a(+/-)Prkar2a(+/-) and Prkar1a(+/-)Prkar2b(+/-) animals, bone lesions formed that looked like those of the Prkar1a(+/-) mice. However, better overall bone organization and mineralization and fewer FDL lesions were found in both double heterozygote groups, indicating a partial restoration of the immature bone structure observed in Prkar1a(+/-) mice. Further investigation indicated increased osteogenesis and higher new bone formation rates in both Prkar1a(+/-)Prkar2a(+/-) and Prkar1a(+/-)Prkar2b(+/-) mice with some minor differences between them. The observations were confirmed with a variety of markers and studies. PKA activity measurements showed the expected PKA-II decrease in both double heterozygote groups. Thus, haploinsufficiency for either of PKA-II regulatory subunits improved bone phenotype of mice haploinsufficient for Prkar1a, in support of the hypothesis that the PRKAR2A and PRKAR2B regulatory subunits were in part responsible for the bone phenotype of Prkar1a(+/-) mice. PMID:26246497

  19. 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.

  20. Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

    PubMed Central

    Issinger, O G; Beier, H; Speichermann, N; Flokerzi, V; Hofmann, F

    1980-01-01

    Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme. Images Fig. 1. Fig. 2. PMID:6246882

  1. Interactions between protein kinase C and arachidonic acid in the gonadotropin response to salmon and chicken gonadotropin-releasing hormone-II in goldfish.

    PubMed

    Chang, J P; Van Goor, F; Neumann, C M

    1994-03-01

    Previous studies have shown that, in goldfish, the gonadotropin (GTH) response to salmon GTH-releasing hormone (sGnRH) is partly mediated by arachidonic acid (AA) metabolism via the lipoxygenase enzyme system, whereas protein kinase C (PKC) participates in both sGnRH- and chicken (c)GnRH-II-induced GTH secretion. In this study, the interactions between AA- and PKC-dependent pathways in mediating the long-term GnRH stimulation of GTH release were further investigated using dispersed goldfish pituitary cell cultures in static incubation. Treatments with AA or the PKC activator tetradecanoylphorbol acetate (TPA) increased GTH release. The GTH responses to AA and TPA were additive. The lipoxygenase inhibitor nordihydroguairetic acid (NDGA) and the PKC inhibitor H7 selectively reduced AA- and TPA-stimulated GTH release, respectively. These findings suggest that the GTH responses to stimulation by AA- and PKC-dependent signaling pathways are independent of one another. In other experiments, the GTH response to cGnRH-II was unaffected by NDGA but was abolished by H7. In contrast, sGnRH-induced GTH release was attenuated by NDGA and H7. Furthermore, in the presence of both NDGA and H7, the GTH response to sGnRH was abolished. These data suggest that sGnRH stimulation of GTH secretion involves both AA- and PKC-dependent mechanisms; in contrast, cGnRH-II action is not dependent on AA metabolism. The pathway by which AA might be mobilized in response to a GnRH challenge was also investigated by pharmacological manipulations. The diacylglcerol (DG) lipase inhibitor, U-57908, did not decrease sGnRH- and cGnRH-II-induced GTH secretion. On the other hand, the phospholipase A2 (PLA2) inhibitors, bromophenacyl bromide (BPB), chloroquine, and quinacrine, reduced sGnRH-elicited, but not cGnRH-II-stimulated GTH release. The addition of AA reversed the inhibitory action of BPB on sGnRH-elicited GTH release. In addition, the GTH response to AA was additive to the cGnRH-II-induced, but

  2. β-Amyloid Impairs AMPA Receptor Trafficking and Function by Reducing Ca2+/Calmodulin-dependent Protein Kinase II Synaptic Distribution*

    PubMed Central

    Gu, Zhenglin; Liu, Wenhua; Yan, Zhen

    2009-01-01

    A fundamental feature of Alzheimer disease (AD) is the accumulation of β-amyloid (Aβ), a peptide generated from the amyloid precursor protein (APP). Emerging evidence suggests that soluble Aβ oligomers adversely affect synaptic function, which leads to cognitive failure associated with AD. The Aβ-induced synaptic dysfunction has been attributed to the synaptic removal of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs); however, it is unclear how Aβ induces the loss of AMPARs at the synapses. In this study we have examined the potential involvement of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a signaling molecule critical for AMPAR trafficking and function. We found that the synaptic pool of CaMKII was significantly decreased in cortical neurons from APP transgenic mice, and the density of CaMKII clusters at synapses was significantly reduced by Aβ oligomer treatment. In parallel, the surface expression of GluR1 subunit as well as AMPAR-mediated synaptic response and ionic current was selectively decreased in APP transgenic mice and Aβ-treated cultures. Moreover, the reducing effect of Aβ on AMPAR current density was mimicked and occluded by knockdown of CaMKII and blocked by overexpression of CaMKII. These results suggest that the Aβ-induced change in CaMKII subcellular distribution may underlie the removal of AMPARs from synaptic membrane by Aβ. PMID:19240035

  3. Mechanism of inhibition of Raf-1 by protein kinase A.

    PubMed Central

    Häfner, S; Adler, H S; Mischak, H; Janosch, P; Heidecker, G; Wolfman, A; Pippig, S; Lohse, M; Ueffing, M; Kolch, W

    1994-01-01

    The cytoplasmic Raf-1 kinase is essential for mitogenic signalling by growth factors, which couple to tyrosine kinases, and by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate, which activate protein kinase C (PKC). Signalling by the Raf-1 kinase can be blocked by activation of the cyclic AMP (cAMP)-dependent protein kinase A (PKA). The molecular mechanism of this inhibition is not precisely known but has been suggested to involve attenuation of Raf-1 binding to Ras. Using purified proteins, we show that in addition to weakening the interaction of Raf-1 with Ras, PKA can inhibit Raf-1 function directly via phosphorylation of the Raf-1 kinase domain. Phosphorylation by PKA interferes with the activation of Raf-1 by either PKC alpha or the tyrosine kinase Lck and even can downregulate the kinase activity of Raf-1 previously activated by PKC alpha or amino-terminal truncation. This type of inhibition can be dissociated from the ability of Raf-1 to associate with Ras, since (i) the isolated Raf-1 kinase domain, which lacks the Ras binding domain, is still susceptible to inhibition by PKA, (ii) phosphorylation of Raf-1 by PKC alpha alleviates the PKA-induced reduction of Ras binding but does not prevent the downregulation of Raf-1 kinase activity by PKA and (iii) cAMP agonists antagonize transformation by v-Raf, which is Ras independent. Images PMID:7935389

  4. Hydrophobic motif site-phosphorylated protein kinaseII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy.

    PubMed

    Das, Falguni; Ghosh-Choudhury, Nandini; Mariappan, Meenalakshmi M; Kasinath, Balakuntalam S; Choudhury, Goutam Ghosh

    2016-04-01

    PKCβII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCβII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCβII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCβII, dominant negative PKCβII, and PKCβII hydrophobic motif phosphorylation-deficient mutant, we found that PKCβII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCβII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCβII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCβII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCβII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCβII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy. PMID:26739493

  5. Inhibitory effects of KN-93, an inhibitor of Ca2+ calmodulin-dependent protein kinase II, on light-regulated root gravitropism in maize

    NASA Technical Reports Server (NTRS)

    Feldman, L. J.; Hidaka, H.

    1993-01-01

    Light is essential for root gravitropism in Zea mays L., cultivar Merit. It is hypothesized that calcium mediates this light-regulated response. KN-93, an inhibitor of calcium/calmodulin kinase II (CaMK II), inhibits light-regulated root gravitropism but does not affect light perception. We hypothesize that CaMK II, or a homologue, operates late in the light/gravity signal transduction chain. Here we provide evidence suggesting a possible physiological involvement of CaMK II in root gravitropism in plants.

  6. Production of platelet-activating factor is a component of the angiotensin II-protein kinase C activation pathway in bovine adrenocortical cells.

    PubMed

    Pelosin, J M; Keramidas, M; Chambaz, E M

    1991-08-15

    Lyso-platelet-activating factor (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) enzyme activity was characterized for the first time in bovine adrenocortical tissue. It was found to be associated with the microsomal membrane fraction, in which it exhibited a specific activity of 0.4 nmol/min per mg of protein and catalytic properties similar to those described in other cell types. The adrenocortical acetyltransferase activity was increased by 2-3-fold on incubation of the preparation with purified protein kinase C (PKC) under phosphorylating condition. This activation was optimal after 5 min of incubation and paralleled an increase in PKC-catalysed 32P incorporation into microsomal proteins. Both acetyltransferase activation and protein phosphorylation were dependent on the presence of Ca2+ and phospholipids, and were blocked in the presence of the potent PKC inhibitor H-7. In the intact adrenocortical cell, angiotensin II and a potent phorbol ester (phorbol 12-myristate 13-acetate) were able to rapidly induce an increase in the biosynthesis of PAF, which was mostly released into the extracellular medium. These data suggest that bovine adrenocortical lyso-PAF acetyltransferase may be regulated by a PKC-dependent activation pathway, whereas no evidence for an additional adrenocorticotropin/cyclic AMP-dependent stimulation process was obtained in this cell type. Bovine adrenocortical cell membrane preparations were shown to possess high-affinity PAF-binding sites (Kd approximately 0.5 nM). Altogether, these observations suggest that PAF production and release may play a role in the autocrine or paracrine control of adrenocortical cell activation. PMID:1883337

  7. 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. PMID:27226575

  8. Long Wavelength Monitoring of Protein Kinase Activity

    PubMed Central

    Oien, Nathan P.; Nguyen, Luong T.; Jernigan, Finith E.; Priestman, Melanie A.

    2014-01-01

    A family of long wavelength protein kinase fluorescent reporters is described in which the probing wavelength is pre-programmed using readily available fluorophores. These agents can assess protein kinase activity within the optical window of tissue, as exemplified by monitoring endogenous cAMP-dependent protein kinase activity (1) in erythrocyte lysates and (2) in intact erythrocytes using a light-activatable reporter. PMID:24604833

  9. Mammalian Target of Rapamycin (mTOR) Tagging Promotes Dendritic Branch Variability through the Capture of Ca2+/Calmodulin-dependent Protein Kinase II α (CaMKIIα) mRNAs by the RNA-binding Protein HuD.

    PubMed

    Sosanya, Natasha M; Cacheaux, Luisa P; Workman, Emily R; Niere, Farr; Perrone-Bizzozero, Nora I; Raab-Graham, Kimberly F

    2015-06-26

    The fate of a memory, whether stored or forgotten, is determined by the ability of an active or tagged synapse to undergo changes in synaptic efficacy requiring protein synthesis of plasticity-related proteins. A synapse can be tagged, but without the "capture" of plasticity-related proteins, it will not undergo long lasting forms of plasticity (synaptic tagging and capture hypothesis). What the "tag" is and how plasticity-related proteins are captured at tagged synapses are unknown. Ca(2+)/calmodulin-dependent protein kinase II α (CaMKIIα) is critical in learning and memory and is synthesized locally in neuronal dendrites. The mechanistic (mammalian) target of rapamycin (mTOR) is a protein kinase that increases CaMKIIα protein expression; however, the mechanism and site of dendritic expression are unknown. Herein, we show that mTOR activity mediates the branch-specific expression of CaMKIIα, favoring one secondary, daughter branch over the other in a single neuron. mTOR inhibition decreased the dendritic levels of CaMKIIα protein and mRNA by shortening its poly(A) tail. Overexpression of the RNA-stabilizing protein HuD increased CaMKIIα protein levels and preserved its selective expression in one daughter branch over the other when mTOR was inhibited. Unexpectedly, deleting the third RNA recognition motif of HuD, the domain that binds the poly(A) tail, eliminated the branch-specific expression of CaMKIIα when mTOR was active. These results provide a model for one molecular mechanism that may underlie the synaptic tagging and capture hypothesis where mTOR is the tag, preventing deadenylation of CaMKIIα mRNA, whereas HuD captures and promotes its expression in a branch-specific manner. PMID:25944900

  10. 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.

  11. Involvement of the Ca2+/calmodulin-dependent protein kinase II pathway in the Ca2+-mediated regulation of the capacitative Ca2+ entry in Xenopus oocytes.

    PubMed Central

    Matifat, F; Fournier, F; Lorca, T; Capony, J P; Brûlé, G; Collin, T

    1997-01-01

    Activation of the phosphoinositide transduction pathway induces capacitative Ca2+ entry in Xenopus oocytes. This can also be evoked by intracellular injection of Ins(1,4.5)P3, external application of thapsigargin and/or incubation in a Ca2+-free medium. Readmission of Ca2+ to voltage-clamped, thapsigargin-treated Xenopus oocytes triggers Ca2+-dependent Cl- current variations that reflect capacitative Ca2+ entry. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by specific peptides markedly increased the amplitude of the transients, suggesting an involvement of the CaMKII pathway in the regulation of capacitative Ca2+ entry. Biochemical studies provide evidence for the activation of CaMKII in response to the development of capacitative Ca2+ entry. In effect, a CaMKII assay in vivo allows us to postulate that readmission of Ca2+ to thapsigargin-treated oocytes can induce a burst of CaMKII activity. Finally, analysis of the Cl- transient kinetics at high resolution of time suggests that CaMKII inhibition blocks the onset of the inactivation process without affecting the activation rate. We therefore postulate that CaMKII might participate in a negative feedback regulation of store-depletion-evoked Ca2+ entry in Xenopus oocytes. PMID:9078272

  12. A role for the CaM Kinase II related anchoring protein (αkap) in maintaining the stability of nicotinic Acetylcholine Receptors

    PubMed Central

    Mouslim, Chakib; Aittaleb, Mohamed; Hume, Richard I.; Akaaboune, Mohammed

    2012-01-01

    αkap, a muscle specific anchoring protein encoded within the Camk2a gene is thought to play a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations. Here we demonstrate a novel function of αkap in stabilizing nicotinic acetylcholine receptors (AChR). Knockdown of αkap expression with shRNA significantly enhanced the degradation of AChR α-subunits (AChRα), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that αkap promoted AChRα stability by a ubiquitin-dependent mechanism. In the absence of αkap, AChRα was heavily ubiquitinated and the number of AChRα was increased by proteasome inhibitors. However, in the presence of αkap, AChRα was less ubiquitinated and proteasome inhibitors had almost no effect on AChRα accumulation. The major sites of AChRα ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChRα stability in the absence of αkap. These results provide an unexpected mechanism by which αkap controls receptor trafficking onto the surface of muscle cells, and thus the maintenance of postsynaptic receptor density and synaptic function. PMID:22496563

  13. The effects of intraganglionic injection of calcium/calmodulin-dependent protein kinase II inhibitors on pain-related behavior in diabetic neuropathy.

    PubMed

    Jelicic Kadic, A; Boric, M; Kostic, S; Sapunar, D; Puljak, L

    2014-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the transmission of nociceptive input in diabetic neuropathy. The aim of this study was to test whether intraganglionic (i.g.) injection of CaMKII inhibitors may alleviate pain-related behavior in diabetic rats. Diabetes was induced in Sprague-Dawley rats using 55 mg/kg streptozotocin intraperitoneally. Two weeks after diabetes induction, CaMKII inhibitors myristoil-AIP and KN93 were injected directly into the right L5 dorsal root ganglion (DRG). Behavioral testing with mechanical and thermal stimuli was performed before induction of diabetes, the day preceding the injection, as well as 2 and 24h after the i.g. injection. The expression of total CaMKII and its alpha isoform in DRG neurons was analyzed using immunohistochemistry. CaMKII inhibitors attenuated pain-related behavior in a modality-specific fashion. Attenuation of nociceptive behavior was accompanied with a corresponding decrease of CaMKII alpha expression in DRG neurons on the side of injection. A significant decrease of CaMKII alpha expression was seen in small- and medium-sized neurons. In conclusion, our study provides evidence that CaMKII inhibitors are potential pharmacological agents that should be further explored for treatment of diabetic neuropathy symptoms. PMID:24161721

  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. Plant protein kinase substrates identification using protein microarrays.

    PubMed

    Ma, Shisong; Dinesh-Kumar, Savithramma P

    2015-01-01

    Protein kinases regulate signaling pathways by phosphorylating their targets. They play critical roles in plant signaling networks. Although many important protein kinases have been identified in plants, their substrates are largely unknown. We have developed and produced plant protein microarrays with more than 15,000 purified plant proteins. Here, we describe a detailed protocol to use these microarrays to identify plant protein kinase substrates via in vitro phosphorylation assays on these arrays. PMID:25930701

  16. Fibronectin phosphorylation by ecto-protein kinase

    SciTech Connect

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru )

    1988-12-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  17. 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.

  18. Affinity-purified CCAAT-box-binding protein (YEBP) functionally regulates expression of a human class II major histocompatibility complex gene and the herpes simplex virus thymidine kinase gene

    SciTech Connect

    Zeleznik-Le, N.J.; Azizkhan, J.C.; Ting, J.P.Y. )

    1991-03-01

    Efficient major histocompatibility complex class II gene expression requires conseved protein-binding promoter elements, including X and Y elements. The authors affinity purified an HLA-DRA Y-element (CCAAT)-binding protein (YEBP) and used it to reconstitute Y-depleted HLA-DRA in vitro transcription. This directly demonstrates a positive functional role for YEBP in HLA-DRA transcription. The ability of YEBP to regulate divergent CCAAT elements was also assessed; YEBP was found to partially activate the thymidine kinase promoter. This functional analysis of YEBP shows that this protein plays an important role in the regulation of multiple genes.

  19. 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

  20. Dynamic architecture of a protein kinase

    PubMed Central

    McClendon, Christopher L.; Kornev, Alexandr P.; Gilson, Michael K.; Taylor, Susan S.

    2014-01-01

    Protein kinases are dynamically regulated signaling proteins that act as switches in the cell by phosphorylating target proteins. To establish a framework for analyzing linkages between structure, function, dynamics, and allostery in protein kinases, we carried out multiple microsecond-scale molecular-dynamics simulations of protein kinase A (PKA), an exemplar active kinase. We identified residue–residue correlated motions based on the concept of mutual information and used the Girvan–Newman method to partition PKA into structurally contiguous “communities.” Most of these communities included 40–60 residues and were associated with a particular protein kinase function or a regulatory mechanism, and well-known motifs based on sequence and secondary structure were often split into different communities. The observed community maps were sensitive to the presence of different ligands and provide a new framework for interpreting long-distance allosteric coupling. Communication between different communities was also in agreement with the previously defined architecture of the protein kinase core based on the “hydrophobic spine” network. This finding gives us confidence in suggesting that community analyses can be used for other protein kinases and will provide an efficient tool for structural biologists. The communities also allow us to think about allosteric consequences of mutations that are linked to disease. PMID:25319261

  1. 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.

  2. DNA-dependent protein kinase interacts functionally with the RNA polymerase II complex recruited at the human immunodeficiency virus (HIV) long terminal repeat and plays an important role in HIV gene expression.

    PubMed

    Tyagi, Shilpi; Ochem, Alex; Tyagi, Mudit

    2011-07-01

    DNA-dependent protein kinase (DNA-PK), a nuclear protein kinase that specifically requires association with DNA for its kinase activity, plays important roles in the regulation of different DNA transactions, including transcription, replication and DNA repair, as well as in the maintenance of telomeres. Due to its large size, DNA-PK is also known to facilitate the activities of other factors by providing the docking platform at their site of action. In this study, by running several chromatin immunoprecipitation assays, we demonstrate the parallel distribution of DNA-PK with RNA polymerase II (RNAP II) along the human immunodeficiency virus (HIV) provirus before and after activation with tumour necrosis factor alpha. The association between DNA-PK and RNAP II is also long-lasting, at least for up to 4 h (the duration analysed in this study). Knockdown of endogenous DNA-PK using specific small hairpin RNAs expressed from lentiviral vectors resulted in significant reduction in HIV gene expression and replication, demonstrating the importance of DNA-PK for HIV gene expression. Sequence analysis of the HIV-1 Tat protein revealed three potential target sites for phosphorylation by DNA-PK and, by using kinase assays, we confirmed that Tat is an effective substrate of DNA-PK. Through peptide mapping, we found that two of these three potential phosphorylation sites are recognized and phosphorylated by DNA-PK. Mutational studies on the DNA-PK target sites of Tat further demonstrated the functional significance of the Tat-DNA-PK interaction. Thus, overall our results clearly demonstrate the functional interaction between DNA-PK and RNAP II during HIV transcription. PMID:21450944

  3. Axodendritic contacts onto calcium/calmodulin-dependent protein kinase type II-expressing neurons in the barn owl auditory space map.

    PubMed

    Rodriguez-Contreras, Adrian; Liu, Xiao-Bo; DeBello, William M

    2005-06-01

    In the owl midbrain, a map of auditory space is synthesized in the inferior colliculus (IC) and conveyed to the optic tectum (OT). Ascending auditory information courses through these structures via topographic axonal projections. Little is known about the molecular composition of projection neurons or their postsynaptic targets. To visualize axodendritic contacts between identified cell types, we used double-label immunohistochemistry, in vivo retrograde tracing, in vitro anterograde tracing, high-resolution confocal microscopy, three-dimensional reconstruction and fly-through visualization. We discovered a major class of IC neurons that strongly expressed calcium/calmodulin-dependent protein kinase type II, alpha subunit (CaMKII). The distribution of these cells within the IC was mostly restricted to the external nucleus of the IC (ICX), in which the auditory space map is assembled. A large proportion of ICX-OT projection neurons were CaMKII positive. In addition to being the principal outputs, CaMKII cells were in direct contact with axonal boutons emanating from the main source of input to ICX, the lateral shell of the central nucleus of the inferior colliculus (ICCls). Numerous sites of putative synaptic contact were found on the somata, proximal dendrites, and distal dendrites. Double-label immunoelectron microscopy confirmed the existence of synapses between ICCls axons and the dendrites of CaMKII cells. Collectively, our data indicate that CaMKII ICX neurons are a cellular locus for the computation of auditory space-specific responses. Because the ICCls-ICX projection is physically altered during experience-dependent plasticity, these results lay the groundwork for probing microanatomical rearrangements that may underlie plasticity and learning. PMID:15944389

  4. Central Rho kinase inhibition restores baroreflex sensitivity and angiotensin II type 1 receptor protein imbalance in conscious rabbits with chronic heart failure.

    PubMed

    Haack, Karla K V; Gao, Lie; Schiller, Alicia M; Curry, Pamela L; Pellegrino, Peter R; Zucker, Irving H

    2013-03-01

    The small GTPase RhoA and its associated kinase ROCKII are involved in vascular smooth muscle cell contraction and endothelial NO synthase mRNA destabilization. Overactivation of the RhoA/ROCKII pathway is implicated in several pathologies, including chronic heart failure (CHF), and may contribute to the enhanced sympathetic outflow seen in CHF as a result of decreased NO availability. Thus, we hypothesized that central ROCKII blockade would improve the sympathovagal imbalance in a pacing rabbit model of CHF in an NO-dependent manner. CHF was induced by rapid ventricular pacing and characterized by an ejection fraction of ≤45%. Animals were implanted with an intracerbroventricular cannula and osmotic minipump (rate, 1 μL/h) containing sterile saline, 1.5 µg/kg per day fasudil (Fas, a ROCKII inhibitor) for 4 days or Fas+100 µg/kg per day Nω-Nitro-l-arginine methyl ester hydrochloride, a NO synthase inhibitor. Arterial baroreflex control was assessed by intravenous infusion of sodium nitroprusside and phenylephrine. Fas infusion significantly lowered resting heart rate by decreasing sympathetic and increasing vagal tone. Furthermore, Fas improved baroreflex gain in CHF in an NO-dependent manner. In CHF Fas animals, the decrease in heart rate in response to intravenous metoprolol was similar to Sham and was reversed by Nω-Nitro-l-arginine methyl ester hydrochloride. Fas decreased angiotensin II type 1 receptor and phospho-ERM protein expression and increased endothelial NO synthase expression in the brain stem of CHF animals. These data strongly suggest that central ROCKII activation contributes to cardiac sympathoexcitation in the setting of CHF and that central Fas restores vagal and sympathetic tone in an NO-dependent manner. ROCKII may be a new central therapeutic target in the setting of CHF. PMID:23283363

  5. cGMP-Dependent Protein Kinase Type II Knockout Mice Exhibit Working Memory Impairments, Decreased Repetitive Behavior, and Increased Anxiety-like Traits

    PubMed Central

    Wincott, Charlotte M.; Abera, Sinedu; Vunck, Sarah A.; Choi, Yoon; Titcombe, Roseann F.; Antoine, Shannon O.; Tukey, David S.; DeVito, Loren M.; Hofmann, Franz; Hoeffer, Charles A.; Ziff, Edward B.

    2015-01-01

    Neuronal activity regulates AMPA receptor trafficking, a process that mediates changes in synaptic strength, a key component of learning and memory. This form of plasticity may be induced by stimulation of the NMDA receptor which, among its activities, increases cyclic guanosine monophosphate through the nitric oxide synthase pathway. cGMP-dependent protein kinase type II (cGKII) is ultimately activated via this mechanism and AMPA receptor subunit GluA1 is phosphorylated at serine 845. This phosphorylation contributes to the delivery of GluA1 to the synapse, a step that increases synaptic strength. Previous studies have shown that cGKII-deficient mice display striking spatial learning deficits in the Morris Water Maze compared to wild-type littermates as well as lowered GluA1 phosphorylation in the postsynaptic density of the prefrontal cortex (Serulle, Zhang, Ninan, Puzzo, McCarthy, Khatri, Arancio, and Ziff, 2007; Wincott, Kim, Titcombe, Tukey, Girma, Pick, Devito, Hofmann, Hoeffer, and Ziff, 2013). In the current study, we show that cGKII knockout mice exhibit impaired working memory as determined using the prefrontal cortex-dependent Radial Arm Maze (RAM). Additionally, we report reduced repetitive behavior in the Marble Burying task (MB), and heightened anxiety-like traits in the Novelty Suppressed Feeding Test (NSFT). These data suggest that cGKII may play a role in the integration of information that conveys both anxiety-provoking stimuli as well as the spatial and environmental cues that facilitate functional memory processes and appropriate behavioral response. PMID:24752151

  6. Transient Overexpression of α-Ca2+/Calmodulin-Dependent Protein Kinase II in the Nucleus Accumbens Shell Enhances Behavioral Responding to Amphetamine

    PubMed Central

    Loweth, Jessica A.; Singer, Bryan F.; Baker, Lorinda K.; Wilke, Georgia; Inamine, Hidetoshi; Bubula, Nancy; Alexander, John K.; Carlezon, William A.; Neve, Rachael L.; Vezina, Paul

    2010-01-01

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to contribute to the expression of psychostimulant sensitization by regulating dopamine (DA) overflow from DA neuron terminals in the nucleus accumbens (NAcc). The present experiments explored the contribution of CaMKII in NAcc neurons postsynaptic to these terminals where it is known to participate in a number of signaling pathways that regulate responding to psychostimulant drugs. Exposure to amphetamine transiently increased αCaMKII levels in the shell but not the core of the NAcc. Thus, HSV (herpes simplex viral) vectors were used to transiently overexpress αCaMKII in NAcc neurons in drug-naive rats, and behavioral responding to amphetamine was assessed. Transiently overexpressing αCaMKII in the NAcc shell led to long-lasting enhancement of amphetamine-induced locomotion and self-administration manifested when αCaMKII levels were elevated and persisting long after they had returned to baseline. Enhanced locomotion was not observed after infection in the NAcc core or sites adjacent to the NAcc. Transient elevation of NAcc shell αCaMKII levels also enhanced locomotor responding to NAcc AMPA and increased phosphorylation levels of GluR1 (Ser831), a CaMKII site, both soon and long after infection. Similar increases in pGluR1 (Ser831) were observed both soon and long after exposure to amphetamine. These results indicate that the transient increase in αCaMKII observed in neurons of the NAcc shell after viral-mediated gene transfer and likely exposure to amphetamine leads to neuroadaptations in AMPA receptor signaling in this site that may contribute to the long-lasting maintenance of behavioral and incentive sensitization by psychostimulant drugs like amphetamine. PMID:20089902

  7. Regulation of Voltage-Gated Ca2+ Currents by Ca2+/Calmodulin-dependent Protein Kinase II in Resting Sensory Neurons

    PubMed Central

    Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-En; Hogan, Quinn H.

    2014-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca2+ channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca2+ currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced depolarization-induced ICa by 16 – 30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent. PMID:25064143

  8. Regulation of voltage-gated Ca(2+) currents by Ca(2+)/calmodulin-dependent protein kinase II in resting sensory neurons.

    PubMed

    Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-En; Hogan, Quinn H

    2014-09-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca(2+) channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca(2+) currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced depolarization-induced ICa by 16-30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent. PMID:25064143

  9. 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

  10. Lack of protein kinase C-α leads to impaired urine concentrating ability and decreased aquaporin-2 in angiotensin II-induced hypertension.

    PubMed

    Thai, Tiffany L; Blount, Mitsi A; Klein, Janet D; Sands, Jeff M

    2012-07-01

    Regulation of water and urea transport in the inner medullary collecting duct is essential for urine concentration. Aquaporin (AQP)2 water channels and urea transporter (UT)-A1 are inserted into the apical membrane upon phosphorylation of the channels to allow the transcellular movement of water and urea. Since ANG II activates PKC in many cell types, we tested the hypothesis that ANG II-induced regulation of water and urea transport is mediated by PKC. Osmotic minipumps delivered ANG II to wild-type (WT) or PKC-α(-/-) mice for 7 days. Inner medullas were harvested, and protein abundance was determined by immunoblot. ANG II increased systolic blood pressure to a similar degree in WT and PKC-α(-/-) mice. ANG II had no effect on the urine output of WT mice but increased that of PKC-α(-/-) mice. In accordance with observed differences in urine output, AQP2 abundance was unchanged in ANG II-treated WT animals but was decreased in PKC-α(-/-) mice. No change in membrane accumulation was seen. Phosphorylation of the cAMP-induced transcription factor CREB was decreased in PKC-α(-/-) mice in response to ANG II with no change in overall CREB abundance. ANG II did not alter the abundance of UT-A1 protein in WT or PKC-α(-/-) mice. Phosphorylation and overall abundance of tonicity-responsive enhancer-binding protein, a transcription factor that regulates UT-A1, were also unaltered by ANG II in either group. We conclude that PKC-α protects against ANG II-induced decreases in urine concentrating ability by maintaining AQP2 levels through CREB phosphorylation. PMID:22492943

  11. Metabolic Control of Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Caspase-2 Suppression by the B55β/Protein Phosphatase 2A (PP2A)*

    PubMed Central

    Huang, Bofu; Yang, Chih-Sheng; Wojton, Jeffrey; Huang, Nai-Jia; Chen, Chen; Soderblom, Erik J.; Zhang, Liguo; Kornbluth, Sally

    2014-01-01

    High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca2+/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr393/Ser395) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55β targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway. PMID:25378403

  12. Intracellular translocation of calmodulin and Ca2+/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes

    PubMed Central

    Gangopadhyay, Jaya Pal; Ikemoto, Noriaki

    2010-01-01

    We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca2+ leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes [Hamada et al., 2009]. The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in the nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca2+/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca2+ transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca2+ transients results in the appearance of trains of Ca2+ spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca2+ events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional sites to activate HT program. PMID

  13. Intracellular translocation of calmodulin and Ca{sup 2+}/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes

    SciTech Connect

    Gangopadhyay, Jaya Pal; Ikemoto, Noriaki; Department of Neurology, Harvard Medical School, Boston, MA 02115

    2010-05-28

    We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca{sup 2+} leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 ). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in the nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca{sup 2+}/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca{sup 2+} transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca{sup 2+} transients results in the appearance of trains of Ca{sup 2+} spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca{sup 2+} events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional

  14. Regulation of Axonal Transport by Protein Kinases.

    PubMed

    Gibbs, Katherine L; Greensmith, Linda; Schiavo, Giampietro

    2015-10-01

    The intracellular transport of organelles, proteins, lipids, and RNA along the axon is essential for neuronal function and survival. This process, called axonal transport, is mediated by two classes of ATP-dependent motors, kinesins, and cytoplasmic dynein, which carry their cargoes along microtubule tracks. Protein kinases regulate axonal transport through direct phosphorylation of motors, adapter proteins, and cargoes, and indirectly through modification of the microtubule network. The misregulation of axonal transport by protein kinases has been implicated in the pathogenesis of several nervous system disorders. Here, we review the role of protein kinases acting directly on axonal transport and discuss how their deregulation affects neuronal function, paving the way for the exploitation of these enzymes as novel drug targets. PMID:26410600

  15. 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.

  16. Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

    PubMed

    Antunes, Tayze T; Callera, Glaucia E; He, Ying; Yogi, Alvaro; Ryazanov, Alexey G; Ryazanova, Lillia V; Zhai, Alexander; Stewart, Duncan J; Shrier, Alvin; Touyz, Rhian M

    2016-04-01

    Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (P<0.05). Acetylcholine-induced vasorelaxation was reduced in Ang II-infused TRPM7Δkinase mice, an effect associated with Akt and endothelial nitric oxide synthase downregulation. Vascular cell adhesion molecule-1 expression was increased in Ang II-infused TRPM7 kinase-deficient mice. TRPM7 kinase targets, calpain, and annexin-1, were activated by Ang II in WT but not in TRPM7Δkinase mice. Echocardiographic and histopathologic analysis demonstrated cardiac hypertrophy and left ventricular dysfunction in Ang II-treated groups. In TRPM7 kinase-deficient mice, Ang II-induced cardiac functional and structural effects were amplified compared with WT counterparts. Our data demonstrate that in TRPM7Δkinase mice, Ang II-induced hypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension

  17. Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region.

    PubMed

    Lei, J; Tang, X; Chambers, T C; Pohl, J; Benian, G M

    1994-08-19

    Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated. PMID:8063727

  18. 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

  19. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  20. A Complex Containing RNA Polymerase II, Paf1p, Cdc73p, Hpr1p, and Ccr4p Plays a Role in Protein Kinase C Signaling

    PubMed Central

    Chang, Meiping; French-Cornay, Delores; Fan, Hua-ying; Klein, Hannah; Denis, Clyde L.; Jaehning, Judith A.

    1999-01-01

    Yeast contains at least two complex forms of RNA polymerase II (Pol II), one including the Srbps and a second biochemically distinct form defined by the presence of Paf1p and Cdc73p (X. Shi et al., Mol. Cell. Biol. 17:1160–1169, 1997). In this work we demonstrate that Ccr4p and Hpr1p are components of the Paf1p-Cdc73p-Pol II complex. We have found many synthetic genetic interactions between factors within the Paf1p-Cdc73p complex, including the lethality of paf1Δ ccr4Δ, paf1Δ hpr1Δ, ccr4Δ hpr1Δ, and ccr4Δ gal11Δ double mutants. In addition, paf1Δ and ccr4Δ are lethal in combination with srb5Δ, indicating that the factors within and between the two RNA polymerase II complexes have overlapping essential functions. We have used differential display to identify several genes whose expression is affected by mutations in components of the Paf1p-Cdc73p-Pol II complex. Additionally, as previously observed for hpr1Δ, deleting PAF1 or CDC73 leads to elevated recombination between direct repeats. The paf1Δ and ccr4Δ mutations, as well as gal11Δ, demonstrate sensitivity to cell wall-damaging agents, rescue of the temperature-sensitive phenotype by sorbitol, and reduced expression of genes involved in cell wall biosynthesis. This unusual combination of effects on recombination and cell wall integrity has also been observed for mutations in genes in the Pkc1p-Mpk1p kinase cascade. Consistent with a role for this novel form of RNA polymerase II in the Pkc1p-Mpk1p signaling pathway, we find that paf1Δ mpk1Δ and paf1Δ pkc1Δ double mutants do not demonstrate an enhanced phenotype relative to the single mutants. Our observation that the Mpk1p kinase is fully active in a paf1Δ strain indicates that the Paf1p-Cdc73p complex may function downstream of the Pkc1p-Mpk1p cascade to regulate the expression of a subset of yeast genes. PMID:9891041

  1. [Mitogen-activated protein kinases in atherosclerosis].

    PubMed

    Bryk, Dorota; Olejarz, Wioletta; Zapolska-Downar, Danuta

    2014-01-01

    Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases) intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase), JNK (c-Jun N-terminal kinase) and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis. PMID:24491891

  2. Inhibition of endogenous heat shock protein 70 attenuates inducible nitric oxide synthase induction via disruption of heat shock protein 70/Na(+) /H(+) exchanger 1-Ca(2+) -calcium-calmodulin-dependent protein kinase II/transforming growth factor β-activated kinase 1-nuclear factor-κB signals in BV-2 microglia.

    PubMed

    Huang, Chao; Lu, Xu; Wang, Jia; Tong, Lijuan; Jiang, Bo; Zhang, Wei

    2015-08-01

    Inducible nitric oxide synthase (iNOS) critically contributes to inflammation and host defense. The inhibition of heat shock protein 70 (Hsp70) prevents iNOS induction in lipopolysaccharide (LPS)-stimulated macrophages. However, the role and mechanism of endogenous Hsp70 in iNOS induction in microglia remains unclear. This study addresses this issue in BV-2 microglia, showing that Hsp70 inhibition or knockdown prevents LPS-induced iNOS protein expression and nitric oxide production. Real-time PCR experiments showed that LPS-induced iNOS mRNA transcription was blocked by Hsp70 inhibition. Further studies revealed that the inhibition of Hsp70 attenuated LPS-stimulated nuclear translocation and phosphorylation of nuclear factor (NF)-κB as well as the degradation of inhibitor of κB (IκB)-α and phosphorylation of IκB kinase β (IKKβ). This prevention effect of Hsp70 inhibition on IKKβ-NF-κB activation was found to be dependent on the Ca(2+) /calcium-calmodulin-dependent protein kinase II (CaMKII)/transforming growth factor β-activated kinase 1 (TAK1) signals based on the following observations: 1) chelation of intracellular Ca(2+) or inhibition of CaMKII reduced LPS-induced increases in TAK1 phosphorylation and 2) Hsp70 inhibition reduced LPS-induced increases in CaMKII/TAK1 phosphorylation, intracellular pH value, [Ca(2+) ]i , and CaMKII/TAK1 association. Mechanistic studies showed that Hsp70 inhibition disrupted the association between Hsp70 and Na(+) /H(+) exchanger 1 (NHE1), which is an important exchanger responsible for Ca(2+) influx in LPS-stimulated cells. These studies demonstrate that the inhibition of endogenous Hsp70 attenuates the induction of iNOS, which likely occurs through the disruption of NHE1/Hsp70-Ca(2+) -CaMKII/TAK1-NF-κB signals in BV-2 microglia, providing further insight into the functions of Hsp70 in the CNS. PMID:25691123

  3. 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

  4. Calcium/calmodulin-dependent protein kinase II regulates cyclooxygenase-2 expression and prostaglandin E2 production by activating cAMP-response element-binding protein in rat peritoneal macrophages

    PubMed Central

    Zhou, Xueyuan; Li, Junying; Yang, Wenxiu

    2014-01-01

    Prostaglandin E2 (PGE2) is an important inducer of inflammation, which is also closely linked to the progress of tumours. In macrophages, PGE2 production is regulated by arachidonic acid release and cyclooxygenase-2 (COX-2) expression. In the present study, we found that COX-2 expression can be achieved by activating Ca2+/Calmodulin (CaM)-dependent protein kinase II (CaMKII) and cAMP-response element-binding protein (CREB) in rat peritoneal macrophages. Our results indicated that lipopolysaccharide and PMA could elicit the transient increase of the concentration of intracellular free calcium ions ([Ca2+]i), which induced activation of CaMKs with the presence of CaM. The subtype of CaMKs, CaMKII, then triggered the activation of CREB, which elevated COX-2 expression and PGE2 production in a chronological order. These results suggested that Ca2+/CaM-dependent CaMKII plays an important role in mediating COX-2 expression and PGE2 production by activating CREB in macrophages. The study also provides more useful information to clarify the mechanism of calcium regulation of PGE2 production, which plays an essential role in inflammation and cancers. PMID:24773364

  5. Rapid activation of hippocampal casein kinase II during long-term potentiation.

    PubMed Central

    Charriaut-Marlangue, C; Otani, S; Creuzet, C; Ben-Ari, Y; Loeb, J

    1991-01-01

    Several studies suggest that protein kinase C and type II Ca2+/calmodulin-dependent protein kinase are activated during induction of long-term potentiation (LTP). We now report that casein kinase II (CK-II), which is present in high concentration in the hippocampus, is also activated in the CA1 region during LTP. CK-II activity increased within 2 min after a train of high-frequency electrical stimulations and reached a maximum (2-fold increase) 5 min later before returning to baseline value. The stimulated protein kinase activity, which was blocked by a selective antagonist of N-methyl-D-aspartate receptors, exhibited specific properties of CK-II, including phosphorylation of the specific substrates of CK-II, marked inhibition by a low heparin concentration, and the use of GTP as a phosphate donor. CK-II activity was also selectively and rapidly augmented in another form of LTP produced by bath application of tetraethylammonium; this LTP (called LTPk) is Ca2+ dependent but N-methyl-D-aspartate independent. Phosphorylation of casein that was not inhibited by heparin (i.e., casein kinase I) remained unchanged. We suggest that an increase in CK-II activity is important in LTP induction. Images PMID:1946443

  6. Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II[S

    PubMed Central

    Cingolani, Francesca; Casasampere, Mireia; Sanllehí, Pol; Casas, Josefina; Bujons, Jordi; Fabrias, Gemma

    2014-01-01

    Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine-1-phosphate (S1P) in cellular processes. Dihydroceramide desaturase (Des1), the last enzyme in the de novo synthesis of ceramide (Cer), regulates the balance between dihydroceramides (dhCers) and Cers. Both SKs and Des1 have interest as therapeutic targets. Here we show that SKI II is a noncompetitive inhibitor (Ki = 0.3 μM) of Des1 activity with effect also in intact cells without modifying Des1 protein levels. Molecular modeling studies support that the SKI II-induced decrease in Des1 activity could result from inhibition of NADH-cytochrome b5 reductase. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF543, reduced cell proliferation with accumulation of cells in the G0/G1 phase. SKI II, but not PF543, induced autophagy. These overall findings should be taken into account when using SKI II as a pharmacological tool, as some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites. PMID:24875537

  7. 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.

  8. 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

  9. 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

  10. 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"…

  11. The protein interaction landscape of the human CMGC kinase group.

    PubMed

    Varjosalo, Markku; Keskitalo, Salla; Van Drogen, Audrey; Nurkkala, Helka; Vichalkovski, Anton; Aebersold, Ruedi; Gstaiger, Matthias

    2013-04-25

    Cellular information processing via reversible protein phosphorylation requires tight control of the localization, activity, and substrate specificity of protein kinases, which to a large extent is accomplished by complex formation with other proteins. Despite their critical role in cellular regulation and pathogenesis, protein interaction information is available for only a subset of the 518 human protein kinases. Here we present a global proteomic analysis of complexes of the human CMGC kinase group. In addition to subgroup-specific functional enrichment and modularity, the identified 652 high-confidence kinase-protein interactions provide a specific biochemical context for many poorly studied CMGC kinases. Furthermore, the analysis revealed a kinase-kinase subnetwork and candidate substrates for CMGC kinases. Finally, the presented interaction proteome uncovered a large set of interactions with proteins genetically linked to a range of human diseases, including cancer, suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways. PMID:23602568

  12. Casein kinase II stimulates rat liver mitochondrial glycerophosphate acyltransferase activity.

    PubMed

    Onorato, Thomas M; Haldar, Dipak

    2002-09-01

    Rat liver mitochondrial glycerophosphate acyltransferase (mtGAT) possesses 14 consensus sites for casein kinase II (CKII) phosphorylation. To study the functional relevance of phosphorylation to the activity of mtGAT, we treated isolated rat liver mitochondria with CKII and found that CKII stimulated mtGAT activity approximately 2-fold. Protein phosphatase-lambda treatment reversed the stimulation of mtGAT by CKII. Labeling of both solubilized and non-solubilized mitochondria with CKII and [gamma-32P]ATP resulted in a 32P-labeled protein of 85kDa, the molecular weight of mtGAT. Our findings suggest that CKII stimulates mtGAT activity by phosphorylation of the acyltransferase. The significance of this observation with respect to hormonal control of the enzyme is discussed. PMID:12207885

  13. 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

  14. SUMOylation regulates the SNF1 protein kinase

    PubMed Central

    Simpson-Lavy, Kobi J.; Johnston, Mark

    2013-01-01

    The AMP-activated protein kinase (AMPK) is a major stress sensor of mammalian cells. AMPK’s homolog in the yeast Saccharomyces cerevisiae, the SNF1 protein kinase, is a central regulator of carbon metabolism that inhibits the Snf3/Rgt2-Rgt1 glucose sensing pathway and activates genes involved in respiration. We present evidence that glucose induces modification of the Snf1 catalytic subunt of SNF1 with the small ubiquitin-like modifier protein SUMO, catalyzed by the SUMO (E3) ligase Mms21. Our results suggest that SUMOylation of Snf1 inhibits its function in two ways: by interaction of SUMO attached to lysine 549 with a SUMO-interacting sequence motif located near the active site of Snf1, and by targeting Snf1 for destruction via the Slx5-Slx8 (SUMO-directed) ubiquitin ligase. These findings reveal another way SNF1 function is regulated in response to carbon source. PMID:24108357

  15. Protein kinases as drug targets in cancer.

    PubMed

    Arslan, Mehmet Alper; Kutuk, Ozgur; Basaga, Huveyda

    2006-11-01

    Identification of the key roles of protein kinases in signaling pathways leading to development of cancer has caused pharmacological interest to concentrate extensively on targeted therapies as a more specific and effective way for blockade of cancer progression. This review will mainly focus on inhibitors targeting these key components of cellular signaling by employing a technology-based point of view with respect to ATP- and non-ATP-competitive small molecule inhibitors and monoclonal antibodies of selected protein kinases, particularly, mammalian target of rapamycin (mTOR), BCR-ABL, MEK, p38 MAPK, EGFR PDGFR, VEGFR, HER2 and Raf. Inhibitors of the heat shock protein Hsp90 are also included in a separate section, as this protein plays an essential role for the maturation/proper activation of cancer-related protein kinases. In the following review, the molecular details of the mode of action of these inhibitors as well as the emergence of drug resistance encountered in several cases are discussed in light of the structural, molecular and clinical studies conducted so far. PMID:17100568

  16. Following a protein kinase activity using a field-effect transistor device.

    PubMed

    Freeman, Ronit; Gill, Ron; Willner, Itamar

    2007-09-01

    The specific phosphorylation of a peptide-functionalized ion-sensitive field-effect transistor device by casein kinase II in the presence of ATP enables the electronic readout of the protein kinase activity; treatment of the phosphorylated surface with alkaline phosphatase results in the regeneration of the active sensing surface. PMID:17700878

  17. Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr.

    PubMed Central

    Sahyoun, N; McDonald, O B; Farrell, F; Lapetina, E G

    1991-01-01

    A neuron-specific Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-1b) that is enriched in brain tissue. The phosphorylation reaction achieves a stoichiometry of about 1 and involves a serine residue near the carboxyl terminus of the substrate. Both CaM kinase Gr and cAMP-dependent protein kinase, but not CaM kinase II, phosphorylate identical or contiguous serine residues in Rap-1b. The rate of phosphorylation of Rap-1b by CaM kinase Gr is enhanced following autophosphorylation of the protein kinase. Other low molecular weight GTP-binding proteins belonging to the Ras superfamily, including Rab-3A, Rap-2b, and c-Ha-ras p21, are not phosphorylated by CaM kinase Gr. The phosphorylation of Rap-1b itself can be reversed by an endogenous brain phosphoprotein phosphatase. These observations provide a potential connection between a neuronal Ca2(+)-signaling pathway and a specific low molecular weight GTP-binding protein that may regulate neuronal transmembrane signaling, vesicle transport, or neurotransmitter release. Images PMID:1901412

  18. 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.

  19. Identification of four plastid-localized protein kinases.

    PubMed

    Richter, Andreas S; Gartmann, Hans; Fechler, Mona; Rödiger, Anja; Baginsky, Sacha; Grimm, Bernhard

    2016-06-01

    In chloroplasts, protein phosphorylation regulates important processes, including metabolism, photosynthesis, gene expression, and signaling. Because the hitherto known plastid protein kinases represent only a fraction of existing kinases, we aimed at the identification of novel plastid-localized protein kinases that potentially phosphorylate enzymes of the tetrapyrrole biosynthesis (TBS) pathway. We screened publicly available databases for proteins annotated as putative protein kinase family proteins with predicted chloroplast localization. Additionally, we analyzed chloroplast fractions which were separated by sucrose density gradient centrifugation by mass spectrometry. We identified four new candidates for protein kinases, which were confirmed to be plastid localized by expression of GFP-fusion proteins in tobacco leaves. A phosphorylation assay with the purified kinases confirmed the protein kinase activity for two of them. PMID:27214872

  20. Pro-survival effects of 17β-estradiol on osteocytes are mediated by nitric oxide/cGMP via differential actions of cGMP-dependent protein kinases I and II.

    PubMed

    Marathe, Nisha; Rangaswami, Hema; Zhuang, Shunhui; Boss, Gerry R; Pilz, Renate B

    2012-01-01

    Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD. PMID:22117068

  1. Characterization of nuclear protein kinases of Xenopus laevis oocytes

    SciTech Connect

    Leiva, L.; Gonzalez, C.; Allende, C.; Allende, J.

    1986-05-01

    Xenopus laevis oocytes contain large nuclei (germinal vesicles) that can be isolated in very pure form and which permit the study of enzymatic activities present in these organelles. Incubation of pure oocyte nuclear homogenates with /sup 32/P in a buffered solution containing 5 mM MgCl/sub 2/ results in the phosphorylation of a large number of proteins by endogenous protein kinases. This phosphorylation is not affected by the addition of cyclic nucleotides or calcium ion and calmodulin. On the other hand the nuclear kinases are considerably stimulated by spermine and spermidine and strongly inhibited by heparin (10 ..mu..g/ml). Addition of exogenous protein substrates shows that the major oocyte kinases are very active with casein and phosvitin as substrates but do not phosphorylate histones or protamines. DEAE-Sephadex chromatography of the nuclear extract fractionates the casein phosphorylating activity in two main peaks. The first peak is not retained on the column equilibrated with 0.1 M NH/sub 2/SO/sub 4/ and uses exclusively ATP as phosphate donor and is insensitive to polyamines or heparin. The second peak which corresponds to 70% of the casein phosphorylation elutes at 0.27 M NH/sub 2/SO/sub 4/ and uses both ATP and GTP as phosphate donors and is greatly stimulated by polyamines and completely inhibited by 10 ..mu..g/ml heparin. On this evidence the authors conclude that the major protein kinase peak corresponds to casein kinase type II which has been found in mammalian nuclei.

  2. 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. PMID:27151216

  3. Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome

    PubMed Central

    Uhle, Stefan; Medalia, Ohad; Waldron, Richard; Dumdey, Renate; Henklein, Peter; Bech-Otschir, Dawadschargal; Huang, Xiaohua; Berse, Matthias; Sperling, Joseph; Schade, Rüdiger; Dubiel, Wolfgang

    2003-01-01

    The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co-purify with CSN. Immunoprecipi tation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immuno precipitates with CSN from HeLa cells. CK2 binds to ΔCSN3(111–403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun–Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation. PMID:12628923

  4. Osmotic stress signaling via protein kinases.

    PubMed

    Fujii, Hiroaki; Zhu, Jian-Kang

    2012-10-01

    Plants face various kinds of environmental stresses, including drought, salinity, and low temperature, which cause osmotic stress. An understanding of the plant signaling pathways that respond to osmotic stress is important for both basic biology and agriculture. In this review, we summarize recent investigations concerning the SNF1-related protein kinase (SnRK) 2 kinase family, which play central roles in osmotic stress responses. SnRK2s are activated by osmotic stress, and a mutant lacking SnRK2s is hypersensitive to osmotic stress. Many questions remain about the signaling pathway upstream and downstream of SnRK2s. Because some SnRK2s also functions in the abscisic acid (ABA) signaling pathway, which has recently been well clarified, study of SnRK2s in ABA signaling can provide clues regarding their roles in osmotic stress signaling. PMID:22828864

  5. 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

  6. Protein kinase A signalling in Schistosoma mansoni cercariae and schistosomules.

    PubMed

    Hirst, Natasha L; Lawton, Scott P; Walker, Anthony J

    2016-06-01

    Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A regulates multiple processes in eukaryotes by phosphorylating diverse cellular substrates, including metabolic and signalling enzymes, ion channels and transcription factors. Here we provide insight into protein kinase A signalling in cercariae and 24h in vitro cultured somules of the blood parasite, Schistosoma mansoni, which causes human intestinal schistosomiasis. Functional mapping of activated protein kinase A using anti-phospho protein kinase A antibodies and confocal laser scanning microscopy revealed activated protein kinase A in the central and peripheral nervous system, oral-tip sensory papillae, oesophagus and excretory system of intact cercariae. Cultured 24h somules, which biologically represent the skin-resident stage of the parasite, exhibited similar activation patterns in oesophageal and nerve tissues but also displayed striking activation at the tegument and activation in a region resembling the germinal 'stem' cell cluster. The adenylyl cyclase activator, forskolin, stimulated somule protein kinase A activation and produced a hyperkinesia phenotype. The biogenic amines, serotonin and dopamine known to be present in skin also induced protein kinase A activation in somules, whereas neuropeptide Y or [Leu(31),Pro(34)]-neuropeptide Y attenuated protein kinase A activation. However, neuropeptide Y did not block the forskolin-induced somule hyperkinesia. Bioinformatic investigation of potential protein associations revealed 193 medium confidence and 59 high confidence protein kinase A interacting partners in S. mansoni, many of which possess putative protein kinase A phosphorylation sites. These data provide valuable insight into the intricacies of protein kinase A signalling in S. mansoni and a framework for further physiological investigations into the roles of protein kinase A in schistosomes, particularly in the context of interactions between the parasite and the host. PMID:26777870

  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. Structure-Based Design of Type II Inhibitors Applied to Maternal Embryonic Leucine Zipper Kinase.

    PubMed

    Johnson, Christopher N; Adelinet, Christophe; Berdini, Valerio; Beke, Lijs; Bonnet, Pascal; Brehmer, Dirk; Calo, Frederick; Coyle, Joseph E; Day, Phillip J; Frederickson, Martyn; Freyne, Eddy J E; Gilissen, Ron A H J; Hamlett, Christopher C F; Howard, Steven; Meerpoel, Lieven; Mevellec, Laurence; McMenamin, Rachel; Pasquier, Elisabeth; Patel, Sahil; Rees, David C; Linders, Joannes T M

    2015-01-01

    A novel Type II kinase inhibitor chemotype has been identified for maternal embryonic leucine zipper kinase (MELK) using structure-based ligand design. The strategy involved structural characterization of an induced DFG-out pocket by protein-ligand X-ray crystallography and incorporation of a slender linkage capable of bypassing a large gate-keeper residue, thus enabling design of molecules accessing both hinge and induced pocket regions. Optimization of an initial hit led to the identification of a low-nanomolar, cell-penetrant Type II inhibitor suitable for use as a chemical probe for MELK. PMID:25589926

  9. 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. PMID:27056740

  10. Detection of protein kinase activity by renaturation in sodium dodecyl sulfate-polyacrylamide gels

    SciTech Connect

    Anostario, M. Jr.; Harrison, M.L.; Geahlen, R.L.

    1986-05-01

    The authors have developed a procedure for identifying protein kinase activity in protein samples following electrophoresis on SDS-polyacrylamide gels. Proteins are allowed to renature directly in the gel by removal of detergent. The gel is then incubated with (..gamma..-/sup 32/P)ATP to allow renatured protein kinases to autophosphorylate or to phosphorylate various substrates which can be incorporated into the gel. The positions of the radiolabeled proteins can then be detected by autoradiography. With this technique, using purified catalytic subunit of cAMP-dependent protein kinase, enzyme concentrations as low as 0.01 ..mu..g can be detected on gels containing 1.0 mg/ml casein. The procedure is also applicable for the determination of active subunits of multisubunit protein kinases. For example, when the two subunits of casein kinase II are separated by SDS-polyacrylamide gel electrophoresis and allowed to renature, only the larger ..cap alpha.. subunit shows activity. This procedure can also be used to detect and distinguish kinases present in heterogeneous mixtures. Starting with a particulate fraction from LSTRA, a murine T cell lymphoma, several distinct enzymes were detected, including a 30,000 Dalton protein with protein-tyrosine kinase activity. This same enzyme has also been detected in T lymphocytes and other T lymphoid cell lines.

  11. Cl- Channels in CF: Lack of Activation by Protein Kinase C and cAMP-Dependent Protein Kinase

    NASA Astrophysics Data System (ADS)

    Hwang, Tzyh-Chang; Lu, Luo; Zeitlin, Pamela L.; Gruenert, Dieter C.; Huganir, Richard; Guggino, William B.

    1989-06-01

    Secretory chloride channels can be activated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase in normal airway epithelial cells but not in cells from individuals with cystic fibrosis (CF). In excised, inside-out patches of apical membrane of normal human airway cells and airway cells from three patients with CF, the chloride channels exhibited a characteristic outwardly rectifying current-voltage relation and depolarization-induced activation. Channels from normal tissues were activated by both cAMP-dependent protein kinase and protein kinase C. However, chloride channels from CF patients could not be activated by either kinase. Thus, gating of normal epithelial chloride channels is regulated by both cAMP-dependent protein kinase and protein kinase C, and regulation by both kinases is defective in CF.

  12. Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B

    PubMed Central

    Jiang, Bing-Hua; Aoki, Masahiro; Zheng, Jenny Z.; Li, Jian; Vogt, Peter K.

    1999-01-01

    The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase), strongly activates myogenic differentiation. Inhibition of endogenous PI 3-kinase activity with the specific inhibitor LY294002, or with dominant-negative mutants of PI 3-kinase, interferes with myotube formation and with the expression of muscle-specific proteins. Here we demonstrate that a downstream target of PI 3-kinase, serine-threonine kinase Akt, plays an important role in myogenic differentiation. Expression of constitutively active forms of Akt dramatically enhances myotube formation and expression of the muscle-specific proteins MyoD, creatine kinase, myosin heavy chain, and desmin. Transdominant negative forms of Akt inhibit myotube formation and the expression of muscle-specific proteins. The inhibition of myotube formation and the reduced expression of muscle-specific proteins caused by the PI 3-kinase inhibitor LY294002 are completely reversed by constitutively active forms of Akt. Wild-type cellular Akt effects a partial reversal of LY294002-induced inhibition of myogenic differentiation. This result suggests that Akt can substitute for PI 3-kinase in the stimulation of myogenesis; Akt may be an essential downstream component of PI 3-kinase-induced muscle differentiation. PMID:10051597

  13. Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle.

    PubMed

    Trappanese, Danielle M; Sivilich, Sarah; Ets, Hillevi K; Kako, Farah; Autieri, Michael V; Moreland, Robert S

    2016-06-01

    Vascular smooth muscle contraction is primarily regulated by phosphorylation of myosin light chain. There are also modulatory pathways that control the final level of force development. We tested the hypothesis that protein kinase C (PKC) and mitogen-activated protein (MAP) kinase modulate vascular smooth muscle activity via effects on MAP kinase phosphatase-1 (MKP-1). Swine carotid arteries were mounted for isometric force recording and subjected to histamine stimulation in the presence and absence of inhibitors of PKC [bisindolylmaleimide-1 (Bis)], MAP kinase kinase (MEK) (U0126), and MKP-1 (sanguinarine) and flash frozen for measurement of MAP kinase, PKC-potentiated myosin phosphatase inhibitor 17 (CPI-17), and caldesmon phosphorylation levels. CPI-17 was phosphorylated in response to histamine and was inhibited in the presence of Bis. Caldesmon phosphorylation levels increased in response to histamine stimulation and were decreased in response to MEK inhibition but were not affected by the addition of Bis. Inhibition of PKC significantly increased p42 MAP kinase, but not p44 MAP kinase. Inhibition of MEK with U0126 inhibited both p42 and p44 MAP kinase activity. Inhibition of MKP-1 with sanguinarine blocked the Bis-dependent increase of MAP kinase activity. Sanguinarine alone increased MAP kinase activity due to its effects on MKP-1. Sanguinarine increased MKP-1 phosphorylation, which was inhibited by inhibition of MAP kinase. This suggests that MAP kinase has a negative feedback role in inhibiting MKP-1 activity. Therefore, PKC catalyzes MKP-1 phosphorylation, which is reversed by MAP kinase. Thus the fine tuning of vascular contraction is due to the concerted effort of PKC, MAP kinase, and MKP-1. PMID:27053523

  14. Identification of Protein Kinase Substrates by the Kinase-Interacting Substrate Screening (KISS) Approach.

    PubMed

    Amano, Mutsuki; Nishioka, Tomoki; Yura, Yoshimitsu; Kaibuchi, Kozo

    2016-01-01

    Identifying the substrates of protein kinases to understand their modes of action has been undertaken by various approaches and remains an ongoing challenge. Phosphoproteomic technologies have accelerated the accumulation of data concerning protein phosphorylation and have uncovered vast numbers of phosphorylation sites in vivo. In this unit, a novel in vitro screening approach for protein kinase substrates is presented, based on protein-protein interaction and mass spectrometry-based phosphoproteomic technology. © 2016 by John Wiley & Sons, Inc. PMID:27580705

  15. Protein kinases are potential targets to treat inflammatory bowel disease

    PubMed Central

    Yang, Lei; Yan, Yutao

    2014-01-01

    Protein kinases play a crucial role in the pathogenesis of inflammatory bowel disease (IBD), the two main forms of which are ulcerative colitis and Crohn’s disease. In this article, we will review the mechanisms of involvement of protein kinases in the pathogenesis of and intervention against IBD, in terms of their effects on genetics, microbiota, mucous layer and tight junction, and the potential of protein kinases as therapeutic targets against IBD. PMID:25374761

  16. 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

  17. 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…

  18. 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

  19. The selectivity of protein kinase inhibitors: a further update

    PubMed Central

    Bain, Jenny; Plater, Lorna; Elliott, Matt; Shpiro, Natalia; Hastie, C. James; Mclauchlan, Hilary; Klevernic, Iva; Arthur, J. Simon C.; Alessi, Dario R.; Cohen, Philip

    2007-01-01

    The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70–80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)–raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes. PMID:17850214

  20. Phosphorylation of the insect immunophilin FKBP46 by the Spodoptera frugiperda homolog of casein kinase II.

    PubMed

    Steplewski, A; Ebel, W; Planey, S L; Alnemri, E S; Robertson, N M; Litwack, G

    2000-04-01

    Immunophilins are a family of conserved proteins found in both prokaryotes and eukaryotes, that exhibit peptidylprolyl isomerase (PPIase) activity. Members of this family bind to immunosuppressive drugs and on this basis are divided into two classes: FKBPs bind to FK506 and rapamycin, while cyclophilins bind to cyclosporin A. In this paper, we report on insect immunophilin FKBP46 and its associated kinase. The insect FKBP46 belongs to the high-molecular-weight immunophilins and shares many characteristic features with its mammalian counterparts, but its functional role remains unclear. Here, we show that FKBP46 is phosphorylated by a protein kinase present in the nucleus of both insect Spodoptera frugiperda (Sf9) and human Jurkat cells. This protein kinase is immunoreactive with polyclonal antiserum raised against Drosophila melanogaster casein kinase II (CKII). We have cloned, overexpressed and characterized a new member of the CKII family derived from Spodoptera frugiperda cells. Recombinant Sf9 CKII alpha subunit shares 75% identity to human, chicken and Drosophila melanogaster homologs, whereas the Sf9 CKII beta subunit is 77% identical to rat, chicken and human. Moreover, we demonstrate that the insect immunophilin FKBP46 can be phosphorylated by human and Sf9 casein kinase II. Finally, we show that FKBP46 interacts with DNA, and this interaction is not prevented by phosphorylation. PMID:10767538

  1. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network.

    PubMed Central

    Alconada, A; Bauer, U; Hoflack, B

    1996-01-01

    We have studied the intracellular trafficking of the envelope glycoprotein I (gpI) of the varicella-zoster virus, a human herpes virus whose assembly is believed to occur in the trans-Golgi network (TGN) and/or in endocytic compartments. When expressed in HeLa cells in the absence of additional virally encoded factors, this type-I membrane protein localizes to the TGN and cycles between this compartment and the cell surface. The expression of gpI promotes the recruitment of the AP-1 Golgi-specific assembly proteins onto TGN membranes, strongly suggesting that gpI, like the mannose 6-phosphate receptors, can leave the TGN in clathrin-coated vesicles for subsequent transport to endosomes. Its return from the cell surface to the TGN also occurs through endosomes. The transfer of the gpI cytoplasmic domain onto a reporter molecule shows that this domain is sufficient to confer TGN localization. Mutational analysis of this domain indicates that proper subcellular localization and cycling of gpI depend on two different determinants, a tyrosine-containing tetrapeptide related to endocytosis sorting signals and a cluster of acidic amino acids containing casein kinase II phosphorylatable residues. Thus, the VZV gpI and the mannose 6-phosphate receptors, albeit localized in different intracellular compartments at steady-state, follow similar trafficking pathways and share similar sorting mechanisms. Images PMID:8947032

  2. Electrogenerated Chemiluminescence Bioassay of Two Protein Kinases Incorporating Peptide Phosphorylation and Versatile Probe.

    PubMed

    Liu, Xia; Dong, Manman; Qi, Honglan; Gao, Qiang; Zhang, Chengxiao

    2016-09-01

    A sensitive electrogenerated chemiluminescence (ECL) bioassay was developed for the detection of two protein kinases incorporating the peptide phosphorylation and a versatile ECL probe. Cyclic adenosine monophosphate-dependent protein kinase (PKA) and casein kinase II (CK2) were used as proof-of-concept targets while a PKA-specific peptide (CLRRASLG) and a CK2-specific peptide (CRRRADDSDDDDD) were used as the recognition substrates. Taking advantage of the ability of protein A binding with the Fc region of a variety of antibodies with high affinity, a ruthenium derivative-labeled protein A was utilized as a versatile ECL probe for bioassay of multiple protein kinases. A specific peptide substrate toward target protein kinase was first self-assembled on the surface of gold electrode and then serine in the specific peptide on the electrode was phosphorylated by target protein kinase in the presence of adenosine-5'-triphosphate. After recognition of the phosphorylated peptide by monoclonal antiphosphoserine antibody, the versatile ECL probe was specifically bound to the antiphosphoserine antibody on the electrode surface. The ECL bioassay was developed successfully in the individual detection of PKA and CK2 with detection limit of 0.005 U/mL and 0.004 U/mL, respectively. In addition, the ECL bioassay was applied to quantitative analysis of the kinase inhibitors and monitoring drug-triggered kinase activation in cell lysates. Moreover, an ECL imaging bioassay using electron-multiplying charged coupled device as detector on the gold electrode array was developed for the simultaneous detection of PKA and CK2 activity from 0.01 U/mL to 0.4 U/mL, respectively, at one time. This work demonstrates that the ingenious design and use of a versatile ECL probe are promising to simultaneous detection of multiple protein kinases and screening of kinase inhibitor. PMID:27518533

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. Protein Kinases: Emerging Therapeutic Targets in Chronic Lymphocytic Leukemia

    PubMed Central

    Balakrishnan, Kumudha; Gandhi, Varsha

    2014-01-01

    Introduction Although protein kinases are primary targets for inhibition in hematological malignancies, until recently their contribution to chronic lymphocytic leukemia (CLL) was poorly understood. Insights into B cell receptor (BCR) signaling and its role in regulating key cellular functions have shed light on candidate protein kinases that are aberrantly activated in CLL. In this regard, protein kinases are now considered as potential drug targets in CLL. Area covered This review has covered signaling pathways and associated protein kinases in CLL and the kinase inhibitors currently available in preclinical and clinical investigations. Individual protein kinases that are abnormally active in CLL and the functional consequences of their inhibition are discussed. Expert opinion A growing body of evidence suggests that protein kinases are druggable targets for patients with CLL. The emergence of novel and bio-available kinase inhibitors and their promising clinical activity in CLL underscore the oncogenic role of kinases in leukemogenesis. Further investigations directed towards their role as single agents or in combinations may provide insight into understanding the substantial role of kinase mediated signal transduction pathways and their inhibition in B- CLL. PMID:22409342

  5. Phosphorylation of Simian Cytomegalovirus Assembly Protein Precursor (pAPNG.5) and Proteinase Precursor (pAPNG1): Multiple Attachment Sites Identified, Including Two Adjacent Serines in a Casein Kinase II Consensus Sequence

    PubMed Central

    Plafker, Scott M.; Woods, Amina S.; Gibson, Wade

    1999-01-01

    The assembly protein precursor (pAP) of cytomegalovirus (CMV), and its homologs in other herpesviruses, functions at several key steps during the process of capsid formation. This protein, and the genetically related maturational proteinase, is distinguished from the other capsid proteins by posttranslational modifications, including phosphorylation. The objective of this study was to identify sites at which pAP is phosphorylated so that the functional significance of this modification and the enzyme(s) responsible for it can be determined. In the work reported here, we used peptide mapping, mass spectrometry, and site-directed mutagenesis to identify two sets of pAP phosphorylation sites. One is a casein kinase II (CKII) consensus sequence that contains two adjacent serines, both of which are phosphorylated. The other site(s) is in a different domain of the protein, is phosphorylated less frequently than the CKII site, does not require preceding CKII-site phosphorylation, and causes an electrophoretic mobility shift when phosphorylated. Transfection/expression assays for proteolytic activity showed no gross effect of CKII-site phosphorylation on the enzymatic activity of the proteinase or on the substrate behavior of pAP. Evidence is presented that both the CKII sites and the secondary sites are phosphorylated in virus-infected cells and plasmid-transfected cells, indicating that these modifications can be made by a cellular enzyme(s). Apparent compartmental differences in phosphorylation of the CKII-site (cytoplasmic) and secondary-site (nuclear) serines suggest the involvement of more that one enzyme in these modifications. PMID:10516011

  6. Conservation of structural fluctuations in homologous protein kinases and its implications on functional sites.

    PubMed

    Kalaivani, Raju; de Brevern, Alexandre G; Srinivasan, Narayanaswamy

    2016-07-01

    Our aim is to explore the similarities in structural fluctuations of homologous kinases. Gaussian Network Model based Normal Mode Analysis was performed on 73 active conformation structures in Ser/Thr/Tyr kinase superfamily. Categories of kinases with progressive evolutionary divergence, viz. (i) Same kinase with many crystal structures, (ii) Within-Subfamily, (iii) Within-Family, (iv) Within-Group, and (v) Across-Group, were analyzed. We identified a flexibility signature conserved in all kinases involving residues in and around the catalytic loop with consistent low-magnitude fluctuations. However, the overall structural fluctuation profiles are conserved better in closely related kinases (Within-Subfamily and Within-family) than in distant ones (Within-Group and Across-Group). A substantial 65.4% of variation in flexibility was not accounted by variation in sequences or structures. Interestingly, we identified substructural residue-wise fluctuation patterns characteristic of kinases of different categories. Specifically, we recognized statistically significant fluctuations unique to families of protein kinase A, cyclin-dependent kinases, and nonreceptor tyrosine kinases. These fluctuation signatures localized to sites known to participate in protein-protein interactions typical of these kinase families. We report for the first time that residues characterized by fluctuations unique to the group/family are involved in interactions specific to the group/family. As highlighted for Src family, local regions with differential fluctuations are proposed as attractive targets for drug design. Overall, our study underscores the importance of consideration of fluctuations, over and above sequence and structural features, in understanding the roles of sites characteristic of kinases. Proteins 2016; 84:957-978. © 2016 Wiley Periodicals, Inc. PMID:27028938

  7. Reconstitution of LHC phosphorylation by a protein kinase isolated from spinach thylakoids

    SciTech Connect

    Hind, G.; Coughlan, S.

    1986-01-01

    Protein kinase activity is responsible for phosphorylating the (LHC) light-harvesting chlorophyll a/b protein complex of photosystem II, leading to its migration in the thylakoid membrane, the fractional redistribution of excitation energy between photosystems II and I, and the phenomenon of state transition. Previous work from this laboratory described the purification to homogeneity of a thylakoid protein kinase which catalyzes the phosphorylation of isolated LHC at 1-10% of a rate estimated for this enzyme and substrate when resident together in the thylakoid membrane. In this communication, we report rates of LHC phosphorylation that are close to physiological, in a system comprised of isolated purified protein kinase (LHCK) and native LHC. 9 refs., 1 fig., 2 tabs.

  8. Protein kinase C, an elusive therapeutic target?

    PubMed Central

    Mochly-Rosen, Daria; Das, Kanad; Grimes, Kevin V

    2013-01-01

    Preface Protein kinase C (PKC) has been a tantalizing target for drug discovery ever since it was first identified as the receptor for the tumor promoter phorbol ester in 19821. Although initial therapeutic efforts focused on cancer, additional diseases, including diabetic complications, heart failure, myocardial infarction, pain and bipolar disease were targeted as researchers developed a better understanding of the roles that PKC’s eight conventional and novel isozymes play in health and disease. Unfortunately, both academic and pharmaceutical efforts have yet to result in the approval of a single new drug that specifically targets PKC. Why does PKC remain an elusive drug target? This review will provide a short account of some of the efforts, challenges and opportunities in developing PKC modulators to address unmet clinical needs. PMID:23197040

  9. 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.

  10. The extended protein kinase C superfamily.

    PubMed Central

    Mellor, H; Parker, P J

    1998-01-01

    Members of the mammalian protein kinase C (PKC) superfamily play key regulatory roles in a multitude of cellular processes, ranging from control of fundamental cell autonomous activities, such as proliferation, to more organismal functions, such as memory. However, understanding of mammalian PKC signalling systems is complicated by the large number of family members. Significant progress has been made through studies based on comparative analysis, which have defined a number of regulatory elements in PKCs which confer specific location and activation signals to each isotype. Further studies on simple organisms have shown that PKC signalling paradigms are conserved through evolution from yeast to humans, underscoring the importance of this family in cellular signalling and giving novel insights into PKC function in complex mammalian systems. PMID:9601053

  11. Human protein kinase CK2 genes.

    PubMed

    Wirkner, U; Voss, H; Lichter, P; Pyerin, W

    1994-01-01

    We have analyzed the genomic structure of human protein kinase CK2. Of the presumably four genes, the gene encoding the regulatory subunit beta and a processed (pseudo)gene of the catalytic subunit alpha have been characterized completely. In addition, a 18.9 kb-long central part of the gene encoding the catalytic subunit alpha has been characterized. The subunit beta gene spans 4.2 kb and is composed of seven exons. Its promoter region shows several features of a "housekeeping gene" and shares common features with the promoter of the regulatory subunit of cAMP-dependent protein kinase. Conforming to the genomic structure, the beta gene transcripts form a band around 1.1 kb. The central part of the subunit alpha gene contains eight exons comprising bases 102 to 824 of the translated region. Within the introns, 16 Alu repeats were identified, some of which arranged in tandems. The structure of both human CK2 coding genes, alpha and beta, is highly conserved. Several introns are located at corresponding positions in the respective genes of the nematode Caenorhabditis elegans. The processed alpha (pseudo)gene has a complete open reading frame and is 99% homologous to the coding region of the CK2 alpha cDNA. Although the gene has a promoter-like upstream region, no transcript could be identified so far. The genomic clones were used for localization in the human genome. The beta gene was mapped to locus 6p21, the alpha gene to locus 20p13 and the alpha (pseudo)gene to locus 11p15. There is no evidence for additional alpha or beta loci in the human genome. PMID:7735323

  12. 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.

  13. 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

  14. Hormone signaling linked to silkmoth sex pheromone biosynthesis involves Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of the insect PAT family protein Bombyx mori lipid storage droplet protein-1(BmLsd)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The structurally-related members of the PAT family of proteins, which are so name based on similarity amongst perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47), are cytoplasmic lipid droplet (LD)-associated proteins charac...

  15. A mechanism for the inactivation of Ca2+/calmodulin-dependent protein kinase II during prolonged seizure activity and its consequence after the recovery from seizure activity in rats in vivo.

    PubMed

    Yamagata, Y; Imoto, K; Obata, K

    2006-07-01

    Seizure is a form of excessive neuronal excitation and seizure-induced neuronal damage has profound effects on the prognosis of epilepsy. In various seizure models, the inactivation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) occurs during seizure activity preceding neuronal cell death. CaMKII is a multifunctional protein kinase enriched in the brain and involved in various ways the regulation of neuronal activity. CaMKII inactivation during seizure activity may modify neuronal cell survival after seizure. However, the mechanism for CaMKII inactivation and its consequence after seizure recovery remain to be elucidated yet. In the present study, we employed a prolonged seizure model by systemic injection of kainic acid into rats and biochemically examined the activity state of CaMKII. In status epilepticus induced by kainic acid, not only the inactivation of CaMKII in brain homogenate, but also a shift in the distribution of CaMKII protein from the soluble to particulate fraction occurred in both hippocampus and parietal cortex. The particulate CaMKII showed a large decrease in the specific activity and a concurrent large increase in the autophosphorylation ratio at Thr-286 (alpha) and at Thr-287 (beta). In contrast, the soluble CaMKII showed normal or rather decreased specific activity and autophosphorylation ratio. After 24 h of recovery from kainic acid-induced status epilepticus, all such changes had disappeared. On the other hand, the total amount of CaMKII was decreased by 35% in hippocampus and 20% in parietal cortex, but the existing CaMKII was indistinguishable from those of controls in terms of the autonomous activity ratio, specific activity and autophosphorylation ratio. Thus, CaMKII inactivation in kainic acid-induced status epilepticus seems to be derived not from simple degradation of the enzyme, but from the formation of the autophosphorylated, inactivated and sedimentable CaMKII. Such a form of CaMKII may be important during pathological

  16. 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

  17. 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.

  18. Microheterogeneity of adenosine cyclic monophosphate-dependent protein kinases from mouse brain and heart.

    PubMed Central

    Malkinson, A M; Gharrett, A J; Hogy, L

    1978-01-01

    1. DEAE-cellulose chromatography of mouse brain cytosol indicated the presence of only the type II isoenzyme of cyclic AMP-dependent protein kinase. Mouse heart cytosol contained approximately equal amounts of the type I and type II isoenzymes. 2. Both brain and heart type II isoenzymes reassociated after a transient exposure to cyclic AMP, but the heart type I isoenzyme remained dissociated. 3. Elution of brain cytosol continuously exposed to cyclic AMP resolved multiple peaks of protein kinase and cyclic AMP-binding activities. A single peak of kinase and multiple peaks of cyclic AMP-binding activities were found under the same conditions with heart cytosol. Various control experiments suggested that the heterogeneity within the brain type II isoenzymic class had not been caused by proteolysis. 4. Kinetic experiments with unfractionated brain cytosol showed that the binding of cyclic AMP, the dissociation of cyclic AMP from protein and the rate of heat denaturation of the cyclic AMP-binding activity gave results consistent with the presence of multiple binding species. 5. It concluded that the type II isoenzymic peak obtained by DEAE-cellulose chromatography of mouse brain cytosol represents a class of enzymes containing multiple regulatory and catalytic subunits. The two heart cytosol isoenzymes contain a common catalytic subunit. The degree of protein kinase 'microheterogeneity", defined as the presence of multiple regulatory and/or catalytic subunits within a single isoenzymic class, appears to be tissue-specific. PMID:217338

  19. The toxic effects of Bisphenol A on the mouse spermatocyte GC-2 cell line: the role of the Ca2+-calmodulin-Ca2+/calmodulin-dependent protein kinase II axis.

    PubMed

    Qian, Wenyi; Wang, Yixin; Zhu, Jingying; Mao, Changfei; Wang, Qiang; Huan, Fei; Cheng, Jie; Liu, Yanqing; Wang, Jun; Xiao, Hang

    2015-11-01

    Bisphenol A (BPA), an endocrine-disrupting chemical (EDC), is known to induce male reproductive toxicity in rodents. However, its toxic effects on the germ cells are still poorly understood. It has been proposed that Ca(2+) homeostasis and Ca(2+) sensors, including calmodulin (CaM) and calmodulin-dependent protein kinase II (CaMKII), play critical roles in spermatogenesis. Therefore, in the present study, we aimed to investigate whether a perturbation in Ca(2+)-CaM-CaMKII signaling was involved in the BPA-induced injury to mouse spermatocyte GC-2spd (ts) (GC-2) cells. Our results showed that BPA (range from 0.2 to 20 μM) induced obvious GC-2 cell injury, including decreased cell viability, the release of mitochondrial cytochrome c and the activation of caspase-3. However, these processes could be partially abrogated by pretreatment with a Ca(2+) chelator (BAPTA/AM), a CaM antagonist (W7) or a CaMKII inhibitor (KN93). These results, taken together, indicate that BPA exposure contributes to male germ cell injury, which may be partially mediated through a perturbation in Ca(2+)/CaM/CaMKII signaling and the mitochondrial apoptotic process. PMID:26096086

  20. Angiotensin II type 2 receptor-interacting protein 3a suppresses proliferation, migration and invasion in tongue squamous cell carcinoma via the extracellular signal-regulated kinase-Snai2 pathway

    PubMed Central

    ZHAO, TINGTING; HE, QIANTING; LIU, ZHONGHUA; DING, XUEQIANG; ZHOU, XIAOFENG; WANG, ANXUN

    2016-01-01

    Our previous studies demonstrated that the downregulation of microtubule-associated tumor suppressor 1/angiotensin II type 2 receptor-interacting protein (MTUS1/ATIP) is associated with poor differentiation and prognosis in tongue squamous cell carcinoma (TSCC), and that ATIP1 exerts an antiproliferative effect on TSCC. The aim of the present study was to further investigate the anticancer effect of MTUS1/ATIP3a in TSCC. It was observed that UM1 cells (a TSCC cell line with high migration and invasion ability) exhibited lower expression of ATIP3a compared with UM2 cells (a TSCC cell line with lower migration and invasion ability). Restoration of ATIP3a expression in UM1 cells exerted antiproliferative effects and inhibited migration and invasion, whereas knockdown of ATIP3a promoted proliferation, migration and invasion in UM2 cells. Restoration of ATIP3a expression inhibited the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and the expression of Snai2 and vimentin in UM1 cells, whereas knockdown of ATIP3a promoted the phosphorylation of ERK1/2 and the expression of Snai2 and vimentin in UM2 cells. Therefore, MTUS1/ATIP3a was found to suppress the proliferation, migration and invasion of TSCC cells via the ERK1/2-Snai2 pathway. PMID:26870214

  1. Cytoskeletal protein kinases: titin and its relations in mechanosensing.

    PubMed

    Gautel, Mathias

    2011-07-01

    Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca(2+)-calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other "MLCKs", is not Ca(2+)-calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation. PMID:21416260

  2. Phosphodiesterase 5 inhibition ameliorates angiontensin II-induced podocyte dysmotility via the protein kinase G-mediated downregulation of TRPC6 activity.

    PubMed

    Hall, Gentzon; Rowell, Janelle; Farinelli, Federica; Gbadegesin, Rasheed A; Lavin, Peter; Wu, Guanghong; Homstad, Alison; Malone, Andrew; Lindsey, Thomas; Jiang, Ruiji; Spurney, Robert; Tomaselli, Gordon F; Kass, David A; Winn, Michelle P

    2014-06-15

    The emerging role of the transient receptor potential cation channel isotype 6 (TRPC6) as a central contributor to various pathological processes affecting podocytes has generated interest in the development of therapeutics to modulate its function. Recent insights into the regulation of TRPC6 have revealed PKG as a potent negative modulator of TRPC6 conductance and associated signaling via its phosphorylation at two highly conserved amino acid residues: Thr(69)/Thr(70) (Thr(69) in mice and Thr(70) in humans) and Ser(321)/Ser(322) (Ser(321) in mice and Ser(322) in humans). Here, we tested the role of PKG in modulating TRPC6-dependent responses in primary and conditionally immortalized mouse podocytes. TRPC6 was phosphorylated at Thr(69) in nonstimulated podocytes, but this declined upon ANG II stimulation or overexpression of constitutively active calcineurin phosphatase. ANG II induced podocyte motility in an in vitro wound assay, and this was reduced 30-60% in cells overexpressing a phosphomimetic mutant TRPC6 (TRPC6T70E/S322E) or activated PKG (P < 0.05). Pretreatment of podocytes with the PKG agonists S-nitroso-N-acetyl-dl-penicillamine (nitric oxide donor), 8-bromo-cGMP, Bay 41-2772 (soluble guanylate cyclase activator), or phosphodiesterase 5 (PDE5) inhibitor 4-{[3',4'-(methylenedioxy)benzyl]amino}[7]-6-methoxyquinazoline attenuated ANG II-induced Thr(69) dephosphorylation and also inhibited TRPC6-dependent podocyte motility by 30-60%. These data reveal that PKG activation strategies, including PDE5 inhibition, ameliorate ANG II-induced podocyte dysmotility by targeting TRPC6 in podocytes, highlighting the potential therapeutic utility of these approaches to treat hyperactive TRPC6-dependent glomerular disease. PMID:24740790

  3. Regulation of mitochondrial protein import by cytosolic kinases.

    PubMed

    Schmidt, Oliver; Harbauer, Angelika B; Rao, Sanjana; Eyrich, Beate; Zahedi, René P; Stojanovski, Diana; Schönfisch, Birgit; Guiard, Bernard; Sickmann, Albert; Pfanner, Nikolaus; Meisinger, Chris

    2011-01-21

    Mitochondria import a large number of nuclear-encoded proteins via membrane-bound transport machineries; however, little is known about regulation of the preprotein translocases. We report that the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM complex), is phosphorylated by cytosolic kinases-in particular, casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes biogenesis of the TOM complex by phosphorylation of two key components, the receptor Tom22 and the import protein Mim1, which in turn are required for import of further Tom proteins. Inactivation of CK2 decreases the levels of the TOM complex and thus mitochondrial protein import. PKA phosphorylates Tom70 under nonrespiring conditions, thereby inhibiting its receptor activity and the import of mitochondrial metabolite carriers. We conclude that cytosolic kinases exert stimulatory and inhibitory effects on biogenesis and function of the TOM complex and thus regulate protein import into mitochondria. PMID:21215441

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. 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

  6. Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

    PubMed Central

    Lizcano, Jose M; Alrubaie, Saif; Kieloch, Agnieszka; Deak, Maria; Leevers, Sally J; Alessi, Dario R

    2003-01-01

    An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well d

  7. 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. PMID:23436195

  8. Protein Kinase C Pharmacology: Refining the Toolbox

    PubMed Central

    Wu-Zhang, Alyssa X.; Newton, Alexandra C.

    2014-01-01

    SYNOPSIS Protein kinase C (PKC) has been in the limelight since the discovery three decades ago that it acts as a major receptor for the tumor-promoting phorbol esters. Phorbol esters, with their potent ability to activate two of the three classes of PKC isozymes, have remained the best pharmacological tool for directly modulating PKC activity. However, with the discovery of other phorbol ester-responsive proteins, the advent of various small-molecule and peptide modulators, and the need to distinguish isozyme-specific activity, the pharmacology of PKC has become increasingly complex. Not surprisingly, many of the compounds originally touted as direct modulators of PKC have subsequently been shown to hit many other cellular targets and, in some cases, not even directly modulate PKC. The complexities and reversals in PKC pharmacology have led to widespread confusion about the current status of the pharmacological tools available to control PKC activity. Here, we aim to clarify the cacophony in the literature regarding the current state of bona fide and discredited cellular PKC modulators, including activators, small-molecule inhibitors, and peptides, and also address the use of genetically-encoded reporters and of PKC mutants to measure the effects of these drugs on the spatiotemporal dynamics of signaling by specific isozymes. PMID:23662807

  9. Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation.

    PubMed Central

    Inoguchi, T; Battan, R; Handler, E; Sportsman, J R; Heath, W; King, G L

    1992-01-01

    In the present study, we have measured protein kinase C (PKC) specific activities and total diacylglycerol (DAG) level in the aorta and heart of rats, which showed that after 2 weeks of streptozotocin (STZ)-induced diabetes, membranous PKC specific activity and total DAG content were increased significantly by 88% and 40% in the aorta and by 21% and 72% in the heart, respectively. Hyperglycemia was identified as being a causal factor since elevated glucose levels increased DAG levels in cultured aortic endothelial and smooth muscle cells. Analysis by immunoblotting revealed that only alpha and beta II PKC isoenzymes are detected in these two tissues and vascular cells among those studied. In STZ-induced diabetic rats, beta II isoenzyme is preferentially increased in both aorta and heart, whereas PKC alpha did not change significantly. The increases in membranous PKC specific activity and DAG level are observed in both spontaneous diabetes-prone diabetic BB rats as well as in STZ-induced diabetic BB and Sprague-Dawley rats, which persisted for up to 5 weeks. After 2 weeks of diabetes without treatment, the normalization of blood glucose levels for up to 3 weeks with islet cell transplants in STZ-induced diabetic BB rats reversed the biochemical changes only in the heart, but not in the aorta. These results suggest that PKC activity and DAG level may be persistently activated in the macrovascular tissues from diabetic animals and indicate a possible role for these biochemical parameters in the development of diabetic chronic vascular complications. Images PMID:1438315

  10. Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation.

    PubMed

    Inoguchi, T; Battan, R; Handler, E; Sportsman, J R; Heath, W; King, G L

    1992-11-15

    In the present study, we have measured protein kinase C (PKC) specific activities and total diacylglycerol (DAG) level in the aorta and heart of rats, which showed that after 2 weeks of streptozotocin (STZ)-induced diabetes, membranous PKC specific activity and total DAG content were increased significantly by 88% and 40% in the aorta and by 21% and 72% in the heart, respectively. Hyperglycemia was identified as being a causal factor since elevated glucose levels increased DAG levels in cultured aortic endothelial and smooth muscle cells. Analysis by immunoblotting revealed that only alpha and beta II PKC isoenzymes are detected in these two tissues and vascular cells among those studied. In STZ-induced diabetic rats, beta II isoenzyme is preferentially increased in both aorta and heart, whereas PKC alpha did not change significantly. The increases in membranous PKC specific activity and DAG level are observed in both spontaneous diabetes-prone diabetic BB rats as well as in STZ-induced diabetic BB and Sprague-Dawley rats, which persisted for up to 5 weeks. After 2 weeks of diabetes without treatment, the normalization of blood glucose levels for up to 3 weeks with islet cell transplants in STZ-induced diabetic BB rats reversed the biochemical changes only in the heart, but not in the aorta. These results suggest that PKC activity and DAG level may be persistently activated in the macrovascular tissues from diabetic animals and indicate a possible role for these biochemical parameters in the development of diabetic chronic vascular complications. PMID:1438315

  11. Identification and characterization of chloroplast casein kinase II from Oryza sativa (rice).

    PubMed

    Lu, Qingtao; Ding, Shunhua; Reiland, Sonja; Rödiger, Anja; Roschitzki, Bernd; Xue, Peng; Gruissem, Wilhelm; Lu, Congming; Baginsky, Sacha

    2015-01-01

    Plastid casein kinase II is an important regulator of transcription, posttranscriptional processes, and, most likely, different metabolic functions in dicotyledonous species. Here we report the identification and characterization of pCKII from the monocotyledonous species Oryza sativa. OspCKII activity was enriched from isolated rice chloroplasts using heparin-Sepharose chromatography, in which it co-elutes with the transcriptionally active chromosome (TAC) and several ribosomal proteins. Inclusion mass scanning of the kinase-active fraction identified the gene model for OspCKII. Transient expression of GFP fused to the 184 N-terminal amino acids of the OspCKII sequence in rice confirmed the chloroplastic localization of the kinase. OspCKII activity shows the characteristic features of casein kinase II, such as the utilization of GTP as phosphate donor, inhibition by low concentrations of heparin and poly-lysine, and utilization of the canonical pCKII motif E-S-E-G-E in the model substrate RNP29. Phosphoproteome analysis of a protein extract from rice leaves combined with a meta-analysis with published phosphoproteomics data revealed differences in the target protein spectrum between rice and Arabidopsis. Consistently, several pCKII phosphorylation sites in dicotyledonous plants are not conserved in monocots and algae, suggesting that details of pCKII regulation in plastids have changed during evolution. PMID:25316064

  12. Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    SciTech Connect

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-07-12

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

  13. 1-[N, O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), an inhibitor of calcium-dependent camodulin protein kinase II, inhibits both insulin- and hypoxia-stimulated glucose transport in skeletal muscle.

    PubMed Central

    Brozinick, J T; Reynolds, T H; Dean, D; Cartee, G; Cushman, S W

    1999-01-01

    Previous studies have indicated a role for calmodulin in hypoxia-and insulin-stimulated glucose transport. However, since calmodulin interacts with multiple protein targets, it is unknown which of these targets is involved in the regulation of glucose transport. In the present study, we have used the calcium-dependent calmodulin protein kinase II (CAMKII) inhibitor 1-[N, O-bis-(5-isoquinolinesulphonyl) -N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) to investigate the possible role of this enzyme in the regulation of glucose transport in isolated rat soleus and epitrochlearis muscles. KN-62 did not affect basal 2-deoxyglucose transport, but it did inhibit both insulin- and hypoxia-stimulated glucose transport activity by 46 and 40% respectively. 1-[N,O-Bis-(1, 5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-04), a structural analogue of KN-62 that does not inhibit CAMKII, had no effect on hypoxia-or insulin-stimulated glucose transport. Accordingly, KN-62 decreased the stimulated cell-surface GLUT4 labelling by a similar extent as the inhibition of glucose transport (insulin, 49% and hypoxia, 54%). Additional experiments showed that KN-62 also inhibited insulin- and hypoxia-stimulated transport by 37 and 40% respectively in isolated rat epitrochlearis (a fast-twitch muscle), indicating that the effect of KN-62 was not limited to the slow-twitch fibres of the soleus. The inhibitory effect of KN-62 on hypoxia-stimulated glucose transport appears to be specific to CAMKII, since KN-62 did not inhibit hypoxia-stimulated 45Ca efflux from muscles pre-loaded with 45Ca, or hypoxia-stimulated glycogen breakdown. Additionally, KN-62 affected neither insulin-stimulated phosphoinositide 3-kinase nor Akt activity, suggesting that the effects of KN-62 are not due to non-specific effects of this inhibitor on these regions of the insulin-signalling cascade. The results of the present study suggest that CAMKII might have a distinct role in insulin- and hypoxia

  14. Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2

    PubMed Central

    Konopatskaya, Olga; Matthews, Sharon A.; Harper, Matthew T.; Gilio, Karen; Cosemans, Judith M. E. M.; Williams, Christopher M.; Navarro, Maria N.; Carter, Deborah A.; Heemskerk, Johan W. M.; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W.

    2016-01-01

    Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion. PMID:21527521

  15. Tonoplast-Bound Protein Kinase Phosphorylates Tonoplast Intrinsic Protein 1

    PubMed Central

    Johnson, Kenneth D.; Chrispeels, Maarten J.

    1992-01-01

    Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [32P]orthophosphate and in vitro by incubating purified tonoplasts with γ-labeled [32P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca2+-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:16653198

  16. [Kinetic studies of protein kinase A in rat liver during late sepsis].

    PubMed

    Jin, Y W; Yang, S L; Hsu, H K; Wu, S N; Liu, M S

    1992-09-01

    . In the case of type-II protein kinase A (eluted at high ionic strength), the Vmax, S0.5, and n values for ATP, histone, and c-AMP were unchanged during late sepsis. The results of the present study indicate that the activities and kinetic characteristics of type I protein kinase A in rat liver are modified during late sepsis. Since protein kinase A is known to regulate glucose metabolism through adrenergic receptor mediation, these findings may have a pathophysiological significance in the understanding of hepatic glucose dyshomeostasis during sepsis.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1294761

  17. Mitogen activated protein kinase at the nuclear pore complex

    PubMed Central

    Faustino, Randolph S; Maddaford, Thane G; Pierce, Grant N

    2011-01-01

    Abstract Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD-98059, two MAP kinase antagonists as well as with SB-202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N-terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function. PMID:20497490

  18. 3′, 5′-Cyclic Adenosine 5′-Monophosphate Response Element-Dependent Transcriptional Regulation of the Secretogranin II Gene Promoter Depends on Gonadotropin-Releasing Hormone-Induced Mitogen-Activated Protein Kinase Activation and the Transactivator Activating Transcription Factor 3

    PubMed Central

    Xie, Jianjun; Roberson, Mark S.

    2008-01-01

    Previous studies demonstrated that GnRH-induced secretogranin II (SgII) promoter regulation required a consensus cAMP response element (CRE) and protein kinase A/CRE binding protein. The present studies examined the role of additional components of the GnRH signaling network on SgII promoter activity with particular attention devoted to CRE-dependent gene regulation. Disruption of the SgII CRE by mutagenesis resulted in inhibition of GnRH agonist (GnRHa) induction of this promoter in αT3-1 cells. Pharmacological and dominant-negative inhibition of the ERK and c-Jun N-terminal kinase (JNK) signaling pathways revealed that GnRHa-induced SgII promoter activity required functional JNK and ERK modules. Combined inhibition of both pathways nearly abolished GnRHa-induced SgII promoter activity. Specific induction of the ERK cascade alone using overexpression of Raf-CAAX was not sufficient to activate the SgII gene promoter. In contrast, overexpression of the catalytic domain of the more pleiotropic MAPK activator, MAPK/ERK kinase-1, was sufficient to induce SgII promoter activity. The effect(s) of mitogen-activated protein/ERK kinase-1 on SgII promoter activity was CRE dependent and was reversed by the combined pharmacological inhibition of both JNK and ERK modules. CRE DNA binding studies demonstrated the recruitment of activating transcription factor (ATF)-3 and c-Jun to the CRE after administration of GnRHa to αT3-1 cells. Specific small interfering RNA knockdown of ATF3 reduced ATF3 DNA binding and the effect of GnRHa on the SgII promoter. These studies support the conclusion that MAPK signaling and ATF3 action are essential for full SgII promoter activation by GnRHa through a canonical CRE. Moreover, we suggest that within the GnRH signaling network, CRE-dependent gene regulation in general may be mediated primarily through the immediate early response gene ATF3. PMID:17962349

  19. Protein kinase A alterations in adrenocortical tumors.

    PubMed

    Espiard, S; Ragazzon, B; Bertherat, J

    2014-11-01

    Stimulation of the cAMP pathway by adrenocorticotropin (ACTH) is essential for adrenal cortex maintenance, glucocorticoid and adrenal androgens synthesis, and secretion. Various molecular and cellular alterations of the cAMP pathway have been observed in endocrine tumors. Protein kinase A (PKA) is a central key component of the cAMP pathway. Molecular alterations of PKA subunits have been observed in adrenocortical tumors. PKA molecular defects can be germline in hereditary disorders or somatic in sporadic tumors. Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) can be observed in patients with ACTH-independent Cushing's syndrome (CS) due to primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic cortisol-secreting adrenocortical adenomas. Germline gene duplication of the catalytic subunits Cα (PRKACA) has been observed in patients with PPNAD. Furthermore, exome sequencing revealed recently activating somatic mutations of PRKACA in about 40% of cortisol-secreting adrenocortical adenomas. In vitro and in vivo functional studies help in the progress to understand the mechanisms of adrenocortical tumors development due to PKA regulatory subunits alterations. All these alterations are observed in benign oversecreting tumors and are mimicking in some way cAMP pathway constitutive activation. On the long term, unraveling these alterations will open new strategies of pharmacological treatment targeting the cAMP pathway in adrenal tumors and cortisol-secretion disorders. PMID:25105543

  20. 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.

  1. Mitogen-Activated Protein Kinase Kinase 3 Is Required for Regulation during Dark-Light Transition.

    PubMed

    Lee, Horim

    2015-07-01

    Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition. PMID:26082029

  2. Ca2+/calmodulin dependent protein kinase from Mycobacterium smegmatis ATCC 607.

    PubMed

    Sharma, S; Giri, S; Khuller, G K

    1998-06-01

    A soluble Ca2+/calmodulin dependent protein kinase has been partially purified (approximately 400 fold) from Mycobacterium smegmatis ATCC 607 using several purification steps like ammonium sulphate precipitation (30-60%), Sepharose CL-6B gel filtration, DEAE-cellulose and finally calmodulin-agarose affinity chromatography. On SDS-PAGE, this enzyme preparation showed a major protein band of molecular mass 35 kD and its activity was dependent on calcium, calmodulin and ATP when measured under saturating histone IIs (exogenous substrate) concentration. Phosphorylation of histone IIs was inhibited by W-7 (calmodulin inhibitor) and KN-62 (CaM-kinase inhibitor) with IC50 of 1.5 and 0.25 microm respectively, but was not affected by inhibitors of PKA (Sigma P5015) and PKC (H-7). All these results confirm that purified enzyme is Ca2+/calmodulin dependent protein kinase of M. smegmatis. The protein kinase of M. smegmatis demonstrated a narrow substrate specificity for both exogenous as well as endogenous substrates. These results suggest that purified CaM-kinase must be involved in regulating specific function(s) in this organism. PMID:9655195

  3. 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.

  4. 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

  5. Protein kinase C sensitizes olfactory adenylate cyclase.

    PubMed

    Frings, S

    1993-02-01

    Effects of neurotransmitters on cAMP-mediated signal transduction in frog olfactory receptor cells (ORCs) were studied using in situ spike recordings and radioimmunoassays. Carbachol, applied to the mucosal side of olfactory epithelium, amplified the electrical response of ORCs to cAMP-generating odorants, but did not affect unstimulated cells. A similar augmentation of odorant response was observed in the presence of phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC). The electrical response to forskolin, an activator of adenylate cyclase (AC), was also enhanced by PDBu, and it was attenuated by the PKC inhibitor Goe 6983. Forskolin-induced accumulation of cAMP in olfactory tissue was potentiated by carbachol, serotonin, and PDBu to a similar extent. Potentiation was completely suppressed by the PKC inhibitors Goe 6983, staurosporine, and polymyxin B, suggesting that the sensitivity of olfactory AC to stimulation by odorants and forskolin was increased by PKC. Experiments with deciliated olfactory tissue indicated that sensitization of AC was restricted to sensory cilia of ORCs. To study the effects of cell Ca2+ on these mechanisms, the intracellular Ca2+ concentration of olfactory tissue was either increased by ionomycin or decreased by BAPTA/AM. Increasing cell Ca2+ had two effects on cAMP production: (a) the basal cAMP production was enhanced by a mechanism sensitive to inhibitors of calmodulin; and (b) similar to phorbol ester, cell Ca2+ caused sensitization of AC to stimulation by forskolin, an effect sensitive to Goe 6983. Decreasing cell Ca2+ below basal levels rendered AC unresponsive to stimulation by forskolin. These data suggest that a crosstalk mechanism is functional in frog ORCs, linking the sensitivity of AC to the activity of PKC. At increased activity of PKC, olfactory AC becomes more responsive to stimulation by odorants, forskolin, and cell Ca2+. Neurotransmitters appear to use this crosstalk mechanism to regulate olfactory

  6. Protein kinase A alterations in endocrine tumors.

    PubMed

    Yu, B; Ragazzon, B; Rizk-Rabin, M; Bertherat, J

    2012-09-01

    Various molecular and cellular alterations of the cyclic adenosine monophosphate (cAMP) pathway have been observed in endocrine tumors. Since protein kinase A (PKA) is a central key component of the cAMP pathway, studies of the alterations of PKA subunits in endocrine tumors reveal new aspects of the mechanisms of cAMP pathway alterations in human diseases. So far, most alterations have been observed for the regulatory subunits, mainly PRKAR1A and to a lower extent, PRKAR2B. One of the best examples of such alteration today is the multiple neoplasia syndrome Carney complex (CNC). The most common endocrine gland manifestations of CNC are pituitary GH-secreting adenomas, thyroid tumors, testicular tumors, and ACTH-independent Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD). Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) are observed in about two-third of CNC patients, and also in patients with isolated PPNAD. PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic endocrine tumors. More than 120 different PRKAR1A mutations have been found today. Most of them lead to an unstable mutant mRNA, which will be degraded by nonsense mediated mRNA decay. In vitro and in vivo functional studies are in progress to understand the mechanisms of endocrine tumor development due to PKA regulatory subunits inactivation. PRKAR1A mutations stimulate in most models PKA activity, mimicking in some way cAMP pathway constitutive activation. Cross-talks with other signaling pathways summarized in this review have been described and might participate in endocrine tumorigenesis. PMID:22752956

  7. In silico design of protein kinase inhibitors: successes and failures.

    PubMed

    Dubinina, Galina G; Chupryna, Oleksandr O; Platonov, Maxim O; Borisko, Petro O; Ostrovska, Galina V; Tolmachov, Andriy O; Shtil, Alexander A

    2007-03-01

    Protein kinases are among the most exploited targets in modern drug discovery due to key roles these enzymes play in human diseases including cancer. The in silico approach, an important part of rational design of protein kinase inhibitors, is founded on vast information about 3D structures of these enzymes. This review summarizes general structural features of the kinase inhibitors and the studies applied toward a large scale chemical database for virtual screening. Analyzed are the ways of validating the modern docking tools and their combinations with different scoring functions. In particular, we discuss the kinase flexibility as a reason for failures of the docking procedure. Finally, evidence is provided for the main patterns of kinase-inhibitor interactions and creation of the hinge-region-directed 2D filters. PMID:17348826

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

    PubMed Central

    Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inês CR; 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

    2014-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.02860.001 PMID:24948515

  9. 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

    2014-01-01

    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. PMID:24948515

  10. Activation of protein kinase C induces mitogen-activated protein kinase dephosphorylation and pronucleus formation in rat oocytes.

    PubMed

    Lu, Qing; Smith, Gary D; Chen, Da-Yuan; Han, Zhi-Ming; Sun, Qing-Yuan

    2002-07-01

    Mammalian oocytes are arrested at metaphase of the second meiotic division (MII) before fertilization. When oocytes are stimulated by spermatozoa, they exit MII stage and complete meiosis. It has been suggested that an immediate increase in intracellular free calcium concentration and inactivation of maturation promoting factor (MPF) are required for oocyte activation. However, the underlying mechanism is still unclear. In the present study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, and their interplay in rat oocyte activation. We found that MAP kinase became dephosphorylated in correlation with pronucleus formation after fertilization. Protein kinase C activators, phorbol 12-myriatate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8), triggered dephosphorylation of MAP kinase and pronucleus formation in a dose-dependent and time-dependent manner. Dephosphorylation of MAP kinase was also correlated with pronucleus formation when oocytes were treated with PKC activators. Effects of PKC activators were abolished by the PKC inhibitors, calphostin C and staurosporine, as well as a protein phosphatase blocker, okadaic acid (OA). These results suggest that PKC activation may cause rat oocyte pronucleus formation via MAP kinase dephosphorylation, which is probably mediated by OA-sensitive protein phosphatases. We also provide evidence supporting the involvement of such a process in fertilization. PMID:12080000

  11. 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.

  12. Dynamics of Protein Kinases: Insights from Nuclear Magnetic Resonance

    PubMed Central

    Xiao, Yao; Liddle, Jennifer C.; Pardi, Arthur; Ahn, Natalie G.

    2015-01-01

    CONSPECTUS Protein kinases are ubiquitous enzymes with critical roles in cellular processes and pathology. As a result, researchers have studied their activity and regulatory mechanisms extensively. Thousands of X-ray structures give snapshots of the architectures of protein kinases in various states of activation and ligand binding. However, the extent of and manner by which protein motions and conformational dynamics underlie the function and regulation of these important enzymes is not well understood. Nuclear magnetic resonance (NMR) methods provide complementary information about protein conformation and dynamics in solution. However, until recently, the large size of these enzymes prevented researchers from using these methods with kinases. Developments in transverse relaxation-optimized spectroscopy (TROSY)-based techniques and more efficient isotope labeling strategies are now allowing researchers to carry out NMR studies on full-length protein kinases. In this Account, we describe recent insights into the role of dynamics in protein kinase regulation and catalysis that have been gained from NMR measurements of chemical shift changes and line broadening, residual dipolar couplings, and relaxation. These findings show strong associations between protein motion and events that control kinase activity. Dynamic and conformational changes occurring at ligand binding sites and other regulatory domains of these proteins propagate to conserved kinase core regions that mediate catalytic function. NMR measurements of slow time scale (microsecond to millisecond) motions also reveal that kinases carry out global exchange processes that synchronize multiple residues and allosteric interconversion between conformational states. Activating covalent modifications or ligand binding to form the Michaelis complex can induce these global processes. Inhibitors can also exploit the exchange properties of kinases by using conformational selection to form dynamically quenched

  13. Leishmania Infection Engages Non-Receptor Protein Kinases Differentially to Persist in Infected Hosts

    PubMed Central

    Zhang, Naixin; Kima, Peter E.

    2016-01-01

    Protein kinases play important roles in the regulation of cellular activities. In cells infected by pathogens, there is an increasing appreciation that dysregulated expression of protein kinases promotes the success of intracellular infections. In Leishmania-infected cells, expression and activation of protein kinases, such as the mitogen-activated protein kinases, kinases in the PI3-kinase signaling pathway, and kinases in the NF-κB-signaling pathway, are modulated in some manner. Several recent reviews have discussed our current understanding of the roles of these kinases in Leishmania infections. Apart from the kinases in the pathways enumerated above, there are other host cell protein kinases that are activated during the Leishmania infection of mammalian cells whose roles also appear to be significant. This review discusses recent observations on the Abl family of protein kinases and the protein kinase regulated by RNA in Leishmania infections. PMID:27148265

  14. Giant protein kinases: domain interactions and structural basis of autoregulation.

    PubMed Central

    Kobe, B; Heierhorst, J; Feil, S C; Parker, M W; Benian, G M; Weiss, K R; Kemp, B E

    1996-01-01

    The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I. Images PMID:9003756

  15. 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

  16. The crystal structure of choline kinase reveals a eukaryotic protein kinase fold

    SciTech Connect

    Peisach, D.; Gee, P.; Kent, K.; Xu, Z.

    2010-03-08

    Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 {angstrom} crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.

  17. Protein Kinase Cδ mediates the activation of Protein Kinase D2 in Platelets

    PubMed Central

    Bhavanasi, Dheeraj; Kim, Soochong; Goldfinger, Lawrence E.; Kunapuli, Satya P.

    2011-01-01

    Protein Kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCμ, PKD2 and PKCν in humans). It is known that PKCs activate PKD, but the relative expression of isoforms of PKD or the specific PKC isoform/s responsible for its activation in platelets is not known. This study is aimed at investigating the pathway involved in activation of PKD in platelets. We show that PKD2 is the major isoform of PKD that is expressed in human as well as murine platelets but not PKD1 or PKD3. PKD2 activation induced by AYPGKF was abolished with a Gq inhibitor YM-254890, but was not affected by Y-27632, a RhoA/p160ROCK inhibitor, indicating that PKD2 activation is Gq-, but not G12/13-mediated Rho-kinase dependent. Calcium-mediated signals are also required for activation of PKD2 as dimethyl BAPTA inhibited its phosphorylation. GF109203X, a pan PKC inhibitor abolished PKD2 phosphorylation but Go6976, a classical PKC inhibitor had no effect suggesting that novel PKC isoforms are involved in PKD2 activation. Importantly, Rottlerin, a non-selective PKCδ inhibitor, inhibited AYPGKF-induced PKD2 activation in human platelets. Similarly, AYPGKF- and Convulxin-induced PKD2 phosphorylation was dramatically inhibited in PKCδ-deficient platelets, but not in PKCθ– or PKCε–deficient murine platelets compared to that of wild type platelets. Hence, we conclude that PKD2 is a common signaling target downstream of various agonist receptors in platelets and Gq-mediated signals along with calcium and novel PKC isoforms, in particular, PKCδ activate PKD2 in platelets. PMID:21736870

  18. Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin.

    PubMed

    Sun, F; Hug, M J; Bradbury, N A; Frizzell, R A

    2000-05-12

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR. PMID:10799517

  19. 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

  20. The human cytomegalovirus UL97 protein is a protein kinase that autophosphorylates on serines and threonines.

    PubMed Central

    He, Z; He, Y S; Kim, Y; Chu, L; Ohmstede, C; Biron, K K; Coen, D M

    1997-01-01

    The product of the human cytomegalovirus (CMV) UL97 gene, which controls ganciclovir phosphorylation in virus-infected cells, is homologous to known protein kinases but diverges from them at a number of positions that are functionally important. To investigate UL97, we raised an antibody against it and overexpressed it in baculovirus-infected insect cells. Recombinant baculovirus expressing full-length UL97 directed the phosphorylation of ganciclovir in insect cells, which was abolished by a four-codon deletion that confers ganciclovir resistance to CMV. When incubated with [gamma-32P]ATP, full-length UL97 was phosphorylated on serine and threonine residues. Phosphorylation was severely impaired by a point mutation that alters lysine-355 in a motif that aligns with subdomain II of protein kinases. However, phosphorylation was impaired much less severely by the four-codon deletion. A UL97 fusion protein expressed from recombinant baculovirus was purified to near homogeneity. It too was phosphorylated upon incubation with [gamma-32P]ATP in vitro. This phosphorylation, which was abolished by the lysine 355 mutation, was optimal at high NaCl and high pH. The activity required either Mn2+ or Mg2+, with a preference for Mn2+, and utilized either ATP or GTP as a phosphate donor, with Kms of 2 and 4 microM, respectively. The phosphorylation rate was first order with protein concentration, consistent with autophosphorylation. These data strongly argue that UL97 is a serine/threonine protein kinase that autophosphorylates and suggest that the four-codon deletion affects its substrate specificity. PMID:8985364

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

    SciTech Connect

    S Eathiraj; R Palma; M Hirschi; E Volckova; E Nakuci; J Castro; C Chen; T Chan; D France; M Ashwell

    2011-12-31

    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.

  2. Alpha-isoform of Ca2+/calmodulin-dependent kinase II autophosphorylation is required for memory consolidation-specific transcription.

    PubMed

    von Hertzen, Laura S J; Giese, K Peter

    2005-08-22

    Autophosphorylation of the alpha-isoform of Ca2+/calmodulin-dependent kinase II switches the kinase into an autonomous activity mode. This molecular switch is important for hippocampal long-term memory formation, which requires de novo gene transcription and protein synthesis. Here, we have studied whether auto-phosphorylation of the alpha-isoform of Ca2+/calmodulin-dependent kinase II is required for gene transcription induced in the hippocampus by contextual fear conditioning. We have shown that upregulation of a nonassociative transcript, the serum and glucocorticoid-induced kinase-1 messenger RNA, is normal in alpha-isoform of Ca2+/calmodulin-dependent kinase II autophosphorylation-deficient mutant mice, whereas upregulation of an associative transcript, the nerve growth factor-inducible gene B messenger RNA, is impaired. Thus, we suggest that autophosphorylation of the alpha-isoform of Ca2+/calmodulin-dependent kinase II is a biochemical switch that regulates association-specific consolidation processes. PMID:16056150

  3. Protein kinase C mediated phosphorylation blocks juvenile hormone action.

    PubMed

    Kethidi, Damu R; Li, Yiping; Palli, Subba R

    2006-03-01

    Juvenile hormones (JH) regulate a wide variety of developmental and physiological processes in insects. Although the biological actions of JH are well documented, the molecular mechanisms underlying JH action are poorly understood. We studied the molecular basis of JH action using a JH response element (JHRE) identified in the promoter region of JH esterase gene cloned from Choristoneura fumiferana, which is responsive to JH and 20-hydroxyecdysone (20E). In Drosophila melanogaster L57 cells, the JHRE-regulated reporter gene was induced by JH I, JH III, methoprene, and hydroprene. Nuclear proteins isolated from L57 cells bound to the JHRE and exposure of these proteins to ATP resulted in a reduction in their DNA binding. Either JH III or calf intestinal alkaline phosphatase (CIAP) was able to restore the binding of nuclear proteins to the DNA. In addition, protein kinase C inhibitors increased and protein kinase C activators reduced the binding of nuclear proteins to the JHRE. In transactivation assays, protein kinase C inhibitors induced the luciferase gene placed under the control of a minimal promoter and the JHRE. These data suggest that protein kinase C mediated phosphorylation prevents binding of nuclear proteins to juvenile hormone responsive promoters resulting in suppression of JH action. PMID:16448742

  4. Peptide biosensors for the electrochemical measurement of protein kinase activity.

    PubMed

    Kerman, Kagan; Song, Haifeng; Duncan, James S; Litchfield, David W; Kraatz, Heinz-Bernhard

    2008-12-15

    The kinase activities are elucidated using the novel redox-active cosubstrate adenosine 5'-[gamma-ferrocene] triphosphate (Fc-ATP), which enables the kinase-catalyzed transfer of a redox active gamma-phosphate-Fc to a hydroxyamino acid. In this report, a versatile electrochemical biosensor is developed for monitoring the activity and inhibition of a serine/threonine kinase, casein kinase 2 (CK2), and protein tyrosine kinases, Abl1-T315I and HER2, in buffered solutions and in cell lysates. The method is based on the labeling of a specific phosphorylation event with Fc, followed by electrochemical detection. The electrochemical response obtained from the "ferrocenylated" peptides enables monitoring the activity of the kinase and its substrate, as well as the inhibition of small molecule inhibitors on protein phosphorylation. Kinetic information was extracted from the electrochemical measurements for the determination of K(m) and V(m) values, which were in agreement with those previously reported. Kinase reactions were also performed in the presence of well-defined inhibitors of CK2, 4,5,6,7-tetrabromo-2-azabenzimidazole, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, and E-3-(2,3,4,5-tetrabromophenyl)acrylic acid as well as the nonspecific kinase inhibitors, staurosporine and N-benzoylstaurosporine. On the basis of the dependency of the Fc signal on inhibitor concentration, K(i) of the inhibitors was estimated, which were also in agreement with the literature values. The performance of the biosensor was optimized including the kinase reaction, incubation with Fc-ATP, and the small molecule inhibitors. Peptide modified electrochemical biosensors are promising candidates for cost-effective in vitro kinase activity and inhibitor screening assays. PMID:18989981

  5. TFIIH kinase places bivalent marks on the carboxyl-terminal domain of RNA polymerase II

    PubMed Central

    Akhtar, M. Sohail; Heidemann, Martin; Tietjen, Joshua; Zhang, David; Chapman, Rob D.; Eick, Dirk; Ansari, Aseem Z.

    2009-01-01

    SUMMARY Post-translational modifications of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) specify a molecular recognition code that is deciphered by proteins involved in RNA biogenesis. The CTD is comprised of a repeating heptapeptide (Y1S2P3T4S5P6S7). Recently, phosphorylation of Serine7 was shown to be important for co-transcriptional processing of two snRNAs in mammalian cells. Here, we report that Kin28/Cdk7, a subunit of the evolutionarily conserved TFIIH complex, is a Ser7 kinase. The ability of Kin28/Cdk7 to phosphorylate Ser7 is particularly surprising because this kinase functions at promoters of protein-coding genes, rather than being restricted to promoter-distal regions of snRNA genes. Kin28/Cdk7 is also known to phosphorylate Ser5 residues of the CTD at gene promoters. Taken together, our results implicate the TFIIH kinase in placing bivalent Ser5 and Ser7 marks early in gene transcription. These bivalent CTD marks, in concert with cues within nascent transcripts, specify the co-transcriptional engagement of the relevant RNA processing machinery. PMID:19450536

  6. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.

    PubMed

    González-Fernández, Lauro; Macías-García, Beatriz; Loux, Shavahn C; Varner, Dickson D; Hinrichs, Katrin

    2013-06-01

    Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways. PMID:23595906

  7. Novel adenosine 3 prime ,5 prime -cyclic monophosphate dependent protein kinases in a marine diatom

    SciTech Connect

    Lin, P.P.C.; Volcani, B.E. )

    1989-08-08

    Two novel adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg{sup 2+} and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser({sup 32}P)-Ser-Asn-Ala-Arg and have an apparent M{sub r} of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M{sub r} of about 78,000 is photolabeled with 8-azido({sup 32}P)cAMP and is also phosphorylated with ({gamma}-{sup 32}P)ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids.

  8. Pyruvate Kinase M2 Regulates Gene Transcription by Acting as A Protein Kinase

    PubMed Central

    Gao, Xueliang; Wang, Haizhen; Jenny, J. Yang; Liu, Xiaowei; Liu, Zhi-Ren

    2012-01-01

    Summary Pyruvate kinase isoform M2 (PKM2) is a glycolysis enzyme catalyzing conversion of phosphoenolpyruvate (PEP) to pyruvate with transferring a phosphate from PEP to ADP. We report here that PKM2 localizes to the cell nucleus. The levels of nuclear PKM2 correlate with cell proliferation. PKM2 activates transcription of MEK5 by phosphorylating stat3 at Y705. In vitro phosphorylation assays show that PKM2 is a protein kinase using PEP as phosphate donor. ADP competes with the protein substrate binding, indicating that the substrate may bind to the ADP site of PKM2. Our experiments suggest that PKM2 dimer is an active protein kinase, while the tetramer is an active pyruvate kinase. Expression a PKM2 mutant that exists as a dimer promotes cell proliferation, indicating that protein kinase activity of PKM2 plays a role in promoting cell proliferation. Our study reveals an important link between metabolism alteration and gene expression during tumor transformation and progression. PMID:22306293

  9. Partial purification of a spinach thylakoid protein kinase that can phosphorylate light-harvesting chlorophyll a/b proteins

    SciTech Connect

    Clark, R.D.; Hind, G.; Bennett, J.

    1985-01-01

    Protein phosphorylation in plant tissues is particularly marked in chloroplasts, protein kinase activity being associated with the outer envelope, the soluble stromal fraction, and the thylakoid membrane. Furthermore, thylakoid-bound activity probably includes several distinct kinases, as suggested by studies of divalent cation specificity and thermal lability carried out with intact thylakoids and by subfractionation of solubilized membranes. Illumination of thylakoids, particularly with red light, promotes the rapid and extensive phosphorylation of the light-harvesting chlorophyll a/b complex (LHCII) on a threonine residue near the amino terminus of the protein. This phosphorylation is thought to be involved in regulating the distribution of absorbed quanta between photosystems II and I and is modulated by the redox state of the thylakoid plastoquinone pool. Neither of the thylakoid kinases reported to date was capable of phosphorylating purified LHCII in vitro or of incorporating phosphate into threonyl residues of exogenous substrates, that some LHCII phosphorylation was catalyzed by a preliminary fraction led workers to suggest that at least one other kinase remained to be isolated. Here, the authors report the solubilization and partial purification of a protein kinase from spinach thylakoids that is capable of phosphorylating LHCII in vitro, and they show that the specific site of phosphorylation is very nearly the same as, if not identical with, the site phosphorylated in organello.

  10. MyRIP anchors protein kinase A to the exocyst complex.

    PubMed

    Goehring, April S; Pedroja, Benjamin S; Hinke, Simon A; Langeberg, Lorene K; Scott, John D

    2007-11-01

    The movement of signal transduction enzymes in and out of multi-protein complexes coordinates the spatial and temporal resolution of cellular events. Anchoring and scaffolding proteins are key to this process because they sequester protein kinases and phosphatases with a subset of their preferred substrates. The protein kinase A-anchoring family of proteins (AKAPs), which target the cAMP-dependent protein kinase (PKA) and other enzymes to defined subcellular microenvironments, represent a well studied group of these signal-organizing molecules. In this report we demonstrate that the Rab27a GTPase effector protein MyRIP is a member of the AKAP family. The zebrafish homolog of MyRIP (Ze-AKAP2) was initially detected in a two-hybrid screen for AKAPs. A combination of biochemical, cell-based, and immunofluorescence approaches demonstrate that the mouse MyRIP ortholog targets the type II PKA holoenzyme via an atypical mechanism to a specific perinuclear region of insulin-secreting cells. Similar approaches show that MyRIP interacts with the Sec6 and Sec8 components of the exocyst complex, an evolutionarily conserved protein unit that controls protein trafficking and exocytosis. These data indicate that MyRIP functions as a scaffolding protein that links PKA to components of the exocytosis machinery. PMID:17827149