Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cDNA clone corresponding to the Dictyostelium myosin heavy chain kinase (MHCK) gene was isolated using antibodies specific to the purified enzyme. Sequence analysis of the cDNA revealed that the Dictyostelium MHCK possesses all of the domains characteristic of members of the protein kinase C family. The amino-terminal region of the MHCK contains the cysteine-rich motif with an internal duplication that is present in all known protein kinase C species. This domain precedes sequences that are highly homologous to protein kinase catalytic domains. The carboxyl-terminal region contains a cluster of 23 serine and threonine residues that may represent the autophosphorylation domain of the Dictyostelium MHCK. These results, along with previous studies that indicate that this enzyme has very restrictive substrate specificity, incorporates approximately 20 mol of phosphate per mol of kinase through an autophosphorylation reaction, and is expressed only during development, suggest that the Dictyostelium MHCK is a distinct member of the protein kinase C family and imply that this kinase family, which may include members with very specific cellular functions, may be even more heterogeneous than previously thought.
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PMID:Membrane-bound Dictyostelium myosin heavy chain kinase: a developmentally regulated substrate-specific member of the protein kinase C family. 132 27

Nerve growth factor (NGF) binds to two structurally unrelated transmembrane proteins on the surface of PC-12 cells, a 75-kDa glycoprotein with a short cytoplasmic sequence, and the trk protooncogene (pp140c-trk), a protein tyrosine kinase activated by NGF. Immediately after binding to cells, NGF induces changes in serine/threonine phosphorylation of several proteins. We have explored the relative roles of these two NGF binding proteins in mediating the activation of two intracellular kinases that may be responsible for some of these phosphorylations. The raf-1 protooncogene is a serine/threonine kinase activated by several growth factors and oncogenic proteins. Treatment of PC-12 cells with NGF increases the serine and threonine phosphorylation of raf-1 in an anti-raf-1 immunoprecipitate kinase assay. This increased phosphorylation observed in vitro is dose-dependent and transient and is accompanied by the NGF-dependent shift in the mobility of immunoblotted raf-1 on SDS sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an effect thought to reflect phosphorylation. NGF-dependent activation of raf-1 is not dependent on protein kinase C, since prolonged exposure to phorbol esters under conditions that cause down-regulation of cellular protein kinase C activity has no effect on the NGF response. Expression of pp140c-trk in 3T3 fibroblasts (3T3-c-trk), as evidenced by cross-linking of 125I-NGF to the 140-kDa protein, permits the NGF-dependent activation of raf-1 kinase, detected in the immunoprecipitate kinase assay, anti-raf immunoblot shift on gel electrophoresis, and incorporation of [32P]orthophosphate into the raf-1 protein. The concentration dependence of raf-1 activation is identical in 3T3-c-trk and PC-12 cells, despite the absence of the 75-kDa NGF binding protein in 3T3-c-trk cells. NGF is without effect in untransfected 3T3 cells or in Chinese hamster ovary cells overexpressing p75, although raf-1 is present in these cells. Similarly, the NGF-dependent activation of mitogen-activated protein (MAP) kinase is detected in 3T3-c-trk cells, but not in untransfected 3T3 or Chinese hamster ovary cells overexpressing p75. As described for raf-1 activation, the NGF dose responses for MAP kinase activation in 3T3-c-trk and PC-12 cells are virtually superimposable. These data indicate that the activation of these two serine/threonine kinases by NGF is mediated solely by binding to and activating the pp140c-trk receptor.
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PMID:Nerve growth factor stimulates the activities of the raf-1 and the mitogen-activated protein kinases via the trk protooncogene. 132 11

Purified bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) showed two bands with subunit M(r) of 58,000 and 54,000 when analysed by SDS/PAGE. Both the 58,000- and 54,000-M(r) forms were phosphorylated by cyclic AMP-dependent protein kinase (PKA) and by protein kinase C (PKC) in vitro. Phosphorylation by PKA decreased the apparent Km of PFK-2 for one of its substrates, fructose 6-phosphate, while phosphorylation by PKC did not correlate with any change in PFK-2 activity. The differences between the 58,000- and 54,000-M(r) forms were studied by electroblotting, peptide mapping and microsequencing. Residues 451-510, which correspond to exon 15 in the rat and contain phosphorylation sites for PKA (Ser-466) and PKC (Thr-475), were absent from the 54,000-M(r) form. Peptide mapping after phosphorylation by [gamma-32P]MgATP and PKC showed a phosphorylated peptide containing Thr-475, which was present in the 58,000-M(r) form but not in the 54,000-M(r) form. The fact that the latter form was phosphorylated by PKC and PKA suggests that other phosphorylation sites for PKA and PKC are located outside the region encoded by exon 15. Finally, analysis of RNA from bovine heart showed that the tissue contains two PFK-2/FBPase-2 mRNAs, only one of which was recognized by a probe specific to the region coding for Ser-466 and Thr-475. Taken together, these findings demonstrate that the 58,000- and 54,000-M(r) forms of bovine heart PFK-2/FBPase-2 result from alternative splicing of the same primary transcript.
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PMID:The two forms of bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase result from alternative splicing. 132 30

Nerve growth factor-stimulated mitogen-activated protein kinase (pp42/44MAP) kinase was characterized by sequential column chromatography on DEAE-Sephacel, phenyl-Sepharose CL4B, and S-200. The kinase displayed an apparent molecular mass of 42 kDa and reacted with an antiphosphotyrosine antibody. Peptide mapping of myelin basic protein revealed the presence of one phosphopeptide that was phosphorylated on Thr-97. pp42/44MAP kinase activity was dependent on Mg2+ and inhibited by K252a both in vitro and in vivo. Nerve growth factor-stimulated kinase activation was diminished by down-regulation of protein kinase C with 200 nM 12-phorbol 13-myristate acetate or with staurosporine (1 nM), a protein kinase C inhibitor. Genistein, a protein tyrosine kinase inhibitor, blocked nerve growth factor-mediated neurite extension as well as diminished activation of pp42/44MAP kinase. Our data demonstrate that activation of this kinase system by nerve growth factor displays a requirement for both protein kinase C as well as protein tyrosine kinase. In addition, other agents that are capable of promoting neurite outgrowth in PC12 cells, such as fibroblast growth factor or dibutyryl cyclic AMP, do so independently of activating this kinase system.
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PMID:pp42/44MAP kinase is a component of the neurogenic pathway utilized by nerve growth factor in PC12 cells. 132 67

The binding of a variety of agonists to their receptors leads to the breakdown of membrane phospholipids and the formation of intracellular second messengers. Hydrolysis of inositol phospholipids by phospholipase C results in the formation of two second messengers, inositol-1,4,5-trisphosphate which mobilizes intracellular calcium and the neutral lipid diacylglycerol (DAG) which binds to and activates protein kinase C (PKC). PKC is actually a family of homologous serine/threonine protein kinases which play a central role in regulation of growth, differentiation and secretion reactions in a variety of cell types. In addition to these feedforward roles of PKC, it is thought to play an important feedback role, regulating early events in signal transduction. To explore these feedback functions we have examined the effect of PKC inhibitors on second messenger formation in thrombin-stimulated human platelets (a rapidly responding system) and the effect of PKC overexpression on second messenger formation and mitogenesis in rat fibroblasts (a system where sustained signaling occurs). Treatment of platelets with inhibitors of PKC potentiates DAG mass formation in response to thrombin while prior activation of PKC with phorbol esters blocks DAG mass formation, consistent with PKC playing a negative feedback role, inhibiting inositol phospholipid breakdown. DAG can also be formed by the sequential hydrolysis of phosphatidylcholine by phospholipase D and phosphatidic acid phosphohydrolase. This is a minor reaction in the rapidly responding platelet system, but may play a role in sustained signaling events. We have found that fibroblasts which overexpress the beta 1 isozyme of PKC display greatly enhanced DAG formation and phospholipase D activation in response to phorbol ester treatment. Upon stimulation of fibroblasts with thrombin, phospholipase D activation is also enhanced by PKC overexpression while formation of inositol phosphates is suppressed. These data suggest that PKC may act as a switch, terminating inositol phospholipid hydrolysis and activating the hydrolysis of phosphatidylcholine. Furthermore, we have observed a strong correlation between activation of phospholipase D and mitogenesis, suggesting an important role for this enzyme in long-term cellular responses to activation.
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PMID:Regulation of phospholipid hydrolysis and second messenger formation by protein kinase C. 132 4

In cultured vascular smooth muscle cells, angiotensin II (Ang II) stimulated a cytosolic protein kinase activity toward myelin basic protein (MBP) in a time- and dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate also increased the MBP kinase activity. Downregulation of protein kinase C by prolonged treatment of the cells with phorbol 12,13-dibutyrate markedly attenuated the Ang II- and PMA-induced MBP kinase activation. The Ang II- and PMA-stimulated MBP kinase activities were resolved almost equally into two distinct fractions on Mono-Q HR5/5 column chromatography (kinase 1 and kinase 2). The kinase assay in polyacrylamide gel revealed that apparent molecular masses of kinase 1 and kinase 2 were 40 and 45 kd, respectively. Microtubule-associated protein 2 also served as a substrate for both the kinases. Immunoblot analysis with an antiphosphotyrosine antibody suggested that both the kinases were tyrosine-phosphorylated during the action of Ang II. Phosphoamino acid analysis revealed that Ang II and PMA induced phosphorylation of both the kinases on serine/threonine as well as tyrosine residues. Phosphopeptide mapping patterns of kinase 1 and kinase 2 isolated from Ang II-stimulated cells were almost identical with those from PMA-stimulated cells. These results indicate that in vascular smooth muscle cells Ang II activates two species of MBP/microtubule-associated protein 2 kinases mainly through the protein kinase C-signaling pathway and suggest that tyrosine and serine/threonine phosphorylation may be involved in this process.
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PMID:Angiotensin II stimulates two myelin basic protein/microtubule-associated protein 2 kinases in cultured vascular smooth muscle cells. 132 34

We have demonstrated that the alpha 2,3 sialyltransferase (alpha 2,3 ST) from C6 cultured glioma cells was inhibited in vivo by W-7 and related Ca2+/Calmodulin (Ca/CaM) antagonists while protein kinase C effectors had no effect. Dephosphorylation of alpha 2,3 ST by the wide specificity alkaline phosphatase led to inactivation indicating that the enzyme is phosphorylated. The serine/threonine protein phosphatase inhibitors okadaic acid and Calyculin A led also to an inhibition of alpha 2,3 ST activity. In addition, Ca/CaM antagonists and phosphatase inhibitors led both to an inhibition of a alpha 2,3 sialoglycoprotein from C6 glioma cells as demonstrated with lectin affinity blotting. A concerted regulatory mechanism with phosphorylation/dephosphorylation of alpha 2,3 ST is then postulated.
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PMID:Study of O-glycan sialylation in C6 cultured glioma cells: regulation of a beta-galactoside alpha 2,3 sialyltransferase activity by Ca2+/calmodulin antagonists and phosphatase inhibitors. 132 69

Recent evidence suggests that the cytoplasmic domains of platelet glycoprotein (GP) IIb-IIIa are involved in the agonist-initiated transformation of this integrin into a receptor for fibrinogen. To identify intracellular reactions that regulate the receptor function of GP IIb-IIIa, membrane-impermeable agonists and antagonists were introduced into the platelet by permeabilizing the plasma membrane with the pore-forming complement proteins C5b-9. Platelet responses were then analyzed by flow cytometry. Non-lytic concentrations of C5b-9 caused permeabilization of the platelet plasma membrane, as determined by uptake of a water-soluble fluorescent tracer dye. The complement pores were large enough to permit the entry of fluorescein isothiocyanate (FITC)-labeled oligopeptides in a size-dependent manner. Under conditions of low external Ca2+, C5b-9 treatment per se did not activate GP IIb-IIIa, as measured by binding of the activation-dependent antibody FITC-PAC1. However, FITC-PAC1 binding to C5b-9-permeabilized platelets was stimulated by a thrombin receptor agonist acting at the cell surface and by guanosine 5'-O-(thiotriphosphate), a membrane-impermeable activator of G proteins. Permeabilization also permitted the entry of cyclic AMP and the peptide, RFARKGALRQKNV, a pseudo-substrate inhibitor of protein kinase C. Each of these inhibited agonist-induced FITC-PAC1 binding to permeabilized platelets but not to intact platelets. Agonist-induced GP IIb-IIIa activation in permeabilized platelets was also inhibited by tyrphostin-23, a protein tyrosine kinase inhibitor. Thus, C5b-9 can be used to permeabilize the plasma membrane to permit the selective entry of small peptides and other bioactive compounds into permeabilized platelets. Results obtained with these platelets indicate that GP IIb-IIIa receptor function is regulated by a network of signaling reactions involving G proteins, serine/threonine kinases, and tyrosine kinases.
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PMID:Regulation of glycoprotein IIb-IIIa receptor function studied with platelets permeabilized by the pore-forming complement proteins C5b-9. 132 24

Erythropoietin stimulation of erythroid cells induces a rapid increase in c-myc and decrease in c-myb mRNA levels. The signal pathway to c-myc requires activation of protein kinase C. We now report that erythropoietin down-regulates expression of c-myb via a discrete, serine/threonine-specific phosphatase-dependent pathway. The protein kinase C-blocker H7 completely prevents the c-myc response to erythropoietin, but has no effect on the c-myb response. In contrast, the phosphatase blocker okadaic acid prevents the c-myb response but not the c-myc response. This effect of okadaic acid on the c-myb response is concentration-dependent. Both the protein kinase C-dependent signal to c-myc and the phosphatase-dependent signal to c-myb regulate gene expression by a transcriptional arrest mechanism operative within the first intron of the respective protooncogenes. In contrast, the chemical inducer of differentiation, dimethyl sulfoxide, regulates expression of c-myc and c-myb without activation of these phosphatase- and kinase-dependent pathways.
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PMID:Activation of two discrete signaling pathways by erythropoietin. 132 29

c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.
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PMID:Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts. 132 60


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