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

The site-specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N-bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71-122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97-Thr-Gly-Ala-Ser-Gly-Ser(PO4)-Phe-Lys105, supports the contention that basic amino acid residues C-terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.
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PMID:Identification of the phosphoserine residue in histone H1 phosphorylated by protein kinase C. 313 56

Protein kinase C, purified to near homogeneity from the brain, has been tested toward a variety of synthetic peptide substrates including different phosphorylatable residues. While it proved totally inactive toward the tyrosyl peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly, as well as toward several more or less acidic seryl peptides, it phosphorylates with a Ca2+/phospholipid-dependent mechanism, at seryl and/or threonyl residues, many basic peptides, some of which are also good substrates for cAMP-dependent protein kinase (A-kinase). Among the peptides tested, however, the best substrate for protein kinase C, with kinetic constants comparable to those of histones, is the nonapeptide Gly-Ser-Arg6-Tyr, which is not a substrate for A-kinase. Moreover, although the peptide Pro-Arg5-Ser-Ser-Arg-Pro-Val-Arg is a good substrate for both kinases, its derivative with ornitines replacing arginines is phosphorylated only by protein kinase C. Some typical substrates of A-kinase on the other hand, like the peptides Phe-Arg2-Leu-Ser-Ile-Ser-Thr-Glu-Ser and Arg2-Ala-Ser-Val-Ala, are phosphorylated by protein kinase C rather slowly and with unfavourable kinetic constants. It is concluded that, while both protein kinase C and A-kinase need basic groups close to the phosphorylatable residues, their primary structure determinants are quite distinct.
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PMID:Distinct structural requirements of Ca2+/phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase as evidenced by synthetic peptide substrates. 315 99

The synthetic lipopeptide Pam3Cys-Ser-Ser-Asn-Ala, an analogue of the N-terminal part of bacterial lipoprotein, constitutes a potent macrophage and B lymphocyte activator. In the macrophage cell-line P388D1 Pam3Cys-Ser-Ser-Asn-Ala stimulated phosphoinositol turnover, whereas in small resting lymphocytes no enhanced turnover was observed. Upon lipopeptide stimulation, a translocation of PKC from the cytosol to the plasma membrane was found in the cell line P388D1 but not in lymphocytes. Substituting lipopeptide for diacylglycerol shows that Pam3Cys-Ser-Ser-Asn-Ala leads to an activation of protein kinase C at Ca2+ concentrations of 0.5 mM. Thus, mitogenic lipopeptides constitute novel tools for investigating the molecular mechanism of transmembrane signaling in leukocyte activation.
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PMID:Phosphatidylinositol metabolism and protein kinase C activation in leukocytes by lipopeptides. 316 41

Soluble extracts prepared from quiescent Swiss mouse 3T3 cells that had been briefly exposed to various mitogens exhibited a 2- to 3-fold elevation in phosphorylating activities toward ribosomal protein S6 and a synthetic peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (RRLSSLRA), patterned after a phosphorylation site sequence from S6. Optimal activation of the phosphorylating activity occurred within 15-20 min of exposure of the cells to platelet-derived growth factor (10 ng/ml), epidermal growth factor (100 nM), and insulin (100 nM), and 2-5 min after 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nM) treatment. Fractionation of the cytosolic extracts from mitogen- or TPA-treated cells on Sephacryl S-300, TSK-400, and DEAE-Sephacel columns gave results suggesting that a single stimulated kinase accounted for the enhanced S6 and RRLSSLRA phosphorylating activities. The mitogen-activated kinase had an apparent Mr of about 85,000 as determined with Sephacryl S-300, but eluted with an apparent Mr of 26,000 from a TSK-400 high pressure liquid chromatography column. The S6 kinase was also stimulated in cytosols from insulin-like growth factor 1- (100 nM), vasopressin- (250 nM), prostaglandin F2 alpha- (250 nM), and 10% fetal calf serum-treated cells but not from quiescent cells exposed to beta-transforming growth factor (2 ng/ml). TPA, vasopressin and prostaglandin F2 alpha appeared to stimulate this kinase via a protein kinase C-dependent mechanism, since the responses to these hormones, but not to platelet-derived growth factor, epidermal growth factor, and insulin, were lost in protein kinase C-depleted cells.
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PMID:Mitogen-activated S6 kinase is stimulated via protein kinase C-dependent and independent pathways in Swiss 3T3 cells. 330 94

We studied the effects of protein kinase C (PKC) activators on LH glycosylation and release and the effect of 17 beta-estradiol on PKC activator-induced LH release. Rat anterior pituitary cells were incubated for 4 h with diluent, GnRH, and the PKC activators, phorbol 12-myristate 13-acetate (PMA), L-alpha-1,2-dioctanoyl glycerol (C8), and 1-oleoyl-2-acetyl-glycerol. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine ([14C]A) and [3H]glucosamine ([3H]GA), respectively, into total (medium + cell) immunoprecipitable LH. Immunoreactive LH (IRLH) was measured by RIA. PMA (10(-9) M) and 1-oleoyl-2-acetyl-glycerol (50-200 microM) had no significant effects. PMA at 10(-7) M elevated (P less than 0.01) medium IRLH, medium and total [3H]GA-LH, and medium but not total [14C]A-LH. PMA at 10(-7) M increased (P less than 0.01) uptake and incorporation of [3H]GA, but not [14C]A, into total pituitary protein. C8 increased both medium IRLH and total [3H]GA-LH (P less than 0.01) without altering total [14C]A-LH. Two hundred micromolar C8 increased medium concentrations of [3H]GA-LH (P less than 0.01) and [14C]A-LH (P less than 0.05). C8 (50-200 microM) had no detectable effects on uptake and incorporation of precursors into protein. GnRH (1 nM) enhanced (P less than 0.01) both medium IRLH and total [3H]GA-LH, but had no effect on total [14C]A-LH. Pretreatment of pituitary cells with 17 beta-estradiol (6 X 10(-10) M) greatly enhanced LH release induced by C8. In conclusion, PMA and C8, like GnRH, stimulated both LH glycosylation and release. These results suggest that PKC may regulate both LH release and glycosylation and may be important in estrogen modulation of LH release.
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PMID:Stimulation by phorbol ester and diacylglycerol of luteinizing hormone glycosylation and release by rat anterior pituitary cells. 331 14

Ca2+/phospholipid-dependent protein kinase (protein kinase C) and trypsin-activated protein kinase C (protein kinase M) phosphorylated the synthetic peptide R1-A13 (Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala-Ser-Thr-Ser-Lys-Ala) which contains both cAMP- and insulin-regulated phosphorylation sites in rat liver ribosomal protein S6 [Wettenhall, R. E. H. & Morgan, F. J. (1984) J. Biol. Chem. 259, 2084-2091]. Both enzymes showed essentially the same kinetic properties; V and apparent Km were determined to be 0.16 mumol min-1 mg-1 and 30 microM, respectively. At first, tryptic phosphopeptides were prepared at the early stage of phosphorylation and purified by high-performance liquid chromatography (HPLC). Through these analyses, four radioactive peptides were isolated. When protein kinase C was employed, phosphorylation was observed on all four peptides in a Ca2+/phospholipid-dependent manner. Irrespective of the protein kinase employed, phosphate incorporation into these peptides increased linearly with time; the peptide concentration did not affect the ratio of phosphate distribution into these four peptides. Analysis of amino acid composition and phosphoamino acid of radioactive peptides obtained after extensive phosphorylation showed that phosphates were incorporated into Ser-4, Ser-5, Ser-9 and Ser-11. The latter three serine residues were major phosphorylated sites. When rat liver 40-S ribosomal subunits were employed as substrate for protein kinases C and M, a radioactive protein with Mr,app = 31,000, which corresponded to S6 protein, was detected on an autoradiogram of a sodium dodecyl sulfate/polyacrylamide slab gel. The rate of phosphorylation with protein kinase M was twice as fast as that with protein kinase C. The elution profile of radioactive tryptic peptides in HPLC suggest that phosphorylation occurred on the sites in S6 protein corresponding to Ser-5, Ser-9 and Ser-11 as major sites and Ser-4 as the minor one. These results indicate that protein kinase C has an ability to recognize at least four sites derived from hormone-dependent phosphorylation sites in ribosomal protein S6 irrespective of the mode of activation of this enzyme.
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PMID:Comparative studies on phosphorylation of synthetic peptide analogue of ribosomal protein S6 and 40-S ribosomal subunits between Ca2+/phospholipid-dependent protein kinase and its protease-activated form. 331 52

A phosphoprotein expressed in rat brain is closely related to the 80 kDa substrate of protein kinase C present in 3T3 cells. The protein kinase C substrates from both sources migrate identically on two-dimensional gel electrophoresis and give similar phosphopeptide fragments when digested with protease. Using a series of chromatographic steps, including DEAE-cellulose chromatography, Sephadex G150 gel filtration and reverse phase fast protein liquid chromatography, this phosphoprotein was purified 3800-fold from rat brain. The preparation appears homogenous by one- and two-dimensional gel electrophoresis, is an effective substrate of protein kinase C and contains a high proportion of the acidic amino acids glutamate and aspartate, and of alanine.
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PMID:Purification of a phosphoprotein from rat brain closely related to the 80 kDa substrate of protein kinase C identified in Swiss 3T3 fibroblasts. 336 Jan 39

The epidermal growth factor (EGF) receptor is a substrate for phosphorylation by the calcium- and phospholipid-dependent protein kinase (protein kinase C) at Thr654. The hypothesis that this phosphorylation is causally related to the regulation of the functional properties of the EGF receptor was tested by substitution of Thr654 with an alanine residue. Activation of protein kinase C using phorbol ester caused a decrease in the high affinity binding of EGF to Chinese hamster ovary cells expressing wild-type [Thr654]EGF receptors. Similar results were obtained with cells expressing mutated [Ala654]EGF receptors. The regulation of the protein kinase activity of the EGF receptor by protein kinase C was examined using a synthetic peptide substrate for tyrosine phosphorylation. Protein kinase C caused a Ca2+-dependent decrease in the tyrosine-protein kinase activity of the wild-type [Thr654]EGF receptor. In contrast, no inhibition of the tyrosine-protein kinase activity of the mutated [Ala654]EGF receptor caused by protein kinase C was detected. In further experiments, the desensitization of EGF action caused by the activation of protein kinase C was examined by investigating the regulation of the transferrin receptor by EGF. Phorbol ester was observed to cause the desensitization of signaling by the wild-type [Thr654] and mutated [Ala654]EGF receptors. These data are consistent with a role for the phosphorylation of EGF receptor Thr654 in the regulation of the receptor tyrosine-protein kinase activity. However, the inhibition of the high affinity binding of EGF to cell-surface receptors caused by protein kinase C does not require Thr654. It is concluded that independent mechanisms account for the regulation by protein kinase C of the EGF receptor affinity and tyrosine-protein kinase activity.
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PMID:Independent mechanisms account for the regulation by protein kinase C of the epidermal growth factor receptor affinity and tyrosine-protein kinase activity. 337 75

The 87-kDa protein, a major specific substrate for protein kinase C, has been purified 500-fold to apparent homogeneity from bovine forebrain supernatant. The purification procedure included batch adsorption to DE-52 (DEAE-cellulose), (NH4)2SO4 precipitation, and chromatography on DEAE-Sephacel, Bio-Gel HTP (hydroxylapatite), Sephacryl S-400, and fast protein liquid chromatography ProRPC. The amino acid composition was notable for its high proportion of alanine (28.6 mol%) and its enrichment in glutamate/glutamine (18.1 mol%), glycine (12.6 mol%), and proline (11.3 mol%). The partial specific volume was 0.702 ml/g; the Stokes radius and sedimentation coefficient were 85 A and 2.11 S, respectively. Although the relative molecular mass of the protein on NaDodSO4/8% PAGE was 87-90 kDa, the molecular mass as determined from the above values was 68 kDa. The frictional ratio was 3.2, and the axial ratio was 60, indicating that the 87-kDa protein is an extremely elongated monomer. The purified 87-kDa protein was phosphorylated by purified protein kinase C to a stoichiometry of 2.2 mol of 32P per mol of 87-kDa protein (calculated using a value of 68 kDa for the molecular mass). Phosphorylation was exclusively on serine residues.
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PMID:The 87-kDa protein, a major specific substrate for protein kinase C: purification from bovine brain and characterization. 347 78

We have investigated the role of phosphorylation in the endocytosis of the human transferrin receptor (TR) by replacing its phosphorylation site, Ser24, with Ala through site-directed mutagenesis of the TR cDNA. The TR Ala24 mutant expressed in mouse 3T3 cells was not phosphorylated, even following stimulation of protein kinase C by phorbol ester. However, in spite of this defect the mutant was efficiently endocytosed and recycled back to the plasma membrane with kinetics similar to those of TR and a control mutant TR Ala63. Thus, these results confirm earlier results by Davis et al. (1986, J. Biol. Chem., 261-9034-9041) that Ser24 of human TR is the phosphorylation site for protein kinase C but do not support a role of this modification as a signal for TR endocytosis and recycling.
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PMID:Phosphorylation of the human transferrin receptor by protein kinase C is not required for endocytosis and recycling in mouse 3T3 cells. 347 28


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