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At least 19 recordsLinked to original sources
PubMed1988

Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites.

Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting the Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C.

Amino Acid Sequence↗
PubMed1995

Phosphorylation of casein kinase II by p34cdc2. Identification of phosphorylation sites using phosphorylation site mutants in vitro.

The alpha and beta subunits of casein kinase II are dramatically phosphorylated in cells that are arrested in mitosis (Litchfield, D. W., Lüscher, B., Lozeman, F. J., Eisenman, R. N., and Krebs, E.G. (1992) J. Biol. Chem. 267, 13943-13951). Comparative phosphopeptide mapping experiments indicated that the mitotic phosphorylation sites on the alpha subunit of casein kinase II can be phosphorylated in vitro by p34cdc2. In the present study, we have demonstrated that a glutathione S-transferase fusion protein encoding the C-terminal 126 amino acids of the alpha subunit is phosphorylated by p34cdc2 at the same sites as intact casein kinase II, indicating that the mitotic phosphorylation sites are localized within the C-terminal domain of alpha. Four residues within this domain, Thr-344, Thr-360, Ser-362, and Ser-370, conform to the minimal consensus sequence for p34cdc2 phosphorylation. Synthetic peptides corresponding to regions of alpha that contain each of these residues are phosphorylated by p34cdc2 at these sites. Furthermore, alterations in the phosphorylation of the glutathione S-transferase proteins encoding the C-terminal domain of alpha are observed when any of the four residues are mutated to alanine. When all four residues are mutated to alanine, the fusion protein is no longer phosphorylated by p34cdc2 at any of the sites that are phosphorylated in mitotic cells. These results indicate that Thr-344, Thr-360, Ser-362, and Ser-370 are the sites on the alpha subunit of casein kinase II that are phosphorylated in mitotic cells.

Amino Acid Sequence↗
PubMed1986

Phosphorylation of the transforming protein of Rous sarcoma virus: direct demonstration of phosphorylation of serine 17 and identification of an additional site of tyrosine phosphorylation in p60v-src of Prague Rous sarcoma virus.

We provide direct evidence that serine 17 is the major site of serine phosphorylation in p60v-src, the transforming protein of Rous sarcoma virus, and in its cellular homolog, p60c-src. The amino acid composition of the tryptic peptide containing the major site of serine phosphorylation in p60v-src was deduced by peptide map analysis of the protein labeled biosynthetically with a variety of radioactive amino acids. Manual Edman degradation revealed that the phosphorylated serine in this peptide was the amino terminal residue. These data are consistent only with the phosphorylation of serine 17. The major site of serine phosphorylation in chicken p60c-src, the cellular homolog of p60v-src, is contained in a tryptic peptide identical to that containing serine 17 in p60v-src of Schmidt Ruppin Rous sarcoma virus of subgroup A. Serine 17 is therefore also phosphorylated in p60c-src. The p60v-src protein encoded by Prague Rous sarcoma virus was found to contain two sites of tyrosine phosphorylation. The previously unrecognized site of tyrosine phosphorylation may be tyrosine 205 or possibly tyrosine 208. Treatment of Prague Rous sarcoma virus-infected cells with vanadyl ions stimulated the protein kinase activity of p60v-src and increased the phosphorylation of tyrosine 416 but not the phosphorylation of the additional site of tyrosine phosphorylation.

Amino Acid Sequence↗
PubMed1987

Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase. Phosphorylation sites and effects of phosphorylation.

Smooth muscle heavy meromyosin (HMM) is phosphorylated by the Ca2+-activated phospholipid-dependent protein kinase, i.e. protein kinase C, at three sites on each 20,000-dalton light chain. Phosphorylation of three sites also is observed with isolated 20,000-dalton light chain and HMM subfragment 1. The phosphorylation sites are serine 1, serine 2, and threonine 9. Threonine is phosphorylated most rapidly followed by either serine 1 or 2. Phosphorylation of the third site occurs only on prolonged incubation. Phosphorylation is a random process. HMM phosphorylated at two sites per light chain by protein kinase C can be dephosphorylated, as shown using two phosphatase preparations. Increasing levels of phosphorylation of HMM by protein kinase C causes a progressive inhibition of the subsequent rate of phosphorylation of serine 19 by myosin light chain kinase and causes a progressive inhibition of actin-activated ATPase activity of HMM, prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of HMM for actin rather than a change in Vmax. Previous results with HMM and protein kinase C (Nishikawa, M., Sellers, J. R., Adelstein, R. S., and Hidaka, H. (1984) J. Biol. Chem. 259, 8808-8814) examined effects induced by phosphorylation of the threonine residues. Our results confirm these and consider also the influence of higher levels of phosphorylation by protein kinase C.

Amino Acid Sequence↗
PubMed1998

Phosphorylation of inositol 1,4,5-trisphosphate receptors by cAMP-dependent protein kinase. Type I, II, and III receptors are differentially susceptible to phosphorylation and are phosphorylated in intact cells.

The ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP3) receptors was examined. The receptors either were immunopurified from cell lines and then phosphorylated with purified PKA or were phosphorylated in intact cells after activating intracellular cAMP formation. The former studies showed that the type I receptor was a good substrate for PKA (0.65 mol Pi incorporated/mol receptor), whereas type II and III receptors were phosphorylated relatively weakly. The latter studies showed that despite these differences, each of the receptors was phosphorylated in intact cells in response to forskolin or activation of neurohormone receptors. Detailed examination of SH-SY5Y neuroblastoma cells, which express >/=99% type I receptor, revealed that minor increases in cAMP concentration were sufficient to cause maximal phosphorylation. Thus, VIP and pituitary adenylyl cyclase activating peptide (acting through Gs-coupled pituitary adenylyl cyclase activating peptide-I receptors) were potent stimuli of type I receptor phosphorylation, and remarkably, even slight increases in cAMP concentration induced by carbachol (acting through Gq-coupled muscarinic receptors) or other Ca2+ mobilizing agents were sufficient to cause phosphorylation. Finally, PKA enhanced InsP3-induced Ca2+ mobilization in a range of permeabilized cell types, irrespective of whether the type I, II, or III receptor was predominant. In summary, these data show that all InsP3 receptors are phosphorylated by PKA, albeit with marked differences in stoichiometry. The ability of both Gs- and Gq-coupled cell surface receptors to effect InsP3 receptor phosphorylation by PKA suggests that this process is widespread in mammalian cells and provides multiple routes by which the cAMP signaling pathway can influence Ca2+ mobilization.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.

The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration.

Amino Acid Sequence↗
PubMed1996

Phosphorylation of proteasomes in mammalian cells. Identification of two phosphorylated subunits and the effect of phosphorylation on activity.

The proteasome, a multimeric protease, plays an important role in nonlysosomal pathways of intracellular protein degradation. This study was undertaken to determine which subunits of mammalian proteasomes are phosphorylated and to investigate the possible role of phosphorylation in regulating proteasome activity and the association with regulatory components. Rat-1 fibroblasts were grown in the presence of [32P]phosphate and proteasomes were immunoprecipitated from cell lysates with proteasome-specific polyclonal antibodies. Subsequent analysis by two-dimensional polyacrylamide gel electrophoresis showed two radiolabeled proteasome subunits which were identified using monoclonal antibodies as C8 and C9. Treatment of human embryonic lung cells (L-132), under identical conditions, also showed the same two phosphorylated subunits. Phosphoamino acid analysis revealed phosphoserine to be present in both C8 and C9. Examination of the sequence of C9 showed a potential cGMP-dependent phosphorylation site (-Arg3-Arg-Tyr-Asp-Ser-Arg8-), whilst C8 contains several potential casein kinase II phosphorylation sites. Following immunoprecipitation by a monoclonal antibody and dephosphorylation by acid phosphatase, proteasomes were observed to have significantly lower activities when compared to phosphorylated proteasomes, implying that phosphorylation may be an important mechanism of regulating proteasome function. Free proteasomes were separated by gel-filtration from those complexed with regulatory complexes to form the 26S proteinase. The ratio of phosphorylation of C8 and C9 was found to be very similar in the two complexes but the level of phosphorylation was higher in the 26S proteinase than in free proteasomes.

Acid Phosphatase↗
PubMed2000

Platelet secretion induced by phorbol esters stimulation is mediated through phosphorylation of MARCKS: a MARCKS-derived peptide blocks MARCKS phosphorylation and serotonin release without affecting pleckstrin phosphorylation.

Previous experiments suggest that actin disassembly, perhaps at a specific site, is required for platelet secretion. Platelet stimulation by phorbol 12-myristate 13-acetate (PMA) induced pleckstrin phosphorylation, platelet aggregation, and secretion. Inhibition of protein kinase C (PKC) is accompanied by inhibition of pleckstrin phosphorylation and serotonin secretion. Here, we demonstrate the presence of myristoylated alanine-rich C kinase substrate (MARCKS), another PKC substrate, in platelets and its phosphorylation during PMA stimulation. MARCKS is known to bind actin and to cross-link actin filaments; the latter is inhibited by PKC-induced MARCKS phosphorylation. MARCKS phosphorylation and serotonin release from permeabilized platelets have the same time course and were blocked by a peptide (MPSD) with the amino acid sequence corresponding to the phosphorylation site domain of MARCKS. Pleckstrin and myosin light chain phosphorylation was not modified. A peptide (Ala-MPSD) in which the four serine residues of MPSD were substituted by alanines was ineffective. These results provide the first evidence that MARCKS may play a role in platelet secretion. Moreover, pleckstrin phosphorylation has a different time course than that of MARCKS or serotonin release and was not modified when MARCKS phosphorylation and serotonin release were inhibited, suggesting that pleckstrin is either not directly involved in secretion or that it might only be involved upstream in the cascade of events leading to exocytosis.

Amino Acid Sequence↗
PubMed1981

Phosphorylation of rat ascites tumor non-histone chromatin proteins. Differential phosphorylation by two cyclic nucleotide-independent protein kinases and comparison to in vivo phosphorylation.

The substrate specificity of two cyclic nucleotide-independent protein kinases purified from both rat ascites tumor cells and calf thymus has been examined. The protein kinases, designated casein kinase I and II, were purified to near homogeneity and are free of contaminating protein substrates. Non-histone chromatin proteins, purified from Novikoff ascites tumor cells and free of protein kinase and activity, were phosphorylated with casein kinases I and II and the spectrum of labeled polypeptides determined. Phosphorylation of non-histone chromatin proteins with casein kinase I from either source, resulted in the same pattern of 32P-labeled polypeptides when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Similarly, casein kinase II from either source phosphorylated an identical set of polypeptides. The distribution of 32P-labeled non-histone chromatin proteins phosphorylated by casein kinase I differed from that labeled by casein kinase II suggesting that each protein kinase phosphorylates a specific subset of non-histone chromatin proteins. The differences in protein substrate specificity of casein kinase I and casein kinase II was confirmed by two-dimensional polyacrylamide gel electrophoresis. Non-histone chromatin proteins, labeled with 32P in vivo, were analyzed by the same methods and the distribution of labeled polypeptides compared to in vitro phosphorylation patterns. A number of prominent in vitro phosphorylated polypeptides appear identical with non-histone chromatin proteins labeled with 32P in vivo. These results suggest that casein kinases I and II may function in vivo in the phosphorylation of non-histone chromatin proteins.

Animals↗
PubMed1993

Phosphorylation of serine 208 in the human vitamin D receptor. The predominant amino acid phosphorylated by casein kinase II, in vitro, and identification as a significant phosphorylation site in intact cells.

The human 1,25-dihydroxyvitamin D3 receptor (hVDR), like other members of the steroid/thyroid receptor superfamily, has been observed to undergo rapid phosphorylation. We report here that the hVDR is a substrate for casein kinase II (CK-II), a regulatory enzyme of significance in the function of nuclear proteins. Intact hVDRs produced by in vitro transcription/translation or in a baculovirus overexpression system served as efficient substrates for purified bovine CK-II, and the magnitude of this phosphorylation was not affected by the addition of 1,25-dihydroxyvitamin D3. CK-II-catalyzed phosphorylation of truncated hVDRs suggested that phosphorylated residues may occur between Arg121 and Asp232, including the region of hVDR which we have previously demonstrated to contain a major site(s) of phosphorylation in intact cells (Jones, B.B., Jurutka, P.W., Haussler, C.A., Haussler, M.R., and Whitfield, G.K. (1991) Mol. Endocrinol. 5, 1137-1146). Site-directed mutagenesis of serine/threonine residues in this region now reveals a site of phosphorylation at Ser208 contained within the sequence -S208 (P)EEDSDD-, a classic CK-II consensus recognition site. Mutation of this serine to a glycine drastically reduces phosphorylation of hVDR by CK-II, in vitro. The Ser208 mutant receptor also shows a dramatic decrease in [32P]orthophosphate incorporation when transfected into COS-7 cells. We therefore propose that phosphorylation of hVDR at Ser208 in target cells is mediated by casein kinase II or a similar enzyme, and that this quantitatively significant post-translational modification is a potential mechanism for the modulation of the activity of hVDR in controlling gene transcription.

Amino Acid Sequence↗
PubMed1994

CD40 signaling pathway: anti-CD40 monoclonal antibody induces rapid dephosphorylation and phosphorylation of tyrosine-phosphorylated proteins including protein tyrosine kinase Lyn, Fyn, and Syk and the appearance of a 28-kD tyrosine phosphorylated protein.

CD40 plays an important role in B cell activation, proliferation, and Ig class switching. The signal transduction pathway mediated by CD40 was studied using monoclonal antibody (mAb) 626.1 to CD40. Burkitt's lymphoma and Epstein-Barr virus-transformed B cell lines and tonsilar B lymphocytes were treated with the anti-CD40 mAb for various lengths of time. The early events triggered by CD40 were examined by monitoring the changes in tyrosine phosphorylation of cellular proteins with anti-phosphotyrosine mAb. Dephosphorylation of specific proteins ranging between 50-110 kD and the appearance of a 28-kD tyrosine phosphorylated protein were seen within 30 s in human B cell lines. The dephosphorylation was reversed and the 28-kD protein was dephosphorylated in cells stimulated for 1 min. In resting B cells, the appearance of the 28-kD phosphoprotein was observed in 30 s after the addition of the anti-CD40 mAb. The tyrosine phosphorylation of this protein persisted. The patterns of protein tyrosine phosphorylation differed from those induced by an anti-immunoglobulin M mAb. The changes in the state of tyrosine phosphorylation induced by the anti-CD40 mAb were obviated by mAb to CD45, a protein tyrosine phosphatase (PTP) or by the addition of sodium orthovanadate, a broad PTP inhibitor. They were also blocked by protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, and PKC and protein serine/threonine kinase inhibitors, H7 and HA1004. In addition, the alteration in the tyrosine phosphorylation of PTKs Lyn, Fyn, and Syk was directly demonstrated. Engagement of CD40 for 30 s induced a transient decrease in tyrosine phosphorylation of these PTKs. These results indicate that the early events in CD40 signaling involve the complex interaction between PTP and protein kinases.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Kinetic analysis of the non-phosphorylated, in vitro phosphorylated, and phosphorylation-site-mutant (Asp8) forms of intact recombinant C4 phosphoenolpyruvate carboxylase from sorghum.

Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8-->Asp) forms of purified recombinant sorghum C4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.31) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent Ki for the negative allosteric effector L-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent Ka for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1 mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and Km (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by L-malate and, most especially, Ka (Glc6P) and Ki (L-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C4-photosynthesis enzyme.

Edible Grain↗
PubMed1982

Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3.

Glycogen synthase kinase-5 (casein kinase-II) phosphorylates glycogen synthase on a serine termed site 5. This residue is just C-terminal to the 3 serines phosphorylated by glycogen synthase kinase-3, which are critical for the hormonal regulation of glycogen synthase in vivo. Although phosphorylation of site 5 does not affect the catalytic activity, it is demonstrated that this modification is a prerequisite for phosphorylation by glycogen synthase kinase-3. Since site 5 is almost fully phosphorylated in vivo under all conditions, the role of glycogen synthase kinase-5 would appear to be a novel one in forming the recognition site for another protein kinase.

Animals↗
PubMed1999

Phosphorylation of tau protein by recombinant GSK-3beta: pronounced phosphorylation at select Ser/Thr-Pro motifs but no phosphorylation at Ser262 in the repeat domain.

Glycogen synthase kinase-3beta (GSK-3beta) has been described as a proline-directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments. However, it has been claimed that GSK-3beta can also phosphorylate the non-proline-directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau from microtubules. We have analyzed the activity of recombinant GSK-3beta and of GSK-3beta preparations purified from tissue, using two-dimensional phosphopeptide mapping, immunoblotting with phosphorylation-sensitive antibodies, and phosphopeptide sequencing. The most prominent phosphorylation sites on tau are Ser396 and Ser404 (PHF-1 epitope), Ser46 and Thr50 in the first insert, followed by a less efficient phosphorylation of other Alzheimer phosphoepitopes (antibodies AT-8, AT-270, etc). We also show that the non-proline-directed activity at KXGS motifs is not due to GSK-3beta itself, but to kinase contaminations in common GSK-3beta preparations from tissues which are activated upon addition of heparin.

Animals↗
PubMed2000

Phosphorylation of steroid receptor coactivator-1. Identification of the phosphorylation sites and phosphorylation through the mitogen-activated protein kinase pathway.

Steroid receptor coactivator-1 (SRC-1) is a member of a coactivator family that enhance the activation of the steroid/nuclear receptor superfamily of ligand-stimulated transcription factors. To study the regulation of SRC-1 by signaling pathways in the cell, the major phosphorylation sites of SRC-1 were identified in COS-1 cells using a combination of in vivo labeling with [(32)P]H(3)PO(4), modified manual Edman degradation, phosphoamino acid analysis, endoproteinase digestion, and mutagenesis of the SRC-1 phosphorylation sites. Seven phosphorylation sites were identified in SRC-1: serine 372, serine 395, serine 517, serine 569, serine 1033, threonine 1179, and serine 1185. All the sites contained consensus sequences for the serine/threonine-proline-directed family of protein kinases, and two sites (serine 395 and threonine 1179) contained a perfect consensus sequence for the mitogen-activated protein kinase family (Erk-1 and Erk-2). Furthermore, Erk-2 phosphorylated threonine 1179 and serine 1185 (and to a lesser extent, serine 395) in vitro, suggesting the importance of this pathway for SRC-1 regulation. Treatment of cells expressing SRC-1 with epidermal growth factor enhanced the ligand-dependent, progesterone receptor-mediated activation of a target reporter gene. These results identify phosphorylation as a regulatory modification of SRC-1 and provide a basis upon which to identify signaling pathways that regulate SRC-1 function and, consequently, modify steroid/nuclear receptor action.

Amino Acid Sequence↗
PubMed1993

Protein kinase C forms a complex with and phosphorylates the GTPase activating protein GAP: phosphorylation by PKC is dependent on tyrosine phosphorylation of GAP and/or a GAP-associated protein.

Protein kinase C (PKC) and the GTPase-activating protein GAP can be detected in immunoprecipitates of mouse epidermis and lung cytosol obtained with either anti-GAP or anti-PKC antisera. The PKC in the immune-complex phosphorylates the coprecipitated GAP protein. Moreover, purified recombinant GAP is phosphorylated in vitro by purified PKC. The efficacy of this phosphorylation appears to depend on the extent of tyrosine phosphorylation of GAP and/or a GAP-associated protein.

Animals↗
PubMed2000

Membrane enzyme systems responsible for the Ca(2+)-dependent phosphorylation of Ser(27), the independent phosphorylation of Tyr(10) and Tyr(7), and the dephosphorylation of these phosphorylated residues in the alpha-chain of H/K-ATPase.

H/K-ATPase preparations (the G1 membrane) from pig stomach contain both kinases and phosphatases and show reversible phosphorylation of Tyr(7), Tyr(10), and Ser(27) residues of the alpha-chain of H/K-ATPase. The Tyr-kinase is sensitive to genistein and quercetin and recognized by anti-c-Src antibody. The Ser-kinase is dependent on Ca(2)(+) (K(0.5) = 0.9 microM), sensitive to a PKC inhibitor, and recognized by antibodies against PKCalpha and PKCbetaII. The addition of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS) caused a dramatic increase in the phosphorylation of added synthetic copolymer substrates and permitted the phosphorylation of maltose-binding proteins fused with the N-terminal domain of alpha-chains. The phosphotyrosine phosphatase was inhibited by vanadate. The phosphoserine phosphatase was inhibited by okadaic acid and by inhibitor-2. The presence of protein phosphatase-1 was immunologically detected. Column chromatographic separation of CHAPS-solubilized G1 membrane and others indicate the apparent molecular weight of the Src-kinase to be approximately 60 kDa, the PKCalpha and/or PKCbII to be approximately 80 kDa, the Tyr-phosphatase to be 200 kDa, and PP-1 to be approximately 35 kDa. These data show that these membrane-bound enzyme systems are in sufficiently close proximity to be responsible for reversible phosphorylation of Tyr(7), Tyr(10), and Ser(27) of the catalytic subunit of membrane H/K-ATPase in parietal cells, the physiological role of which is unknown.

Animals↗
PubMed2006

Angiotensin II-induced MAPK phosphorylation mediated by Ras and/or phospholipase C-dependent phosphorylations but not by protein kinase C phosphorylation in cultured rat vascular smooth muscle cells.

Angiotensin II (Ang II) induces a rapid increase in mitogen-activated protein kinase (MAPK) activity through the Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMCs). In the present study, we examined the effects of the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor GF109203X, and the Ras inhibitor farnesylthiosalicylic acid (FTS) on Ang II-induced activation of p42/p44 MAPKs in cultured VSMCs. Phosphorylation was shown using the Western blot technique with specific phospho-antibodies against MAPK proteins. The PLC inhibitor U73122 abolished the Ang II-induced MAPK activity, while the PKC inhibitor GF109203X only decreased it. There was also an inhibition observed with the Ras inhibitor, FTS on Ang II-induced MAPK activity. These data suggest that Ang II-induced MAPK phosphorylation through the Ang II type 1 receptor could be mediated by Ras and/or PLC-dependent phosphorylations but not by PKC phosphorylation.

Angiotensin II↗