Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphorylation of stathmin, a 19-kDa protein found in many tissues, has been linked to cell differentiation and proliferation. This protein is present in lymphocytes, and both phosphorylation and expression of stathmin are regulated by lymphotropic agents. In this study an antibody specific for stathmin was used to examine phosphorylation in response to PRL. The results suggest that PRL stimulates stathmin phosphorylation in the Nb2 lymphoma and that phosphorylation correlates with PRL-induced cell proliferation. Stathmin expression does not change substantially as PRL-stimulated Nb2 cells move through the cell cycle and enter into the S-phase. Thus, stathmin phosphorylation, but not expression, is regulated by PRL. Activation of protein kinase-C (PKC) in Nb2 cells also induces phosphorylation of stathmin, but PKC does not appear to mediate phosphorylation in response to PRL. The pattern of phosphorylation in response to 12-O-tetradecanoylphorbol-13-acetate differs from that in response to PRL, and down-regulation of PKC does not inhibit PRL-induced phosphorylation or proliferation. In addition to stathmin, PRL increases phosphorylation of a group of stathmin-like proteins. Phosphorylation of these proteins also correlates well with PRL-induced proliferation. Taken together, the results suggest that phosphorylation of stathmin and stathmin-like proteins may mediate some actions of PRL in Nb2 cells. The results further suggest that activation of PKC is not an important early event in PRL-stimulated mitogenesis in Nb2 cells.
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PMID:Prolactin-induced proliferation of the Nb2 T-lymphoma is associated with protein kinase-C-independent phosphorylation of stathmin. 139 41

Op18 is a highly conserved major cytosolic phosphoprotein that has been implicated in signal transduction in a wide variety of cell types. Freshly isolated peripheral blood lymphocytes (PBL) constitutively express low levels of mostly unphosphorylated Op18. After mitogenic stimulation of PBL, Op18 synthesis is induced at a time when cells are entering S-phase. In this study, we have examined the phosphorylation of Op18 in freshly isolated PBL after activation of the T cell receptor by OKT3. Quantitative analysis of Op18 phosphorylation was undertaken by metabolic labeling with 32Pi and PhosphorImager analysis of two-dimensional gels. After 10 or 15 min of activation by OKT3, one of the three major phosphorylated forms of Op18, designated Op18c, increased approximately 10-fold, which represented a most pronounced change among a large number of phosphoproteins analyzed. In time course experiments, increased Op18 phosphorylation to yield Op18c was observed as early as 2 min. Continued OKT3-induced activation for 20 to 72 h resulted in a further increase in phosphorylated Op18 forms, which paralleled new Op18 synthesis and occurred at a time when cells were entering S-phase, as determined by [3H]-thymidine incorporation. Inhibitors of lymphoid proliferation, cyclosporin A and RPM, had no effect on early (less than 15 min) phosphorylation. Addition of calphostin C, a specific inhibitor of protein kinase C, 1 min prior to stimulation of resting T cells with OKT3 completely inhibited further phosphorylation of Op18. Incubation of PBL with calphostin C for 75 min decreased constitutive levels of phosphorylated Op18. In contrast, inhibition of cyclic nucleotide-dependent protein kinases with HA1004 had no effect on Op18 phosphorylation. Activation of cAMP-dependent protein kinase with Forskolin or 8Br-cAMP did not increase Op18 phosphorylation. Our results suggest that Op18 phosphorylation is mediated by protein kinase C activation as an early event in T cell activation through the T cell receptor.
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PMID:Activation of resting peripheral blood lymphocytes through the T cell receptor induces rapid phosphorylation of Op18. 150 Jul 12

p19 is a highly conserved 19-kDa cytosolic protein that undergoes phosphorylation in mammalian cells upon activation of several distinct signal transduction pathways. Its expression is widespread but developmentally regulated. To determine the in vivo phosphorylation site(s) of p19, the protein was purified from bovine brain and resolved into the unphosphorylated form (p19) and a mixture of the two predominant phospho-forms (pp19). Proteolytic fragments of p19 and pp19 were examined by liquid chromatography/mass spectrometry (LC/MS). We detected ion masses corresponding to fragments spanning the entire amino acid sequence as deduced from the cDNA except for those predicted to contain an unmodified amino terminus. Instead, the digests revealed ions corresponding to peptides lacking the initiator methionine and containing an N-acetylated alanine at the amino terminus. The analysis of pp19, but not that of p19, revealed two sets of ions representing peptides whose m/z values differed by 80 atomic mass units, the incremental mass of a phosphate residue. These putative phosphate-bearing peptides were sensitive to alkaline phosphatase treatment. Using combined trypsin and V8 protease digestions, the phosphorylation sites were mapped to Ser-25 and Ser-38, in the peptides Leu-Ile-Leu-Ser*-Pro-Arg and Phe-Pro-Leu-Ser*-Pro-Pro-Lys, respectively. Interestingly, both phosphoserines are in a very similar sequence context, suggesting that a single proline-directed serine protein kinase, possibly p34cdc2, is responsible for phosphorylation of both sites in vivo.
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PMID:Analysis of phosphoprotein p19 by liquid chromatography/mass spectrometry. Identification of two proline-directed serine phosphorylation sites and a blocked amino terminus. 173 1

Prosolin is a major cytosolic phosphoprotein expressed prominently in rapidly proliferating human peripheral lymphocytes but produced at very low levels in resting (G0) PBL. It undergoes rapid phosphorylation upon treatment of growing cells with tumor-producing phorbol esters (TPA) and this phosphorylation event is correlated with a rapid down-regulation of DNA synthesis. In the present report we have studied various agents that, like TPA, act as partial or complete mitogens for G0 PBL and have determined their effect on phosphorylation of prosolin and on DNA synthesis in rapidly proliferating (IL-2-dependent) human PBL. Agents that activate the TCR (OKT3 and PHA), as well as agents that by-pass the receptor but activate biochemical pathways associated with TCR activation (TPA and Ca2(+)-ionophore), all produced rapid phosphorylation of prosolin and prompt down-regulation of DNA synthesis. Four phosphorylated forms of prosolin were produced, indicating activation of a complex phosphorylation pathway. Down-regulation of DNA synthesis did not lead to cell death or to permanent arrest, but was reversed after 24 to 48 h, and was not associated with any reduction in overall protein synthesis. Agents that bind to determinants closely connected to the TCR but without activating it (OKT4 and OKT8) had no effect on either prosolin phosphorylation or DNA synthesis. The results indicate that prosolin is an early target of the protein kinase activities induced by activation of the TCR in proliferating PBL, and suggest that its phosphorylation mediates the TCR signal, transmitting it into a biochemical pathway leading specifically to down-regulation of DNA synthesis. In G0 PBL, in which the negligible expression of prosolin precludes significant production of phosphorylated species, this inhibitory pathway is effectively blocked.
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PMID:T cell receptor activation induces rapid phosphorylation of prosolin, which mediates down-regulation of DNA synthesis in proliferating peripheral lymphocytes. 190 11

Prosolin is a major cytosolic phosphoprotein of proliferating normal PBL. Treatment of growing PBL with phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)) or calcium ionophore (A23187) for 1 h caused phosphorylation of prosolin with the production of up to four prominent phosphorylated forms differing in degree of phosphorylation and/or two-dimensional electrophoretic mobility (peptides B to E). Formation of these phosphopeptides coincided with rapid down-regulation of DNA synthesis. A23187 was particularly effective in inducing phosphorylation of the more highly phosphorylated peptides D and E, suggesting the existence of a (Ca2+)-activated mechanism in their phosphorylation. The T cell leukemia cell lines Jurkat, HuT-78, CCRF-CEM, and Molt-4 showed reduced to absent ability to phosphorylate prosolin peptides rapidly in response to A23187 and also showed diminished down-regulation of DNA synthesis. In leukemic cells treated with both TPA and A23187, peptides B and C were rapidly phosphorylated, but the phosphorylation of peptides D and E seen in normal PBL remained deficient. The T cell leukemic cells appear to have intact a TPA-activated mechanism for phosphorylating prosolin peptides B and C, but share an impairment of a specific Ca2(+)-activated mechanism, possibly a Ca2(+)-dependent protein kinase, required for phosphorylation of prosolin phosphopeptides D and E. The degree of rapid down-regulation of DNA synthesis was correlated with degree of phosphorylation of peptide E in PBL and in three of four T cell leukemic cell lines. Thus, rapid phosphorylation of prosolin may mediate responses to TPA and A23187 in normal proliferating PBL, including down-regulation of DNA synthesis. A deficiency of this pathway in leukemic T cells may impede their response to physiologic growth regulatory signals utilizing this pathway and contribute to unrestrained cell growth.
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PMID:A specific defect of prosolin phosphorylation in T cell leukemic lymphoblasts is associated with impaired down-regulation of DNA synthesis. 211 78

Stathmin is a ubiquitous soluble protein (Mr approximately 19,000; pI approximately 6.2-5.5) whose phosphorylation is associated with the intracellular mechanisms involved in the regulations of cell differentiation and functions by extracellular effectors. It is present in various tissues and cell types and has several nonphosphorylated and increasingly phosphorylated forms, and it is particularly abundant in brain. Very high concentrations of stathmin were also detected in mouse embryo striatal neurons grown in primary culture, whereas stathmin was barely detectable in astrocytes from the same source. Stathmin appeared in neurons as a major substrate for protein phosphorylation and, in particular, for the cyclic AMP (cAMP)-dependent protein kinase, because its phosphorylation was stimulated by cAMP in cell-free preparations and in intact cells by forskolin, a potent activator of adenylate cyclase. During brain ontogenesis, stathmin was first detected at embryonic day 12; its concentration increased until birth and then decreased from postnatal day 10 to adulthood. In parallel, its molecular forms shifted from the least phosphorylated to the more phosphorylated ones. This result may reflect the evolution of the activity of stathmin during development and the subsequent maturation of the brain. In conclusion, our results substantiate the likely role of stathmin as an intracellular relay of extracellular regulations, as they point out its specific importance related to neuronal functions and brain differentiation.
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PMID:Stathmin is a major phosphoprotein and cyclic AMP-dependent protein kinase substrate in mouse brain neurons but not in astrocytes in culture: regulation during ontogenesis. 247 33

Stathmin is a ubiquitous soluble protein (Mr approximately 19,000, pI approximately 6.2-5.5) whose phosphorylation is associated with the intracellular mechanisms involved in the regulations of cell differentiation and functions by extracellular effectors. Its purification from rat brain and the preparation of specific antibodies allowed us to identify a set of immunologically related unphosphorylated (N1, N2) and phosphorylated (P1, P2a, P2b, P3) proteins of decreasing isoelectric points. All these proteins yielded identical silver-stained or 32P-radioactive peptide maps with the protease V8 from Staphylococcus aureus, indicating that they are also structurally related. In vitro phosphorylation with the exogenous catalytic subunit of the cAMP-dependent protein kinase, as well as dephosphorylation with alkaline phosphatase, indicated that P1, P2, and P3 derived from N1 and N2 by progressive phosphorylation. Phosphorylation of individual proteins extracted from semi-preparative two-dimensional polyacrylamide gels demonstrated the existence of two distinct isoforms of stathmin, alpha and beta: N1 and N2 are their respective unphosphorylated forms (alpha O and beta O), whereas proteins P1-P3 could be resolved as at least three increasingly phosphorylated forms of both alpha and beta stathmin (alpha 1, alpha 2, alpha(3) and beta 1, beta 2, beta(3]. In intact pituitary GH4C1 cells, hormones like thyrotropin-releasing hormone and vasoactive intestinal peptide induced a similar conversion from N1 and N2 to P1, P2, and P3. The phosphorylation of both alpha and beta isoforms of stathmin is therefore a physiologically significant response to specific extracellular regulatory agents. In conclusion, stathmin represents a family of at least two distinct protein isoforms, whose respective phosphorylation and expression might play a role in its likely function as an intracellular relay of various converging extracellular signals.
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PMID:Identification of two distinct isoforms of stathmin and characterization of their respective phosphorylated forms. 272 86

Treatment of [32P]phosphate prelabeled intact human A431 epidermoid carcinoma cells with epidermal growth factor (EGF, 100 ng/ml) or 12-O-tetradecanoylphorbol-13-acetate (TPA, 10(-7)M) resulted in a selective enhancement in the phosphorylation of the following soluble acidic proteins: a phosphoprotein with a molecular weight of 17,000 (pp17; similar notation used throughout) pI approximately 5.5); pp27 (pI approximately 5.5); pp34 (pI approximately 6.2); and pp80 (pI approximately 4.5) as detected by two-dimensional gel electrophoresis. EGF or TPA induced a 4- to 6-fold increase in the phosphorylation of pp17 and a 2- to 4-fold increase in the phosphorylation of pp27, pp34, and pp80 within 15 min after treatment of subconfluent A431 cells. Alkali treatment of the gels removed most of the incorporated [32P] phosphate from the phosphoproteins, including pp27, pp34, and pp80; however, the phosphoester bond in pp17 was stable to alkaline hydrolysis since there was no removal of [32P]phosphate from this protein. Treatment of A431 cells with dibutyryl cyclic adenosine 3':5'-monophosphate (1 mM) also increased the phosphorylation of pp17, pp27, and pp34 but not of pp80. Activation of endogenous calcium- and phospholipid-dependent protein kinase C in the cytosol of A431 cells in a cell-free system resulted in the enhanced phosphorylation of pp27, pp34, and pp80 but not of pp17 while exogenous addition of the catalytic subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase to cytosol preparations resulted in the phosphorylation of pp17, pp27, and pp34, but not of pp80. These results demonstrate that at least four soluble acidic proteins are phosphorylated in A431 cells in response to either EGF or TPA in vivo suggesting that these two agents may exert part of their biological effects on A431 cells through a biochemical pathway involving the phosphorylation of several common proteins; moreover, the studies suggest that these four acidic proteins may be substrates in vitro for protein kinase C and/or a cyclic adenosine 3':5'-monophosphate-dependent protein kinase.
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PMID:Effect of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on the phosphorylation of soluble acidic proteins in A431 epidermoid carcinoma cells. 301 83

The effects of differentiating agents on the activity and phosphorylation pattern produced by phospholipid- and Ca2+-dependent protein kinase (PL-Ca-PK) were examined in human promyelocytic leukemia cell line HL-60. Dimethyl sulfoxide (DMSO), retinoic acid (RA), and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] increased the appearance of mature myelocytic (DMSO and RA) or monocytic [1,25(OH)2D3] cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the appearance of adherent macrophage-like cells. Coincident with the appearance of differentiated cells induced by DMSO, RA, and 1,25(OH)2D3 was an increase in PL-Ca-PK activity. In contrast, TPA treatment resulted in the rapid disappearance of PL-Ca-PK and the induction of phospholipid- and Ca2+- (PL-Ca-) independent protein kinase activity. The phosphorylation pattern resulting from endogenous PL-Ca-PK in extracts from cells treated with DMSO, RA, or 1,25(OH)2D3 showed a prominent phosphorylated protein of molecular weight 37,000 (pp37) and 38,000 (pp38) which was related to the appearance of the myelocyte/monocyte phenotype. pp37 and pp38 were also present in TPA-treated cells, but their phosphorylation was no longer dependent on the presence of phospholipid and calcium. Cells treated with DMSO and RA also exhibited a PL-Ca-dependent pp21 which was barely evident in 1,25(OH)2D3-treated cells and thus represented a myeloid cell marker. Also present was a prominent PL-Ca-dependent pp19 which remained unchanged following treatment with DMSO, RA, and 1,25(OH)2D3, but which diminished markedly in TPA-treated cells. On the other hand, TPA-treated cells exhibited a characteristic pp130 which was antigenically related to the actin binding protein, vinculin. These results indicate that there are characteristic PL-Ca-dependent phosphorylated proteins indicative of mature myelocytic and monocytic cells, as well as PL-Ca-independent phosphorylated proteins characteristic of the macrophage-like phenotype.
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PMID:Phospholipid- and Ca2+-dependent protein kinase activity and protein phosphorylation patterns in the differentiation of human promyelocytic leukemia cell line HL-60. 316 11

Oncoprotein 18 (Op18) is a conserved cytosolic protein that is a target for both cell cycle and cell surface receptor-regulated phosphorylation events. The four residues Ser16, Ser25, Ser38, and Ser63 are all subject to cell cycle-regulated phosphorylation. Ser25 and Ser38 are targets for cyclin dependent kinases (CDKs), while Ser16 and Ser63 are phosphorylated by an unidentified protein kinase. We have recently shown that induced expression of a CDK target site-deficient mutant, Op18-S25A,S38A, blocks human cell lines during G2/M transition. In the present report we show that mitosis is associated with complete phosphorylation of the two Op18 CDK target sites Ser25 and Ser38 and that Ser16 and Ser63 are also phosphorylated to a high stoichiometry. To evaluate the function of multisite phosphorylation of Op18, we expressed and analyzed the cell cycle phenotype of different kinase target site-deficient mutants. The data showed that induced expression of the S16A,S63A, S25A,S38A, and S16A,S25A,S38A,S63A mutants all resulted in an indistinguishable phenotype, i.e. immediate G2/M block and subsequent endoreduplication, a given fraction of G2 versus M-phase blocked cells, and a characteristic nuclear morphology of M-blocked cells. This result was unexpected; however, a likely explanation was provided by analysis of Op18 phosphoisomers, which revealed that mutations of the CDK sites interfere with phosphorylation of Ser16 and Ser63. The simplest interpretation of our results is that phosphorylation of Ser16 and Ser63 is essential during G2/M transition and that the phenotype of the S25A,S38A mutant is mediated by the observed block of Ser16/Ser63 phosphorylation.
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PMID:G2/M transition requires multisite phosphorylation of oncoprotein 18 by two distinct protein kinase systems. 777 78


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