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)

Human progesterone receptors (PR) in T47D breast cancer cells are synthesized as two different sized proteins, PR-A [94 kilodaltons (kDa)] and PR-B (120 kDa). Progestin addition to cells (in vivo) causes a 2-fold increase in total phosphorylation of PR and an increase in the apparent mol wt of both PR-A and PR-B on sodium dodecyl sulfate (SDS)-gels. Time-course experiments showed that increased PR phosphorylation that results from hormone addition is a multistep process and involves a rapid increase into total 32P labeling that takes place before the more slowly occurring phosphorylation(s) responsible for the change in electrophoretic mobility of PR on SDS-gels. As an approach to test whether phosphorylation is involved in regulating PR activity, we have examined the effects of cellular modulators of protein phosphorylation on PR-mediated target gene transcription in vivo using a T47D cloned cell line containing a stably transfected mouse mammary tumor virus-chloramphenicol acetyltransferase construct. Treatment with 8-bromo-cAMP (activator of cAMP-dependent protein kinases) or okadaic acid (protein phosphatase-1 and -2A inhibitor) did not stimulate target gene expression in the absence of progestin. When added together with progestin, either compound augmented PR-mediated target gene transcription by 3- to 4-fold. The cyclic nucleotide-dependent protein kinase inhibitor H8 completely blocked target gene responsiveness to hormone. Neither 8-bromo-cAMP, okadaic acid, nor H8 altered the hormone- or DNA-binding activities of PR, as measured in vitro or affected cellular concentrations of PR. These agents, therefore, appeared to selectively modulate PR transcriptional activity. Moreover, none of these compounds altered expression from a control reporter gene, pSV2CAT, indicating that these agents affect PR-mediated processes directly and are not acting through a general effect on transcription. Effects on PR phosphorylation were assessed by measuring 32P labeling of PR in vivo. None of these treatments had a substantial effect on the extent of total 32P labeling of immune isolated PR or on the phosphorylation(s) responsible for PR up-shifts on SDS-gels. This suggests that these agents modulate PR transcriptional activity either through phosphorylation of another protein intimately involved in PR-mediated transcription or through modification of a key site(s) not measurable as a change in total PR phosphorylation or electrophoretic mobility on SDS gels.
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PMID:Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors. 131 49

The isolated modulator subunit of the inactive protein phosphatase-1 is phosphorylated in vitro by casein kinase-1 at two different sites: Ser-86 and Ser-174. The Ser-86 site is a common target for casein kinase-1 and casein kinase-2, but is preferentially phosphorylated by the former enzyme. The Ser-174 site seems to be specific for casein kinase-1, and is phosphorylated at a slower rate. These results give a new insight into the in vitro phosphorylation pattern of the modulator subunit of the phosphatase and provides additional data on the specificity of casein kinase-1.
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PMID:Phosphorylation of the phosphatase modulator subunit (inhibitor-2) by casein kinase-1. Identification of the phosphorylation sites. 131 29

Molecular cloning of glycogen synthase kinase-3 (GSK-3) has demonstrated the existence of a novel form, termed GSK-3 beta, which is highly related to the well characterised GSK-3 alpha protein but derived from a distinct gene. The cDNA cloning also revealed a striking degree of amino acid identity between the two GSK-3 proteins, particularly the beta-form, and the zeste-white3/shaggy (zw3sgg) homeotic gene of Drosophila melanogaster. Abrogation of zw3sgg causes pleiotropic effects on fruitfly development affecting segmental organisation and cell fate determination. In view of the potential importance of GSK-3 beta in mammalian development and the lack of previous characterisation, we have expressed this protein in insect cells using recombinant baculovirus. A rapid purification scheme has been developed yielding essentially pure GSK-3 beta protein in three chromatographic steps. The protein has autonomous protein kinase activity and similar, but not identical, substrate preferences to GSK-3 alpha. Both GSK-3 proteins activate the MgATP-dependent form of protein phosphatase-1 and thus display 'factor A' activity. Since GSK-3 beta exhibits an identical site specificity to GSK-3 alpha with respect to phosphorylation of the proto-oncogene/transcription factors c-jun and c-myc, it is likely that the Drosophila zw3sgg protein kinase has a similar specificity for such transcription factors which may underlie the pleiotropic phenotypes observed when the Drosophila homologue is mutationally inactivated.
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PMID:Baculovirus-mediated expression and characterisation of rat glycogen synthase kinase-3 beta, the mammalian homologue of the Drosophila melanogaster zeste-white 3sgg homeotic gene product. 134 4

It has been recently proposed that DARPP-32 participates, as third messenger, in the mediation of effects induced by dopamine at the cellular level. DARPP-32 is indeed localized almost exclusively on dopaminoceptive neurons bearing the D1 receptor subtype and it is phosphorylated by cAMP-dependent protein kinase. In its phospho-form, DARPP-32 acts as an inhibitor of protein phosphatase-1. In vivo pharmacological treatment with selective D1 agonists and antagonists induces changes in the phosphorylation state of DARPP-32 that can be correlated to changes in cAMP, mediated in turn by D1 and D2 receptors. These data demonstrate that the measurement of the phosphorylation state of DARPP-32 with the back-phosphorylation assay can represent a useful biochemical tool to gain further insight into the sequence of events elicited by specific dopaminergic drugs in vivo.
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PMID:The phosphorylation state of DARPP-32, a third messenger for dopamine, is regulated by in vivo pharmacological treatments. 136 18

PTH receptors on osteoblasts and calcitonin receptors on osteoclasts are coupled to adenylate cyclase. Despite similar transduction mechanisms, these hormones have opposing physiological actions. We investigated the consequences of persistent protein phosphorylation on bone resorption in neonatal mouse calvariae using okadaic acid (OA) and calyculin-A, two inhibitors of protein phosphatase-1 and -2A. These two inhibitors caused different responses in bone at picomolar and low nanomolar concentrations. OA inhibited, in a dose-dependent manner, bone resorption stimulated by PTH, 1,25-Dihydroxyvitamin D3, phorbol ester, and prostaglandin E2 (PGE2). OA did not inhibit the generation of the second messengers cAMP or PGs and did not have nonspecific toxic effects, as measured by protein and RNA synthesis. Thus, OA appeared to mimic the global inhibitory action of calcitonin on bone resorption. Unlike OA, calyculin-A elicited a biphasic dose response. At concentrations of 3.3 nM and greater, calyculin-A inhibited, in a dose-dependent manner, stimulated bone resorption. However, calyculin-A alone, at 0.625 and 2.5 nM, stimulated bone resorption via a PG-independent pathway. In calvariae, OA and calyculin-A increased phosphorylation of a 58- to 60-kilodalton protein. A protein of similar molecular mass was hyperphosphorylated in OA-treated ROS 17/2.8 osteoblast-like cells. We conclude that in addition to hormonal regulation of protein kinase activity, protein dephosphorylation plays a functionally important role in the modulation of bone resorption.
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PMID:Protein phosphatase inhibitors and bone resorption: inhibition by okadaic acid and biphasic actions of calyculin-A. 137 1

Insulin stimulates protein phosphatase-1 and FA, assayed as phosphatase-1 activator, in 3T3-L1 cells. Since other kinases, such as casein kinase-II may also contribute to such FA activity, we assayed casein kinase-II and FA as peptide kinase on extracts from 3T3-L1 cells that had been exposed to insulin for various times. Under such conditions FA, assayed as phosphatase-1 activator, was stimulated 2-3-fold within 1-2 min. Casein kinase-II was stimulated about 2-fold but at a slightly later time (2-3 min) than FA, making it unlikely that casein kinase-II contributes to FA stimulation. Insulin slightly stimulated also the kinase activity of FA towards a synthetic peptide at 2 min, thus confirming the FA activation seen when FA was assayed as activator of phosphatase-1.
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PMID:Stimulation of FA and casein kinase II by insulin in 3T3-L1 cells. 164 65

Improved methodology was used to establish that the phosphorylation of a serine located 10 residues from the N-terminus of glycogen synthase (N10) increases from 0.12 mol.mol-1 to 0.54 mol.mol-1 in vivo in response to adrenalin. The only 'N10 kinase' detected in muscle extracts was casein kinase-1 (CK1), although its activity was unaffected by injection of adrenalin in vivo or by incubation with cyclic-AMP-dependent protein kinase and MgATP in vitro. Prior phosphorylation of the serine residue N7 by phosphorylase kinase increased sixfold the rate of phosphorylation of glycogen synthase by CK1, and altered the specificity of CK1 so that it phosphorylated the serine residue N10 specifically. Stoichiometric phosphorylation of N7 decreased the activity ratio (+/- glucose 6-phosphate) of glycogen synthase from 0.80 to 0.45, and subsequent phosphorylation of N10 to 0.8 mol.mol-1 produced a further decrease to 0.17, demonstrating that N10 phosphorylation inhibits glycogen synthase. The major 'N10 phosphatase' in skeletal muscle extracts was identified as the glycogen-associated form of protein phosphatase-1 (PP1G), accounting for approximately 75% of the N10 phosphatase activity in the extracts and about 90% of the activity in isolated glycogen particles. Phosphorylation of N10, after prior phosphorylation of N7, decreased the rate of dephosphorylation of N7. These results, in conjunction with previous findings, establish that adrenalin inhibits glycogen synthase by increasing the phosphorylation of N7, N10 and three further serines located 30, 34 and 38 residues from the start of the C-terminal CNBr peptide (termed the region C30-C38). They also indicate that increased phosphorylation of N10, the region C30-C38, and perhaps N7, is initiated through the inhibition of PP1G by adrenalin, which results from phosphorylation of its glycogen-targetting subunit by cyclic-AMP-dependent protein kinase [Hubbard, M.J. & Cohen, P. (1989) Eur. J. Biochem. 186, 711-716]. The conclusion that direct phosphorylation of glycogen synthase by cyclic-AMP-dependent protein kinase makes little contribution to inhibition by adrenalin, is at variance with the teachings of the major textbooks of biochemistry.
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PMID:The molecular mechanism by which adrenalin inhibits glycogen synthesis. 165 Dec 42

1. In isolated rat adipocytes, acetyl-CoA carboxylase is inactivated by treatment of the cells with adrenaline or the beta-agonist isoproterenol, but not by the alpha-agonist phenylephrine. The inactivation is stable during purification in the presence of protein phosphatase inhibitors, and is associated with a 30-40% increase in the labelling of enzyme isolated from 32P-labelled cells. 2. Increased phosphorylation occurs within peptide T1, which was identified by sequencing to be the peptide Ser-Ser77-Met-Ser79-Gly-Leu-His-Leu-Val-Lys, containing Ser-77 (phosphorylated by cyclic-AMP-dependent protein kinase) and Ser-79 (phosphorylated by the AMP-activated protein kinase). Analysis of the release of radioactivity as free phosphate during Edman degradation of peptide T1 revealed that all of the phosphate was in Ser-79 in both basal and hormone- or agonist-stimulated cells. Treatment of adipocytes with various agents which activate cyclic-AMP-dependent protein kinase by receptor-independent mechanisms (forskolin, cyclic AMP analogues, isobutylmethylxanthine) also produced inactivation of acetyl-CoA carboxylase and increased phosphorylation at Ser-79. 3. The (Rp)-[thio]phosphate analogue of cyclic AMP, which is an antagonist of binding of cyclic AMP to the regulatory subunit of cyclic-AMP-dependent protein kinase, opposes the effect of adrenaline on phosphorylation and inactivation of acetyl-CoA carboxylase. Together with the effects of isobutylmethylxanthine and the stimulatory cyclic AMP analogues, this strongly indicates that cyclic-AMP-dependent protein kinase is an essential component of the signal transduction pathway, although clearly it does not directly phosphorylate acetyl-CoA carboxylase. 4. As shown by okadaic acid inhibition, greater than 95% of the acetyl-CoA carboxylase phosphatase activity in extracts of rat adipocytes or liver is accounted for by protein phosphatase-2A, with less than 5% attributable to protein phosphatase-1. Inhibition of protein phosphatase-1 via phosphorylation of inhibitor-1 is therefore unlikely to be the mechanism by which cyclic-AMP-dependent protein kinase indirectly increases phosphorylation of acetyl-CoA carboxylase. Various other potential mechanisms are discussed.
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PMID:Roles of the AMP-activated and cyclic-AMP-dependent protein kinases in the adrenaline-induced inactivation of acetyl-CoA carboxylase in rat adipocytes. 168 96

Synthetic peptides based on the threonine phosphorylation site and proposed inhibitory site of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were prepared and analyzed as substrates for cAMP-dependent protein kinase and protein phosphatases-1c, -2Ac (the catalytic subunits of protein phosphatase-1 and 2A, respectively) and -2B, and as inhibitors of protein phosphatase-1c. Studies of the kinetics of phosphorylation of the peptides by cAMP-dependent protein kinase indicated an important role in facilitating phosphorylation for the region COOH-terminal to the phosphorylatable threonyl residue. Studies of the dephosphorylation of the phosphopeptides demonstrated that they were effectively dephosphorylated by protein phosphatase-2A and -2B and poorly dephosphorylated by protein phosphatase-1. The active inhibitory region of phospho-DARPP-32 was analyzed by determining the effects of synthetic phosphopeptides on the activity of protein phosphatase-1c. Phospho-D32-(8-48) and phospho-D32-(8-38) inhibited protein phosphatase-1c with IC50 values of 2 x 10(-8) and 4 x 10(-8) M, respectively, compared with an IC50 of 8 x 10(-9) M for intact phospho-DARPP-32. Phospho-D32-(9-38) was equipotent with phospho-D32-(8-38); however, further NH2-terminal deletions resulted in marked reductions in IC50 values. An analog of an active DARPP-32 phosphopeptide containing a phosphoseryl residue in place of the phosphothreonyl residue also exhibited a much reduced IC50. These data identify the essential inhibitory region of phospho-DARPP-32 as residues 9-38, which contains the phosphorylation site (Thr34). This region exhibits extensive amino acid sequence identity with phosphatase inhibitor-1, a distinct inhibitor of protein phosphatase-1. Kinetic studies of the inhibition of protein phosphatase-1c by phospho-D32-(9-38), a potent inhibitor, as well as by phospho-D32-(10-38), a weak inhibitor, indicated a mixed competitive/noncompetitive mechanism of inhibition, as has been previously found for both intact phospho-DARPP-32 and intact phospho-inhibitor-1. These findings support the hypothesis that a 30-amino acid domain in the NH2-terminal region of phospho-DARPP-32 is sufficient for the inhibition of protein phosphatase-1.
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PMID:Synthetic peptide analogs of DARPP-32 (Mr 32,000 dopamine- and cAMP-regulated phosphoprotein), an inhibitor of protein phosphatase-1. Phosphorylation, dephosphorylation, and inhibitory activity. 217 4

A 10-50-fold, biphasic increase in the rate of 32Pi labeling of eIF-4E was closely correlated with the induction of protein and glycoprotein biosynthesis when resting murine splenic B lymphocytes (B cells) were activated by bacterial lipopolysaccharide or the combination of phorbol 12-myristate 13-acetate and ionomycin. The fraction of eIF-4E which was phosphorylated only increased from 46% in resting cells to 83% in lipopolysaccharide-activated cells. This discrepancy between the increase in the fraction of phosphorylated eIF-4E and the increase in 32Pi labeling suggested that the phosphoryl group of eIF-4E turns over slowly in resting B cells compared with activated cells. The turnover rate for the eIF-4E phosphate moiety in lipopolysaccharide-activated cells was rapid (t1/2 = 2 h) in comparison to the eIF-4E polypeptide chain, which did not turn over detectably in 6 h. Neither protein kinase C nor a cyclic nucleotide-dependent protein kinase appeared to be involved in eIF-4E phosphorylation in B cells, based on the observations that the metabolic labeling of eIF-4E by 32Pi was insensitive to the protein kinase inhibitors H-7 and HA1004, and that maximal labeling occurred after protein kinase C activity was "down-regulated" to very low levels in phorbol 12-myristate 13-acetate/ionomycin-activated cells. Dephosphorylation in vivo was blocked by okadaic acid (IC50 = 200 nM). These results indicate that a rapid phosphorylation-dephosphorylation of eIF-4E is associated with high translation rates during the activation of B cells, and implicate protein phosphatase-1 (or possibly-2A) in the dephosphorylation of the initiation factor.
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PMID:Increased rate of phosphorylation-dephosphorylation of the translational initiation factor eIF-4E correlates with the induction of protein and glycoprotein biosynthesis in activated B lymphocytes. 224 37


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