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)

To examine the role of phosphorylation of the elongation factor eEF-1 in regulation of translation, 32P-labeled 3T3-L1 cells were deprived of serum, then incubated in the presence or absence of 10 nM insulin for 15 min. eEF-1 was purified by affinity chromatography on tRNA-Sepharose and shown to be phosphorylated on the alpha, beta and delta subunits. Phosphorylation of eEF-1alpha was stimulated sixfold in response to insulin, beta was stimulated fourfold and delta was threefold. The rate of elongation assayed with eEF-1 from insulin-stimulated cells was over twofold greater than with eEF-1 from serum-deprived cells. When eEF-1 from insulin-treated cells was subjected to two-dimensional tryptic phosphopeptide mapping, nine phosphopeptides were obtained with the alpha subunit, one with the beta subunit and three with the delta subunit. When compared with phosphopeptide maps of alpha, beta and delta subunits of eEF-1 phosphorylated in vitro by the insulin-stimulated multipotential protein kinase, the maps of the beta and delta subunits were identical. Five phosphopeptides obtained with the alpha subunit in vivo were identical to those obtained with S6 kinase in vitro; the remainder were unique. To examine whether protein kinase C had a role in phosphorylation of eEF-1 in response to insulin, protein kinase C was down-regulated by prolonged exposure of 3T3-L1 cells to 4beta-phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the alpha, beta and delta subunits was stimulated 2.5-fold in response to insulin, with elongation activity stimulated to a similar extent, suggesting that protein kinase C had no effect on stimulation of elongation in response to insulin. Thus, stimulation of eEF-1 activity in response to insulin appears to be mediated primarily by multipotential S6 kinase. This data is consistent with previous studies on stimulation of initiation via phosphorylation of initiation factors by multipotential S6 kinase [Morley, S. J. & Traugh, J. A. (1993) Biochemie (Paris) 95, 985-989].
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PMID:Insulin stimulation of phosphorylation of elongation factor 1 (eEF-1) enhances elongation activity. 949 85

Erythropoietin (EPO) is a hormone, as well as a hematopoietic growth factor, that specifically regulates the proliferation and differentiation of erythroid progenitor cells. Although the membrane-bound receptor for EPO has no intrinsic kinase activity, it triggers the activation of protein kinases via phospholipases A2, C, and D. A cascade of serine and threonine kinases, including Raf-1, MAP kinase and protein kinase C (PKC) is activated following tyrosine phosphorylation. In this study, we have examined whether changes in nuclear PKC and 1,2-diacylglycerol (DAG) are induced following EPO treatment of the murine target cell line, B6SUt.EP. Western blot analysis using isoform-specific antibodies demonstrated the presence of PKC beta II, but not PKC alpha, beta I, gamma, epsilon, delta, eta, or zeta in the nuclei of cells stimulated with EPO. The increase in nuclear beta II levels was accompanied by an immediate rise in DAG mass levels with both of the increases peaking by 1 min. These rapid increases in nuclear DAG and PKC beta II expression suggest a mechanism for EPO-induced changes in gene expression necessary for cell proliferation.
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PMID:Erythropoietin stimulates nuclear localization of diacylglycerol and protein kinase C beta II in B6SUt.EP cells. 952 23

Modulation of protein phosphorylation activities by insulin was investigated in glioma and normal glial cells. Insulin suppressed the in vitro protein phosphorylation of glioma cells in a dose-dependent manner while it stimulated that of meningiomas, neurilemmomas and glial cells. Although gliomas and glial cells contained different species of tyrosyl phosphoproteins before treatment, they expressed similar kinds of tyrosyl phosphoproteins in response to insulin. Insulin increased the activities of casein kinase II and total protein kinase C (PKC) in glioma and normal glial cells. The membrane-bound PKC activity in U373-MG cells was elevated by insulin. The PKC isozymes, including subtypes alpha, beta, delta, epsilon and gamma, were detected in gliomas, but few were found in glial cells. Insulin down regulated the cytosolic PKC-gamma and the membrane-bound PKC-epsilon proteins in gliomas. These results indicate that an altered insulin signaling pathway exists in human gliomas, which might involve differential regulation of PKC isozymes.
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PMID:Effects of insulin on protein phosphorylation and protein kinase C activity in human malignant gliomas. 953 17

Calmodulin-like domain protein kinases (CDPKs) are a family of calcium- but not calmodulin-dependent protein kinases found in a wide variety of plants and in protists. CDPKs are encoded by large multigene families, and to assess whether family members play distinct or redundant roles in vivo, we characterized soybean CDPK isoforms alpha, beta, and gamma, which share 60-80% identity in amino acid sequence. RNA blot analysis showed that the three CDPKs were expressed in most plant tissues examined and in suspension-cultured soybean cells. Recombinant CDPKalpha, -beta, and -gamma phosphorylated peptide substrates containing the four-residue motif R/K-X-X-S/T, but CDPKalpha was the most selective for residues outside of the motif. The CDPKs were inhibited by the general protein kinase inhibitors K252a and staurosporine and by calphostin C, which is an inhibitor of protein kinase C. The calcium-binding properties of each CDPK were distinct. The Kd's for Ca2+ determined by flow dialysis in the absence of substrates were 51, 1.4, and 1.6 micro M for CDPKalpha, -beta, and -gamma, respectively. In the presence of the peptide substrate syntide-2 the Kd of CDPKalpha decreased to 0.6 microM. Also, the sensitivity of this isoenzyme's activity to calcium varied with protein substrate. The concentrations of Ca2+ required for half-maximal activity (K0.5) for each CDPK with syntide-2 as substrate were 0.06, 0.4, and 1 micro M, respectively. These results show that members of the CDPK family differ in biochemical properties and support the hypothesis that each isoform may have a distinct role in calcium signal transduction.
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PMID:Kinetic and calcium-binding properties of three calcium-dependent protein kinase isoenzymes from soybean. 957 65

Lacrimal gland protein secretion is primarily under the control of cholinergic muscarinic and alpha 1-adrenergic receptors. Cholinergic agonists are coupled to the activation of phospholipase C (PLC), which leads to the production of two second messenger molecules: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 increases the cytoplasmic concentration of calcium ([Ca2+]i), and DAG activates protein kinase C (PKC), two events that are thought to trigger protein secretion. Lacrimal gland alpha 1-adrenergic receptors are not coupled to the PLC pathway, although their activation leads to a slight increase in [Ca2+]i(3). We have also shown that unlike the cholinergic receptors, alpha 1-adrenergic receptors are not linked to the activation of phospholipase D in lacrimal gland acini. Thus the transduction pathway(s) used by the alpha 1-adrenergic receptors to trigger lacrimal gland protein secretion remains to be identified. PKC was originally described as a Ca2+ and phospholipid-dependent protein kinase activated by DAG produced by the receptor-mediated breakdown of phosphoinositides. Molecular cloning and biochemical techniques have shown that PKC is a family of closely related enzymes consisting of at least eleven different isoforms that has been divided into three categories: (1) conventional PKCs, including PKC alpha, beta I, -beta II and -gamma isoforms have a Ca2+ and DAG-dependent kinase activity; (2) novel PKCs, including PKC epsilon, -delta, -theta, -nu, and -mu isoforms, are Ca(2+)-independent and DAG-stimulated kinases; (3) atypical PKCs, including PKC zeta, and -iota/lambda isoforms, are Ca2+ and DAG-independent kinases. All PKC isoforms, except PKC mu, have a pseudosubstrate sequence in their N-terminal part that is thought to interact with the catalytic domain to keep the enzyme inactive in resting cells. In previous studies, we showed that lacrimal gland acini express three isoforms of PKC: PKC alpha -delta, and -epsilon. In the present study, we report the identification of two other PKC isoforms, namely PKC mu and -iota/lambda. We show that these isoforms are differentially located and that they translocate differentially in response to phorbol esters and cholinergic agonists. We also show that PKC isoforms differentially control lacrimal gland protein secretion and cholinergic-induced Ca2+ elevation. Part of these results has been recently published.
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PMID:Lacrimal gland functions are differentially controlled by protein kinase C isoforms. 959 15

Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in alpha, beta, and gamma adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant alpha adducin. The mutant alpha adducin was no longer concentrated at the cell membrane at sites of cell-cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant alpha adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant alpha adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.
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PMID:Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons. 967 46

Of the six distinct isoforms of mouse protein phosphatase 2C (PP2C) (alpha, beta-1, beta-2, beta-3, beta-4 and beta-5), PP2C alpha was specifically phosphorylated on the serine residue(s) when expressed in COS7 cells. Analysis of phosphorylation sites using site-directed mutagenesis demonstrated that Ser-375 and/or Ser-377 were phosphorylated in vivo. These serine residues were the sites of phosphorylation by casein kinase II in vitro. Phosphorylation of PP2C alpha was enhanced two-fold by the addition of okadaic acid to the culture medium, but addition of cyclosporin A had no such effect. These results suggest that the expressed PP2C alpha is phosphorylated by a casein kinase II-like protein kinase and dephosphorylated by PP1 and/or PP2A in COS7 cells.
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PMID:Isoform specific phosphorylation of protein phosphatase 2C expressed in COS7 cells. 968 43

Protein kinase C (PKC)-mediated phosphorylation has been implicated in neuronal growth and differentiation [R.S. Turner, R.L. Mazzei, G.J. Raynor, P.R. Girard, J.F. Kuo, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 3143-3147.]. We examined effects of gestational exposure to the neurotoxicant, methylmercury (CH3Hg), on the developmental profile of immunoreactivity (IR) for alpha, beta, gamma and epsilon PKC isoforms and cytosolic PKC activity. Long-Evans dams were dosed on gestational days (GD)6-15 (p.o.) with 0, 1, or 2 mg kg-1 day-1 CH3Hg dissolved in saline. Pups were sacrificed and perfused with buffered paraformaldehyde on post-natal days (PND) 1, 4, 10, 21, 45 and 85. The brains were sectioned sagittally, stained immunohistochemically, and examined throughout the medial to lateral extent. IR in neuronal cell bodies for PKC isoforms alpha, beta, gamma, and epsilon was densest in the olfactory bulb, hippocampus, shell of the inferior colliculus, pons, cerebral, piriform, and cerebellar cortex, whereas axonal staining was prominent in the brainstem, internal capsule, corpus callosum, anterior commissure, fornix and olfactory tract. In controls, the PKC alpha and epsilon IR was highest on PND1-4, decreased dramatically by PND10, and decreased further by PND21. In the neonate, the regional and cellular distributions of alpha and epsilon IR were similar. The PKC gamma IR was greater at post-weaning ages (PND21-85) with the greatest regional density apparent in the hippocampus, cortex, and cerebellum. Only the highest dose of CH3Hg (2 mg kg-1 day-1; GD6-15) produced a persistent decrease in regional alpha and epsilon, but not beta or gamma IR during the post-natal period. These regional and time-dependent changes in PKC isoforms were complemented by the examination of PKC activity in cortex, olfactory bulb, cerebellum and brainstem. Cytosolic PKC activity increased from PND1 to 10 in cortex, olfactory bulb, and cerebellum. On PND21, PKC activity decreased in the cortex and olfactory bulb, but remained high in the cerebellum. By contrast, PKC activity in the brainstem was highest on PND1 and 4 and decreased dramatically by PND21. CH3Hg (2 mg kg-1 day-1) significantly decreased PKC activity on PND1 and 4 in the cortex. The present results characterize the cellular and regional ontogeny of PKC isoenzymes alpha, beta, gamma and epsilon, and indicate that developmental exposure to CH3Hg can alter the ontogeny of specific isoforms and regional PKC activity.
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PMID:Effects of gestational methylmercury exposure on immunoreactivity of specific isoforms of PKC and enzyme activity during post-natal development of the rat brain. 970 89

A single addition of ATP (20-1000 microM) to cultures of HL-60 cells resulted here in permanent, Ca(2+)-independent inhibition of cellular proliferation, evident 48 h following treatment. Extracellular ATP (ATPo) was maximally effective at 250 microM giving 90 +/- 1.5% growth inhibition. Up to a concentration of 250 microM ATPo, growth inhibition is solely attributable to ATPo, while at higher ATPo concentrations adenosine generated from ATPo hydrolysis contributes to this effect. The order of potency for growth inhibition was ATP = ADP > AMP > adenosine. Suramin, a P2 receptor antagonist, attenuated growth inhibition by ATP and ADP, indicative of P2 receptor involvement. Equipotency of ATP and ADP excludes the involvement of either an ecto-protein kinase or a P2X7 receptor in growth inhibition. Neither UTP (P2Y2 agonist) nor alpha, beta-methyleneATP (P2X1 agonist) inhibited growth, indicating that such inhibition is mediated by a previously undescribed P2 receptor on HL-60 cells.
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PMID:Growth inhibition of HL-60 cells by extracellular ATP: concentration-dependent involvement of a P2 receptor and adenosine generation. 975 40

The c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH2 termini (alpha, beta, and gamma isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7alpha isoforms lack an NH2-terminal extension that is present in the other MKK7 isoforms. This NH2-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7alpha isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7beta and MKK7gamma isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus was not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli.
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PMID:The MKK7 gene encodes a group of c-Jun NH2-terminal kinase kinases. 989 Oct 90


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