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)

Studies were conducted to determine if norepinephrine activates both protein kinase C and the cyclic AMP-dependent protein kinase in cultured rat astrocytes using phosphoproteins as markers. Norepinephrine was found to decrease 32P incorporation into an acidic 80,000 M(R) protein. A similar response was observed with isoproterenol and cyclic AMP analogs. In contrast, phorbol myristate acetate (PMA) increased 32P incorporation into this protein. Further studies looked at phosphorylation sites on glial fibrillary acidic protein and vimentin using two-dimensional tryptic phosphopeptide maps. The pattern of phosphorylation of these two proteins by norepinephrine resembles that of 8-bromo cyclic AMP and isoproterenol, and not that of PMA. Additionally, the effect of norepinephrine on the phosphorylation of GFAP and vimentin was blocked by alprenolol. One difference noted between norepinephrine and isoproterenol was the phosphorylation of an 18,000 M(R) protein. Norepinephrine increased, and isoproterenol decreased, 32P incorporation into this protein; however, the mechanism which mediates the norepinephrine effect remains to be determined. Overall, these studies indicate that the most prominent phosphorylation events mediated by norepinephrine are the consequence of the activation of cyclic AMP-dependent protein kinase.
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PMID:Norepinephrine-mediated protein phosphorylation in astrocytes. 132 20

Phosphorylation of glial fibrillary acidic protein (GFAP) induces disassembly of the filaments. An amino-terminal fragment of bovine GFAP (G-Hf) was produced by lysylendopeptidase digestion. G-Hf formed ribbon-like filaments in the presence of GFAP even in low ionic strength, whereas the fragment itself did not form any structures. Only one (PK3) of the five V8 protease fragments of G-Hf accelerated GFAP assembly to the same degree as G-Hf did, whereas the other fragments did not. When PK3 was cleaved into two fragments, it lost the assembly-accelerating property. The sequence of PK3 was determined as RRRVTSATRRSYVSSSE, which corresponded to residues 3-19 of porcine GFAP. It was concluded that PK3 contains a sequence indispensable for GFAP assembly and that neither PK1 (RRRVTS) nor PK2 (ATRRSYVSSSE) included all of the sequence. A single phosphorylation of PK3 by cyclic AMP-dependent protein kinase diminished its assembly-accelerating property. The phosphorylation site was determined as Ser-12 of porcine GFAP. It was shown that single phosphorylation of the amino-terminal head domain, which contains an indispensable sequence for GFAP assembly, might be sufficient for GFAP disassembly.
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PMID:Assembly regulatory domain of glial fibrillary acidic protein. A single phosphorylation diminishes its assembly-accelerating property. 142 73

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation of glial fibrillary acidic protein and vimentin by cytoskeletal-associated intermediate filament protein kinase activity in astrocytes. 172 39

Both the protein kinase C (PK-C) activator, phorbol 12-myristate 13-acetate (PMA), and the cyclic AMP-dependent protein kinase (PK-A) activator, 8-bromo-cyclic AMP (8-BR), have been shown to increase 32P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8-BR results in the morphological transformation of flat, polygonal-shaped cells into stellate, process-bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8-BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32P incorporation into all the peptide fragments that were phosphorylated by 8-BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8-BR treatment was restricted to serine residues in the N-terminal domain of these proteins. Studies were also conducted to compare the two-dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8-BR with those produced when the proteins were phosphorylated with purified PK-C or PK-A. PK-C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK-C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phorbol myristate acetate and 8-bromo-cyclic AMP-induced phosphorylation of glial fibrillary acidic protein and vimentin in astrocytes: comparison of phosphorylation sites. 201 62

Monoclonal antibody YC10 showed specificity for the phosphorylated form of human, bovine and porcine glial fibrillary acidic proteins (GFAPs) and negligible reactivity towards the dephosphorylated form of the GFAPs. Analysis of species specificity and of the epitope, determined using synthetic phosphopeptides, indicated that this antibody recognized the local phosphorylation-site sequence Thr-phosphoSer-Ala-Ala-Arg-Arg (residues 7-12 of GFAP). Making use of this antibody we developed a non-radioactive method to measure protein kinase activities. After incubation of a protein kinase with non-radioactive ATP in ninety-six wells coated with the synthetic peptide Arg-Arg-Arg-Val-Thr-Ser-Ala-Ala-Arg-Arg-Ser-Cys (residues 3-13 of GFAP), the phosphorylated product was detected by using this mouse antibody and peroxidase-labeled goat anti-mouse IgG. This method proved to be equally as sensitive as the radioactive method for the measurement of protein kinase activities and was less affected by concentrations of ATP present in the reaction mixture.
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PMID:A monoclonal antibody to the phosphorylated form of glial fibrillary acidic protein: application to a non-radioactive method for measuring protein kinase activities. 202 46

We have investigated the actions of Ca2(+)-calmodulin (CaM)-dependent protein kinase II on various types of non-epithelial intermediate filament proteins, vimentin, desmin, glial fibrillary acidic protein (GFAP) and neurofilament triplet proteins. Most of these filament proteins could serve as substrates. The effects of phosphorylation on the filamentous structure of vimentin were investigated in sedimentation experiments and by using electron microscopy. The amount of unassembled vimentin increased linearly with increased phosphorylation. However, the extent of the effect of phosphorylation on the potential to polymerize was also affected by the MgCl2 concentration, under conditions for reassembly. The actions of Ca2(+)-CaM-dependent protein kinase II on non-epithelial intermediate filaments under physiological conditions are given attention.
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PMID:Ca2(+)-calmodulin-dependent protein kinase II phosphorylates various types of non-epithelial intermediate filament proteins. 211 9

The expression of c-fos protein in cultured human glial cells derived from the brain and spinal cord was investigated immunocytochemically. Primary cultures of fetal glial cells were maintained in culture for three weeks and deprived of animal sera for 22 h. The glial cell nature of the cells was ascertained by GFAP-immunoreactivity. Incubations with phorbol dibutyrate, 8-Br-cAMP and sodium nitroprusside representing signal transduction pathways of PKC, PKA and cyclic GMP kinase, respectively, were carried out for 60 and 120 min. The control serum-deprived cultures did not display c-fos protein immunoreactivity (c-fos-IR), whereas phorbol dibutyrate incubation for 120 min induced strong c-fos-IR in the nuclei of both brain and spinal cord derived glial cells. Semiquantitative intensity measurements revealed a slight c-fos-IR induction after 8-Br-cAMP as well, but not after sodium nitroprusside. The observations suggest that c-fos protein is involved in PKC and PKA signal transduction in cultured human glial cells.
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PMID:Immunohistochemical detection of c-fos proteins in cultured human glial cells--induction by cyclic AMP and phorbol ester. 212 26

The glial fibrillary acidic protein (GFAP) was found to be phosphorylated in vivo after intracerebral injection of [32P]-orthophosphate, in brain slices, and in a cell free system. The phosphorylated proteins were separated by two-dimensional gel electrophoresis and then transferred to nitrocellulose sheets. Two isoelectric variants of GFAP were immunochemically identified by monoclonal antibodies. Autoradiography demonstrated that only the more acidic isoelectric variant of GFAP was phosphorylated. Phosphoamino acid analysis revealed that under all conditions GFAP was phosphorylated at serine and threonine residues. Incubation of brain slices with [32P]-orthophosphate and the protein kinase C activator phorbol 12-myristate 13-acetate or forskolin, an activator of cyclic AMP-dependent protein kinase, stimulated phosphorylation of GFAP. Likewise phosphorylation of GFAP was also accentuated by calcium/phosphatidylserine/diolein and by exogenous cyclic AMP-dependent kinase in a cell free system. These findings announce that protein kinase C and cyclic-AMP dependent kinase may play physiologic roles in the in situ phosphorylation of GFAP. When isolated cytoskeletal preparations were incubated with [gamma-32P] ATP, GFAP was phosphorylated in vitro by two additional protein kinases, a Ca2++/calmodulin-dependent kinase and an effector-independent kinase. The results of these investigations strongly suggest that phosphorylation of GFAP appears to be regulated by multiple second messenger pathways.
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PMID:Phosphorylation of the glial fibrillary acidic protein. 225 63

The protein kinase-C (PKC) second messenger system contributes to regulation of cell growth and differentiation. This study was undertaken to examine the effects of modulators of the PKC enzyme system on the state of differentiation and proliferation rates of human gliomas in vitro. The administration of the PKC-activating phorbol esters 4-beta-phorbol-12,13-dibutyrate (PDB) and phorbol-12-myristate-13-acetate (PMA) resulted in a dose-related inhibition of growth of human glioma cell lines in vitro as measured by 3H-thymidine uptake. The synthetic nonphorbol PKC activator (SC-9) produced an even more pronounced decrease of 3H-thymidine uptake. Diacylglycerol, an endogenous activator of the system, applied externally, transiently decreased the proliferation, in concordance with its short-lived existence in vivo. Conversely, the administration of 4-alpha-phorbol-12,13-didecanoate (alpha-PDD), a phorbol ester that binds but does not activate the enzyme, had no effect on the proliferation rate. At the dosages that maximally decreased proliferation, there was no evidence of direct glioma cell lysis induced by these agents as measured by a chromium-release assay. Immunocytochemical analysis and cytofluorometric measurement of glial fibrillary acidic protein (GFAP) staining in the treated cultures revealed an increase in GFAP staining over control cultures. In contrast to the response of glioma cells, nonmalignant human adult astrocytes treated with the PKC activators responded by increasing their proliferation rate. The authors postulate that the diametrically opposed effects of PKC activators on nonmalignant astrocytes versus glioma growth may be due to a high intrinsic PKC activity in glioma cells, with resultant down-regulation of enzyme activity following the administration of the pharmacological activators.
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PMID:Inhibition of growth of established human glioma cell lines by modulators of the protein kinase-C system. 200 88

We are characterizing toxicant-induced injury to the nervous system by measuring nervous system, cell-type specific proteins together with accompanying changes in morphology and behavior. In the present study, cerebellar neurotoxicity was assessed in the Gunn rat, an autosomal recessive mutant that exhibits degeneration of Purkinje cells due to hereditary hyperbilirubinemia. Five proteins associated with neuronal or glial cell types were chosen for evaluation as follows: G-substrate, a Purkinje cell-specific phosphoprotein that serves as the endogenous substrate of cyclic GMP-dependent protein kinase; PCPP-260, a Purkinje cell-specific phosphoprotein that serves as an endogenous substrate of cyclic AMP-dependent protein kinase; synapsin I, a synapse-specific phosphoprotein present in all neurons; glial fibrillary acidic protein, an astrocyte-specific protein; and myelin basic protein, a protein unique to myelin. In comparison to heterozygote (Jj) controls, homozygous (jj) rats showed alterations in the amounts of neurotypic and gliotypic proteins in cerebellum that were consistent with the neuropathological effects associated with development of hyperbilirubinemia in the Gunn rat. Decreased cerebellar cyclic GMP, but not cyclic AMP, alterations in indices of motoric competence and increased responsiveness to a nociceptive stimulus also were observed in jj rats. In general, the degree of cerebellar hypoplasia was predictive of the degree of biochemical, morphological or behavioral change observed. The results indicate that neurotypic and gliotyic proteins may be used as biochemical indicators of neurotoxicity.
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PMID:Cerebellar hypoplasia in the Gunn rat is associated with quantitative changes in neurotypic and gliotypic proteins. 241 May 96


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