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)

Possible differentiation mechanisms were investigated in a glioblastoma multiform cell line (GL15) presenting an undifferentiated phenotype with weak glial fibrillary acidic protein (GFAP) and strong vimentin (VIM) expression. Serum-free conditions induced time-dependent increases of GFAP-mRNA and GFAP protein levels, associated with a process-bearing astrocytic morphology. Activation of protein kinase C (PKC) by tumor promoter phorbol 12-myrystate 13-acetate (PMA) induced a rapid morphological differentiation and a decrease in GFAP mRNA, whereas the GFAP level remained unchanged. Such parameters were shown to characterize a physiological differentiation stage in astroglial cultures. Treatment of process-bearing GL15 cells with dibutyryl cyclic AMP (dbcAMP), a protein kinase A (PKA) activator, induced a time-dependent decrease in the GFAP mRNA and GFAP protein levels and reverted morphological changes induced by serum-free conditions. Neither PMA nor dbcAMP influenced the VIM mRNA expression. In GL15 cells, PKC and PKA activation have opposite effects. Understanding the role of these kinases in malignant transformation and in the in vitro differentiation process is of both basic and clinical interest.
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PMID:PKA and PKC activation induces opposite glial fibrillary acidic protein (GFAP) expression and morphology changes in a glioblastoma multiform cell line of clonal origin. 754 74

We have recently shown that expression of specific protein kinase C (PKC) isoforms correlates with cell fate in neural chicken embryo cells. Therefore we investigated the effects of PKC activation by phorbol esters on acquisition of the astrocytic phenotype, using cultured embryonic cortical astrocytes, derived from 15-day-old chick embryos (E15CH), as a model. Short term treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA), which activates PKC-alpha/beta in E15CH, caused association of PKC with the cytoskeleton. In vitro kinase assays of cytoskeleton-associated PKC demonstrated phosphorylation of many cytoskeletal proteins. Phosphorylation was blocked by protein kinase inhibitors (H8), and enhanced by phosphatase inhibitors (calyculin A). Among these PKC substrates, a most prominent 60-kDa protein was identified as vimentin. Assembly of vimentin into the cytoskeleton depends on cell type and state of differentiation. To establish that TPA (PKC) regulates assembly of vimentin into the cytoskeleton of astrocytes, we used pulse-chase (20/5 min) labeling with [35S]methionine, and immunoprecipitations with an anti-vimentin mAb from extractable and cytoskeletal fractions. These studies revealed that 20 min treatment with TPA leads to a 3-fold increase in the rate of newly synthesized full-length vimentin assembly (posttranslational assembly). Furthermore, TPA increased cotranslational assembly of vimentin. The protein kinase A activator forskolin, did not have such effects on vimentin assembly. Long-term TPA treatment, which correlates with a prolonged phospholipase D (PLD) activation, was mitogenic and caused dramatic changes in the morphology of astrocytes. In addition these fibrous, polarized astrocytes had decreased activity of the astrocyte specific enzyme, glutamine synthetase, but had increased abundance of vimentin protein. These studies provide biochemical evidence on acquisition of a different astrocytic phenotype after activation of the PKC/PLD pathway, in the chick embryo. Therefore PKC and PLD activation is pivotal for the acquisition and maintenance of phenotypes in chick embryonic astrocytes.
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PMID:Phorbol esters and PKC signaling regulate proliferation, vimentin cytoskeleton assembly and glutamine synthetase activity of chick embryo cerebrum astrocytes in culture. 755 27

12(S)-HETE, a lipoxygenase metabolite of arachidonic acid, has been demonstrated to induce a reversible retraction of vascular endothelial cells (EC). 12(S)-HETE-induced microvascular EC retraction was blocked by a selective protein kinase C inhibitor, calphostin C, but not by the protein kinase A inhibitor, H8. EC exposed to 12(S)-HETE demonstrated a gradual dissolution of actin microfilaments and a decrease of vinculin-containing focal adhesions. The intermediate filaments, vimentin, also underwent extensive reorganization (i.e., filament bundling and enrichment to the cell filapodia) following 12(S)-HETE treatment. In vivo phosphorylation studies revealed that 12(S)-HETE induced a hyperphosphorylation of several major cytoskeletal proteins including myosin light chain, actin, and vimentin. The increased phosphorylation of these cytoskeletal proteins following 12(S)-HETE stimulation was abolished by calphostin C but not by H8. Confluent EC express alpha v beta 3 in focal adhesions at both the cell body and the cell-cell borders. 12(S)-HETE induced a sequential rearrangement of the alpha v beta 3-containing focal adhesions, resulting in a general decrease in alpha v beta 3 integrin receptors, especially in those retracted EC. 12(S)-HETE-induced rearrangement of alpha v beta 3 was inhibited by calphostin C but not by H8. In contrast to alpha v beta 3, confluent EC enrich alpha 5 beta 1 integrin receptors primarily at the cell-cell borders, colocalizing with extracellular fibronectin and cell cortical microfilaments. 12(S)-HETE treatment also disrupted the cell-border distribution pattern of alpha 5 beta 1 as EC retracted, but no distinct alterations (such as time-related redistribution and quantitative differences) in alpha 5 beta 1 were observed.
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PMID:12(S)-HETE-induced microvascular endothelial cell retraction results from PKC-dependent rearrangement of cytoskeletal elements and alpha V beta 3 integrins. 768 34

The effects of the calcium ionophore, A23187, on human neutrophil activation were studied in relation to the signaling mechanism of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G-kinase). Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton after stimulation with A23187. Over a period of 5 minutes, G-kinase was transiently colocalized with the intermediate filament protein, vimentin. At 3 minutes' stimulation with A23187, colocalization of G-kinase and vimentin was predominantly confined to filaments that extended into the uropod. The time of colocalization of G-kinase and vimentin was reduced in the A23187-stimulated cell from 3 minutes to 1 minute by 8-Br-cGMP. Coincident with colocalization was an increase in cGMP levels and transient phosphorylation of vimentin in adhered A23187-stimulated cells. Phosphorylation of vimentin was maximal after 3 minutes with A23187, and was essentially over at 5 minutes. The time of phosphorylation of vimentin was also reduced from 3 minutes to 1 minute when cells were preincubated with 8-Br-cGMP and then stimulated with A23187, which suggests that cyclic adenosine monophosphate (cAMP)-dependent protein kinase does not phosphorylate vimentin in A23187-treated neutrophils. Phosphorylation of vimentin was not observed in nonactivated cells treated only with 8-Br-cGMP. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation in A23187-treated cells, which provides supportive data that protein kinase C is not the phosphorylating enzyme. These results suggest that vimentin and G-kinase are colocalized in a Ca(2+)-dependent manner in neutrophils, and that vimentin is transiently phosphorylated by G-kinase in response to the colocalization of the two proteins. The transient redistribution of compartmentalized G-kinase represents one type of neutrophil activation mechanism.
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PMID:Cyclic guanosine monophosphate-dependent protein kinase is targeted to intermediate filaments and phosphorylates vimentin in A23187-stimulated human neutrophils. 780 96

The autophosphorylation-dependent protein kinase has been identified as a potent vimentin kinase that incorporates 2 mol of phosphates per mol of protein and generates five major phosphorylation sites in vimentin. Tryptic phosphopeptide mapping by high-performance liquid chromatography followed by sequential manual Edman degradation and direct peptide sequence analysis revealed that Ser-25, Ser-38, Ser-65, and Ser-71 in the amino-terminal domain and Ser-411 in the carboxyl-terminal domain are the phosphorylation sites in vimentin phosphorylated by this kinase, indicating that autophosphorylation-dependent protein kinase is a potent and unique vimentin kinase. Functional study further revealed that phosphorylation of vimentin by autophosphorylation-dependent protein kinase can completely inhibit polymerization and assembly of the cytoskeletal intermediate filament as demonstrated by electron microscopic analysis. Taken together, the results provide initial evidence that the autophosphorylation-dependent protein kinase may function as a vimentin kinase involved in the structure-function regulation of the cytoskeletal system. The results also support the notion that this cyclic nucleotide- and calcium-independent protein kinase may function as a multisubstrate/multifunctional protein kinase involved in the regulation of diverse cell functions.
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PMID:Autophosphorylation-dependent protein kinase phosphorylates Ser25, Ser38, Ser65, Ser71, and Ser411 in vimentin and thereby inhibits cytoskeletal intermediate filament assembly. 783 80

The Mos protein kinase is a serine-/threonine-specific protein kinase with a crucial role in meiotic cell divisions in vertebrates. Several oncogenic derivatives of the c-Mos protein have been discovered in murine retroviruses. These proteins have acquired mutations and exhibit different degrees of protein kinase activity in vitro. In an attempt to understand the factors governing Mos protein kinase activity we have compared the kinase activities of the wild-type c-Mos protein and two v-Mos proteins (strain HT1 and MSV124) after expression in insect cells. Only the 124 v-Mos protein showed kinase activity in vitro as measured by autophosphorylation, vimentin phosphorylation or by phosphorylation and activation of MAP kinase kinase. By domain swapping and site-directed mutagenesis we identified a single point mutation in the 124 v-Mos protein (Arg145-->Gly) which is responsible for its constitutive activity. This residue is located in the alpha-helix C of the kinase domain close to the ATP binding fold and is conserved in all known c-Mos proteins. Introduction of the corresponding mutation into HT1 v-Mos and into murine c-Mos activated both proteins for autophosphorylation, vimentin phosphorylation and for signalling via MAP kinase kinase in vitro. We hypothesize that the Arg145-->Gly mutation found in 124 v-Mos mimicks a conformational change which might be an obligatory step in the activation of c-Mos in vivo.
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PMID:Kinase activities of c-Mos and v-Mos proteins: a single amino acid exchange is responsible for constitutive activation of the 124 v-Mos kinase. 786 39

Purified assembly-competent vimentin, an intermediate filament protein, was obtained from bovine lens in this study. The effects of withangulatin A on vimentin assembly with or without phosphorylation were examined by negative-stain electron microscopy. Soluble tetrameric vimentin was assembled into irregular fibrils with lateral associations or short filaments after pretreatment with 50 or 100 microM withangulatin A, respectively. Incubation of assembled vimentin filaments with withangulatin A at 50 or 100 microM resulted in the formation of aggregates, and the degree of aggregation was concentration dependent. The appearance of vimentin filaments was slightly altered after treatment with cAMP-dependent protein kinase or protein kinase C; however, phosphorylation of filamentous vimentin by the protein kinases in the presence of withangulatin A resulted in higher degrees of aggregation of the filaments, compared with those treated only with the drug. Moreover, the level of phosphorylation of filamentous vimentin by the protein kinases was augmented in the presence of withangulatin A. Experimental results indicated that withangulatin A directly and specifically affects the conformation of the vimentin molecules, thereby resulting in alterations in assembly behavior and reactivity toward cAMP-dependent protein kinase and protein kinase C. The data observed further imply that withangulatin A, which directly causes aggregation of vimentin filaments, is a vimentin intermediate filament-targeting drug.
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PMID:Induction of aggregation and augmentation of protein kinase-mediated phosphorylation of purified vimentin intermediate filaments by withangulatin A. 796 40

To investigate the role of intermediate filament (IF) protein phosphorylation by cdc2 kinase during mitosis, we developed a monoclonal antibody 4A4 recognizing Ser55-phosphorylated vimentin. Western blotting indicated that this antibody reacted with vimentin phosphorylated by cdc2 kinase but not with non-phosphorylated vimentin or with vimentin phosphorylated by other kinases such as cAMP-dependent protein kinase, protein kinase C, or Ca(2+)-calmodulin-dependent protein kinase II. Immunofluorescence and immunoelectron microscopy showed that vimentin Ser55 residues distributed in the entire cytoplasmic vimentin filament system are phosphorylated when the cells enter mitosis and dephosphorylated in cytokinesis. All cell lines examined showed a similar appearance of immunoreactivity with antibody 4A4. Fractionation of mitotic cell extracts on Mono-Q Sepharose revealed a single peak of vimentin Ser55 kinase activity, and the anti-p34cdc2 antibody reacted with the 34 kDa band in the kinase containing fractions. Vimentin Ser55 kinase activities were nil in the interphase cell extract. Immunofluorescent evidence using antibody 4A4 and biochemical analysis using vimentin Ser55 peptide showed that the degree of disassembly of vimentin filament of various cell types at early mitotic phase correlated well with the amount of mitotically activated cdc2 kinase.
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PMID:Visualization and function of vimentin phosphorylation by cdc2 kinase during mitosis. 798 50

The major receptor protein for cyclic GMP (cGMP) in smooth muscle is the cGMP-dependent protein kinase (cGMP kinase). The more abundant I alpha isoform (subunit M(r) congruent to 78,000) of this enzyme mediates the effects of cGMP to relax contracted vascular smooth muscle preparations. In this study, we have addressed the hypothesis that the cGMP kinase is anchored to intracellular proteins which might serve to target cGMP kinase to protein substrates. Using a gel overlay technique, immunoprecipitation, and a fluorescence binding assay for cGMP kinase, we have identified vimentin as a high-affinity and specific binding protein for cGMP kinase. Binding of cGMP kinase to vimentin is reversible and stoichiometric (one cGMP kinase dimer/vimentin dimer) with a KD of approximately 49 nM. The site of high-affinity binding between cGMP kinase and vimentin did not appear to be localized to the catalytic domain of the kinase since vimentin phosphorylated by cGMP kinase and peptide substrates for cGMP kinase did not compete for high-affinity binding. Neither the proteolytically-derived 69-kDa catalytic fragment nor the 8-kDa N-terminal fragment bound vimentin with high affinity, suggesting that the cGMP kinase dimer was necessary for the interaction. Vimentin was readily phosphorylated in vitro with the dimer, but not the monomeric 69-kDa catalytic fragment even though the monomeric 69-kDa fragment was catalytically active toward other substrates such as histone F2b and peptides. This suggests that the high-affinity interaction between cGMP kinase and vimentin occurs at the N-terminal region, thus allowing the interaction between the phosphorylation site of vimentin and the catalytic site of cGMP kinase to occur.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:High-affinity binding and localization of the cyclic GMP-dependent protein kinase with the intermediate filament protein vimentin. 802 8

Exposure of 3T3 fibroblasts to the phosphatase inhibitor, calyculin-A, induces marked morphological changes and the formation of an aggregate of actin and myosin connected to the nucleus by intermediate filaments (Hirano, K., L. Chartier, R. G. Taylor, R. E. Allen, N. Fusetani, H. Karaki, D. J. Hartshorne: J. Muscle Res. Cell Motil. 13, 341-353 (1992)). Vimentin was isolated from this complex and shown to be phosphorylated. At least 4 phosphorylation sites were indicated. These sites were distinct from those phosphorylated by the cAMP-dependent protein kinase. Limited proteolysis was used to define the domains in which phosphorylation occurred. Vimentin was isolated from 32P-labeled calyculin-A-treated cells and digested with thrombin and alpha-chymotrypsin. Proteolysis with thrombin limited the phosphorylation to either the central core or C-terminal domain. Proteolysis with alpha-chymotrypsin indicated that the multiple phosphorylation sites were restricted to the C-terminal domain of vimentin.
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PMID:Phosphorylation of vimentin in the C-terminal domain after exposure to calyculin-A. 826 79


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