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)

Protein phosphorylation in intact S49 mouse lymphoma cells was studied by using high-resolution two-dimensional gel electrophoresis of proteins labelled with [35S]methionine or [32P]Pi. In wild-type cells substrates for cyclic AMP-stimulatable phosphorylation exhibited high basal phosphorylation; in mutant cells deficient in activities of either cyclic AMP-dependent protein kinase or adenylate cyclase, basal phosphorylation of most of these substrates was negligible. Analysis of tryptic phosphopeptides from proteins labelled with [32P]Pi in wild-type cells suggested that identical sites were phosphorylated under conditions of both basal and hormonally elevated concentrations of cyclic AMP. These results argue that most basal phosphorylation is a consequence of partial activation of cyclic AMP-dependent protein kinase and that this activation is attributable to basal concentrations of cyclic AMP. For the intermediate filament protein vimentin, basal phosphorylation was largely at a site distinct from that stimulated by increased cyclic AMP, and basal phosphorylation was not markedly different in mutant and wild-type cells. Vimentin phosphorylated at both sites was not observed. Cyclic AMP treatment resulted in enhanced phosphorylation at the cyclic AMP-specific site and decreased phosphorylation at the cyclic AMP-independent site.
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PMID:Basal phosphorylation of cyclic AMP-regulated phosphoproteins in intact S49 mouse lymphoma cells. 298 11

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

Our previous studies have shown that vimentin can serve as a substrate for the v-Mos protein kinase in vitro. Furthermore, the amount of vimentin molecules in Moloney murine sarcoma virus (Mo-MuSV) transformed cells is decreased relative to uninfected cells and a lower molecular weight form is observed in these v-mos transformed cells (Singh & Arlinghaus, Virology 173: 144-156, 1989). Vimentin filaments are hyperphosphorylated and disassemble when cells enter mitosis. Here, we show that vimentin was coprecipitated with p85gag-mos from mitotic cell extracts by two different anti-Mos antibodies that do not crossreact with vimentin. However, we were unable to detect vimentin/v-Mos complexes interphase extracts, possibly due to lack of soluble vimentin. p37env-mos was also found to associate with purified bovine lens vimentin in vitro. The significance of these findings with regard to v-mos-induced cellular transformation is discussed.
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PMID:Association of v-Mos with soluble vimentin in vitro and in transformed cells. 833 43

Vimentin and glial fibrillary acidic protein (GFAP) are intermediate filament proteins expressed in the cytoplasm of various types of cells. The head domains of these proteins are phosphorylated by various protein kinases. Site- and phosphorylation-specific antibodies which recognize a phosphorylated serine/threonine residue in the head domains and its flanking sequence provide a useful tool to monitor and visualize protein kinase activities in single cells.
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PMID:Visualization of protein kinase activities in single cells by antibodies against phosphorylated vimentin and GFAP. 899 82

PKN is a serine/threonine protein kinase with a catalytic domain homologous to the protein kinase C family and unique N-terminal leucine zipper-like sequences. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that the regulatory domain of PKN interacted with vimentin. We then examined whether PKN would phosphorylate vimentin in vitro. Vimentin proved to be an excellent substrate for PKN, and the phosphorylation of vimentin by PKN occurred in the head domain with the result of a nearly complete inhibition of its filament formation in vitro. Similar results were also obtained with another type III intermediate filament protein, glial fibrillary acidic protein (GFAP). These results raise the possibility that PKN may regulate filament structures of vimentin and GFAP by domain-specific phosphorylation.
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PMID:Domain-specific phosphorylation of vimentin and glial fibrillary acidic protein by PKN. 917 63

We have characterized the expression and activity of the cell cycle regulatory machinery and the organization of the cytoskeleton of the p16(Ink4a)-deficient astrocytoma cell line, U343 MG-a (U343), following retinoic acid (RA) treatment. RA causes cell cycle arrest at low cell density and significant morphological changes in U343 cells, reflected by reorganization of the intermediate filament, GFAP, and actin. RA-induced cell cycle arrest is also associated with induction of p27Kip1 expression, inhibition of cdk2-associated kinase activity and alteration of the phosphorylation state of the pRB-family proteins. We next determined the effect of inducing expression of the cyclin dependent kinase inhibitors (CKI's), p16(Ink4a), p21Cip1/Waf1 or p27Kip1 on the proliferation and morphology of these malignant astrocytoma cells in the absence and presence of RA. Induction of p16, p21 or p27, using the tetracycline repressor system, potently inhibits proliferation of U343 cells. However, rather than resembling RA-treated cells, CKI-induced U343 cells become flat with abundant cytoplasm and perinuclear vacuolization. CKI-induced morphological alterations are accompanied by a significant reorganization of glial filaments within the cytoplasm. Interestingly, when U343 cells are growth arrested by p16, p21 or p27 induction and treated simultaneously with RA, a dramatic morphological change occurs, cells acquiring multiple long, tapering processes reminiscent of primary astrocytes. This rearrangement is accompanied by reorganization of GFAP, vimentin and actin. Vimentin specifically relocalizes to the tips of the long processes which form. The arrangement of intermediate filaments in these cells is, in fact, indistinguishable from their arrangement in primary human astrocytes. These data demonstrate that when a strong proliferative block, produced by CKI expression, occurs in conjunction with the morphogenic signals generated by RA, these p16-deficient malignant astrocytoma cells are induced to phenotypically resemble normal astrocytes.
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PMID:Retinoic acid and the cyclin dependent kinase inhibitors synergistically alter proliferation and morphology of U343 astrocytoma cells. 936 21

Using immobilized GST-Raf-1 as bait, we have isolated the intermediate filament protein vimentin as a Raf-1-associated protein. Vimentin coimmunoprecipitated and colocalized with Raf-1 in fibroblasts. Vimentin was not a Raf-1 substrate, but was phosphorylated by Raf-1-associated vimentin kinases. We provide evidence for at least two Raf-1-associated vimentin kinases and identified one as casein kinase 2. They are regulated by Raf-1, since the activation status of Raf-1 correlated with the phosphorylation of vimentin. Vimentin phosphorylation by Raf-1 preparations interfered with its polymerization in vitro. A subset of tryptic vimentin phosphopeptides induced by Raf-1 in vitro matched the vimentin phosphopeptides isolated from v-raf-transfected cells labeled with orthophosphoric acid, indicating that Raf-1 also induces vimentin phosphorylation in intact cells. In NIH 3T3 fibroblasts, the selective activation of an estrogen-regulated Raf-1 mutant induced a rearrangement and depolymerization of the reticular vimentin scaffold similar to the changes elicited by serum treatment. The rearrangement of the vimentin network occurred independently of the MEK/ERK pathway. These data identify a new branch point in Raf-1 signaling, which links Raf-1 to changes in the cytoskeletal architecture.
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PMID:The Raf-1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments. 1102 85

Vimentin, one of type III intermediate filament (IF) proteins, is expressed not only in mesenchymal cells but also in most types of tumor cells. In the present study, we introduced several types of vimentin mutated at putative phosphorylation sites in its amino-terminal head domain into type III IF-negative T24 cells. Site-specific mutation induced the formation of an unusually long bridge-like IF structure between the unseparated daughter cells, although these mutants formed the filament network similar to wild type in interphase cells. Together with sites phosphorylated by Rho-kinase and protein kinase C (PKC), vimentin-Ser72, which can not be phosphorylated by any known vimentin kinase, was one of the mutation sites essential for this phenotype. We further demonstrated that vimentin-Ser72 was phosphorylated specifically at the cleavage furrow during cytokinesis. These observations suggest the existence of a novel protein kinase responsible for vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. We propose that Rho-kinase, PKC, and an unidentified vimentin-Ser72 kinase may play important roles in vimentin filament separation during cytokinesis.
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PMID:Protein kinases required for segregation of vimentin filaments in mitotic process. 1142 Jun 99


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