Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

12(S)-HETE, a lipoxygenase metabolite of arachidonic acid induced a nondestructive and reversible endothelial cell (EC) retraction. 12(S)-HETE induced EC retraction was inhibited by protein kinase C inhibitors calphostin C and staurosporine but not by the protein kinase A inhibitor H8. The role of EC integrins alpha v beta 3 and alpha 5 beta 1 in 12(S)-HETE induced EC retraction was investigated. In confluent EC cultures, alpha v beta 3 is localized to focal adhesions at both the cell body and cell-cell borders and is colocalized with vinculin-containing focal adhesions. In contrast, alpha 5 beta 1 is primarily enriched at the cell-cell borders, demonstrating codistribution with cell cortical microfilaments and extracellular fibronectin. Both receptors were functional in mediating cell-cell or cell-matrix interactions based on the observations that specific antibodies inhibited EC adhesion to intact subendothelial matrix and disrupted the monolayer integrity. 12(S)-HETE induced a multistep, temporally defined redistribution of the alpha v beta 3-containing focal adhesions, leading to an eventual decrease in alpha v beta 3 plaques in the retracted ECs. This effect of 12(S)-HETE was inhibited by calphostin C but not by H8. The alterations of alpha v beta 3-containing focal adhesions preceded the development of EC retraction. 12(S)-HETE also enhanced EC alpha v beta 3 surface expression as revealed by immunofluorescence, flow cytometry, and digitized image analysis. 12(S)-HETE-induced alpha v beta 3 rearrangement (i.e., decreased focal adhesion localization and enhanced surface expression) did not result from altered mRNA transcription (as revealed by semi-quantitative RT-PCR analysis) or protein translation (as revealed by Western blotting). In contrast to its effect on alpha v beta 3, 12(S)-HETE did not demonstrate a temporally related, well-defined effect on the distribution pattern and the surface expression of alpha 5 beta 1, although the cell-cell border staining pattern of alpha 5 beta 1 was disrupted due to EC retraction. It is concluded that 12(S)-HETE-induced decrease of alpha v beta 3 localization to focal adhesions may contribute to the development of EC retraction and that 12(S)-HETE induced increase in alpha v beta 3 surface expression may promote adhesion of inflammatory leukocytes as well as tumor cells to endothelium.
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PMID:Protein kinase C-dependent effects of 12(S)-HETE on endothelial cell vitronectin receptor and fibronectin receptor. 768 91

We studied the participation of the protein kinase C pathway in thrombin-induced cytoskeletal alterations in confluent cultured bovine corneal endothelial (BCE) cells. Cultured BCE cells were exposed to alpha-thrombin (0.1-10 U/ml for 15-60 min) and the distribution of F-actin and vinculin plaques was examined using immunofluorescent staining and electron microscopy. Phorbol 12-myristate 13-acetate (PMA, 10 nM for 15 min), the broad spectrum protein kinase inhibitors staurosporine (10 nM) and H-7 (10 nM), and highly specific PKC inhibitor calphostin C (10 nM) were used to evaluate the role of PKC/phosphorylation in this phenomenon. HA-1004 (10 nM) was used as a negative control for these inhibitors. In a parallel experiment, PKC activity of cytosol and membrane of BCE cells was also evaluated. In control samples, F-actin was distributed mainly at the periphery of cells, where it formed dense peripheral bundles; vinculin plaques were also present at the cell boundary. Exposure of BCE cells to thrombin changed the distribution of F-actin and vinculin into a diffuse pattern; a similar alteration was also induced by incubation with PMA. These phenomena were blocked by incubation with H-7, staurosporine and calphostin C. Both cytosolic and membrane PKC activity was increased after 5 to 30 min exposure of alpha-thrombin and returned to the control level after 1 h. alpha-Thrombin induces alteration in the cytoskeleton of BCE cells, and this message is transduced at least in part by PKC dependent pathways. PKC/phosphorylation may thus play an important role in physiological processes that involve alterations of the cytoskeleton.
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PMID:Thrombin induced cytoskeletal change in cultured bovine corneal endothelial cells mediated via protein kinase C pathway. 772 Apr 4

Vascular endothelial cells are constantly in contact with oxyradicals and must be especially well equipped to resist their toxic effects and generate appropriate physiological responses. Despite the importance of oxyradicals in the physiopathology of the vascular endothelium, the mechanisms regulating the oxidative response of endothelial cells are poorly understood. In the present study, we observed that H2O2 in concentrations that induced severe fragmentation of F-actin in fibroblasts rather induced a reorganization of F-actin in primary cultures of human umbilical vein endothelial cells (HUVECs) that was characterized by the accumulation of stress fibers, the recruitment of vinculin to focal adhesions, and the loss of membrane ruffles, H2O2 also induced in these cells a strong (10- to 14-fold) activation of the p38 mitogen-activated protein (MAP) kinase, which resulted in activation of MAP kinase-activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). The MAP kinases extracellular-regulated kinase, and c-Jun N-terminal kinase/stress-activated protein kinase were only slightly increased by these treatments. Inhibiting p38 activity with the highly specific inhibitor SB203580 blocked the H2O2-induced endothelial microfilament responses. Moreover, fibroblasts acquired an endothelium-like SB203580-sensitive actin response when HSP27 concentration was increased by gene transfection to the same high level as found in HUVECs. The results indicate that activation of p38 MAP kinase in cells such as endothelial cells, which naturally express high level of HSP27, plays a central role in modulating microfilament responses to oxidative stress. Consequently, the p38 MAP kinase pathway may participate in the several oxyradical-activated functions of the endothelium that are associated with reorganization of microfilament network.
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PMID:Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. 904 59

Vascular endothelial growth factor (VEGF) is a potent chemotactic agent for endothelial cells. Yet the signalling pathways that modulate the motogenic effects of VEGF in vascular endothelial cells are still ill defined. In the present study, we found in primary cultures of human umbilical vein endothelial cells (HUVEC) that VEGF increased cell migration and induced a marked reorganization of the microfilament network that was characterized by the formation of stress fibers and the recruitment of vinculin to focal adhesions. VEGF also stimulated the mitogen activated protein (MAP) kinases ERK (extracellular signal-regulated kinase) and p38 (stress activated protein kinase-2), but not SAPK1/JNK (stress activated protein kinase-1/c-Jun NH2-terminal kinase). Activation of p38 resulted in activation of MAP kinase activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). Inhibiting the VEGF-induced activation of ERK with PD098059 did not influence actin organization or cell migration but totally inhibited the VEGF-induced incorporation of thymidine into DNA. Inhibition of p38 activity by the specific inhibitor SB203580 led to an inhibition of HSP27 phosphorylation, actin reorganization and cell migration. The results indicate that the p38 pathway conveys the VEGF signal to microfilaments inducing rearrangements of the actin cytoskeleton that regulate cell migration. By modulating cell migration, p38 may thus be an important regulator of angiogenesis.
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PMID:p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. 939 75

SPARC, a counteradhesive matricellular protein, inhibits endothelial cell adhesion and proliferation, but the pathways through which these activities are blocked are not known. In this study, we used inhibitors of major signaling proteins to identify mediators through which SPARC exerts its counteradhesive and antiproliferative functions. Pretreatments with the general protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, protected against the inhibitory effect of SPARC on bovine aortic endothelial (BAE) cell spreading by more than 60%. Similar pretreatments with PTK inhibitors significantly blocked the diminishment of focal adhesions by SPARC in confluent BAE cell monolayers, as determined by the formation of actin stress-fibers and the distribution of vinculin in focal adhesion plaques. Inhibition of endothelial cell cycle progression by SPARC and a calcium-binding SPARC peptide, however, was not affected by PTK inhibitors. Inhibition of DNA synthesis by SPARC was not reversed by inhibitors of the activity of protein kinase C (PKC), or of cAMP-dependent protein kinase (PKA), but was sensitive to pertussis (and to a lesser extent, cholera) toxin. The counteradhesive effect of SPARC on endothelial cells is, therefore, mediated through a tyrosine phosphorylation-dependent pathway, whereas its antiproliferative function is dependent, in part, on signal transduction via a G protein-coupled receptor.
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PMID:SPARC inhibits endothelial cell adhesion but not proliferation through a tyrosine phosphorylation-dependent pathway. 971 51

Activation of protein kinase C (PKC) in many cell types results in cytoskeletal reorganization associated with cell proliferation. We previously described a new cell cycle-regulated myristylated PKC substrate, SSeCKS (pronounced essex), that interacts with the actin cytoskeleton [Lin et al., 1995, 1996]. SSeCKS shares significant homology with Gravin, which encodes kinase scaffolding functions for PKC and PKA [Nauert et al., 1997]. This article describes the cellular effects of ectopically expressing SSeCKS in untransformed NIH3T3 fibroblasts. Because the constitutive overexpression of SSeCKS is toxic [Lin et al., 1995], we developed cell lines with tetracycline (tet)-regulated SSeCKS expression. The induction of SSeCKS (removal of tet) caused significant cell flattening and the elaboration of an SSeCKS-associated cortical cytoskeletal matrix resistant to Triton X-100 extraction. Flattened cells were growth-arrested and marked by the formation of cellular projections and the temporary loss of actin stress fibers and vinculin-associated adhesion plaques. SSeCKS overexpression did not affect steady-state levels of actin, vinculin, or focal adhesion kinase (FAK) but did increase integrin-independent FAK tyrosine phosphorylation. Stress fiber loss was coincident with induced SSeCKS expression, strongly suggesting a direct effect. Cytochalasin, and to a lesser extent nocodazole, inhibited SSeCKS-induced cell flattening, however, only cytochalasin affected the shape of pre-flattened cells, suggesting a greater dependence on microfilaments, rather than microtubules. By contrast, only nocodazole caused retraction of the filopodia-like processes. These data indicate a role for SSeCKS in modulating both cytoskeletal and signaling pathways. Thus, we propose to expand SSeCKS scaffolding functions to include the ability to control actin-based cytoskeletal architecture, as well as mitogenic signal pathways.
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PMID:Control of cytoskeletal architecture by the src-suppressed C kinase substrate, SSeCKS. 974 95

Activation of protein kinase C is a key signal transduction event in mesangial cell dedifferentiation and proliferation, yet little is known about downstream substrates or their roles in normal or diseased glomeruli. SSeCKS, a novel protein kinase C substrate originally isolated as a src-suppressed negative mitogenic regulator in fibroblasts, controls actin-based cytoskeletal architecture and scaffolds key signaling kinases such as protein kinase C and protein kinase A. Based on the morphologic similarity between SSeCKS-overexpressing fibroblasts and stellate mesangial cells, we hypothesized that SSeCKS might play a role in mesangial cell morphology in a protein kinase C-dependent manner. Immunoblotting, in situ staining and northern blotting detected abundant expression of SSeCKS in human and rodent mesangial cells and glomerular parietal cells but not in renal tubular epithelia. Immunofluorescence analysis showed enrichment of SSeCKS in mesangial cell podosomes and along a cytoskeletal network distinct from F-actin. Activation of protein kinase C by phorbol ester resulted in a rapid serine phosphorylation of SSeCKS and its subsequent translocation to perinuclear sites, coincident with the retraction of stellate processes. These effects were blocked by concentrations of bis-indolylmaleimide that selectively inhibit protein kinase C. Finally, ablation of SSeCKS expression using retroviral anti-sense vectors induced (1) an elongated, fibroblastic cell morphology, (2) production of thick, longitudinal stress fibers and (3) repositioning of vinculin-associated focal complexes away from the cell edges. These data suggest a role for SSeCKS as a downstream mediator of protein kinase C-controlled, actin-based mesangial cell cytoskeletal architecture.
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PMID:Involvement of the protein kinase C substrate, SSeCKS, in the actin-based stellate morphology of mesangial cells. 988 89

PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the alpha5beta1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways.
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PMID:The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells. 1002 29

Human megakaryoblastic leukemia Meg-01 cells were attached to fibronectin (FN)-coated substratum, on which remarkable spreading and cytoplasmic elongation was induced by treatment with a protein kinase inhibitor, staurosporine (stp). This effect was inhibited by RGDS and was also not seen on FN-lacking substratum. The extended cytoplasm had swollen terminals and nodes, which contained GpIIb and beta-thromboglobulin, occasionally included alpha granules, and tended to form particles (2-5 microm) after rupture of the narrowed cytoplasm. Among other protein kinase modulators tested, only K252a promoted the elongation, while calphostin, herbimycin, TPA, and calyculin suppressed it. The cells began to migrate soon after addition of stp, with attachment to the substratum held at some sites during the migration. This tethered movement seemed to cause the cytoplasmic elongation and the rupture into particles. The elongation was retarded by pretreating the cells with cytochalasin A and Clostridium C3 toxin but not with demecolcine. Actin microfilaments in the stp-treated Meg-01 cells accumulated in the filopodia and periphery of the extended cytoplasm, in which vinculin was colocalized as adhesion plaques. The microtubules were longitudinally oriented through the cytoplasmic extension and showed no ring profile in the nodes and particles. Thus, stp in the presence of FN appears to stimulate reorganization of actin-based cytoskeleton and formation of focal contacts in Meg-01 cells. This leads to the activation of cell adhesion and motility, and then cytoplasmic elongation and rupture into particles.
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PMID:Cytoplasmic elongation and rupture in megakaryoblastic leukemia cells via activation of adhesion and motility by staurosporine on fibronectin-bound substratum. 1019 57


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