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:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

As cells adhere to extracellular matrix proteins, several focal adhesion proteins become tyrosine phosphorylated. One of the most prominent of these has been identified as the tyrosine kinase p125FAK (focal adhesion kinase, FAK). An interaction between FAK and members of the Src family tyrosine kinases p59fyn, pp60v-src, and activated pp60c-src (527F) has been demonstrated, raising the possibility that these kinases may regulate FAK activity. To explore the role of Src family kinases in focal adhesions and in the regulation of FAK activity, we isolated fibroblasts from transgenic mice that lack either pp60c-src, p59fyn, or pp62c-yes. These primary fibroblasts, and those of a control mouse, were passaged numerous times and resulted in spontaneously immortalized cell lines without the addition of transforming agents. After confirming the absence of the appropriate nonreceptor tyrosine kinases in the fyn-, src- and yes- fibroblasts, the ability of these fibroblasts to form focal adhesions and stress fibers was assessed by immunofluorescence microscopy and found to be comparable to that of normal fibroblasts. We investigated phosphotyrosine levels in response to adhesion to fibronectin and identified the pp60src substrate p130 as the one major protein with reduced levels of tyrosine phosphorylation in the cells lacking p59fyn and pp62c-yes, and particularly in those lacking pp60c-src. We examined FAK phosphorylation and kinase activity and found that there were no significant differences between these cells.
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PMID:An examination of focal adhesion formation and tyrosine phosphorylation in fibroblasts isolated from src-, fyn-, and yes- mice. 758 9

Effects of various types of protein kinase inhibitor on the adhesion and spreading of BALB/c mouse 3T3 cells and on the phosphorylation and stability of focal adhesion kinase (FAK) in the cells were studied. Inhibitors of protein tyrosine kinases, methyl 2,5-dihydroxycinnamate and herbimycin A, inhibited tyrosine-phosphorylation of FAK and the adhesion of 3T3 cells to fibronectin. Among inhibitors of serine/threonine kinases tested, calphostin C, a specific inhibitor of protein kinase C, inhibited cell spreading rather than cell adhesion, and it induced the decrease of intracellular FAK within 30 min. Inhibitors of tyrosine kinase, A kinase, G kinase, and myosin light chain kinase did not induce such a rapid and specific decrease of FAK. When calphostin C (20 microM) was added to sub-confluent monolayer cultures, serine-phosphorylation of FAK was inhibited by 67% within 2 h, and decrease in the amount of FAK and rounding up of the cells began after 4 h. Label-chase experiments indicated that about 60% of 35S-labeled FAK degraded within 1-2 h after addition of calphostin C to monolayer cultures. These results indicated that serine-phosphorylation of FAK induced by protein kinase C was important in the regulation of metabolic stability of FAK.
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PMID:Possible role of protein kinase C in the regulation of intracellular stability of focal adhesion kinase in mouse 3T3 cells. 758 52

Our previous work demonstrated that 12(S)-HETE, a lipoxygenase metabolite of arachidonic acid, promoted B16 amelanotic melanoma (B16a) cell spreading on fibronectin. In the current study, we investigated the biochemical mechanisms of the 12(S)-HETE induced response. 12(S)-HETE treatment resulted in a time-dependent increase in B16a cell spreading on fibronectin, which was blocked by either calphostin C or by genistein but not by H8. Two hours following cell plating, both spontaneous and 12(S)-HETE promoted cell spreading reached their maximum (nearly 100%). Spontaneous cell spreading was inhibited by the select 12-lipoxygenase inhibitor, BHPP, whose inhibitory effect could be overcome by increasing doses of exogenous 12(S)-HETE. 12(S)-HETE-treated B16a cells plated on either fibronectin or cultured on their own extracellular matrix demonstrated increased vinculin and tyrosine-phosphorylated proteins, which were colocalized at focal adhesions. The increase in vinculin localization to focal adhesions appeared to be a post-transcriptional process, since 12(S)-HETE treatment did not alter the overall protein level of vinculin in tumor cells, but resulted in a specific enrichment of vinculin to focal adhesions. Pretreatment of B16a cells with either calphostin C or genistein abolished 12(S)-HETE-increased formation of vinculin- and phosphotyrosine-containing focal adhesions. Immunoblotting using antiphosphotyrosine antibody 4G10 demonstrated, following 12(S)-HETE stimulation, an increased tyrosine phosphorylation of several proteins in focal adhesions; most prominently, a approximately 155 kd protein, a 120-130 kd protein cluster, a 76 kd protein, and a 42/44 kd complex. Immunoprecipitation with anti-phosphotyrosine antibody PY20 revealed increased tyrosine phosphorylation, post 12(S)-HETE stimulation, of proteins migrating at 120, 76, and 42/44 kd, of which the 120 kd protein co-migrated with pp125FAK. Immunoprecipitation with anti-FAK antibody BC-3 followed by immunoblotting with anti-phosphotyrosine antibody RC20H demonstrated a time-dependent hyperphosphorylation of pp125FAK. The present study suggests that 12(S)-HETE promoted melanoma cell spreading on fibronectin involves tyrosine phosphorylation of pp125FAK and protein kinase C- and tyrosine kinase-dependent focal adhesion formation.
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PMID:Melanoma cell spreading on fibronectin induced by 12(S)-HETE involves both protein kinase C- and protein tyrosine kinase-dependent focal adhesion formation and tyrosine phosphorylation of focal adhesion kinase (pp125FAK). 759 7

The interaction of cells with components of the extracellular matrix through their integrin receptors results in the stimulation of tyrosine phosphorylation of several proteins, suggesting that these receptors play a key role in signal transduction. Here we report that antibody-mediated ligation and clustering of alpha 3 beta 1 and alpha 6 beta 1/alpha 6 beta 4 integrins resulted in the stimulation of tyrosine phosphorylation of proteins that are specific for each heterodimer. Thus, ligation and clustering of the alpha 3 beta 1 integrin on human prostate carcinoma cells (PC-3) and human umbilical vein endothelial cells (HUVEC) with anti-alpha 3 antibodies resulted in the stimulation of tyrosine phosphorylation of a 55 kDa protein. In contrast, ligation and clustering of the alpha 6 beta 1 integrin on these cells with anti-alpha 6 antibody resulted in the dramatic stimulation of tyrosine phosphorylation of a 90 kDa protein in addition to a 52 kDa protein, and ligation and clustering of alpha 5 beta 1 on HUVEC did not result in the apparent stimulation of tyrosine phosphorylation of any proteins. Clustering with anti-beta 1 antibodies triggered the tyrosine phosphorylation of all of these proteins, whereas ligation and clustering of PC-3 cells with an anti-beta 4 antibody resulted in the tyrosine phosphorylation of a distinct 62 kDa protein. Since the PC-3 cells express both alpha 6 beta 1 and alpha 6 beta 4, these data suggest that these two receptors can transduce distinct signals. All of the phosphorylations could be inhibited by treating the cells with Genistein, a tyrosine kinase inhibitor. Antibody-mediated ligation and clustering of integrins on the two types of cells did not result in the stimulation of tyrosine phosphorylation of pp125 focal adhesion kinase, although this was observed upon cell attachment and spreading on fibronectin, laminin and anti-alpha 3 monoclonal antibody. Collectively, these data demonstrate that cross-linking of different integrin heterodimers can stimulate tyrosine kinase activities, leading to the phosphorylation of distinct proteins, which are also different from those observed when cells are allowed to spread on a matrix.
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PMID:Stimulation of tyrosine phosphorylation of distinct proteins in response to antibody-mediated ligation and clustering of alpha 3 and alpha 6 integrins. 762 2

To understand the mechanism for resistance of primary cultures of rat embryo fibroblasts (REFs) to oncogene-induced transformation, we studied the transforming ability of a recombinant retrovirus, ZSV, containing v-src and neo genes in REFs and in the rat cell line F2408. The susceptibility of REFs to p60v-src transformation was markedly reduced when compared with that of F2408 cells, despite high levels of expression of functional p60v-src tyrosine kinase in the two systems. In hybrid cells obtained by somatic cell fusion between F2408 cells transformed by v-src and uninfected REFs, the transformed phenotype was suppressed despite persistent expression of p60v-src tyrosine kinase. On the other hand, hybrid cells between v-src transformed F2408 cells and uninfected F2408 cells retained the transformed phenotypes. These results indicate that primary cells possess an intracellular function(s) that cause suppression of the transformed phenotype induced by the v-src gene. In ZSV-infected REFs, tyrosine phosphorylation of cellular proteins, including p125 focal adhesion kinase, p70 paxillin and p130 was similar to that in the ZSV-infected F2408 cells, indicating that tyrosine phosphorylation of these proteins is not sufficient for the expression of transformed phenotype. On the other hand, cellular fibronectin and one of integrin receptors were downregulated in the ZSV-transformed F2408 cells but not in ZSV-infected REFs, suggesting that fibronectin and/or its receptor might play a role in suppressing v-src transformation in primary rat cells.
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PMID:Suppression of v-Src transformation in primary rat embryo fibroblasts. 762 40

The organisation of the actin cytoskeleton was examined in H9c2 and human intestinal smooth muscle cells adherent on fibronectin or thrombospondin-1. Whereas cells adherent on fibronectin adopted a polygonal shape and rapidly assembled prominent stress fibres and focal contacts, cells adherent on thrombospondin-1 assumed a more irregular morphology with large lamellae containing radial actin microspikes. Focal contacts were not detected in cells adherent on thrombospondin-1, as determined by indirect immunofluorescence staining for vinculin and other focal contact components. Instead, the radial microspikes stained positively for the actin-bundling protein, 55 kDa/fascin, and myosins. In cells adherent on fibronectin, 55 kDa/fascin immunoreactivity was diffuse and tended to be concentrated in the perinuclear region. In long-term adherent cells cultured in serum-containing medium, 55 kDa/fascin was detected in membrane ruffles, in stress fibres and in the perinuclear region. The microspikes formed within 40 minutes of plating cells on thrombospondin-1 and remained present when cells were treated with sodium orthovandate and hydrogen peroxide to increase intracellular phosphotyrosine levels. Indeed, although vanadate-treated cells tended to retract, the microspikes became more prominent and showed an increased intensity of staining for fascin. Under these conditions, a proportion of the microspikes did not appear to be in contact with the substratum: these spikes stained weakly for focal adhesion kinase, talin and vinculin. Cells treated with genistein also spread and formed fascin-containing microspikes which tended to be more slender than those of control cells. In contrast, cells adherent on fibronectin displayed a complex rearrangement of the actin cytoskeleton and a transient enrichment of 55 kDa/fascin-containing structures at the cell surface when treated with sodium orthovanadate and hydrogen peroxide. These observations indicate that cell interactions with fibronectin or thrombospondin-1 send distinct organisational signals to the actin cytoskeleton and may offer a mechanistic framework for further investigations of the anti-adhesive properties of thrombospondin-1.
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PMID:Formation of stable microspikes containing actin and the 55 kDa actin bundling protein, fascin, is a consequence of cell adhesion to thrombospondin-1: implications for the anti-adhesive activities of thrombospondin-1. 765 18

A second protein-tyrosine kinase (PTK) of the focal adhesion kinase (FAK) subfamily, cell adhesion kinase beta (CAK beta), was identified by cDNA cloning. The rat CAK beta is a 115.7-kDa PTK that contains N- and C-terminal domains of 418 and 330 amino acid residues besides the central kinase domain. The rat CAK beta has a homology with mouse FAK over their entire lengths except for the extreme N-terminal 88 residues and shares 45% overall sequence identity (60% identical in the catalytic domain), which indicates that CAK beta is a protein structurally related to but different from FAK. The CAK beta gene is less evenly expressed in a variety of rat organs than the FAK gene. Anti-CAK beta antibody immunoprecipitated a 113-kDa protein from rat brain, 3Y1 fibroblasts, and COS-7 cells transfected with CAK beta cDNA. The tyrosine-phosphorylated state of CAK beta was not reduced on trypsinization, nor enhanced in response to plating 3Y1 cells onto fibronectin. CAK beta localized to sites of cell-to-cell contact in COS-7 transfected with CAK beta cDNA, in which FAK was found at the bottom of the cells. Thus, CAK beta is a PTK possibly participating in the signal transduction regulated by cell-to-cell contacts.
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PMID:Cloning and characterization of cell adhesion kinase beta, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily. 767 54

The very late activated Ag (VLA) molecules not only mediate T cell adhesions, but also provide costimulation in a TCR/CD3-dependent manner. However, little is known about the signals mediated by the ligation of VLA molecules. Previous work from our laboratory identified a 105-kDa protein that is predominantly phosphorylated on tyrosine residue upon engagement of VLA-4 in a human T lymphoblastic cell line, H9, and in peripheral T cells. In the present study, we have shown that the A and B epitope of VLA-4 plays a key role in VLA-4-mediated T cell costimulation. Moreover, we have demonstrated that the solid phase cross-linking of VLA-4 using Ab (against A and B) or the CS-1 region of fibronectin, stimulated tyrosine phosphorylation of 140-, 120-, 80- to 70-, 60- to 55-, 50-, and 45-kDa proteins in addition to the 105-kDa protein. In contrast, Ab ligation of the C epitope of VLA-4 mainly induced tyrosine phosphorylation of pp105, weakly induced other protein tyrosine phosphorylation, and additionally induced only minimal T cell costimulation. Using immunoblotting, we have identified some of the tyrosine-phosphorylated proteins to be phospholipase C gamma (pp140), pp125 focal adhesion kinase (pp120), paxillin (pp70 and pp50), p59fyn/p56lck (pp60-55), and mitogen-activated protein kinase (pp45). Since solid phase cross-linking of VLA-4 by B2 epitope-specific Ab induced T cell costimulation most strongly via the CD3 pathway, our results suggested that the above tyrosine-phosphorylated proteins may play an important role in VLA-4-mediated T cell costimulatory signaling events.
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PMID:Role of the VLA-4 molecule in T cell costimulation. Identification of the tyrosine phosphorylation pattern induced by the ligation of VLA-4. 767 11

We provide evidence for both matrix-dependent and pp60v-src tyrosine kinase-dependent modulation of cell migration via tyrosine phosphorylation of pp125FAK, a focal adhesion kinase, thought to be involved in integrin-mediated signaling. Enhanced pp125FAK tyrosine phosphorylation and cell spreading was associated with decreased migration. Cells plated on type I collagen were less spread and exhibited lower levels of pp125FAK tyrosine phosphorylation and faster migration rates compared with cells on fibronectin that were well spread, which exhibited enhanced levels of pp125FAK tyrosine phosphorylation and slower migration rates. Inside-out signaling via expression of pp60v-src or its kinase-negative mutant caused a decrease in cell migration by changing the extent of pp125FAK tyrosine phosphorylation to above or below the levels obtained with control cells plated on fibronectin. Hence, pp125FAK tyrosine phosphorylation appears to play a role in the signaling cascade pathway involved in regulation of extracellular matrix-modulated, integrin-mediated cell migration.
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PMID:Modulation of cell spreading and migration by pp125FAK phosphorylation. 767 74

Increasing evidence indicates that the integrin family of cell adhesion receptors can transduce biochemical signals from the extracellular matrix to the cell interior to modulate cell behavior. We have investigated the role of protein kinase C in alpha 5 beta 1 integrin-mediated signal transduction in Chinese hamster ovary cells. Up-regulation of protein kinase C activity by phorbol esters was found to enhance cell adhesion, spreading, and migration on a fibronectin substrate, without affecting the cell surface expression or fibronectin binding of the alpha 5 beta 1 integrin, whereas inhibition of protein kinase C activity by calphostin C inhibited these functions as well in unstimulated cells. In addition, we observed that protein kinase C activity in the cell membrane fraction transiently increases preceding cell spreading on fibronectin, but not on polylysine, additionally implying a specific role of protein kinase C in alpha 5 beta 1 integrin-mediated spreading on fibronectin. Experiments with phorbol esters and calphostin C also suggested that protein kinase C activity is required for enhanced phosphorylation of the focal adhesion kinase, pp125fak, in cells plated on fibronectin. Protein kinase C-alpha was not, however, found to directly act on pp125fak, suggesting that other mechanisms are involved in this phenomenon.
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PMID:Activation of protein kinase C precedes alpha 5 beta 1 integrin-mediated cell spreading on fibronectin. 769 9


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