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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)

Insulin signaling results in rapid changes to the cell cytoskeleton, and it has recently been shown that insulin stimulates the dephosphorylation of the cytoskeletal-associated tyrosine kinase, focal adhesion kinase (pp125(FAK)). We report here that mutation of two tryptic cleavage sites (Lys164 and Lys582 --> Asn; 2N) in the insulin receptor alpha-subunit results in a cell-line (CHO.2N-10) with altered morphology associated with an increase in cell size, a decrease in cell adhesiveness, and a decrease in pp125(FAK) tyrosine phosphorylation in the absence of insulin (45.2 +/- 9.7% compared to nontransfected Chinese hamster ovary (CHO) cells). In contrast to pp125(FAK), paxillin phosphorylation was similar in all cell lines despite lower levels (61.0 +/- 10.4% compared to CHO cells) of paxillin protein in CHO.2N-10 cells. We observed comparable protein levels of pp125(FAK) and the structural focal adhesion protein, vinculin, in all cell lines. Despite underphosphorylation of pp125(FAK) in the basal state, insulin stimulation of CHO.2N-10 cells still resulted in dephosphorylation of pp125(FAK). CHO.2N-10 and CHO.T (overexpress wild-type insulin receptor) cells have similar insulin binding characteristics, insulin-stimulated autokinase and peptide phosphorylation, and insulin-stimulated pp185/IRS-1 phosphorylation. Our results suggest that the insulin receptor may play an important role in cell-matrix interactions, such as modulating cell adhesion and inducing cell architecture changes.
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PMID:Reduced cell attachment and phosphorylation of focal adhesion kinase associated with expression of a mutant insulin receptor. 891 May 46

Paxillin is a 68-kD focal adhesion phosphoprotein that interacts with several proteins including members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal proteins vinculin and the tyrosine kinase, focal adhesion kinase (FAK). This suggests a function for paxillin as a molecular adaptor, responsible for the recruitment of structural and signaling molecules to focal adhesions. The current study defines the vinculin- and FAK-interaction domains on paxillin and identifies the principal paxillin focal adhesion targeting motif. Using truncation and deletion mutagenesis, we have localized the vinculin-binding site on paxillin to a contiguous stretch of 21 amino acids spanning residues 143-164. In contrast, maximal binding of FAK to paxillin requires, in addition to the region of paxillin spanning amino acids 143-164, a carboxyl-terminal domain encompassing residues 265-313. These data demonstrate the presence of a single binding site for vinculin, and at least two binding sites for FAK that are separated by an intervening stretch of 100 amino acids. Vinculin- and FAK-binding activities within amino acids 143-164 were separable since mutation of amino acid 151 from a negatively charged glutamic acid to the uncharged polar residue glutamine (E151Q) reduced binding of vinculin to paxillin by >90%, with no reduction in the binding capacity for FAK. The requirement for focal adhesion targeting of the vinculin- and FAK-binding regions within paxillin was determined by transfection into CHO.K1 fibroblasts. Significantly and surprisingly, paxillin constructs containing both deletion and point mutations that abrogate binding of FAK and/or vinculin were found to target effectively to focal adhesions. Additionally, expression of the amino-terminal 313 amino acids of paxillin containing intact vinculin- and FAK-binding domains failed to target to focal adhesions. This indicated other regions of paxillin were functioning as focal adhesion localization motifs. The carboxyl-terminal half of paxillin (amino acids 313-559) contains four contiguous double zinc finger LIM domains. Transfection analyses of sequential carboxyl-terminal truncations of the four individual LIM motifs and site-directed mutagenesis of LIM domains 1, 2, and 3, as well as deletion mutagenesis, revealed that the principal mechanism of targeting paxillin to focal adhesions is through LIM3. These data demonstrate that paxillin localizes to focal adhesions independent of interactions with vinculin and/or FAK, and represents the first definitive demonstration of LIM domains functioning as a primary determinant of protein subcellular localization to focal adhesions.
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PMID:Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding. 892 90

Calreticulin is an ubiquitous and highly conserved high capacity Ca(2+)-binding protein that plays a major role in Ca2+ storage within the lumen of the ER. Here, using L fibroblast cell lines expressing different levels of calreticulin, we show that calreticulin plays a role in the control of cell adhesiveness via regulation of expression of vinculin, a cytoskeletal protein essential for cell-substratum and cell-cell attachments. Both vinculin protein and mRNA levels are increased in cells overexpressing calreticulin and are downregulated in cells expressing reduced level of calreticulin. Abundance of actin, talin, alpha 5 and beta 1 integrins, pp125 focal adhesion kinase, and alpha-catenin is not affected by the differential calreticulin expression. Overexpression of calreticulin increases both cell-substratum and cell-cell adhesiveness of L fibroblasts that, most surprisingly, establish vinculin-rich cell-cell junctions. Upregulation of calreticulin also affects adhesion-dependent phenomena such as cell motility (which decreases) and cell spreading (which increases). Downregulation of calreticulin brings about inverse effects. Cell adhesiveness is Ca2+ regulated. The level of calreticulin expression, however, has no effect on either the resting cytoplasmic Ca2+ concentration or the magnitude of FGF-induced Ca2+ transients. Calreticulin, however, participates in Ca2+ homeostasis as its level of expression affects cell viability at low concentrations of extracellular Ca2+. Consequently, we infer that it is not the Ca2+ storage function of calreticulin that affects cell adhesiveness. Neither endogenous calreticulin nor overexpressed green fluorescent protein-calreticulin construct can be detected outside of the ER. Since all of the adhesion-related effects of differential calreticulin expression can be explained by its regulation of vinculin expression, we conclude that it is the ER-resident calreticulin that affects cellular adhesiveness.
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PMID:Calreticulin modulates cell adhesiveness via regulation of vinculin expression. 899 Nov 1

The versatility of integrin functions is mediated by engagement of a number of proteins that assemble with integrins. Among them, paxillin is one of the important molecules interacting with a variety of signaling molecules and cytoskeletal building blocks. We report here that paxillin is not a single molecule with a unique physiological property. We identified two human paxillin isoforms, beta and gamma. These isoforms have distinct amino acid insertions; each consists of a distinct exon, at the same site of previously reported paxillin (paxillin alpha). Several proteins were co-precipitated with paxillin, and we found that beta bound to focal adhesion kinase but weakly to vinculin, and gamma bound to vinculin but only weakly to focal adhesion kinase, although both bound equally to talin. No additional proteins were found to bind to beta and gamma over those binding to alpha. Unlike the alpha isoform, beta and gamma mRNAs were not detected in normal tissues, but several cancer cells expressed both alpha and beta proteins simultaneously. All three isoform proteins were expressed in promonocytic cells with ratios comparable with each other, and the expression patterns were altered during differentiation of floating promonocytic cells into adherent macrophage-like cells. Therefore, each isoform of paxillin exhibits distinct expression and different biochemical as well as physiological properties and thereby appears to act as a distinct module involved in different functions of integrins.
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PMID:Monocyte cells and cancer cells express novel paxillin isoforms with different binding properties to focal adhesion proteins. 905 45

Assembly of a fibronectin (FN) matrix is a multistep process which influences a number of cellular functions including intracellular cytoskeletal organization and signaling responses. We have previously reported on a recombinant FN (recFN), FN delta III1-7, which differs from native FN in its rate of fibril formation. To determine the intracellular consequences of a delay in assembly, we compared the distribution of cytoskeletal proteins during the formation of native and recFN matrices by immunofluorescence at various time points. CHO alpha 5 cell cytoskeleton was reorganized in response to both native and recFN matrix formation. Assembly of native FN induced a rapid reorganization of actin into stress fibers and colocalization of alpha 5 beta 1 integrin, focal adhesion kinase (FAK), vinculin, and paxillin to regions of cell-matrix contact. alpha 5 beta 1 integrins and FAK are also clustered upon binding of FN delta III1-7 to cells but actin reorganization and focal adhesion formation are delayed and appear to be dependent on the formation of FN delta III1-7 fibrils. These results suggest that the structural framework of the matrix plays an important role in the ability of FN to initiate intracellular responses.
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PMID:Coordinated regulation of fibronectin fibril assembly and actin stress fiber formation. 917 3

We have investigated the signal transduction pathway of the G-protein mu-opioid receptor upstream of phospholipase D (PLD) and protein kinase C-epsilon (PKC-epsilon) activation in postmitotic E6CH chick embryo cortical neurons. The mu-opioid receptor and PLD-PKC-epsilon functional coupling depends on upstream tyrosine kinase activation. We now report that the mu-opioid agonists specifically stimulated tyrosine phosphorylation and activation of the focal adhesion kinase (FAK) in a time-dependent manner. We also demonstrate that met-enkephalin, a mu-opioid agonist in E6CH cultures, significantly increases tyrosine phosphorylation of another Src kinase substrate, the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin led to drastic changes in subcellular localization, an estimated 2-fold enrichment in the cytosol. Similarly, opioids stimulated a sustained tyrosine phosphorylation of vinculin, a protein enriched in focal adhesion sites. These data provide novel evidence that opioid receptor intracellular signaling engages the specific activation of tyrosine kinase FAK and regulates the neuronal cytoskeleton during central nervous system morphogenesis.
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PMID:mu-Opioids activate tyrosine kinase focal adhesion kinase and regulate cortical cytoskeleton proteins cortactin and vinculin in chick embryonic neurons. 936 24

In previous studies, we have shown that smooth muscle cells and myofibroblast subpopulations of the perivascular stem villous sheath of the human placenta contain focal adhesion plaques and talin immunoreactivity. The close association of these cells to elastic and collagen fibres have led to the assumption of a functional myofibroelastic unit within the perivascular stem villous sheath. Interactions between the extracellular matrix and smooth muscle cells depend on a variety of structural protein assemblies. In the present study, we examined, by immunocytochemistry, whether the molecular assembly of extracellular matrix proteins and molecules of focal adhesions, known to be essential for signal transduction in smooth muscle cells, are also found in smooth muscle cells of the perivascular stem villous sheath of the human placenta. Vascular and extravascular smooth muscle cells were immunoreactive for alpha-actinin, vinculin, paxillin and tensin, the integrin chains alpha1 and beta1, and the basement membrane components laminin and heparan/-chondroitin sulfate proteoglycan perlecan. pp125(FAK) did not react. In the extracellular matrix of blood vessel walls and the perivascular stem villous sheath, we found immunoreactivity of fibronectin and collagen types I, VI and undulin (collagen type XIV). From our data we conclude that within the perivascular stem villous sheath, there exists a system of signal transduction molecules, indicating a cross talk between the smooth muscle cells of this sheath and their surrounding extracellular matrix.
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PMID:Molecular anatomy of the perivascular sheath in human placental stem villi: the contractile apparatus and its association to the extracellular matrix. 936 35

Endothelial cell (EC) gap formation and barrier function are subject to dual regulation by (1) axial contractile forces, regulated by myosin light chain kinase activity, and (2) tethering forces, represented by cell-cell and cell-substratum adhesions. We examined whether focal adhesion plaque proteins (vinculin and talin) and focal adhesion kinase, p125FAK (FAK), represent target regulatory sites involved in thrombin-mediated EC barrier dysfunction. Histologically, thrombin produced dramatic rearrangement of EC actin, vinculin, and FAK in parallel with the evolution of gap formation and barrier dysfunction. Vinculin and talin were in vitro substrates for phosphorylation by EC PKC, a key effector enzyme involved in thrombin-induced EC barrier dysfunction. Although vinculin and talin were phosphorylated in situ under basal conditions in 32P-labeled EC, thrombin failed to alter the basal level of phosphorylation of these proteins. Phosphotyrosine immunoblotting showed that neither vinculin nor talin was significantly phosphorylated in situ on tyrosine residues in unstimulated ECs, and this was not further increased after thrombin. In contrast, both thrombin and the thrombin receptor-activating peptide (TRAP) produced an increase in FAK phosphotyrosine levels (corrected for immunoreactive FAK content) present in EC immunoprecipitates. Ionomycin, which produces EC barrier dysfunction in a myosin light chain kinase-independent manner, was used to increase intracellular Ca2+ and evaluate the Ca2+ sensitivity of this observation. In contrast to thrombin, ionomycin effected a dramatic decrease in the phosphotyrosine-to-immunoreactive FAK ratios, suggesting distinct effects of the two agents on FAK phosphorylation and function. These data indicate that modulation of cell tethering via phosphorylation of focal adhesion proteins is complex, agonist-specific, and may be a relevant mechanism of EC barrier dysfunction in permeability models that do not depend on an increase in myosin 20-kD regulatory light chain phosphorylation.
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PMID:Thrombin-mediated focal adhesion plaque reorganization in endothelium: role of protein phosphorylation. 937 19

The bovine papillomavirus type 1 (BPV-1) E6 oncoprotein can transform fibroblasts and induce anchorage-independent growth and disassembly of the actin stress fibers. We have previously shown that the E6 protein interacts with the focal adhesion protein, paxillin, suggesting a direct role of E6 in the disruption of the actin cytoskeleton. We have now mapped the E6 binding sites on paxillin to the LD motif repeats region, which has been implicated in mediating paxillin binding to two other focal adhesion proteins, vinculin and the focal adhesion kinase. The five LD motif repeats identified in paxillin do not contribute equally to its interaction with E6. The first LD repeat is most critical for paxillin binding to E6 both in vitro and in vivo. Furthermore, the binding of recombinant wild-type E6 protein to paxillin blocked the interaction of several cellular proteins with paxillin, including vinculin and the focal adhesion kinase. A mutant E6 protein (H105) which does not bind to paxillin had no effect on the binding of these cellular proteins to paxillin. These data suggest that E6 disruption of the actin stress fibers occurs through blocking the interaction of paxillin with its cellular effectors such as vinculin and the focal adhesion kinase.
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PMID:The bovine papillomavirus E6 protein binds to the LD motif repeats of paxillin and blocks its interaction with vinculin and the focal adhesion kinase. 940 31

The rat L6 skeletal muscle cell line was used to study expression of the dystrophin-containing glycoprotein complex and its interaction with the integrin system involved in the cell-matrix adhesion reaction. A complex of dystrophin and its associated proteins was fully expressed in L6 myotubes, from which anti-dystrophin or anti-alpha-sarcoglycan co-precipitated integrin alpha 5 beta 1 and other focal adhesion-associated proteins vinculin, talin, paxillin, and focal adhesion kinase. Immunostaining and confocal microscopy revealed that dystrophin, alpha-sarcoglycan, integrin alpha 5 beta 1, and vinculin exhibited overlapping distribution in the sarcolemma, especially at focal adhesion-like, spotty structures. Adhesion of cells to fibronectin- or collagen type I-coated dishes resulted in induction of tyrosine phosphorylation of alpha- and gamma-sarcoglycans but not beta-sarcoglycan. The same proteins were also tyrosine-phosphorylated when L6 cells in suspension were exposed to Arg-Gly-Asp-Ser peptide. All of these tyrosine phosphorylations were inhibited by herbimycin A. On the other hand, treatment of L6 myotubes with alpha- and gamma-sarcoglycan antisense oligodeoxynucleotides resulted in complete disappearance of alpha- and gamma-sarcoglycans and in significant reduction of levels of the associated focal adhesion proteins, which caused about 50% reduction of cell adhesion. These results indicate the existence of bidirectional communication between the dystrophin-containing complex and the integrin adhesion system in cultured L6 myocytes.
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PMID:Bidirectional signaling between sarcoglycans and the integrin adhesion system in cultured L6 myocytes. 943 Jun 99


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