Activation of protein kinase C is required for the stable attachment of adherent platelets to collagen but is not needed for the initial rapid adhesion under flow conditions.
(65/1396)
We have investigated the role of protein kinase C (PKC) in the initial events of alpha(2)beta(1)-integrin-mediated platelet adhesion to collagen under flow conditions. Although adhesion caused activation of PKC, as evidenced by pleckstrin phosphorylation, the PKC inhibitors GF 109203X and Go 6976 had no effect on adhesion, even though they prevented pleckstrin phosphorylation. The initial kinetics and extent of platelet adhesion to collagen (<5 seconds) and tyrosine phosphorylation of p125(FAK) and p72(syk) were not influenced by the PKC inhibitors, whereas adhesion to polylysine was prevented. These results indicate that adhesion to collagen and polylysine involve different mechanisms and requirements for PKC activation. Pretreatment with GF 109203X destabilized collagen-adherent platelets, accelerating their detachment, which was associated with tyrosine dephosphorylation of p125(FAK). Thus, although PKC activation was not required for rapid platelet adhesion to collagen, it appears to play an important role in stabilizing the attachment of adherent platelets to collagen. We also examined the effect of PKC activation by the phorbol ester phorbol 12-myristate 13-acetate (PMA) on platelet adhesion to collagen. PMA at 100 nmol/L strongly potentiated adhesion and tyrosine phosphorylation of p125(FAK) and p72(syk) and activated beta(1)-integrins, as determined by increased exposure of the 15/7 epitope. The PMA-stimulated adhesion was partially blocked by an anti-alpha(2)beta(1) antibody, was completely inhibited by GF 109203X, and was not correlated with the extent of pleckstrin phosphorylation. Therefore, strong PKC activation may lead to inside-out signaling, enhancing the role of beta(1)-integrins in adhesion. Pleckstrin phosphorylation does not appear to be involved in the initial phase of basic or PMA-stimulated adhesion but may help stabilize the adherent platelets. (+info)
The role of focal adhesion kinase binding in the regulation of tyrosine phosphorylation of paxillin.
(66/1396)
Focal adhesion kinase (FAK) and paxillin are focal adhesion-associated, phosphotyrosine-containing proteins that physically interact. A previous study has demonstrated that paxillin contains two binding sites for FAK. We have further characterized these two binding sites and have demonstrated that the binding affinity of the carboxyl-terminal domain of FAK is the same for each of the two binding sites. The presence of both binding sites increases the affinity for FAK by 5-10-fold. A conserved paxillin sequence called the LD motif has been implicated in FAK binding. We show that mutations in the LD motifs in both FAK-binding sites are required to dramatically impair FAK binding in vitro. A paxillin mutant containing point mutations in both FAK-binding sites was characterized. The mutant exhibited reduced levels of phosphotyrosine relative to wild type paxillin in subconfluent cells growing in culture, following cell adhesion to fibronectin and in src-transformed fibroblasts. These results suggest that paxillin must bind FAK for maximal phosphorylation in response to cell adhesion and that FAK may function to direct tyrosine phosphorylation of paxillin in the process of transformation by the src oncogene. (+info)
Adhesion to fibronectin promotes the activation of the p125(FAK)/Zap-70complex in human T cells.
(67/1396)
The beta1 integrins are a family of heterodimeric adhesion receptors involved in cell-to-cell contacts and cell-to-extracellular matrix interactions. Through their adhesive role, integrins participate in transduction of outside/inside signals and contribute to trigger a multitude of cellular events such as differentiation, cell activation, and motility. The fibronectin integrin receptors, alpha4beta1 and alpha5beta1, can function as costimulatory molecules in T-cell receptor (TCR)-dependent T-cell activation. In the current study the Jurkat T-cell line was used as a model system to investigate the TCR-independent role of cell adhesion to fibronectin in the activation of Zap-70, a central molecule in the signalling events in T cells. Upon adhesion to plastic immobilized fibronectin but not to bovine serum albumin (BSA) the phosphorylation of p125FAK, a protein kinase that localizes to focal adhesion sites, was induced. Moreover, clustering of fibronectin receptors led to the detection of a p125FAK/Zap-70 complex. Finally, while the complex between fak-B, another protein kinase localized to focal adhesion sites, and Zap-70 was detected in cells plated either on BSA or on fibronectin, the formation of the p125FAK/Zap-70 complex appeared specifically induced following fibronectin-mediated integrin clustering. These data suggest the existence of a high degree of specificity when the members of the beta1 integrin family mediate signalling pathways in T cells. (+info)
Requirement for focal adhesion kinase in tumor cell adhesion.
(68/1396)
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase and a major phosphotyrosine-containing protein. FAK is found in cell-matrix attachment sites (focal adhesions), and is activated on integrin-ligand binding and by other signaling pathways. Several roles have been proposed for FAK; here we report a novel function. We observed abundant FAK protein in all human melanoma cell lines tested except COLO839, a line that grows predominantly in suspension and was derived from peripheral blood. Five adherent lines, isolated from solid metastases in the same patient as COLO839, did express FAK. We derived four adherent sublines from COLO839. These did express FAK, even when plated on bacteriological plastic, to which they did not adhere. Thus, substrate attachment was not required for FAK expression. Three of the adherent sublines were then grown in the presence of antisense oligonucleotides to the initial FAK coding sequence. All showed substantially reduced FAK expression and, interestingly, the cells largely detached from the substrate while continuing to grow. Similar results were obtained with an independent melanoma line, DX3. Thus, FAK expression appears to be required by melanoma cells for substrate adhesion. (+info)
Tyrosine phosphorylation of alpha-actinin in activated platelets.
(69/1396)
The integrin alpha(IIb)beta(3) mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human alpha-actinin sequences deposited in the Swiss Protein Database. alpha-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with alpha-actinin and anti-phosphotyrosine antibodies. The distribution of alpha-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained alpha-actinin. The purified alpha-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. alpha-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with alpha-actinin. Taken together, these data demonstrate that a fraction of alpha-actinin is tyrosine-phosphorylated in activated platelets. (+info)
Expression of DFak56, a Drosophila homolog of vertebrate focal adhesion kinase, supports a role in cell migration in vivo.
(70/1396)
Focal adhesion kinase (FAK) is a highly conserved, cytoplasmic tyrosine kinase that has been implicated in promoting cell migration and transmission of antiapoptotic signals in vertebrate cells. In cultured cells, integrin engagement with the extracellular matrix promotes the recruitment of FAK to focal contacts and increases in its phosphotyrosine content and kinase activity, suggesting FAK is an intracellular mediator of integrin signaling. We have identified a Drosophila FAK homolog, DFak56, that is 33% identical to vertebrate FAK, with the highest degree of homology in domains critical for FAK function, including the kinase and focal adhesion targeting domains, and several protein-protein interaction motifs. Furthermore, when expressed in NIH 3T3 cells, DFak56 both localizes to focal contacts and displays the characteristic elevation of phosphotyrosine content in response to plating the cells on fibronectin. During embryogenesis, DFak56 is broadly expressed, and it becomes elevated in the gut and central nervous system at later stages. Consistent with a role in cell migration, we also observe that DFak56 is abundant in the border cells of developing egg chambers before the onset of, and during, their migration. (+info)
Regulation of T cell receptor- and CD28-induced tyrosine phosphorylation of the focal adhesion tyrosine kinases Pyk2 and Fak by protein kinase C. A role for protein tyrosine phosphatases.
(71/1396)
The T cell receptor (TCR)-CD3 complex and the costimulatory molecule CD28 are critical for T cell function. Both receptors utilize protein tyrosine kinases (PTKs) for the phosphorylation of various signaling molecules, a process that is critical for the function of both receptors. The PTKs of the focal adhesion family, Pyk2 and Fak, have been implicated in the signaling of TCR and CD28. We show here evidence for the regulation of TCR- and CD28-induced tyrosine phosphorylation of the focal adhesion PTKs by protein kinase C (PKC). Thus, treating Jurkat T cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) rapidly and strongly reversed receptor-induced tyrosine phosphorylation of the focal adhesion PTKs. In contrast, PMA did not affect TCR-induced tyrosine phosphorylation of CD3zeta or the PTKs Fyn and Zap-70. However, PMA induced a strong and rapid dephosphorylation of the linker molecule for activation of T cells. PMA failed to induce the dephosphorylation of proteins in PKC-depleted cells or in cells pretreated with the PKC inhibitor Ro-31-8220, confirming the role of PKC in mediating the PMA effect on receptor-induced protein tyrosine phosphorylation. The involvement of protein tyrosine phosphatases (PTPases) in mediating the dephosphorylation of the focal adhesion PTKs was confirmed by the failure of PMA to dephosphorylate Pyk2 in cells pretreated with the PTPase inhibitor orthovanadate. These results implicate PKC in the regulation of receptor-induced tyrosine phosphorylation of the focal adhesion PTKs in T cells. The data also suggest a role for PTPases in the PKC action. (+info)
Tissue inhibitor of metalloproteinase-1 inhibits apoptosis of human breast epithelial cells.
(72/1396)
The signaling pathways critical for cell survival are mediated in part by the composition and integrity of the extracellular matrix and the action of its components on specific cell adhesion receptors. Withdrawal of anchorage-dependent epithelial cells from their association with ECM results in apoptotic cell death. Consistently, the matrix metalloproteinases (MMPs) or their inhibitors (TIMPs) have been suggested to regulate apoptosis. In this report, we investigated whether bcl-2 inhibition of apoptosis involves regulation of TIMP expression. We have found that bcl-2 overexpression induces TIMP-1 expression in breast epithelial cell lines (MCF10A, MCF10AneoT.TG3B, and MCF-7), whereas it has no effect on TIMP-2 expression. We demonstrated that TIMP-1 inhibits cell death induced by hydrogen peroxide, Adriamycin, or X-ray irradiation. In addition, TIMP-1 overexpression inhibits apoptosis after the loss of cell adhesion (anoikis) in MCF10A cells, suggesting that the antiapoptotic activity of TIMP-1 does not depend on its ability to stabilize cell-matrix interactions. We also showed that TIMP-1 overexpression is associated with constitutive activation of focal adhesion kinase, a signaling molecule known to be critical for the cell survival pathway. (+info)