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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In pressure-overloaded myocardium, our recent study demonstrated cytoskeletal assembly of c-Src and other signaling proteins which was partially mimicked in vitro using adult feline cardiomyocytes embedded in three-dimensional (3D) collagen matrix and stimulated with an integrin-binding Arg-Gly-Asp (RGD) peptide. In the present study, we improved this model further to activate c-Src and obtain a full assembly of the focal adhesion complex (FAC), and characterized c-Src localization and integrin subtype(s) involved. RGD dose response experiments revealed that c-Src activation occurs subsequent to its cytoskeletal recruitment and is accompanied by p130Cas cytoskeletal binding and focal adhesion kinase (FAK) Tyr925 phosphorylation. When cardiomyocytes expressing hexahistidine-tagged c-Src via adenoviral gene delivery were used for RGD stimulation, the expressed c-Src exhibited relocation: (i) biochemical analysis revealed c-Src movement from the detergent-soluble to the -insoluble cytoskeletal fraction and (ii) confocal microscopic analysis showed c-Src movement from a nuclear/perinuclear to a sarcolemmal region. RGD treatment also caused sarcolemmal co-localization of FAK and vinculin. Characterization of integrin subtypes revealed that beta3, but not beta1, integrin plays a predominant role: (i) expression of cytoplasmic domain of beta1A integrin did not affect the RGD-stimulated FAC formation and (ii) both pressure-overloaded myocardium and RGD-stimulated cardiomyocytes exhibited phosphorylation of beta3 integrin at Tyr773/785 sites but not beta1 integrin at Thr788/789 sites. Together these data indicate that RGD treatment in cardiomyocytes causes beta3 integrin activation and c-Src sarcolemmal localization, that subsequent c-Src activation is accompanied by p130Cas binding and FAK Tyr925 phosphorylation, and that these events might be crucial for growth and remodeling of hypertrophying adult cardiomyocytes.
J Mol Cell Cardiol 2003 Jun
PMID:Focal complex formation in adult cardiomyocytes is accompanied by the activation of beta3 integrin and c-Src. 1278 85

We previously reported that fully assembled basement membranes are nonpermissive to smooth muscle cell (SMC) replication and that perlecan (PN), a basement membrane heparan sulfate proteoglycan, is a dominant effector of this response. We report here that SMC adhesion to basement membranes, and perlecan in particular, up-regulate the expression of focal adhesion kinase-related nonkinase (FRNK), a SMC-specific endogenous inhibitor of FAK, which subsequently suppresses FAK-mediated, ERK1/2-dependent growth signals. Up-regulation of FRNK by perlecan is actively and continuously regulated. Relative to the matrix proteins studied, the effects are unique to perlecan, because plating of SMCs on several other basement membrane proteins is associated with low levels of FRNK and corresponding high levels of FAK and ERK1/2 phosphorylation and SMC growth. Perlecan supports SMC adhesion, although there is reduced cell spreading compared with fibronectin (FN), laminin (LN), or collagen type IV (IV). Despite the reduction in cell spreading, we report that perlecan-induced up-regulation of FRNK is independent of cell shape changes. Growth inhibition by perlecan was rescued by overexpressing a constitutively active FAK construct, but overexpressing kinase-inactivated mutant FAK or FRNK attenuated fibronectin-stimulated growth. These data indicate that perlecan functions as an endogenously produced inhibitor of SMC growth at least in part through the active regulation of FRNK expression. FRNK, in turn, may control SMC growth by downregulating FAK-dependent signaling events.
Mol Biol Cell 2003 May
PMID:Perlecan up-regulation of FRNK suppresses smooth muscle cell proliferation via inhibition of FAK signaling. 1280 66

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.
Mol Endocrinol 2003 Nov
PMID:Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways. 1290 54

We investigated the focal adhesion proteins paxillin and Fak, and the cell-cell adhesion protein cadherin in developing zebrafish (Danio rerio) embryos. Cadherins are expressed in presomitic mesoderm where they delineate cells. The initiation of somite formation coincides with an increase in the phosphorylation of Fak, and the accumulation of Fak, phosphorylated Fak, paxillin, and fibronectin at nascent somite boundaries. In the notochord, cadherins are expressed on cells during intercalation, and phosphorylated Fak accumulates in circumferential rings where the notochord cells contact laminin in the perichordal sheath. Subsequently, changes in the orientations of collagen fibers in the sheath suggest that Fak-mediated adhesion allows longitudinal expansion of the notochord, but not lateral expansion, resulting in notochord elongation. Novel observations showed that focal adhesion kinase and paxillin concentrate at sites of cell-cell adhesion in the epithelial enveloping layer and may associate with actin cytoskeleton at epithelial junctions containing cadherins. Fak is phosphorylated at these epithelial junctions but is not phosphorylated on Tyr397, implicating a noncanonical mechanism of regulation. These data suggest that Fak and paxillin may function in the integration of cadherin-based and integrin-based cell adhesion during the morphogenesis of the early zebrafish embryo.
Mol Biol Cell 2003 Aug
PMID:Activity and distribution of paxillin, focal adhesion kinase, and cadherin indicate cooperative roles during zebrafish morphogenesis. 1292 47

Unlike most other matrix metalloproteinases (MMPs) MMP-19 is expressed in undifferentiated basal keratinocytes of healthy human skin. The human keratinocyte cell line HaCaT, which like basal keratinocytes constitutively expresses MMP-19, down-regulated the expression of MMP-19 at high calcium concentrations. Calcium-regulation occurred through E-cadherin mediated cell-cell contacts because neutralizing anti-E-cadherin antibodies restored MMP-19 expression in high calcium. Overexpression of MMP-19 in HaCaT cells (HaCaT-WT) increased cellular proliferation, as well as migration and adhesion on type I collagen. This was due to proteolysis of the insulin-like growth factor (IGF) binding protein-3 by MMP-19, which augmented signaling through the IGF-I receptor, as evidenced by its increased autophosphorylation. Conversely, these effects were not observed in cells transfected with MMP-2 or a catalytically inactive MMP-19 mutant. As further proof that increased IGF-signaling promoted adhesion and migration in HaCaT-WT cells, we reproduced these effects by treating parental HaCaT with IGF-I. We observed dephosphorylation of the focal adhesion kinase in HaCaT-WT as well as IGF-I-treated HaCaT cells, suggesting that inactivating focal adhesion kinase is a mechanism by which IGF-I enhances adhesion. Furthermore, IGF-I-triggered motility on type I collagen was mediated by MMP activity, which, however, was distinct from MMP-19. Considering the coexpression of IGFBP-3 and MMP-19 in the skin, we conclude that MMP-19 is a likely candidate to be the major IGFBP-3 degrading MMP in the quiescent epidermis. This activity might have widespread consequences for the behavior of epidermal keratinocytes.
Mol Biol Cell 2003 Nov
PMID:Matrix metalloproteinase 19 regulates insulin-like growth factor-mediated proliferation, migration, and adhesion in human keratinocytes through proteolysis of insulin-like growth factor binding protein-3. 1293 69

beta1,4-Galactosyltransferase I (GalT) participates in both glycoconjugates and cellular interactions. GalT's role in lamellipodia formation and cell migration on laminin is associated with a transient phosphorylation of focal adhesion kinase (FAK) and a consequent reorganization of the actin cytoskeleton and focal adhesions. We transfected wild type FAK and different FAK mutants into NIH3T3 cell line, measured GalT gene expression by Northern blot hybridization, and evaluated its activity. It was found that wtFAK and FAKY576F up-regulated GalT gene expression and its surface activity, while FAKY397F down-regulated them. At the same time, we used ricinus communis agglutinin (RCA)-I lectin staining to demonstrate its binding reactions. We found that wtFAK and FAKY576F bound stronger, while FAKY397F bound weaker than the control. By flow cytometry analysis, it was found that FAK promoted G1/S transition and enhanced the expression of cyclin D1 while FAKY397F inhibited these steps compared with the control NIH3T3 cells. G1/S checkpoint regulation proteins control GalT mRNA transcription. The results indicate that FAK regulated the expression of GalT and its activity in NIH3T3 cells may contribute to the effect of FAK on the cell-cycle.
Mol Cell Biochem 2003 Oct
PMID:Regulation of the expression and activity of beta1,4-galactosyltransferase I by focal adhesion kinase. 1457 71

We have investigated a role for the amino-terminal FERM-like domain of the focal adhesion kinase (FAK) as a negative regulator of its own activity and phosphorylation state. Deletion of the first 375 amino acids from the amino terminus of FAK increases its catalytic activity in vitro, its phosphorylation when expressed in mammalian cells, and the phosphorylation of a FAK substrate, paxillin. Deletion mutants are phosphorylated in suspension, suggesting that they are no longer regulated by adhesion. The amino terminus of FAK can interact with the kinase domain of FAK in vitro and in vivo, suggesting that it might act as an autoinhibitor of FAK activity. The amino terminus of FAK can act in trans to inhibit FAK phosphorylation when expressed in mammalian cells or to directly inhibit FAK activity in vitro. Expression of the amino terminus of FAK inhibits cell cycle progression in CHO cells, consistent with its inhibition of FAK phosphorylation and function in trans. A glutathione S-transferase fusion protein containing the cytoplasmic tail of the beta1 integrin stimulates FAK activity in vitro, suggesting that FAK could be regulated by molecular interactions with the amino terminus. Based on these and previous data, we propose a working model for activation of FAK in cell adhesion.
Mol Cell Biol 2003 Nov
PMID:Regulation of focal adhesion kinase by its amino-terminal domain through an autoinhibitory interaction. 1458 64

Multiple mechanisms are involved in the resistance of cancer cells to cisplatin, including the expression of multidrug resistance-associated protein (MRP) and enhanced DNA repair. Here, we report findings to show that oligosaccharide changes in alpha5beta1 integrin are associated with cisplatin resistance in a head and neck squamous cell carcinoma cell line, HSC-2. Cisplatin-resistant HSC-2 (HSC-2/CR) cells were established by stepwise treatment with various concentrations of cisplatin. The oligosaccharides containing beta1, 6-N-acetylglucosamine (beta1-6GlcNAc) branching, detected by leukoagglutinating phytohemagglutinin (L(4)-PHA) lectin blot, were found to be dramatically decreased in alpha5beta1 integrin immunoprecipitated from HSC-2/CR cells. To better understand the mechanisms underlying cisplatin resistance and oligosaccharide alteration, we analyzed the downstream signaling of alpha5beta1 integrin, one of the target glycoproteins of beta1-6GlcNAc transferase [UDP-GlcNAc:alpha-D-mannoside beta1, 6-N-acetylglucosaminyltransferase (GnT-V)]. Cell adhesion to fibronectin and phosphorylation of focal adhesion kinase (FAK), which are associated with alpha5beta1 integrin and involved in a cell survival signaling, were found to be increased in the cisplatin-resistant cells. Enhancement of the inhibition of cell adhesion and FAK phosphorylation also support the above data in GnT-V transfectants of HSC-2 cells. Interestingly, the differences in sensitivity to cisplatin and FAK phosphorylation between cisplatin-sensitive and -resistant cells were completely abolished by treatment with a neutral antibody of alpha5beta1 integrin. These results suggest that modification of oligosaccharides of alpha5beta1 integrin represents one of the possible mechanisms of drug resistance in head and neck cancer cells.
Mol Cancer Ther 2003 Nov
PMID:Involvement of oligosaccharide changes in alpha5beta1 integrin in a cisplatin-resistant human squamous cell carcinoma cell line. 1461 94

Our previous studies on the transmembrane domain of human integrin subunits have shown that a conserved basic amino acid in both subunits of integrin heterodimers is positioned in the plasma membrane in the absence of interacting proteins. To investigate the possible functional role of the lipid-embedded lysine in the mouse integrin beta1 subunit, this amino acid was replaced with leucine, and the mutated beta1 subunit (beta1A(K756L)) was stably expressed in beta1-deficient GD25 cells. The extracellular domain of beta1A(K756L) integrins possesses a competent conformation for ligand binding as determined by the ability to mediate cell adhesion, and by the presence of the monoclonal antibody 9EG7 epitope. However, the spreading of GD25-beta1A(K756L) cells on fibronectin and laminin-1 was impaired, and the rate of migration of GD25-beta1A(K756L) cells on fibronectin was reduced compared with GD25-beta1A cells. Phosphorylation of tyrosines in focal adhesion kinase (FAK) and the Y416 in c-Src in response to beta1A(K756L)-mediated adhesion was similar to that induced by wild-type beta1. The tyrosine phosphorylation level of paxillin, a downstream target of FAK/Src, was unaffected by the beta1 mutation, whereas tyrosine phosphorylation of CAS was strongly reduced. The results demonstrate that CAS is a target for phosphorylation both by FAK-dependent and -independent pathways after integrin ligation. The latter pathway was inhibited by wortmannin and LY294002, implicating that it required an active phosphatidylinositol 3-kinase. Furthermore, the K756L mutation in the beta1 subunit was found to interfere with beta1-induced activation of Akt. The results from this study identify phosphatidylinositol 3-kinase as an early component of a FAK-independent integrin signaling pathway triggered by the membrane proximal part of the beta1 subunit.
Mol Biol Cell 2004 Jun
PMID:The integrin beta1 subunit transmembrane domain regulates phosphatidylinositol 3-kinase-dependent tyrosine phosphorylation of Crk-associated substrate. 1503 38

The late stages of human breast cancer development are poorly understood complex processes associated with the expression of genes by cancers that promote specific tumorigenic activities, such as angiogenesis. Here, we describe the identification of periostin as a mesenchyme-specific gene whose acquired expression by human breast cancers leads to a significant enhancement in tumor progression and angiogenesis. Undetectable in normal human breast tissues, periostin was found to be overexpressed by the vast majority of human primary breast cancers examined. Tumor cell lines engineered to overexpress periostin showed a phenotype of accelerated growth and angiogenesis as xenografts in immunocompromised animals. The underlying mechanism of periostin-mediated induction of angiogenesis was found to derive in part from the up-regulation of the vascular endothelial growth factor receptor Flk-1/KDR by endothelial cells through an integrin alpha(v)beta(3)-focal adhesion kinase-mediated signaling pathway. These findings demonstrate the presence of a novel mechanism by which tumor angiogenesis is acquired with the expression of a mesenchyme-specific gene as a crucial step in late stages of tumorigenesis.
Mol Cell Biol 2004 May
PMID:Acquired expression of periostin by human breast cancers promotes tumor angiogenesis through up-regulation of vascular endothelial growth factor receptor 2 expression. 1508 92


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