v-Src rescues actin-based cytoskeletal architecture and cell motility and induces enhanced anchorage independence during oncogenic transformation of focal adhesion kinase-null fibroblasts

The ability of the focal adhesion kinase (FAK) to integrate signals from extracellular matrix and growth factor receptors requires the integrity of Tyr397, a major autophosphorylation site that mediates the Src homology 2-dependent binding of Src family kinases. However, the precise roles played by FAK in specific Src-induced pathways, especially as they relate to oncogenic transformation, remain unclear. Here, we investigate the role of FAK in v-Src-induced oncogenic transformation by transducing temperature-sensitive v-Src (ts72v-Src) into p53-null FAK+/+ or FAK-/- mouse embryo fibroblasts (MEF). At the permissive temperature (PT), ts72v-Src induced abundant tyrosine phosphorylation, morphological transformation and cytoskeletal rearrangement in FAK-/- MEF, including the restoration of cell polarity, typical focal adhesion complexes, and longitudinal F-actin stress fibers. v-Src rescued the haptotactic, linear directional, and invasive motility defects of FAK-/- cells to levels found in FAK+/+ or FAK+/+-[ts72v-Src] cells, and, in the case of monolayer wound healing motility, there was an enhancement. Src activation failed to increase the high basal tyrosine phosphorylation of the Crk-associated substrate, CAS, found in FAK-/- MEF, indicating that CAS phosphorylation alone is insufficient to induce motility in the absence of FAK- or v-Src-induced cytoskeletal remodeling. Compared with FAK+/+[ts72v-Src] controls, FAK-/-[ts72v-Src] clones exhibited 7-10-fold higher anchorage-independent proliferation that could not be attributed to variations in either v-Src protein level or stability. Re-expression of FAK diminished the colony-forming activities of FAK-/-[ts72v-Src] without altering ts72v-Src expression levels, suggesting that FAK attenuates Src-induced anchorage independence. Our data also indicate that the enhanced Pyk2 level found in FAK-/- MEF plays no role in v-Src-induced anchorage independence. Overall, our data indicate that FAK, although dispensable, attenuates v-Src-induced oncogenic transformation by modulating distinct signaling and cytoskeletal pathways.     

Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src

Tyrosine phosphorylation of CAS (Crk-associated substrate, p130(Cas)) has been implicated as a key signaling step in integrin control of normal cellular behaviors, including motility, proliferation, and survival. Aberrant CAS tyrosine phosphorylation may contribute to cell transformation by certain oncoproteins, including v-Crk and v-Src, and to tumor growth and metastasis. The CAS substrate domain (SD) contains 15 Tyr-X-X-Pro motifs, which are thought to represent the major tyrosine phosphorylation sites and to function by recruiting downstream signaling effectors, including c-Crk and Nck. CAS makes multiple interactions, direct and indirect, with the tyrosine kinases Src and focal adhesion kinase (FAK), and as a result of this complexity, several plausible models have been proposed for the mechanism of CAS-SD phosphorylation. The objective of this study was to provide experimental tests of these models in order to determine the most likely mechanism(s) of CAS-SD tyrosine phosphorylation by FAK and Src. In vitro kinase assays indicated that FAK has a very poor capacity to phosphorylate CAS-SD, relative to Src. However, FAK expression along with Src was found to be important for achieving high levels of CAS tyrosine phosphorylation in COS-7 cells, as well as recovery of CAS-associated Src activity toward the SD. Structure-functional studies for both FAK and CAS further indicated that FAK plays a major role in regulating CAS-SD phosphorylation by acting as a docking or scaffolding protein to recruit Src to phosphorylate CAS, while a secondary FAK-independent mechanism involves Src directly bound to the CAS Src-binding domain (SBD). Our results do not support models in which FAK either phosphorylates CAS-SD directly or phosphorylates CAS-SBD to promote Src binding to this site.     

v-Src induces tyrosine phosphorylation of focal adhesion kinase independently of tyrosine 397 and formation of a complex with Src

The non-receptor tyrosine kinase FAK plays a key role at sites of cellular adhesion. It is subject to regulatory tyrosine phosphorylation in response to a variety of stimuli, including integrin engagement after attachment to extracellular matrix, oncogene activation, and growth factor stimulation. Here we use an antibody that specifically recognizes the phosphorylated form of the putative FAK autophosphorylation site, Tyr(397). We demonstrate that FAK phosphorylation induced by integrins during focal adhesion assembly differs from that induced by activation of a temperature-sensitive v-Src, which is associated with focal adhesion turnover and transformation. Specifically, although v-Src induces tyrosine phosphorylation of FAK, there is no detectable phosphorylation of Tyr(397). Moreover, activation of v-Src results in a net decrease in fibronectin-stimulated phosphorylation of Tyr(397), suggesting possible antagonism between v-Src and integrin-induced phosphorylation. Our mutational analysis further indicates that the binding of v-Src to Tyr(397) of FAK in its phosphorylated form, which is normally mediated, at least in part, by the SH2 domain of Src, is not essential for v-Src-induced cell transformation. We conclude that different stimuli can induce phosphorylation of FAK on distinct tyrosine residues, linking specific phosphorylation events to ensuing biological responses.     

Crk-associated substrate tyrosine phosphorylation sites are critical for invasion and metastasis of SRC-transformed cells

Crk-associated substrate (CAS, p130Cas) is a major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. We recently reported that reexpression of CAS in CAS-deficient mouse embryo fibroblasts transformed by oncogenic Src promoted an invasive phenotype associated with enhanced cell migration through Matrigel, organization of actin into large podosome ring and belt structures, activation of matrix metalloproteinase-2, and elevated tyrosine phosphorylation of the focal adhesion proteins FAK and paxillin. We have now extended these studies to examine the mechanism by which CAS achieves these changes and to evaluate the potential role for CAS in promoting in vivo tumor growth and metastasis. Whereas the presence or absence of CAS did not alter the primary growth of subcutaneous-injected Src-transformed mouse embryo fibroblasts, CAS expression was required to promote lung metastasis following removal of the primary tumor. The substrate domain YxxP tyrosines, the major sites of CAS phosphorylation by Src that mediate interactions with Crk, were found to be critical for promoting both invasive and metastatic properties of the cells. The ability of CAS to promote Matrigel invasion, formation of large podosome structures, and tyrosine phosphorylation of Src substrates, including FAK, paxillin, and cortactin, was also strictly dependent on the YxxP tyrosines. In contrast, matrix metalloproteinase-2 activation was most dependent on the CAS SH3 domain, whereas the substrate domain YxxP sites also contributed to this property. Thus multiple CAS-mediated signaling events are implicated in promoting invasive and metastatic properties of Src-transformed cells.     

Focal Adhesion Targeting: The Critical Determinant of FAK Regulation and Substrate Phosphorylation

The focal adhesion kinase (FAK) is discretely localized to focal adhesions via its C-terminal focal adhesion-targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can regulate tyrosine phosphorylation of downstream substrates, like paxillin. By the use of a mutational strategy, the regions of FAK that are required for cell adhesion-dependent regulation and for inducing tyrosine phosphorylation of paxillin were determined. The results show that the FAT sequence was the single region of FAK that was required for each function. Furthermore, the FAT sequence of FAK was replaced with a focal adhesion-targeting sequence from vinculin, and the resulting chimera exhibited cell adhesion-dependent tyrosine phosphorylation and could induce paxillin phosphorylation like wild-type FAK. These results suggest that subcellular localization is the major determinant of FAK function.     

Inhibition of the catalytic activity of cell adhesion kinase beta by protein-tyrosine phosphatase-PEST-mediated dephosphorylation

Protein-tyrosine phosphatase (PTP)-PEST is a cytoplasmic tyrosine phosphatase that can bind and dephosphorylate the focal adhesion-associated proteins p130(CAS) and paxillin. Focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta)/PYK2/CADTK/RAFTK are protein-tyrosine kinases that can colocalize with, bind to, and induce tyrosine phosphorylation of p130(CAS) and paxillin. Thus, we considered the possibility that these kinases might be substrates for PTP-PEST. Using a combination of substrate-trapping assays and overexpression of PTP-PEST in mammalian cells, CAKbeta was found to be a substrate for PTP-PEST. Both the major autophosphorylation site of CAKbeta (Tyr(402)) and activation loop tyrosine residues, Tyr(579) and Tyr(580), were targeted for dephosphorylation by PTP-PEST. Dephosphorylation of CAKbeta by PTP-PEST dramatically inhibited CAKbeta kinase activity. In contrast, FAK was a poor substrate for PTP-PEST, and treatment with PTP-PEST had no effect on FAK kinase activity. Tyrosine phosphorylation of paxillin, which is greatly enhanced by CAKbeta overexpression, was dramatically reduced upon coexpression of PTP-PEST. Finally, endogenous PTP-PEST and endogenous CAKbeta were found to localize to similar cellular compartments in epithelial and smooth muscle cells. These results suggest that CAKbeta is a substrate of PTP-PEST and that FAK is a poor PTP-PEST substrate. Further, PTP-PEST can negatively regulate CAKbeta signaling by inhibiting the catalytic activity of the kinase.     

Y-27632, an Inhibitor of Rho-Associated Kinases, Prevents Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin Induced by Bombesin: Dissociation from Tyrosine Phosphorylation of p130

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.     

Y-27632, an inhibitor of Rho-associated kinases, prevents tyrosine phosphorylation of focal adhesion kinase and paxillin induced by bombesin: dissociation from tyrosine phosphorylation of p130(CAS)

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.     

Paxillin enables attachment-independent tyrosine phosphorylation of focal adhesion kinase and transformation by RAS

Paxillin and HIC5 are closely related adapter proteins that regulate cell migration and are tyrosine-phosphorylated by focal adhesion kinase (FAK). Paxillin, HIC5, and FAK tyrosine phosphorylation increase upon cell attachment and decrease upon detachment from extracellular matrix. Unexpectedly, we found that although FAK tyrosine phosphorylation in attached cells did not require paxillin, in detached fibroblasts there was remaining FAK tyrosine phosphorylation that required expression of paxillin and was not supported by HIC5. The support of attachment-independent FAK tyrosine phosphorylation required the paxillin LIM domains and suggested that paxillin might facilitate oncogenic transformation. Paxillin but not HIC5 augmented anchorage-independent cell proliferation induced by RAS. Both anchorage-independent FAK tyrosine phosphorylation and RAS-induced colony formation required multiple docking sites on paxillin, including LD4 (docking sites for FAK-Src and GIT1/2-PIX-NCK-PAK complex), LD5, and all four carboxyl-terminal LIM domains (that bind tubulin and PTP-PEST). Analysis using paxillin mutants dissociated domains of paxillin that are required for regulation of cell migration from domains that are required for anchorage-independent cell proliferation and demonstrated essential functions of the paxillin LIM domains that are not found in HIC5 LIM domains. These results highlight the role of paxillin in facilitating attachment-independent signal transduction implicated in cancer.     

The catalytic activity of Src is dispensable for translocation to focal adhesions but controls the turnover of these structures during cell motility.

The Src family of protein tyrosine kinases is involved in transducing signals at sites of cellular adhesion. In particular, the v-Src oncoprotein resides in cellular focal adhesions, where it induces tyrosine phosphorylation of pp125FAK and focal adhesion loss during transformation. v-Src is translocated to cellular focal adhesions by an actin-dependent process. Here we have used mutant v-Src proteins that are temperature-dependent for translocation, but with secondary mutations that render them constitutively kinase-inactive or myristylation-defective, to show that neither v-Src kinase activity nor a myristyl group are required to induce association of v-Src with actin stress fibres and redistribution to sites of focal adhesions at the stress fibre termini. Moreover, switching the constitutively kinase-inactive or myristylation-defective temperature-sensitive v-Src proteins to the permissive temperature resulted in concomitant association with tyrosine-phosphorylated focal adhesion kinase (pp125FAK) and redistribution of both to focal adhesions. However, both catalytic activity and myristylation-mediated membrane association are required to induce dissociation of pp125FAK from v-Src, later degradation of pp125FAK and focal adhesion turnover during transformation and cell motility. These observations provide strong evidence that the role of the tyrosine kinase activity of the Src family at sites of cellular focal adhesions is to regulate the turnover of these structures during cell motility.     

Protein tyrosine phosphatase-dependent proteolysis of focal adhesion complexes in endothelial cell apoptosis

Adenosine and/or homocysteine causes endothelial cell apoptosis, a mechanism requiring protein tyrosine phosphatase (PTPase) activity. We investigated the role of focal adhesion contact disruption in adenosine-homocysteine endothelial cell apoptosis. Analysis of focal adhesion kinase (FAK), paxillin, and vinculin demonstrated disruption of focal adhesion complexes after 4 h of treatment with adenosine-homocysteine followed by caspase-induced proteolysis of FAK, paxillin, and p130(CAS). No significant changes were noted in tyrosine phosphorylation of FAK or paxillin. Pretreatment with the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone prevented adenosine-homocysteine-induced DNA fragmentation and FAK, paxillin, and p130(CAS) proteolysis. Asp-Glu-Val-Asp-ase activity was detectable in endothelial cells after 4 h of treatment with adenosine-homocysteine. The PTPase inhibitor sodium orthovanadate did not prevent endothelial cell retraction or FAK, paxillin, or vinculin redistribution. Sodium orthovanadate did block adenosine-homocysteine-induced FAK, paxillin, and p130(CAS) proteolysis and Asp-Glu-Val-Asp-ase activity. Thus disruption of focal adhesion contacts and caspase-induced degradation of focal adhesion contact proteins occurs in adenosine-homocysteine endothelial cell apoptosis. Focal adhesion contact disruption induced by adenosine-homocysteine is independent of PTPase or caspase activation. These studies demonstrate that disruption of focal adhesion contacts is an early, but not an irrevocable, event in endothelial cell apoptosis.     

v-Src-Induced Modulation of the Calpain-Calpastatin Proteolytic System Regulates Transformation

v-Src-induced oncogenic transformation is characterized by alterations in cell morphology, adhesion, motility, survival, and proliferation. To further elucidate some of the signaling pathways downstream of v-Src that are responsible for the transformed cell phenotype, we have investigated the role that the calpain-calpastatin proteolytic system plays during oncogenic transformation induced by v-Src. We recently reported that v-Src-induced transformation of chicken embryo fibroblasts is accompanied by calpain-mediated proteolytic cleavage of the focal adhesion kinase (FAK) and disassembly of the focal adhesion complex. In this study we have characterized a positive feedback loop whereby activation of v-Src increases protein synthesis of calpain II, resulting in degradation of its endogenous inhibitor calpastatin. Reconstitution of calpastatin levels by overexpression of exogenous calpastatin suppresses proteolytic cleavage of FAK, morphological transformation, and anchorage-independent growth. Furthermore, calpastatin overexpression represses progression of v-Src-transformed cells through the G(1) stage of the cell cycle, which correlates with decreased pRb phosphorylation and decreased levels of cyclins A and D and cyclin-dependent kinase 2. Calpain 4 knockout fibroblasts also exhibit impaired v-Src-induced morphological transformation and anchorage-independent growth. Thus, modulation of the calpain-calpastatin proteolytic system plays an important role in focal adhesion disassembly, morphological transformation, and cell cycle progression during v-Src-induced cell transformation.     

Signaling through focal adhesion kinase

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase implicated in controlling cellular responses to the engagement of cell-surface integrins, including cell spreading and migration, survival and proliferation. Aberrant FAK signaling may contribute to the process of cell transformation by certain oncoproteins, including v-Src. Progress toward elucidating the events leading to FAK activation following integrin-mediated cell adhesion, as well as events downstream of FAK, has come through the identification of FAK phosphorylation sites and interacting proteins. A signaling partnership is formed between FAK and Src-family kinases, leading to tyrosine phosphorylation of FAK and associated ‘docking’ proteins Cas and paxillin. Subsequent recruitment of proteins containing Src homology 2 domains, including Grb2 and c-Crk, to the complex is likely to trigger adhesion-induced cellular responses, including changes to the actin cytoskeleton and activation of the Ras-MAP kinase pathway.     

Alterations in granule matrix and cell surface of focal adhesion kinase-deficient mast cells

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays an important role in many cellular processes and is tyrosine phosphorylated after FcepsilonRI aggregation in mast cells. In mice, null mutation of the fak gene results in a lethal phenotype in which the embryos fail to develop past day 8.5 of gestation. To study the role of FAK in these mast cells, 8.5-day embryos were isolated and placed in culture with IL-3 and stem cell factor (SCF). Although FAK was not required for the development of mast cells in culture, the FAK(-/-) embryo-derived mast cells had several distinct characteristics. Compared with the controls, the mast cells that lack FAK were less metachromatic and by electron microscopy had granules that appeared largely electron lucid, although their histamine content was unchanged. The FAK-deficient mast cells had a reduction in the content of chondroitin/dermatan sulfate, the major glycosaminoglycan component of the granular matrix. The FAK-deficient cells had fewer microvilli that were fused with each other, giving the cell surface a ruffled appearance. There was also a 3-fold increase in the number of cells highly expressing beta(7) integrin. However, signal transduction from the high affinity IgE receptor for the secretion of histamine was similar in the wild-type, heterozygote, and the FAK-deficient cells. The FcepsilonRI-induced tyrosine phosphorylation of paxillin, Crk-associated tyrosine kinase substrate (CAS), and mitogen-activated protein kinase proteins was independent of FAK. These results indicate that FAK plays a role in regulating the glycosaminoglycan content of the secretory granules and influences the cell surface morphology of mast cells.     

Regulation of neutrophil adhesion by pituitary growth hormone accompanies tyrosine phosphorylation of Jak2, p125FAK, and paxillin

Neutrophil adhesion is fundamentally important during the onset of inflammatory responses. The adhesion signaling pathways control neutrophil arrest and extravasation and influence neutrophil shape and function at sites of inflammation. In the present study the intracellular signaling pathways for the adhesion of human neutrophils by pituitary growth hormone (GH) were examined. Pituitary GH triggered the tyrosine phosphorylation of Janus kinase 2 (Jak2) and STAT3 in neutrophils. In addition, pituitary GH treatment resulted in the morphological changes and the tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin. Preincubation with genistein, a tyrosine kinase inhibitor, blocked the GH-stimulated adhesion and Jak2, STAT3, p125FAK, and paxillin phosphorylation. Confocal microscopy revealed that pituitary GH stimulates the focal localization of p125FAK, paxillin, phosphotyrosine, and filamentous actin filament into the membrane rufflings and uropods of human neutrophils. Immunoprecipitation experiments revealed a physical association of Jak2 with p125FAK via STAT3 in vivo. Also an in vitro kinase assay showed an augmentation of p125FAK autophosphorylation as a result of pituitary GH treatment. These results suggest that pituitary GH modulates neutrophil adhesion through tyrosine phosphorylation of Jak2, p125FAK, and paxillin and actin polymerization.     

Hepatocyte growth factor induces ERK-dependent paxillin phosphorylation and regulates paxillin-focal adhesion kinase association.

Hepatocyte growth factor (HGF) modulates cell adhesion, migration, and branching morphogenesis in cultured epithelial cells, events that require regulation of cell-matrix interactions. Using mIMCD-3 epithelial cells, we studied the effect of HGF on the focal adhesion proteins, focal adhesion kinase (FAK) and paxillin and their association. HGF was found to increase the tyrosine phosphorylation of paxillin and to a lesser degree FAK. In addition, HGF induced association of paxillin and activated ERK, correlating with a gel retardation of paxillin that was prevented with the ERK inhibitor U0126. The ability of activated ERK to phosphorylate and induce gel retardation of paxillin was confirmed in vitro in both full-length and amino-terminal paxillin. Several potential ERK phosphorylation sites in paxillin flank the paxillin-FAK association domains, so the ability of HGF to regulate paxillin-FAK association was examined. HGF induced an increase in paxillin-FAK association that was inhibited by pretreatment with U0126 and reproduced by in vitro phosphorylation of paxillin with ERK. The prevention of the FAK-paxillin association with U0126 correlated with an inhibition of the HGF-mediated FAK tyrosine phosphorylation and inhibition of HGF-dependent cell spreading and adhesion. An examination of cellular localization of FAK and paxillin demonstrated that HGF caused a condensation of focal adhesion complexes at the leading edges of cell processes and FAK-paxillin co-localization in these large complexes. Thus, these data suggest that HGF can induce serine/threonine phosphorylation of paxillin most probably mediated directly by ERK, resulting in the recruitment and activation of FAK and subsequent enhancement of cell spreading and adhesion.     

Phosphorylation of focal adhesion kinase at tyrosine 861 is crucial for Ras transformation of fibroblasts

Although elevated expression and increased tyrosine phosphorylation of focal adhesion kinase (FAK) are crucial for tumor progression, the mechanism by which FAK promotes oncogenic transformation is unclear. We have therefore determined the role of FAK phosphorylation at tyrosine 861 in the oncogenic transformation of NIH3T3 fibroblasts. FAK phosphorylation at tyrosine 861 was increased in both constitutively H-Ras-transformed and H-Ras-inducible NIH3T3 cells, in parallel with cell transformation. However, H-Ras-inducible cells transfected with the nonphosphorylatable mutant FAK Y861F showed decreased migration/invasion, focus forming activity and anchorage-independent growth, compared with either wild-type or kinase-defective FAK. In contrast to unaltered FAK/Src activity, the association of FAK and p130(CAS) was decreased in FAK Y861F-transfected cells, and FAK phosphorylation at tyrosine 861 enhanced this association in vitro. Consistently, FAK Y861F-transfected cells were defective in activation of c-Jun NH(2)-terminal kinase and in expression of matrix metalloproteinase-9 during transformation. Taken together, these results strongly suggest that FAK phosphorylation at tyrosine 861 is crucial for H-Ras-induced transformation through regulation of the association of FAK with p130(CAS).     

Differential regulation of cell motility and invasion by FAK

Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK-/- fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK-/- cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK-/- v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK-Src-p130Cas-Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways.     

Regulation and localization of CAS substrate domain tyrosine phosphorylation

Crk-associated substrate (CAS) is a tyrosine kinase substrate implicated in integrin control of cell behavior. Phosphorylation, by Src family kinases, of multiple tyrosine residues in the CAS substrate domain (SD) is a major integrin signaling event that promotes cell motility. In this study, novel phosphospecific antibodies directed against CAS SD phosphotyrosine sites ("pCAS" antibodies) were characterized and employed to investigate the cellular regulation and localization of CAS SD tyrosine phosphorylation. An analysis of CAS and focal adhesion kinase (FAK) variants expressed in CAS- and FAK-deficient cell lines, respectively, indicated that CAS SD tyrosine phosphorylation is substantially achieved by Src family kinases brought into association with CAS through two distinct mechanisms: direct binding to the CAS Src-binding domain and indirect association through a FAK bridge. Cell immunostaining with pCAS antibodies revealed that CAS SD tyrosine phosphorylation occurs exclusively at sites of integrin adhesion including both nascent focal complexes formed at the edges of extending lamellipodia as well as mature focal adhesions underlying the cell body. These findings further document a role for FAK as an important upstream regulator of CAS SD tyrosine phosphorylation and implicate CAS-mediated signaling events in promoting membrane protrusion/lamellipodium extension during cell motility.     

Mitogen-induced,FAK-dependent tyrosine phosphorylation of the SSeCKS scaffolding protein

The ability of mitogens to rapidly induce tyrosine phosphorylation of cellular proteins has been taken as evidence of participation in subsequent signaling pathways. SSeCKS, a major protein kinase C (PKC) substrate with protein scaffolding and tumor suppressive properties, becomes tyrosine phosphorylated in NIH3T3 and rodent embryo fibroblasts after short-term treatment with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or fetal calf serum in the presence of pervanadate, but not by treatment with insulin or insulin-like growth factor-1. The relative phosphotyrosine level on SSeCKS was higher in actively dividing cells than in confluent cultures. Tyrosine phosphorylation of SSeCKS was apparent in cells deficient in Src, Fyn, Yes, or Abl tyrosine kinases or in NIH3T3 cells expressing a temperature-sensitive v-Src allele, but not in FAK-deficient embryo fibroblasts. Purified FAK or Src enzyme failed to directly phosphorylate SSeCKS in vitro. EGF failed to induce SSeCKS tyrosine phosphorylation in FAK-/- fibroblasts, indicating that the EGF receptor is probably not the direct kinase of SSeCKS. Phosphorylation under these conditions was rescued by the transient reexpression of wt-FAK but not FAK mutated at Y397, a major autophosphorylation and SH2-based docking site. Adhesion of FAK+/+ cells to fibronectin failed to significantly induce SSeCKS tyrosine phosphorylation although FAK was activated, suggesting that SSeCKS phosphorylation is mediated through a growth factor receptor-FAK rather than an integrin-FAK pathway. Moreover, PDGF could induce SSeCKS tyrosine phosphorylation in the absence of FAK activation, suggesting a role for FAK SH2-based docking rather than kinase activity. Immunofluorescence analysis showed that in FAK-/- cells, SSeCKS costains along F-actin stress fibers, in contrast to FAK+/+ cells, where most SSeCKS stains at the cell edge and along a cortical cytoskeletal matrix. This correlated with increased coprecipitation of SSeCKS with biotin-phalloidin-bound F-actin from FAK-/- compared to FAK+/+ cell lysates. Similarly, bacterially expressed, unphosphorylated SSeCKS cosedimented with F-actin in ultracentrifugation assays. These data suggest that mitogen-induced, FAK-dependent tyrosine phosphorylation of SSeCKS modulates its binding to the actin-based cytoskeleton, suggesting a role for SSeCKS in mitogen-induced cytoskeletal reorganization.