Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins.

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.     

Complex Formation with Focal Adhesion Kinase: A Mechanism to Regulate Activity and Subcellular Localization of Src Kinases

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60(c-src) or p59(fyn) results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60(c-src) or p59(fyn) to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60(c-src) is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60(c-src) from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60(c-src) to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.     

A Role for the Protein Tyrosine Phosphatase CD45 in Macrophage Adhesion through the Regulation of Paxillin Degradation

CD45 is a protein tyrosine phosphatase expressed on all cells of hematopoietic origin that is known to regulate Src family kinases. In macrophages, the absence of CD45 has been linked to defects in adhesion, however the molecular mechanisms involved remain poorly defined. In this study, we show that bone marrow derived macrophages from CD45-deficient mice exhibit abnormal cell morphology and defective motility. These defects are accompanied by substantially decreased levels of the cytoskeletal-associated protein paxillin, without affecting the levels of other proteins. Degradation of paxillin in CD45-deficient macrophages is calpain-mediated, as treatment with a calpain inhibitor restores paxillin levels in these cells and enhances cell spreading. Inhibition of the tyrosine kinases proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK), kinases that are capable of mediating tyrosine phosphorylation of paxillin, also restored paxillin levels, indicating a role for these kinases in the CD45-dependent regulation of paxillin. These data demonstrate that CD45 functions to regulate Pyk2/FAK activity, likely through the activity of Src family kinases, which in turn regulates the levels of paxillin to modulate macrophage adhesion and migration.     

Microinjection of Protein Tyrosine Phosphatases into Fibroblasts Disrupts Focal Adhesions and Stress Fibers

Microinjection and scrape-loading have been used to load cells in culture with soluble protein tyrosine phosphatases (FTPs). The introduction of protein tyrosine phosphatases into cells caused a rapid (within 5 minutes) decrease in tyrosine phosphorylation of major tyrosine phosphorylated substrates, including the focal adhesion kinase and paxillin. This decrease was detected both by blotting whole cell lysates with anti-phosphotyrosine antibodies and visualizing the phosphotyrosine in focal adhesions by immunofluorescence microscopy. After 30 minutes, many of the cells injected with tyrosine phosphatases revealed disruption of focal adhesions and stress fibers. To determine whether this disruption was due to the dephosphorylation of FAK and its substrates in focal adhesions, we have compared the effects of protein tyrosine phosphatase microinjection with the effects of displacing FAK from focal adhesions by microinjection of a dominant negative FAK construct. Although both procedures resulted in a marked decrease in the level of phosphotyrosine in focal adhesions, disruption of focal adhesions and stress fibers only occurred in cells loaded with exogenous protein tyrosine phosphatases. These results lead us to conclude that although tyrosine phosphorylation regulates focal adhesion and stress fiber stability, this does not involve FAK nor does it appear to involve tyrosine-phosphorylated proteins within focal adhesions. The critical tyrosine phosphorylation event is upstream of focal adhesions, a likely target being in the Rho pathway that regulates the formation of stress fibers and focal adhesions.     

Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness

The ArfGAP paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT)2 has been implicated in regulating cell spreading and motility through its transient recruitment of the p21-activated kinase (PAK) to focal adhesions. The Nck-PAK-PIX-PKL protein complex is recruited to focal adhesions by paxillin upon integrin engagement and Rac activation. In this report, we identify tyrosine-phosphorylated PKL as a protein that associates with the SH3-SH2 adaptor Nck, in a Src-dependent manner, after cell adhesion to fibronectin. Both cell adhesion and Rac activation stimulated PKL tyrosine phosphorylation. PKL is phosphorylated on tyrosine residues 286/392/592 by Src and/or FAK and these sites are required for PKL localization to focal adhesions and for paxillin binding. The absence of either FAK or Src-family kinases prevents PKL phosphorylation and suppresses localization of PKL but not GIT1 to focal adhesions after Rac activation. Expression of an activated FAK mutant in the absence of Src-family kinases partially restores PKL localization, suggesting that Src activation of FAK is required for PKL phosphorylation and localization. Overexpression of the nonphosphorylated GFP-PKL Triple YF mutant stimulates cell spreading and protrusiveness, similar to overexpression of a paxillin mutant that does not bind PKL, suggesting that failure to recruit PKL to focal adhesions interferes with normal cell spreading and motility.     

Integrin adhesions: Who’s on first? What's on second?

Cell migration requires the coordination of adhesion site assembly and turnover. Canonical models for nascent adhesion formation postulate that integrin binding to extracellular matrix (ECM) proteins results in the rapid recruitment of cytoskeletal proteins such as talin and paxillin to integrin cytoplasmic domains. It is thought that integrin-talin clusters recruit and activate tyrosine kinases such as focal adhesion kinase (FAK). However, the molecular connections of this linkage remain unresolved. Our recent findings support an alternative model whereby FAK recruits talin to new sites of β1 integrin-mediated adhesion in mouse embryonic fibroblasts and human ovarian carcinoma cells. This is dependent on a direct binding interaction between FAK and talin and occurs independently of direct talin binding to β1 integrin. Herein, we discuss differences between nascent and mature adhesions, interactions between FAK, talin and paxillin, possible mechanisms of FAK activation and how this FAK-talin complex may function to promote cell motility through increased adhesion turnover.     

Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility

The coordinated interplay of substrate adhesion and deadhesion is necessary for cell motility. Using MCF-7 cells, we found that insulin-like growth factor I (IGF-I) induces the adhesion of MCF-7 to vitronectin and collagen in a dose- and time-dependent manner, suggesting that IGF-I triggers the activation of different integrins. On the other hand, IGF-I promotes the association of insulin receptor substrate 1 with the focal adhesion kinase (FAK), paxillin, and the tyrosine phosphatase SHP-2, resulting in FAK and paxillin dephosphorylation. Abrogation of SHP-2 catalytic activity with a dominant-negative mutant (SHP2-C>S) abolishes IGF-I-induced FAK dephosphorylation, and cells expressing SHP2-C>S show reduced IGF-I-stimulated chemotaxis compared with either mock- or SHP-2 wild-type-transfected cells. This impairment of cell migration is recovered by reintroduction of a catalytically active SHP-2. Interestingly, SHP-2-C>S cells show a larger number of focal adhesion contacts than wild-type cells, suggesting that SHP-2 activity participates in the integrin deactivation process. Although SHP-2 regulates mitogen-activated protein kinase activity, the mitogen-activated protein kinase kinase inhibitor PD-98059 has only a marginal effect on MCF-7 cell migration. The role of SHP-2 as a general regulator of cell chemotaxis induced by other chemotactic agents and integrins is discussed.     

Paxillin modulates squamous cancer cell adhesion and is important in pressure-augmented adhesion

Paxillin is an adapter protein regulating signaling and focal adhesion assembly that has been linked to malignant potential in many malignancies. Overexpression of paxillin has been noted in aggressive tumors. Integrin-mediated binding through the focal adhesion complex is important in metastatic adhesion and is upregulated by extracellular pressure in malignant colonocytes through FAK and Src activation. Neither head and neck cancers nor paxillin have been studied in this regard. We hypothesized that paxillin would play a role in modulating squamous cancer adhesion both at baseline and under conditions of increased extracellular pressure. Using SCC25 tongue squamous cancer cells stably transfected with either an empty selection vector or paxillin expression and selection vectors, we studied adhesion to collagen, paxillin, FAK, and Src expression and phosphorylation in cells maintained for 30 min under ambient or 15 mmHg increased pressure conditions. Paxillin-overexpressing cells exhibited adhesion 121 +/- 2.9% of that observed in vector-only cells (n = 6, P < 0.001) under ambient pressure. Paxillin-overexpression reduced FAK phosphorylation. Pressure stimulated adhesion to 118 +/- 2.3% (n = 6, P < 0.001) of baseline in vector-only cells, similar to its effect in the parental line, and induced paxillin, FAK, and Src phosphorylation. However, increased pressure did not stimulate adhesion or phosphorylate paxillin, FAK, or Src further in paxillin-overexpressing cells. Metastasizing squamous cancer cell adhesiveness may be increased by paxillin-overexpression or by paxillin activation by extracellular pressure during surgical manipulation or growth within a constraining compartment. Targeting paxillin in patients with malignancy and minimal tumor manipulation during surgical resection may be important therapeutic adjuncts.     

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.     

Signaling transduction pathway of angiotensin II in human mesangial cells: mediation of focal adhesion and GTPase activating proteins

Human mesangial cells (HMCs) respond to angiotensin II stimulation, which modulates their physiological activities, i.e., contraction and proliferation. It has been revealed that focal adhesion kinase (FAK) and paxillin participate in the angiotensin II-mediated signaling and cytoskeletal rearrangements at focal adhesion. We investigated the influences of cell adhesion upon angiotensin II effects in HMCs. In adherent cells, both FAK and paxillin were tyrosine phosphorylated by angiotensin II, while the cell detachment completely inhibited the tyrosine phosphorylation of paxillin. Activation of p44/42 mitogen-activated protein (MAP) kinase by angiotensin II was accentuated in suspended cells. Moreover, p190, a member of Rho GTPase activating protein (GAP), and RasGAP were coprecipitated with paxillin in adherent cells and angiotensin II stimulation reduced the formation of paxillin-p190 and paxillin-RasGAP complexes. These results suggest that the formation of focal adhesion complexes accelerated by accumulation of mesangial matrices may inhibit the proliferation of HMCs by modulating MAP kinase activity and be related to mesangial cell depletion.     

GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin

The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins for the ADP-ribosylation factor family of small GTP-binding proteins, but also serve as adaptors to link signaling proteins to distinct cellular locations. One role for GIT proteins is to link the PIX family of Rho guanine nucleotide exchange factors and their binding partners, the p21-activated protein kinases, to remodeling focal adhesions by interacting with the focal adhesion adaptor protein paxillin. We here identified the C-terminal domain of GIT1 responsible for paxillin binding. Combining structural and mutational analyses, we show that this region folds into an anti-parallel four-helix domain highly reminiscent to the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Our results suggest that the GIT1 FAT-homology (FAH) domain and FAT bind the paxillin LD4 motif quite similarly. Since only a small fraction of GIT1 is bound to paxillin under normal conditions, regulation of paxillin binding was explored. Although paxillin binding to the FAT domain of FAK is regulated by tyrosine phosphorylation within this domain, we find that tyrosine phosphorylation of the FAH domain GIT1 is not involved in regulating binding to paxillin. Instead, we find that mutations within the FAH domain may alter binding to paxillin that has been phosphorylated within the LD4 motif. Thus, despite apparent structural similarity in their FAT domains, GIT1 and FAK binding to paxillin is differentially regulated.     

Differential regulation of components of the focal adhesion complex by heregulin: role of phosphatase SHP-2.

Heregulin (HRG) has been implicated in the progression of breast cancer cells to a malignant phenotype, a process that involves changes in cell motility and adhesion. Here we demonstrate that HRG differentially regulates the site-specific phosphorylation of the focal adhesion components focal adhesion kinase (FAK) and paxilin in a dose-dependent manner. HRG at suboptimal doses (0.01 and 0.1 nM) increased adhesion of cells to the substratum, induced phosphorylation of FAK at Tyr-577, -925, and induced formation of well-defined focal points in breast cancer cell line MCF-7. HRG at a dose of 1 nM, increased migratory potential of breast cancer cells, selectively dephosphorylated FAK at Tyr-577, -925, and paxillin at Tyr-31. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by HRG stimulation. FAK associated with HER2 only in response to 0.01 nM HRG. In contrast, 1 nM HRG induced activation and increased association of tyrosine phosphatase SHP-2 with HER2 but decreased association of HER2 with FAK. Expression of dominant-negative SHP-2 blocked HRG-mediated dephosphorylation of FAK and paxillin, leading to persistent accumulation of mature focal points. Our results suggest that HRG differentially regulates signaling from focal adhesion complexes through selective phosphorylation and dephosphorylation and that tyrosine phosphatase SHP-2 has a role in the HRG signaling.     

The association of ASAP1, an ADP ribosylation factor-GTPase activating protein,with focal adhesion kinase contributes to the process of focal adhesion assembly

ASAP1 (ADP ribosylation factor [ARF]- GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60(Src). Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). Glutathione S-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK.     

Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase.

Focal adhesion kinase (pp125FAK or FAK) and paxillin colocalize with integrins in structures called focal adhesions. pp125FAK plays an important role in the transmission of integrin-induced cytoplasmic signals. Paxillin has also been implicated in cell signaling by virtue of its association with the protein tyrosine kinases pp60src and Csk (C-terminal Src kinase) as well as with the adapter/oncoprotein p47gag-crk. In this report we show that endogenous pp125FAK and paxillin form a stable complex both in vivo and in vitro and that this interaction is direct, requiring only pp125FAK and paxillin. The paxillin binding site on pp125FAK has been localized to the carboxy-terminal 148 residues of pp125FAK, but appears to be distinct from the previously identified focal adhesion-targeting sequence also present in the carboxy-terminal domain of pp125FAK. The interaction of paxillin and pp125FAK is independent of the adhesion of cells to the extracellular matrix, as the association can be detected in suspension cells as well as those attached to fibronectin.     

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.     

Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells

The relationship between focal adhesion protein (FAK) activity and loss of cell-matrix contact during apoptosis is not entirely clear nor has the role of FAK in chemically induced apoptosis been studied. We investigated the status of FAK phosphorylation and cleavage in renal epithelial cells during apoptosis caused by the nephrotoxicant dichlorovinylcysteine (DCVC). DCVC treatment caused a loss of cell-matrix contact which was preceded by a dissociation of FAK from the focal adhesions and tyrosine dephosphorylation of FAK. Paxillin was also dephosphorylated at tyrosine. DCVC treatment activated caspase-3 which was associated with cleavage of FAK. However, FAK cleavage occurred after cells had already lost focal adhesions indicating that cleavage of FAK by caspases is not responsible for loss of FAK from focal adhesions. Accordingly, although inhibition of caspase activity with zVAD-fmk blocked activation of caspase-3, FAK cleavage, and apoptosis, it neither affected dephosphorylation nor translocation of FAK or paxillin. However, zVAD-fmk completely blocked the cell detachment caused by DCVC treatment. Orthovanadate prevented DCVC-induced tyrosine dephosphorylation of both FAK and paxillin; however, it did not inhibit DCVC-induced apoptosis and actually potentiated focal adhesion disorganization and cell detachment. Thus, FAK dephosphorylation and loss of focal adhesions are not due to caspase activation; however, caspases are required for FAK proteolysis and cell detachment.     

Dissociation of FAK/p130CAS/c-Src Complex during Mitosis: Role of Mitosis-specific Serine Phosphorylation of FAK

At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. We have found that components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130^CAS (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading.We have found two significant alterations in FAK-mediated signal transduction during mitosis. First, the association of FAK with CAS or c-Src is greatly inhibited, with levels decreasing to 16 and 13% of the interphase levels, respectively. Second, mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells. Mitosis-specific phosphorylation is responsible for the disruption of FAK/CAS binding because dephosphorylation of mitotic FAK in vitro by protein serine/threonine phosphatase 1 restores the ability of FAK to associate with CAS, though not with c-Src. These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading.     

Phosphorylated alpha-actinin and protein-tyrosine phosphatase 1B coregulate the disassembly of the focal adhesion kinase x Src complex and promote cell migration

The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein alpha-actinin at Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) is an alpha-actinin phosphatase. PTP 1B-dependent dephosphorylation of alpha-actinin was seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type alpha-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the alpha-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered by PTP 1B. In addition, we found that phosphorylated alpha-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation at Tyr-397 in FAK triggered by wild-type alpha-actinin and PTP 1B caused a significant increase in cell migration. We propose that phosphorylated alpha-actinin disrupts the FAK x Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel feedback loop involving phosphorylated alpha-actinin and PTP 1B that regulates FAK x Src interaction and cell migration.