Role of Grb7 targeting to focal contacts and its phosphorylation by focal adhesion kinase in regulation of cell migration

We have previously described Grb7 association with focal adhesion kinase (FAK) and its possible roles in cell migration. In this paper, we investigated the mechanisms by which Grb7 and its association with FAK regulate cell migration. We found that deletion of the Grb7 SH2 domain eliminated partial Grb7 localization to focal contacts and its ability to stimulate cell migration. Replacement of the SH2 domain with the focal adhesion targeting sequence from FAK resulted in the focal contacts localization of the chimeric molecule and restored its activity to stimulate cell migration. We also found that Grb7 could be phosphorylated by FAK, which was dependent on the FAK kinase activity but not the presence of the Src family kinases. Cell adhesion also enhanced Grb7 phosphorylation in FAK+/+ cells but not FAK-/- cells, suggesting that Grb7 is a physiological substrate of FAK. Furthermore, both Grb7 and the chimeric molecule did not increase migration of FAK-/- cells, although the chimeric molecule was targeted to the focal contacts. Last, we showed that other Grb7 family members could not stimulate cell migration under similar experimental conditions. Together, these results demonstrate a role for Grb7 targeting to focal contacts and its phosphorylation by FAK in the regulation of cell migration.     

A direct interaction between the large GTPase dynamin-2 and FAK regulates focal adhesion dynamics in response to active Src

Tumor cell migration is supported in part by the cyclic formation and disassembly of focal adhesions (FAs); however, the mechanisms that regulate this process are not fully defined. The large guanosine 5'-triphosphatase dynamin (Dyn) plays an important role in FA dynamics and is activated by tyrosine phosphorylation. Using a novel antibody specific to phospho-dynamin (pDyn-Tyr-231), we found that Dyn2 is phosphorylated at FAs by Src kinase and is recruited to FAs by a direct interaction with the 4.1/ezrin/radizin/moesin domain of focal adhesion kinase (FAK), which functions as an adaptor between Src and Dyn2 to facilitate Dyn2 phosphorylation. This Src-FAK-Dyn2 trimeric complex is essential for FA turnover, as mutants disrupting the formation of this complex inhibit FA disassembly. Importantly, phosphoactivated Dyn2 promotes FA turnover by mediating the endocytosis of integrins in a clathrin-dependent manner. This study defines a novel mechanism of how Dyn2 functions as a downstream effector of FAK-Src signaling in turning over FAs.     

v-Src SH3-enhanced interaction with focal adhesion kinase at beta 1 integrin-containing invadopodia promotes cell invasion

In viral Src (v-Src)-transformed cells, focal adhesion kinase (FAK) associates with v-Src by combined v-Src SH2 and gain-of-function v-Src SH3 domain binding to FAK. Here we assess the significance of the Arg-95 to Trp gain-of-function mutation in the v-Src SH3 domain through comparisons of Src-/- fibroblasts transformed with either Prague C v-Src or a point mutant (v-Src-RT) containing a normal (Arg-95) SH3 domain. Both v-Src isoforms exhibited equivalent kinase activity, enhanced Src-/- cell motility, and stimulated cell growth in both low serum and soft agar. The stability of a v-Src-RT.FAK signaling complex and FAK phosphorylation at Tyr-861 and Tyr-925 were reduced in v-Src-RT- compared with v-Src-transformed cells. v-Src but not v-Src-RT promoted Src-/- cell invasion through a reconstituted Matrigel basement membrane barrier and v-Src co-localized with FAK and beta(1) integrin at invadopodia. In contrast, v-Src-RT exhibited a partial perinuclear and focal contact distribution in Src-/- cells. Adenovirus-mediated FAK overexpression promoted v-Src-RT recruitment to invadopodia, the formation of a v-Src-RT.FAK signaling complex, and reversed the v-Src-RT invasion deficit. Adenovirus-mediated inhibition of FAK blocked v-Src-stimulated cell invasion. These studies establish that gain-of-function v-Src SH3 targeting interactions with FAK at beta(1) integrin-containing invadopodia act to stabilize a v-Src.FAK signaling complex promoting cell invasion.     

Calcium rises locally trigger focal adhesion disassembly and enhance residency of focal adhesion kinase at focal adhesions

Focal adhesion kinase (FAK) activity and Ca(2+) signaling led to a turnover of focal adhesions (FAs) required for cell spreading and migration. We used yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca(2+) sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca(2+) variations at FA sites and FA dynamics. Discrete subcellular Ca(2+) oscillators initiate both propagating and abortive Ca(2+) waves in migrating U87 astrocytoma cells. Ca(2+)-dependent FA disassembly occurs when the Ca(2+) wave reaches individual FAs, indicating that local but not global Ca(2+) increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. The FAK-Ycam recovery half-time (17 s) at FAs was slowed (to 29 s) by Ca(2+) elevation. FAK-related non-kinase domain-Ycam had a faster, Ca(2+)-insensitive recovery half-time (11 s), which is consistent with the effect of Ca(2+) on FAK-Ycam dynamics not being due to a general modification of the dynamics of FA components. Because FAK association at FAs was prolonged by Ca(2+) and FAK autophosphorylation was correlated to intracellular Ca(2+) levels, we propose that local Ca(2+) elevations increase the residency of FAK at FAs, possibly by means of tyrosine phosphorylation of FAK, thereby leading to increased activation of its effectors involved in FA disassembly.     

P130Cas Src-binding and substrate domains have distinct roles in sustaining focal adhesion disassembly and promoting cell migration

The docking protein p130Cas is a prominent Src substrate found in focal adhesions (FAs) and is implicated in regulating critical aspects of cell motility including FA disassembly and protrusion of the leading edge plasma membrane. To better understand how p130Cas acts to promote these events we examined requirements for established p130Cas signaling motifs including the SH3-binding site of the Src binding domain (SBD) and the tyrosine phosphorylation sites within the substrate domain (SD). Expression of wild type p130Cas in Cas -/- mouse embryo fibroblasts resulted in enhanced cell migration associated with increased leading-edge actin flux, increased rates of FA assembly/disassembly, and uninterrupted FA turnover. Variants lacking either the SD phosphorylation sites or the SBD SH3-binding motif were able to partially restore the migration response, while only a variant lacking both signaling functions was fully defective. Notably, the migration defects associated with p130Cas signaling-deficient variants correlated with longer FA lifetimes resulting from aborted FA disassembly attempts. However the SD mutational variant was fully defective in increasing actin assembly at the protruding leading edge and FA assembly/disassembly rates, indicating that SD phosphorylation is the sole p130Cas signaling function in regulating these processes. Our results provide the first quantitative evidence supporting roles for p130Cas SD tyrosine phosphorylation in promoting both leading edge actin flux and FA turnover during cell migration, while further revealing that the p130Cas SBD has a function in cell migration and sustained FA disassembly that is distinct from its known role of promoting SD tyrosine phosphorylation.     

Residues within the first subdomain of the FERM-like domain in focal adhesion kinase are important in its regulation

We have previously described regulation of focal adhesion kinase (FAK) by its amino-terminal FERM-like domain through an autoinhibitory interaction with its kinase domain (Cooper, L. A., Shen, T. L., and Guan, J. L. (2003) Mol. Cell. Biol. 23, 8030-8041). Here we show that the first two subdomains of the FERM-like domain are independently capable of inhibiting phosphorylation of FAK in trans. We characterized several point mutations within the first subdomain of the FERM-like domain and find that mutation of Lys-38 to alanine results in a FAK mutant that is strongly hyperphosphorylated when expressed in mammalian cells, and promotes increased phosphorylation of the FAK substrate paxillin. A second mutation of Lys-78 to alanine results in a FAK mutant that is underphosphorylated, but can be activated by extracellular matrix stimuli. Like deletion of the amino terminus itself the K38A mutation is phosphorylated in suspension. The Delta375 truncation mutant of FAK is strongly phosphorylated both when Tyr-397 is mutated to phenylalanine, and in the presence of the Src inhibitor, PP2, suggesting that removal of the amino terminus can render FAK Src independent. This is in contrast to the K38A mutant that is not phosphorylated in the Y397F background, and which shows decreased phosphorylation in the presence of the Src inhibitor PP2, suggesting that regulation of FAK by Src is a secondary step in its activation. The K38A mutation weakens the interaction between the amino terminus of FAK and its own kinase domain, and disrupts the ability of the amino terminus to inhibit the phosphorylation of FAK in trans. The K38A mutation of FAK also increases the ability of FAK to promote cell cycle progression and cell migration, suggesting that hyperphosphorylation of this mutant can positively affect FAK function in cells. Together, these data strongly suggest a role for the first FAK subdomain of the FERM domain in its normal regulation and function in the cell.     

Calcium oscillations trigger focal adhesion disassembly in human U87 astrocytoma cells

Integrin-associated intracellular Ca(2+) oscillations modulate cell migration, probably by controlling integrin-mediated release of the cell rear during migration. Focal adhesion kinase (FAK), via its tyrosine phosphorylation activity, plays a key role in integrin signaling. In human U87 astrocytoma cells, expression of the dominant negative FAK-related non-kinase domain (FRNK) inhibits the Ca(2+)-sensitive component of serum-dependent migration. We investigated how integrin-associated Ca(2+) signaling might be coupled to focal adhesion (FA) dynamics by visualizing the effects of Ca(2+) spikes on FAs using green fluorescent protein (GFP)-tagged FAK and FRNK. We report that Ca(2+) spikes are temporally correlated with movement and disassembly of FAs, but not their formation. FRNK transfection did not affect generation of Ca(2+) spikes, although cell morphology was altered, with fewer FAs of larger size and having a more peripheral localization being observed. Larger sized FAs in FRNK-transfected cells were not disassembled by Ca(2+) spikes, providing a possible explanation for impaired Ca(2+)-dependent migration in these cells. Stress fiber end movements initiated by Ca(2+) spikes were visualized using GFP-tagged myosin light chain kinase (MLCK). Ca(2+)-associated movements of stress fiber ends and FAs had similar kinetics, suggesting that stress fibers and FAs move in a coordinated fashion. This indicates that increases in Ca(2+) likely trigger disassembly of adhesive structures that involves disruption of integrin-extracellular matrix interactions, supporting a key role for Ca(2+)-sensitive inside-out signaling in cell migration. A rapid increase in tyrosine phosphorylation of FAK was found in response to an elevation in Ca(2+) induced by thapsigargin, and we propose that this represents the initial triggering event linking Ca(2+) signaling and FA dynamics to cell motility.     

Nck-2 interacts with focal adhesion kinase and modulates cell motility

Nck-2 is a ubiquitously expressed adaptor protein comprising primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We report here that Nck-2 interacts with focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase critically involved in the cellular control of motility. Using a mutational strategy, we have found that the formation of the Nck-2-FAK complex is mediated by interactions involving multiple SH2 and SH3 domains of Nck-2. The Nck-2 SH2 domain-mediated interaction with FAK is dependent on phosphorylation of Tyr397, a site that is involved in the regulation of cell motility. A fraction of Nck-2 co-localizes with FAK at cell periphery in spreading cells. Furthermore, overexpression of Nck-2 modestly decreased cell motility, whereas overexpression of a mutant form of Nck-2 containing the SH2 domain but lacking the SH3 domains significantly promoted cell motility. These results identify a novel interaction between Nck-2 and FAK and suggest a role of Nck-2 in the modulation of cell motility.     

In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase

Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with growth factor receptor-bound protein 2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH-dependent, but this does not reflect the pH dependence of Src kinase activity. Circular dichroism and nuclear magnetic resonance data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the microsecond to millisecond time scale.     

The Shb adaptor protein causes Src-dependent cell spreading and activation of focal adhesion kinase in murine brain endothelial cells

The mechanisms leading to focal adhesion kinase (FAK) activation remain obscure. We have investigated the role of the adaptor protein Shb in cell spreading and the regulation of FAK phosphorylation in immortalised brain endothelial (IBE) cells. Fibroblast growth factor 2 (FGF-2) stimulation lead to a direct association between Shb and FAK, which was mediated by the phosphotyrosine binding (PTB) domain of Shb. IBE cells overexpressing wild-type or R522K Shb (with an inactive Src homology 2 (SH2) domain) displayed increased FAK phosphorylation as well as enhanced spreading when seeded on collagen. FGF-2-induced tyrosine phosphorylation of Shb was dependent upon Src activity but independent of FAK activation. The use of Rat-1 fibroblasts overexpressing a temperature sensitive v-Src (tsLA29) confirmed that active Src enhanced Shb phosphorylation. The data indicate that Shb binds directly to FAK and regulates its phosphorylation leading to enhanced cell spreading in a Src-dependent manner.     

Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.     

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.     

Fibroblast growth factor-2 induces the activation of Src through Fes, which regulates focal adhesion disassembly

Cell migration is regulated by focal adhesion (FA) turnover. Fibroblast growth factor-2 (FGF-2) induces FA disassembly in the murine brain capillary endothelial cell line IBE, leading to FGF-2-directed chemotaxis. We previously showed that activation of Src and Fes by FGF-2 was involved in chemotaxis of IBE cells. In this study, we examined the interplay between Src and Fes. FGF-2 treatment decreased the number of FA in IBE cells, but not in cells expressing dominant-negative Fes (denoted KE5-15 cells). FGF-2 induced the activation of Src and subsequent binding to and phosphorylation of Cas in IBE cells, but not in KE5-15 cells. Focal adhesion kinase (FAK) activation and tyrosine phosphorylation by Src were also delayed in KE5-15 cells compared to parental cells. FGF-2 induced activation of Src within FA in IBE cells, but not in KE5-15 cells. Downregulation of Fes or FAK using small interfering RNA diminished Src activation by FGF-2 within FA. These findings suggest that activation of Fes by FGF-2 enhances FAK-dependent activation of Src within FA, promoting FGF-2-induced disassembly of focal adhesions.     

Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.     

Regulation of focal adhesion formation and filopodia extension by the cellular prion protein (PrPC)

While the prion protein (PrP) is clearly involved in neuropathology, its physiological roles remain elusive. Here, we demonstrate PrP functions in cell-substrate interaction in Drosophila S2, N2a and HeLa cells. PrP promotes cell spreading and/or filopodia formation when overexpressed, and lamellipodia when downregulated. Moreover, PrP normally accumulates in focal adhesions (FAs), and its downregulation leads to reduced FA numbers, increased FA length, along with Src and focal adhesion kinase (FAK) activation. Furthermore, its overexpression elicits the formation of novel FA-like structures, which require intact reggie/flotillin microdomains. Altogether, PrP modulates process formation and FA dynamics, possibly via signal transduction involving FAK and Src.     

The v-Src SH3 domain facilitates a cell adhesion-independent association with focal adhesion kinase

Integrins facilitate cell attachment to the extracellular matrix, and these interactions generate cell survival, proliferation, and motility signals. Integrin signals are relayed in part by focal adhesion kinase (FAK) activation and the formation of a transient signaling complex initiated by Src homology 2 (SH2)-dependent binding of Src family protein-tyrosine kinases to the FAK Tyr-397 autophosphorylation site. Here we show that in viral Src (v-Src)-transformed NIH3T3 fibroblasts, an adhesion-independent FAK-Src signaling complex occurs. Co-expression studies in human 293T cells showed that v-Src could associate with and phosphorylate a Phe-397 FAK mutant at Tyr-925 promoting Grb2 binding to FAK in suspended cells. In vitro, glutathione S-transferase fusion proteins of the v-Src SH3 but not c-Src SH3 domain bound to FAK in lysates of NIH3T3 fibroblasts. The v-Src SH3-binding sites were mapped to known proline-X-X-proline (PXXP) SH3-binding motifs in the FAK N- (residues 371-377) and C-terminal domains (residues 712-718 and 871-882) by in vitro pull-down assays, and these sites are composed of a PXXPXXPhi (where Phi is a hydrophobic residue) v-Src SH3 binding consensus. Sequence comparisons show that residues in the RT loop region of the c-Src and v-Src SH3 domains differ. Substitution of c-Src RT loop residues (Arg-97 and Thr-98) for those found in the v-Src SH3 domain (Trp-97 and Ile-98) enhanced the binding of distinct NIH3T3 cellular proteins to a glutathione S-transferase fusion protein of the c-Src (Trp-97 + Ile-98) SH3 domain. FAK was identified as a c-Src (Trp-97 + Ile-98) SH3 domain target in fibroblasts, and co-expression studies in 293T cells showed that full-length c-Src (Trp-97 + Ile-98) could associate in vivo with Phe-397 FAK in an SH2-independent manner. These studies establish a functional role for the v-Src SH3 domain in stabilizing an adhesion-independent signaling complex with FAK.     

Cortactin as a Target for FAK in the Regulation of Focal Adhesion Dynamics

Background

Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.

Principal Findings

Using FAK-null fibroblasts stably reconstituted with green fluorescent protein (GFP) tagged FAK constructs, we find that FAK activity and FAK C-terminal proline-rich region 2 (PRR2) and PRR3 are required for FA turnover and cell motility. Cortactin binds directly to FAK PRR2 and PRR3 sites via its SH3 domain and cortactin expression is important in promoting FA turnover and GFP-FAK release from FAs. FAK-cortactin binding is negatively-regulated by FAK activity and associated with cortactin tyrosine phosphorylation. FAK directly phosphorylates cortactin at Y421 and Y466 and over-expression of cortactin Y421, Y466, and Y482 mutated to phenylalanine (3YF) prevented FAK-enhanced FA turnover and cell motility. However, phospho-mimetic cortactin mutated to glutamic acid (3YE) did not affect FA dynamics and did not rescue FA turnover defects in cells with inhibited FAK activity or with PRR2-mutated FAK that does not bind cortactin.

Conclusions

Our results support a model whereby FAK-mediated FA remodeling may occur through the formation of a FAK-cortactin signaling complex. This involves a cycle of cortactin binding to FAK, cortactin tyrosine phosphorylation, and subsequent cortactin-FAK dissociation accompanied by FA turnover and cell movement.     

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.     

Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.     

Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.