Signaling through focal adhesion kinase

Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130^Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK⁻) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK⁻ cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.     

Multiple connections link FAK to cell motility and invasion

The ability of intracellular signaling networks to orchestrate a complex biological response such as cell motility requires that individual signaling proteins must act as integrators, responding to multiple extracellular inputs and regulating multiple signaling pathway outputs. In this review, we highlight recent findings that place focal adhesion kinase (FAK) in an important receptor-proximal position in the regulation of growth factor and integrin-stimulated cell motility. Emphasis is placed on the molecular mechanisms of FAK activation, connections of FAK to focal contact formation as well as turnover, and the potential that FAK function in promoting cell invasion may be distinct from its role in cell motility.     

Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK.

Integrins play a central role in cellular adhesion and anchorage of the cytoskeleton and participate in the generation of intracellular signals, including tyrosine phosphorylation. We have recently isolated a cDNA encoding a unique, focal adhesion-associated protein tyrosine kinase (FAK) that is a component of an integrin-mediated signal transduction pathway. Here we report the isolation of cDNAs encoding the C-terminal, noncatalytic domain of the FAK kinase, termed FRNK (FAK-related nonkinase). Both the FAK- and FRNK-encoded polypeptides, pp125FAK and p41/p43FRNK, are expressed in normal chicken embryo cells. pp125FAK and p41/p43FRNK were localized to focal adhesions, suggesting that pp125FAK is directed to the focal adhesions by sequences within its C-terminal domain. We also show that the fibronectin-dependent increase in tyrosine phosphorylation of pp125FAK is accompanied by a concomitant posttranslational modification of p41FRNK.     

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.     

A role for focal adhesion kinase in phenylephrine-induced hypertrophy of rat ventricular cardiomyocytes

A variety of agonists including phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Here we report that signals provided by extracellular matrix proteins (ECM) augment the PE-induced hypertrophic response of cardiomyocytes and provide evidence that ECM-dependent signaling is mediated in part by the protein tyrosine kinase, focal adhesion kinase (FAK). Addition of PE to cultured neonatal cardiomyocytes stimulated sarcomeric organization, increased cell size, and induced atrial natriuretic factor in cardiomyocytes plated on the ECM protein laminin or fibronectin. In contrast, cardiomyocytes plated on the non-adhesive substrate gelatin exhibited a reduced capacity to undergo these PE-stimulated hypertrophic changes. In cardiomyocytes cultured on ECM, PE stimulated a rapid increase in tyrosine phosphorylation of focal adhesion proteins including FAK, paxillin, and p130 Crk-associated substrate and subsequent formation of peripheral focal complexes. Inhibition of the PE-induced hypertrophic response by genistein and herbimycin-A indicated a requirement for protein tyrosine kinases in PE signaling. To determine whether activation of FAK is required for PE-induced hypertrophy, a dominant-interfering mutant form of FAK, termed FRNK (FAK-related non-kinase), was ectopically expressed in cardiomyocytes using a replication-defective adenovirus expression system. FRNK expression attenuated PE-stimulated hypertrophy as assessed by cell size, sarcomeric organization, and induction of atrial natriuretic factor. These data indicate that the signal transduction pathways leading to cardiomyocyte hypertrophy are strongly influenced by and/or dependent upon an integrin-mediated signaling process requiring FAK.     

Epidermal growth factor-induced tumor cell invasion and metastasis initiated by dephosphorylation and downregulation of focal adhesion kinase

Upregulated epidermal growth factor (EGF) receptor (EGFR) expression and EGFR-induced signaling have been correlated with progression to invasion and metastasis in a wide variety of carcinomas, but the mechanism behind this is not well understood. We show here that, in various human carcinoma cells that overexpress EGFR, EGF treatment induced rapid tyrosine dephosphorylation of focal adhesion kinase (FAK) associated with downregulation of its kinase activity. The downregulation of FAK activity was both required and sufficient for EGF-induced refractile morphological changes, detachment of cells from the extracellular matrix, and increased tumor cell motility, invasion, and metastasis. Tumor cells with downregulated FAK activity became less adherent to the extracellular matrix. However, once cells started reattaching, FAK activity was restored by activated integrin signaling. Moreover, this process of readhesion and spreading could not be abrogated by further EGF stimulation. Interruption of transforming growth factor alpha-EGFR autocrine regulation with an EGFR tyrosine kinase inhibitor led to a substantial increase in FAK tyrosine phosphorylation and inhibition of tumor cell invasion in vitro. Consistent with this, FAK tyrosine phosphorylation was reduced in cells from tumors growing in transplanted, athymic, nude mice, which have an intact autocrine regulation of the EGFR. We suggest that the dynamic regulation of FAK activity, initiated by EGF-induced downregulation of FAK leading to cell detachment and increased motility and invasion, followed by integrin-dependent reactivation during readhesion, plays a role in EGF-associated tumor invasion and metastasis.     

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.     

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.     

Targeting Pyk2 to beta 1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells

Focal adhesion kinase-null (FAK(-/-) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK(-/-) cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK(-/-) cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a beta 1-integrin-containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK(-/-) cells, exhibit elevated FN-stimulated extracellular signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH(2)-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to beta-integrin-containing focal contact sites via interactions mediated by the FAK-CT domain.     

Focal adhesion kinase facilitates platelet-derived growth factor-BB-stimulated ERK2 activation required for chemotaxis migration of vascular smooth muscle cells

The focal adhesion (FAK) non-receptor protein-tyrosine kinase (PTK) links both extracellular matrix/integrin and growth factor stimulation to intracellular signals promoting cell migration. Here we show that both transient and stable overexpression of the FAK C-terminal domain termed FRNK (FAK-related non-kinase) inhibits serum and platelet-derived growth factor (PDGF)-BB-induced vascular smooth muscle cell (SMC) migration in wound healing and in vitro Boyden Chamber chemotaxis assays, respectively. Expression of FRNK, but not a point mutant of FRNK (FRNK L1034S), disrupted the formation of a complex containing both FAK and the activated PDGF-beta receptor and resulted in reduced tyrosine phosphorylation of endogenous FAK at the Tyr-397 binding site for Src family PTKs. As demonstrated using FAK-deficient and FAK-reconstituted fibroblasts, FAK positively contributed to PDGF-BB-stimulated ERK2/MAP kinase activity, and in SMCs, ERK2/MAP kinase activity was required for PDGF-BB-stimulated chemotaxis. Stable expression of FRNK but not FRNK L1034S expression in SMCs lowered the extent and duration of stimulated ERK2/MAP kinase activation at low but not at high PDGF-BB concentrations. Importantly, stable expression of FRNK in SMCs did not affect SMC morphology or proliferation in culture. Because the increased migration of vascular SMCs in response to extracellular matrix proteins and growth factors contributes to neointima formation, our results show that FAK inhibition by FRNK expression may provide a novel approach to regulate abnormal vascular SMC migration in vivo.     

Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase

Vascular endothelial growth factor (VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury. VEGF-induced endothelial cell migration is a key step in the angiogenic response and is mediated by an accelerated rate of focal adhesion complex assembly and disassembly. In this study, we identified the signaling mechanisms by which VEGF regulates human brain microvascular endothelial cell (HBMEC) integrity and assembly of focal adhesions, complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. We found that VEGF treatment of HBMECs plated on laminin or fibronectin stimulated cytoskeletal organization and increased focal adhesion sites. Pretreating cells with VEGF antibodies or with the specific inhibitor SU-1498, which inhibits Flk-1/KDR receptor phosphorylation, blocked the ability of VEGF to stimulate focal adhesion assembly. VEGF induced the coupling of focal adhesion kinase (FAK) to integrin alphavbeta5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130cas. Additionally, FAK and related adhesion focal tyrosine kinase (RAFTK)/Pyk2 kinases were tyrosine-phosphorylated by VEGF and found to be important for focal adhesion sites. Overexpression of wild type RAFTK/Pyk2 increased cell spreading and the migration of HBMECs, whereas overexpression of catalytically inactive mutant RAFTK/Pyk2 markedly suppressed HBMEC spreading ( approximately 70%), adhesion ( approximately 82%), and migration ( approximately 65%). Furthermore, blocking of FAK by the dominant-interfering mutant FRNK (FAK-related non-kinase) significantly inhibited HBMEC spreading and migration and also disrupted focal adhesions. Thus, these studies define a mechanism for the regulatory role of VEGF in focal adhesion complex assembly in HBMECs via activation of FAK and RAFTK/Pyk2.     

DWnt4 regulates cell movement and focal adhesion kinase during Drosophila ovarian morphogenesis

Cell motility is regulated by extracellular cues and by intracellular factors that accumulate at sites of contact between cells and the extracellular matrix. One of these factors, focal adhesion kinase (FAK), regulates the cycle of focal adhesion formation and disassembly that is required for cell movement to occur. Recently, Wnt signaling has also been implicated in the control of cell movement in vertebrates, but the mechanism through which Wnt proteins influence motility is unclear. We demonstrate that Drosphila Wnt4 is required for cell movement and FAK regulation during ovarian morphogenesis. Dfrizzled2, Disheveled, and protein kinase C are also required. The DWnt4 cell motility pathway is distinct from both the canonical Wnt pathway and the planar polarity pathway. Our data suggest that DWnt4 facilitates motility through regulation of focal adhesions.     

FAK regulates tyrosine phosphorylation of CAS, paxillin, and PYK2 in cells expressing v-Src, but is not a critical determinant of v-Src transformation.

FAK (focal adhesion kinase) is a nonreceptor protein-tyrosine kinase activated by tyrosine phosphorylation following integrin-mediated cell adhesion. Oncogenic Src promotes enhanced and deregulated FAK tyrosine phosphorylation which has been proposed to contribute to altered cell growth and/or morphological properties associated with transformation. In this study, an inducible FAK expression system was used to study the potential role of FAK in v-Src transformation. Our results portray FAK as a major v-Src substrate that also plays a role in recruiting v-Src to phosphorylate substrates CAS (Crk-associated substrate) and paxillin. The FAK Tyr-397 autophosphorylation site was necessary for this scaffolding function, but was not required for v-Src to stably interact with and phosphorylate FAK. FAK was also shown to negatively regulate v-Src mediated phosphorylation of the FAK-related kinase PYK2. Despite these effects, FAK does not play an essential role in targeting v-Src to major cellular substrates including CAS and paxillin. Nor is FAK strictly required to achieve the altered morphological and growth characteristics of v-Src transformed cells.     

Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: a role for paxillin tyrosine phosphorylation.

pp125FAK is a tyrosine kinase that appears to regulate the assembly of focal adhesions and thereby promotes cell spreading on the extracellular matrix. In some cells, the C terminus of pp125FAK is expressed as a separate protein, pp41/43FRNK. We have previously shown that overexpression of pp41/43FRNK inhibits tyrosine phosphorylation of pp125FAK and paxillin and, in addition, delays cell spreading and focal adhesion assembly. Thus, pp41/43FRNK functions as a negative inhibitor of adhesion signaling and provides a tool to dissect the mechanism by which pp125FAK promotes cell spreading. We report here that the inhibitory effects of pp41/43FRNK expression can be rescued by the co-overexpression of wild-type pp125FAK and partially rescued by catalytically inactive variants of pp125FAK. However, coexpression of an autophosphorylation site mutant of pp125FAK, which fails to bind the SH2 domain of pp60c-Src, or a mutant that fails to bind paxillin did not promote cell spreading. In contrast, expression of pp41/43FRNK and pp60c-Src reconstituted cell spreading and tyrosine phosphorylation of paxillin but did so without inducing tyrosine phosphorylation of pp125FAK. These data provide additional support for a model whereby pp125FAK acts as a "switchable adaptor" that recruits pp60c-Src to phosphorylate paxillin, promoting cell spreading. In addition, these data point to tyrosine phosphorylation of paxillin as being a critical step in focal adhesion assembly.     

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.     

FAK is required for the assembly of podosome rosettes

Podosomes are dynamic actin-enriched membrane structures that play an important role in invasive cell motility and extracellular matrix degradation. They are often found to assemble into large rosettelike structures in highly invasive cells. However, the mechanism of this assembly remains obscure. In this study, we identified focal adhesion kinase (FAK) as a key molecule necessary for assembly. Moreover, phosphorylation of p130Cas and suppression of Rho signaling by FAK were found to be important for FAK to induce the assembly of podosome rosettes. Finally, we found that suppression of vimentin intermediate filaments by FAK facilitates the assembly of podosome rosettes. Collectively, our results strongly suggest a link between FAK, podosome rosettes, and tumor invasion and unveil a negative role for Rho signaling and vimentin filaments in podosome rosette assembly.     

Deoxycholic acid differentially regulates focal adhesion kinase phosphorylation: role of tyrosine phosphatase ShP2

Environmental factors, including dietary fats, are implicated in colonic carcinogenesis. Dietary fats modulate secondary bile acids including deoxycholic acid (DCA) concentrations in the colon, which are thought to contribute to the nutritional-related component of colon cancer risk. Here we demonstrate, for the first time, that DCA differentially regulated the site-specific phosphorylation of focal adhesion kinase (FAK). DCA decreased adhesion of HCA-7 cells to the substratum and induced dephosphorylation of FAK at tyrosine-576/577 (Tyr-576/577) and Tyr-925. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by DCA stimulation. Interestingly, we found that c-Src was constitutively associated with FAK and DCA actually activated Src, despite no change in FAK-397 and an inhibition of FAK-576 phosphorylation. DCA concomitantly and significantly increased association of tyrosine phosphatase ShP2 with FAK. Incubation of immunoprecipitated FAK, in vitro, with glutathione-S-transferase-ShP2 fusion protein resulted in tyrosine dephosphorylation of FAK in a concentration-dependent manner. Antisense oligodeoxynucleotides directed against ShP2 decreased ShP2 protein levels and attenuated DCA-induced FAK dephosphorylation. Inhibition of FAK by adenoviral-mediated overexpression of FAK-related nonkinase and gene silencing of Shp2 both abolished DCA's effect on cell adhesion, thus providing a possible mechanism for inside-out signaling by DCA in colon cancer cells. Our results suggest that DCA differentially regulates focal adhesion complexes and that tyrosine phosphatase ShP2 has a role in DCA signaling.     

Characterization of the tyrosine kinases RAFTK/Pyk2 and FAK in nerve growth factor-induced neuronal differentiation

The related adhesion focal tyrosine kinase (RAFTK), a member of the focal adhesion kinase (FAK) family and highly expressed in brain, is a key mediator of various extracellular signals that elevate intracellular Ca(2+) concentration. We investigated RAFTK and FAK signaling upon nerve growth factor (NGF) stimulation of PC12 cells. NGF induced the tyrosine phosphorylation of RAFTK in a time- and dose-dependent manner, whereas no change in the tyrosine phosphorylation of FAK was observed. Chemical inhibition showed that RAFTK phosphorylation was inhibited by blocking phospholipase Cgamma activity or intracellular Ca(2+). Blocking of extracellular Ca(2+) or phosphatidylinositol 3-kinase activity partially reduced the phosphorylation of RAFTK. In addition, disruption of actin polymerization abolished RAFTK phosphorylation, indicating that an intact actin-based cytoskeletal organization is required for RAFTK phosphorylation. The focal adhesion molecule paxillin was co-immunoprecipitated with RAFTK, and its tyrosine phosphorylation was increased in a Ca(2+)-dependent manner upon NGF stimulation. Confocal microscopic analysis demonstrated that RAFTK translocated from the cytoplasm to potential neurite initiation sites at the cell periphery, where RAFTK co-localized with paxillin and bundled actin in the early phase (within 5 min) of NGF stimulation, whereas FAK co-localized with paxillin at "point contacts," which are the primary cell adhesion sites in neuronal cells. Significant distribution of RAFTK was observed in the neurites and growth cones of differentiated PC12 cells. Furthermore, potassium depolarization induced the tyrosine phosphorylation of both RAFTK and paxillin in an intracellular Ca(2+)-dependent manner in the differentiated PC12 cells. Taken together, these results demonstrate that RAFTK is involved in NGF-induced cytoskeletal organization and may play a role in neurite and growth cone function(s).     

Induced Focal Adhesion Kinase (FAK) Expression in FAK-Null Cells Enhances Cell Spreading and Migration Requiring Both Auto- and Activation Loop Phosphorylation Sites and Inhibits Adhesion-Dependent Tyrosine Phosphorylation of Pyk2

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130(Cas), were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAKbeta/RAFTK/CadTK.     

RAFTK/Pyk2 regulates EGF-induced PC12 cell spreading and movement

The protein tyrosine kinase RAFTK, also termed Pyk2, is a member of the focal adhesion kinase (FAK) subfamily. In this report, we show the role of RAFTK in neuroendocrine PC12 cells upon epidermal growth factor (EGF) stimulation. Following EGF treatment, we observed that RAFTK was tyrosine-phosphorylated in a time- and dose-dependent manner, while FAK was constitutively phosphorylated and primarily regulated by cell adhesion. Moreover, we found that RAFTK associated with the phosphorylated EGF receptor (EGFR) upon EGF stimulation. RAFTK phosphorylation was mediated primarily through PLCgamma-IP3-Ca(2+) signaling and partially through PI3-Kinase. Furthermore, overexpression of PRNK, a specific dominant-negative construct of RAFTK, was sufficient to block EGF-induced cell spreading and movement. Paxillin, a key modulator of the actin cytoskeleton and an RAFTK substrate, was also phosphorylated following EGF treatment. EGF induced a dynamic reorganization of RAFTK and paxillin at neuronal adhesion sites, with the specific localization of paxillin at the inner juxtaposition of RAFTK. Additionally, we observed that RAFTK associated with the scaffold protein c-Cbl and mediated its phosphorylation. Our data demonstrate that while FAK mediated cell adhesion, RAFTK was localized at the cytoplasm where it mediated inside-out signaling through intracellular Ca(2+), thus leading to cell spreading and movement upon EGF stimulation.