Focal adhesion kinase-like


Table of contents

Interaction of Fak with Cas

The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, a two-hybrid screen was used to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas (see CAS/CSE1 segregation protein), and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen (Polte, 1995).

p130(Cas) (crk associated substrate) has the structural characteristics of an adapter protein, containing multiple consensus SH2 binding sites, an SH3 domain, and a proline-rich domain. The structure of p130(Cas) suggests that it may act to provide a framework for protein-protein interactions; however, as yet, its functional role in cells is unknown. p130(Cas) is localized to focal adhesions. p130(Cas) associates both in vitro and in vivo with pp125(FAK) (focal adhesion kinase), a kinase implicated in signaling by the integrin family of cell adhesion receptors. p130(Cas) also associates with pp41/43(FRNK) (pp125(FAK)-related, non-kinase), an autonomously expressed form of pp125(FAK) composed of only the C-terminal noncatalytic domain. The association of p130(Cas) with pp125(Fak) and pp41/43(FRNK) is direct, and is mediated by the binding of the SH3 domain of p130(Cas) to a proline-rich sequence present in both the C terminus of pp125(FAK) and in pp41/43(FRNK). p130(Cas) is tyrosine-phosphorylated upon integrin mediated cell adhesion. The association of p130(Cas) with pp125(FAK), a kinase which is activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction (Harte, 1996).

Focal adhesion kinase (FAK) is an important regulator of integrin signaling in adherent cells and accordingly its activity is significantly modulated during mitosis when cells detach from the extracellular matrix. During mitosis, FAK becomes heavily phosphorylated on serine residues concomitant with its inactivation and dephosphorylation on tyrosine. Little is known about the regulation of FAK activity by serine phosphorylation. Two novel sites of serine phosphorylation within the C-terminal domain of FAK have been characterized. Phosphorylation-specific antibodies directed to these sites and against two previously characterized sites of serine phosphorylation were used to study the regulated phosphorylation of FAK in unsynchronized and mitotic cells. Among the four major phosphorylation sites, designated pS1-pS4, phosphorylation of pS1 (Ser722) is unchanged in unsynchronized and mitotic cells. In contrast, pS3 and pS4 (Ser843 and Ser910) exhibit increased phosphorylation during mitosis. In vitro peptide binding experiments provide evidence that phosphorylation of pS1 (Ser722) may play a role in modulating FAK binding to the SH3 domain of the adapter protein p130Cas (Ma, 2001).

Interaction of Fak with Src kinases

Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, a tryptic phosphopeptide mapping approach was used to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. Four tyrosines, 397, 407, 576, and 577, have been identified that are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src (see Drosophila Src oncogene at 64B) in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII -- a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. Maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. These results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex (Calalb, 1995).

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. 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 (Schaller, 1999).

High-efficiency entry of the enteropathogenic bacterium Yersinia pseudotuberculosis into nonphagocytic cells is mediated by the bacterial outer membrane protein invasin. Invasin-mediated uptake requires high affinity binding of invasin to multiple beta1 chain integrin receptors on the host eukaryotic cell. Previous studies using inhibitors have indicated that high-efficiency uptake requires tyrosine kinase activity. In this paper a requirement for focal adhesion kinase (FAK) for invasin-mediated uptake is demonstrated. Overexpression of a dominant interfering form of FAK reduces the amount of bacterial entry. Specifically, the autophosphorylation site of FAK, which is a reported site of c-Src kinase binding, is required for bacterial internalization, as overexpression of a derivative lacking the autophosphorylation site has a dominant interfering effect as well. Cultured cells expressing interfering variants of Src kinase also show reduced bacterial uptake, demonstrating the involvement of a Src-family kinase in invasin-promoted uptake (Alrutz, 1998).

Grb7 is an Src homology (SH) 2-containing and pleckstrin homology domain-containing molecule, which shares significant homology with the C. elegans gene for Mig-10 involved in cell migration during embryogenesis. The SH2 domain of Grb7 can directly interact with FAK through Tyr-397, a major autophosphorylation site in vitro and in vivo. This interaction is cell adhesion-dependent, suggesting that the FAK-Grb7 complex is involved in integrin signaling. Overexpression of Grb7 enhances cell migration toward fibronectin, whereas overexpression of its SH2 domain alone inhibits cell migration. In addition, phosphorylation of FAK or p130(cas) is not affected by the expression of either Grb7 or its SH2 domain alone, suggesting that Grb7 is downstream of FAK and does not compete with Src for binding to FAK in vivo. Taken together, these results suggest that the FAK-Grb7 complex plays a role in cell migration stimulated by integrin signaling through FAK (Han, 1999).

Focal adhesion kinase (FAK) is an important mediator of integrin signaling in the regulation of cell proliferation, survival, migration, and invasion. To understand how FAK contributes to cell invasion, the regulation of matrix metalloproteinases (MMPs) by FAK was explored. v-Src-transformed cells activate a FAK-dependent mechanism that attenuates endocytosis of MT1-MMP. This in turn increases cell-surface expression of MT1-MMP and cellular degradation of extracellular matrix. Further, an interaction between FAK's second Pro-rich motif and endophilin A2's SH3 domain was identified. This interaction served as an autophosphorylation-dependent scaffold to allow Src phosphorylation of endophilin A2 at Tyr315. Tyr315 phosphorylation inhibits endophilin/dynamin interactions, and blockade of Tyr315 phosphorylation promotes endocytosis of MT1-MMP. Together, these results suggest a regulatory mechanism of cell invasion whereby FAK promotes cell-surface presentation of MT1-MMP by inhibiting endophilin A2-dependent endocytosis (Wu, 2005).

Fak interaction with phospholipase C

The nonreceptor tyrosine kinase FAK ("focal adhesion kinase") is a key mediator of integrin signaling events controlling cellular responses to the extracellular matrix, including spreading, migration, proliferation, and survival. Integrin-ligand interactions stimulate FAK tyrosine phosphorylation and activation of FAK signaling functions. Evidence is presented that the FAK autophosphorylation site Tyr-397 mediates a direct interaction with the C-terminal Src homology 2 domain of phospholipase C (PLC)-gamma1 and that this is required for both adhesion-dependent association of the two molecules and increased inositol phosphate production in mouse embryo fibroblasts. Overexpression of FAK and PLC-gamma1 in COS-7 cells increases PLC-gamma1 enzymatic activity and tyrosine phosphorylation, also dependent on FAK Tyr-397. However, FAK appears incapable of directly phosphorylating PLC-gamma1. These observations suggest a role for FAK in recruiting PLC-gamma1 to the plasma membrane at sites of cell-matrix adhesion and there promoting its enzymatic activity, possibly by releasing the repression caused by intramolecular interactions of the PLC-gamma1 Src homology domains and/or by positioning it for phosphorylation by associated Src-family kinases. These findings expand the known signaling functions of FAK and provide mechanistic insight into integrin-stimulation of PLC-gamma1 (X. Zhang, 1999).

Fak interaction with Phosphatidylinositol 3-kinase

Overexpression of focal adhesion kinase (FAK) in Chinese hamster ovary (CHO) cells promotes their migration on fibronectin. This effect is dependent on the phosphorylation of FAK at Tyr-397. This residue is known to serve as a binding site for both Src and phosphatidylinositol 3-kinase (PI3K), implying that either one or both are required for FAK to promote cell migration. The role of PI3K in FAK-promoted cell migration has been studied. The PI3K inhibitors, wortmannin and LY294002, are able to inhibit FAK-promoted migration in a dose-dependent manner. Furthermore, a FAK mutant capable of binding Src but not PI3K was generated by a substitution of Asp residue 395 with Ala. When overexpressed in CHO cells, this differential binding mutant fails to promote cell migration although its association with Src is retained. Together, these results strongly suggest that PI3K binding is required for FAK to promote cell migration and that the binding of Src and p130(Cas) to FAK may not be sufficient for this event (Reiske, 1999).

Fak and phosphatases

The tumor suppressor PTEN (see Drosophila Pten) dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. Expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein results in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation are also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, these components were examined after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity are inhibited by expression of PTEN, whereas EGF receptor autophosphorylation is unaffected. The ability of cells to spread at normal rates is partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation is enhanced as indicated by paxillin staining. The phosphatase domain of PTEN is essential for all of these functions, because PTEN with an inactive phosphatase domain does not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression does not affect c-Jun NH2-terminal kinase (JNK) or PDGF-stimulated Akt. These data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading (Gu, 1998).

The coordinated interplay of substrate adhesion and deadhesion is necessary for cell motility. Insulin-like growth factor I (IGF-I) has been found to induce 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. 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 abolishes IGF-I-induced FAK dephosphorylation, and cells expressing dominant negative SHP2 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, dominant negative SHP-2 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 (Manes, 1999).

Related adhesion focal tyrosine kinase (RAFTK) (also known as PYK2) is a cytoplasmic tyrosine kinase related to the focal adhesion kinase (FAK) p125(FAK). RAFTK is rapidly phosphorylated on tyrosine residues in response to various stimuli, such as tumor necrosis factor-alpha, changes in osmolarity, elevation in intracellular calcium concentration, lysophosphatidic acid, and bradykinin. Overexpression of RAFTK induces activation of c-Jun amino-terminal kinase (also known as stress-activated protein kinase), mitogen-activated protein kinase (MAPK), and p38 MAPK. The present studies demonstrate that RAFTK binds constitutively to the protein tyrosine phosphatase SHPTP1. In contrast to PTP1B, overexpression of wild-type SHPTP1 blocks tyrosine phosphorylation of RAFTK. The results further demonstrate that RAFTK is a direct substrate of SHPTP1 in vitro. Moreover, treatment of PC12 cells with bradykinin is associated with inhibition in tyrosine phosphorylation of RAFTK in the presence of SHPTP1. Furthermore, in contrast to the phosphatase-dead SHPTP1 C453S mutant, overexpression of wild-type SHPTP1 blocks interaction of RAFTK with the SH2-domain of c-Src and inhibits RAFTK-mediated MAPK activation. Significantly, cotransfection of RAFTK with SHPTP1 does not inhibit RAFTK-mediated c-Jun amino-terminal kinase activation. Taken together, these findings suggest that SHPTP1 plays a negative role in PYK2/RAFTK signaling by dephosphorylating RAFTK (Kumar, 1999).

The tumor suppressor PTEN is a phosphatase with sequence homology to tensin. PTEN dephosphorylates phosphatidylinositol 3,4, 5-trisphosphate (PIP3) and focal adhesion kinase (FAK), and it can inhibit cell growth, invasion, migration, and focal adhesions. Molecular interactions of PTEN and FAK were studied in glioblastoma and breast cancer cells lacking PTEN. The PTEN trapping mutant D92A bind wild-type FAK, requiring FAK autophosphorylation site Tyr397. In PTEN-mutated cancer cells, FAK phosphorylation is retained even in suspension after detachment from extracellular matrix, accompanied by enhanced PI 3-K association with FAK and sustained PI 3-K activity, PIP3 levels, and Akt phosphorylation; expression of exogenous PTEN suppresses all five properties. PTEN-mutated cells are resistant to apoptosis in suspension, but most of the cells enter apoptosis after expression of exogenous PTEN or wortmannin treatment. Moreover, overexpression of FAK in PTEN-transfected cells reverses the decreased FAK phosphorylation and PI 3-K activity, and it partially rescues PIP3 levels, Akt phosphorylation, and PTEN-induced apoptosis. These results show that FAK Tyr397 is important in PTEN interactions with FAK, that PTEN regulates FAK phosphorylation and molecular associations after detachment from matrix, and that PTEN negatively regulates the extracellular matrix-dependent PI 3-K/Akt cell survival pathway in a process that can include FAK (Tamura, 1999).

The T cell receptor (TCR)-CD3 complex and the costimulatory molecule CD28 are critical for T cell function. Both receptors utilize protein tyrosine kinases (PTKs) for the phosphorylation of various signaling molecules, a process that is critical for the function of both receptors. The PTKs of the focal adhesion family, Pyk2 and Fak, have been implicated in the signaling of TCR and CD28. Evidence for the regulation of TCR- and CD28-induced tyrosine phosphorylation of the focal adhesion PTKs by protein kinase C (PKC) is shown. Thus, treating Jurkat T cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) rapidly and strongly reverses receptor-induced tyrosine phosphorylation of the focal adhesion PTKs. In contrast, PMA did not affect TCR-induced tyrosine phosphorylation of CD3zeta or the PTKs Fyn and Zap-70. However, PMA induces a strong and rapid dephosphorylation of the linker molecule for activation of T cells. PMA fails to induce the dephosphorylation of proteins in PKC-depleted cells or in cells pretreated with the PKC inhibitor Ro-31-8220, confirming the role of PKC in mediating the PMA effect on receptor-induced protein tyrosine phosphorylation. The involvement of protein tyrosine phosphatases (PTPases) in mediating the dephosphorylation of the focal adhesion PTKs was confirmed by the failure of PMA to dephosphorylate Pyk2 in cells pretreated with the PTPase inhibitor orthovanadate. These results implicate PKC in the regulation of receptor-induced tyrosine phosphorylation of the focal adhesion PTKs in T cells. The data also suggest a role for PTPases in the PKC action (Tsuchida, 2000).

Infection with cagA-positive Helicobacter pylori (H. pylori) is associated with atrophic gastritis, peptic ulcer, and gastric adenocarcinoma. The cagA gene product CagA is translocated from H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation by Src family kinases (SFKs). Tyrosine-phosphorylated CagA binds and activates SHP-2 phosphatase and the C-terminal Src kinase (Csk) while inducing an elongated cell shape termed the 'hummingbird phenotype.' CagA reduces the level of focal adhesion kinase (FAK) tyrosine phosphorylation in gastric epithelial cells. The decrease in phosphorylated FAK is due to SHP-2-mediated dephosphorylation of FAK at the activating phosphorylation sites, not due to Csk-dependent inhibition of SFKs, which phosphorylate FAK. Coexpression of constitutively active FAK with CagA inhibits induction of the hummingbird phenotype, whereas expression of dominant-negative FAK elicits an elongated cell shape characteristic of the hummingbird phenotype. These results indicate that inhibition of FAK by SHP-2 plays a crucial role in the morphogenetic activity of CagA. Impaired cell adhesion and increased motility by CagA may be involved in the development of gastric lesions associated with cagA-positive H. pylori infection (Tsutsumi, 2006).

Table of contents

Focal adhesion kinase-like: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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