Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK.

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.     

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.     

PDGF and FGF induce focal adhesion kinase (FAK) phosphorylation at Ser-910: dissociation from Tyr-397 phosphorylation and requirement for ERK activation

A rapid increase in the tyrosine phosphorylation of focal adhesion kinase (FAK) has been extensively documented in cells stimulated by multiple signaling molecules, but very little is known about the regulation of FAK phosphorylation at serine residues. Stimulation of Swiss 3T3 cells with platelet-derived growth factor (PDGF) promoted a striking increase in the phosphorylation of FAK at Ser-910, as revealed by site-specific antibodies that recognized the phosphorylated state of this residue. FAK phosphorylation at Ser-910 could be distinguished from that at Tyr-397 in terms of dose-response relationships and kinetics. Furthermore, the selective phosphoinositide 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 abrogated FAK phosphorylation at Tyr-397 but did not interfere with PDGF-induced FAK phosphorylation at Ser-910. Conversely, treatment with U0126, a potent inhibitor of MEK-mediated ERK activation, prevented FAK phosphorylation at Ser-910 induced by PDGF but did not interfere with PDGF-induced FAK phosphorylation at Tyr-397. These results were extended using growth factors that either stimulate, fibroblast growth factor (FGF), or do not stimulate (insulin) the ERK pathway activation in Swiss 3T3 cells. FGF but not insulin promoted a striking ERK-dependent phosphorylation of FAK at Ser-910. Our results indicate that FAK phosphorylation at Tyr-397 and FAK phosphorylation at Ser-910 are induced in response to PDGF stimulation through different signaling pathways, namely PI 3-kinase and ERK, respectively.     

Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets

We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125FAK and that pp125FAK molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays. pp125FAK was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIb-IIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP IIb-IIIa. Fibrinogen binding to GP IIb-IIIa was not sufficient to induce pp125FAK phosphorylation because pp125FAK was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125FAK is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP IIb-IIIa. The induction of tyrosine phosphorylation of pp125FAK was inhibited in thrombin- and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125FAK in vivo and stimulation of the phosphorylation of pp125FAK in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125FAK is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.     

Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions

The integrin family of heterodimeric cell surface receptors play critical roles in multiple biological processes by mediating cellular adhesion to the extracellular matrix (ECM). Adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation and elevation of [Ca2+]i. The Focal Adhesion Kinase (FAK or pp125FAK), a protein tyrosine kinase that colocalizes with integrins in cellular focal adhesions, is a prime candidate for a mediator of integrin signaling events. Here we report an analysis of the domain structure of FAK in which we have identified a contiguous stretch of 159 amino acids within the COOH terminus essential for correct subcellular localization. When placed in the context of an unrelated cytosolic protein, this Focal Adhesion Targeting (FAT) sequence functions to efficiently mediate the focal adhesion localization of this fusion protein. Furthermore, this analysis suggests that pp125FAK cannot be activated oncogenically by mutation. This result could be explained if pp125FK either exhibits a narrow substrate specificity or is diametrically opposed by cellular phosphatases or other cellular processes.     

FAK phosphorylation at Ser-843 inhibits Tyr-397 phosphorylation, cell spreading and migration

Multiple stimuli promote the tyrosine phosphorylation and activation of focal adhesion kinase (FAK), which ultimately facilitates migration. Little is known about the effect of adhesion-dependent signals and cytoskeleton organization on the regulation of FAK phosphorylation at serine sites, or about the role of FAK serine phosphorylation in cell migration. Here, we show that FAK phosphorylation at Ser-843 is strikingly increased when adherent cells are removed from the substratum and held in suspension or by treatment of adherent cells with cytochalasin D, conditions that disrupt the F-actin cytoskeleton and promote focal adhesion disassembly. Notably, the increase in Ser-843 phosphorylation was accompanied by a concomitant sharp decrease in Tyr-397 phosphorylation. To further examine the cause-effect relationship between these two phosphorylation sites we generated Ser-843 phosphorylation-deficient and phosphorylation-mimicking FAK mutants. We found that mutation of Ser-843 to aspartic acid (FAK[S843D]) markedly decreased FAK Tyr-397 phosphorylation in integrin-stimulated cells. While the migratory defect of FAK-deficient fibroblasts was rescued by stable re-expression of WT FAK or FAK[S843A], stable re-expression of FAK[S843D] failed to restore the ability of the cells to migrate into the denuded area of a wound. Our results indicate that increased FAK phosphorylation at Ser-843 represses FAK phosphorylation at Tyr-397, thus suggesting a mechanism of cross-talk between these phosphorylation sites that could regulate FAK-mediated cell shape and migration.     

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.     

FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion

 Cell migration is a highly complex process that requires the coordinated formation of membrane protrusion and focal adhesions (FAs). Focal adhesion kinase (FAK), a major signaling component of FAs, is involved in the disassembly process of FAs through phosphorylation and dephosphorylation of its tyrosine residues, but the role of such phosphorylations in nascent FA formation and turnover near the cell front and in cell protrusion is less well understood. In the present study, we demonstrate that, depending on the phosphorylation status of Tyr-925 residue, FAK modulates cell migration via two specific mechanisms. FAK^−/− mouse embryonic fibroblasts (MEFs) expressing nonphosphorylatable Y925F-FAK show increased interactions between FAK and unphosphorylated paxillin, which lead to FA stabilization and thus decreased FA turnover and reduced cell migration. Conversely, MEFs expressing phosphomimetic Y925E-FAK display unchanged FA disassembly rates, show increase in phosphorylated paxillin in FAs, and exhibit increased formation of nascent FAs at the cell leading edges. Moreover, Y925E-FAK cells present enhanced cell protrusion together with activation of the p130^CAS/Dock180/Rac1 signaling pathway. Together, our results demonstrate that phosphorylation of FAK at Tyr-925 is required for FAK-mediated cell migration and cell protrusion.     

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

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

Beta2-integrin, LFA-1, and TCR/CD3 synergistically induce tyrosine phosphorylation of focal adhesion kinase (pp125(FAK)) in PHA-activated T cells

Complete T cell activation requires not only a first signal via TCR/CD3 engagement but also a costimulatory signal through accessory receptors such as CD2, CD28, or integrins. Focal adhesion kinase, pp125(FAK) (FAK), was previously shown to be localized in focal adhesions in fibroblasts and to be involved in integrin-mediated cellular activation. Although signaling through beta1- or beta3-integrins induces tyrosine phosphorylation of FAK, there has been no evidence that activation of T cells through the beta2-integrin, LFA-1, involves FAK. We report here that crosslinking of LFA-1 induces tyrosine phosphorylation of FAK in PHA-activated T cells. Moreover, cocrosslinking with anti-LFA-1 mAb and suboptimal concentration of anti-CD3 mAb markedly increases tyrosine phosphorylation of FAK in an antibody-concentration-dependent and time-kinetics-dependent manner compared with stimulation through CD3 alone, which correlates well with enhanced proliferation of PHA-activated T cells. Furthermore, LFA-1beta costimulation with CD3 induces tyrosine phosphorylation of Syk associated with FAK. These results indicate, for the first time, that signals mediated by LFA-1 can regulate FAK, suggesting that LFA-1-mediated T cell costimulation may be involved in T cell activation at least partially through FAK.     

Growth hormone stimulates tyrosine phosphorylation of focal adhesion kinase (p125(FAK)) and actin stress fiber formation in human osteoblast-like cells, Saos2.

Bone is one of the essential target tissues of growth hormone (GH). In bone remodeling, cell-matrix attachment is important where focal adhesion kinase (FAK) is involved. FAK plays a central role in determining the shape and motility of cells in response to the extracellular matrix stimuli. In the present study, we have demonstrated that GH stimulated tyrosine phosphorylation of FAK in human osteoblast-like cells, Saos2. Moreover, GH rapidly enhanced the formation of actin stress fibers. In Saos2, Jak2 was tyrosine phosphorylated by GH stimulation, and AG490, a Jak2 specific inhibitor, inhibited GH-induced tyrosine phosphorylation of FAK and actin stress fiber reorganization. These results suggest that GH activates FAK via Jak2, and stimulates the formation of actin stress fibers in Saos2. Activation of FAK and actin stress fiber formation induced by GH seem to be important for the physiological role of osteoblast.     

Possible involvement of focal adhesion kinase,p125FAK,in osteoclastic bone resorption

Involvement of tyrosine phosphorylation in osteoclastic bone resorption was examined using osteoclast-like multinucleated cells prepared from co-cultures of mouse osteoblastic cells and bone marrow cells in the presence of 1α,25-dihydroxyvitamin D₃. When osteoclast-like cells were plated on culture dishes in the presence of 10% fetal bovine serum, they were sharply stained in their peripheral region by anti-phosphotyrosine antibody. Western blot analysis revealed that 115-to 130-kD proteins were tyrosine-phosphorylated in osteoclast-like cells. Using immunoprecipitation and immunoblotting, one of the proteins with 115–130 kD was identified as focal adhesion kinase (p125^FAK), a tyrosine kinase, which is localized in focal adhesions. Immunostaining with anti-p 125^FAK antibody revealed that p125^FAK was mainly localized at the periphery of osteoclast-like cells. Herbimycin A, a tyrosine kinase inhibitor, not only suppressed tyrosine phosphorylation of p125^FAK but also changed the intracellular localization of p125^FAK and disrupted a ringed structure of F-actin-containing podosomes in osteoclast-like cells. Antisense oligodeoxynucleotides to p125^FAK inhibited dentine resorption by osteoclast-like cells, whereas sense oligodeoxynucleotides did not. These results suggest that p125^FAK is involved in osteoclastic bone resorption and that tyrosine phosphorylation of p125^FAK is critical for regulating osteoclast function.     

Phosphorylation of focal adhesion kinase tyrosine 397 critically mediates gastrin-releasing peptide's morphogenic properties

We have proposed that gastrin-releasing peptide (GRP) and its receptor (GRP-R) are morphogens that when aberrantly re-expressed in colon cancer promote tumor cell differentiation and retard metastasis. Because circumstantial evidence suggested that these properties were mediated via focal adhesion kinase (FAK), the purpose of this study was to elucidate the role of GRP-induced activation of this enzyme on properties fundamental to metastasis including cell attachment, motility, and deformability. To do this, we studied 293 cells, a non-malignant epithelial cell line that we show expresses GRP and GRPR. To dissect out the role of FAK, 293 cells were modified to inducibly express the dominant negative enzyme FAK-related non-kinase (FRNK) under control of a Tet-On (i.e., doxycycline-sensitive) promoter. Under serum-free conditions, GRP acting in an autocrine manner caused FAK to be phosphorylated at Y397; and this could be completely inhibited either by incubating with the specific GRP-R antagonist D-Phe(6)(bombesin) methyl ester, or by upregulating FRNK using doxycycline. To measure cell attachment, we designed a cone-plate viscometer that recorded the shear stress required to detach cells from their underlying matrix. To assess motility, confluent cells were wounded and behavior assessed by time-lapse photography. To measure deformability, we recorded the ability of cells to be completely drawn into a micropipette <50% the size of the non-deformed cell. Control 293 cells adhered more avidly to their underlying matrix, rapidly remodeled wounded tissues without any increase in overall proliferation, and were less distensible than cells treated with antagonist or doxycycline. Thus, these findings suggest that expression of GRP/GRPR in cancer inhibits metastasis by enhancing cell attachment to the matrix, regulating motility in the context of remodeling, and decreasing deformability.     

A lymphoma protein with an in vitro site of tyrosine phosphorylation homologous to that in pp60src

The major in vitro substrate for a tyrosine protein kinase in the particulate fraction of the lymphoma cell line LSTRA is a protein of molecular weight of 58,000 (pp58) (Casnellie, J. E. Harrison, M. L., Pike, L. J., Hellstrom, K. E., and Krebs, E. G. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 282-286). In order to determine if this protein was related to pp60src, the transformation-specific protein from Rous sarcoma virus, partial proteolysis maps of in vitro 32P-labeled pp58 and pp60src were prepared using Staphylococcus aureus V8 protease and papain. The maps were clearly different, indicating the pp58 is distinct from pp60src. However characterization of the tryptic fragment containing the single site of in vitro tyrosine phosphorylation in pp58 has revealed that the amino acid sequence around this site is extremely homologous to, if not identical with the sequence around the site of tyrosine phosphorylation in pp60src.     

A role for phosphoinositides in tyrosine phosphorylation of p125 focal adhesion kinase in rat pancreatic acini

Previous studies have shown that different agonists increase tyrosine phosphorylation of the focal adhesion related proteins p125(FAK), p130(Cas), and paxillin in different cell types and that tyrosine phosphorylation depends on the integrity of the actin cytoskeleton. Because phosphoinositides are important for the maintenance of the cytoskeleton, the role of phosphoinositides in the tyrosine phosphorylation of these proteins in response to occupancy of m3 muscarinic and CCK(A) receptors has been investigated in pancreatic acini. Addition of carbachol or CCK-8 to pancreatic acini resulted in rapid increases in the tyrosine phosphorylation of p125(FAK), p130(Cas), and paxillin. Pretreatment of pancreatic acini with LY294002 or wortmannin resulted in a concentration-dependent inhibition of tyrosine phosphorylation of p125(FAK), p130(Cas), and paxillin stimulated by carbachol or CCK-8. Carbachol- or CCK-8-stimulated tyrosine phosphorylation of these proteins was not inhibited by rapamycin, PD 98059 or SB 203580, and thus it was dissociated from the activation of p70 S6 or MAP kinases. These results indicate that m3 muscarinic and CCK(A) receptor-mediated increase in p125(FAK), p130(Cas), and paxillin tyrosine phosphorylation in pancreatic acini depends on the ability of these cells to synthesise phosphoinositides.     

p56(lck) Controls phosphorylation of filamin (ABP-280) and regulates focal adhesion kinase (pp125(FAK))

Transformation of cells by src -like kinases leads to altered cell morphology associated with the disassembly of focal contacts and concomitant increase in tyrosine phosphorylation of pp125(FAK) x p56(lck) is a lymphocyte-specific member of the src family of protein tyrosine kinases that associates with cell surface glycoproteins such as CD4 and CD8. It phosphorylates and activates pp125(FAK) and increases its autokinase activity, thus pretreatment of pp125(FAK) with protein kinase C (PKC) markedly attenuates its phosphorylation and activation, suggesting a potential regulatory pathway of pp125(FAK) activation in focal contacts. p56(lck) further phosphorylates and activates actin binding protein (ABP-280; filamin) and controls its association with cell surface receptors such as beta-2 integrins, actin filament cross-linking, and possibly lipid membrane insertion.     

Distribution and Y397 phosphorylation of focal adhesion kinase on follicular development in the mouse ovary

Several protein tyrosine kinases (PTKs) are identified as follicle survival factors that suppress apoptosis in granulosa cells. Focal adhesion kinase (FAK/PTK2) interacts with numerous signaling partners and is important for cell adhesion, survival and other vital processes in which FAK autophosphorylation at Y397 (pY397 FAK) is critical for activating signaling pathways. Despite its important roles in apoptosis, the expression and function of FAK in the ovaries remain unknown. Here, we describe FAK expression, including pY397 FAK, in normal healthy mouse ovaries and its association with follicular development and/or atresia. Normal healthy mouse ovaries were used for western blot (n > 60) and immunohistochemical (n > 180) analyses. Western blot results in immature and mature mice revealed that total FAK and pY397 FAK were highly expressed in the ovary and immunohistochemistry results in 3-week-old mice showed they were localized to granulosa cells of ovarian follicles, especially preantral follicles. In 3-week-old mice treated with 5 IU pregnant mare serum gonadotropin (for obtaining homogenous populations of growing or atretic follicles), western blotting revealed that follicular atresia progression involved decreased phosphorylation of Y397 at 72 and 96 h after treatment, particularly in granulosa cells of atretic follicles, as shown by immunohistochemistry results at 72 h after treatment. Moreover, immunostaining patterns of FAK and cleaved caspase-3 were negatively correlated in serial sections of 3-week-old mouse ovaries. These results suggest that FAK is most active in ovarian follicle granulosa cells and that its phosphorylation at Y397 is histologically meaningful in follicular development in normal healthy ovaries.     

Cell density regulates tyrosine phosphorylation and localization of focal adhesion kinase

We have investigated tyrosine phosphorylation of cellular proteins at different cell densities. A tyrosine-phosphorylated protein of 120 kDa was detected when cells were plated sparsely. The phosphorylation level of the protein gradually declined as the cells were plated at higher densities or when the sparsely plated cells approached confluence. This density-dependent phosphorylation was also associated with cell attachment since it disappeared when the cells were detached from plates or when the cells were cultured in suspension. Immunoblotting and immunoprecipitation analyses with specific antibodies revealed that the 120-kDa protein corresponded to the focal adhesion kinase (FAK) and the protein level of FAK was not altered at different cell densities. In vitro kinase assays demonstrated that the kinase activity of FAK decreased with increasing cell densities in parallel with its dephosphorylation. Cell density also affects localization of FAK associated with rearrangement of actin stress fibers. At low cell densities, FAK and actin stress fiber are distributed around the periphery of cells while they are dispersed over the ventral surface in high-density cells. Finally, the density-regulated tyrosine phosphorylation and localization of FAK appeared to be mediated by an insoluble factor produced by high-density cells.