Fibronectin fragment activation of proline-rich tyrosine kinase PYK2 mediates integrin signals regulating collagenase-3 expression by human chondrocytes through a protein kinase C-dependent pathway

Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the alpha5beta1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the alpha5beta1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCdelta inhibitor rottlerin. Furthermore, PKCdelta translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after cotransfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves FN-f stimulation of the alpha5beta1 integrin and activation of the nonreceptor tyrosine kinase PYK2 by PKC, most likely PKCdelta     

Hyperosmotic stress induces rapid focal adhesion kinase phosphorylation at tyrosines 397 and 577. Role of Src family kinases and Rho family GTPases

Hyperosmotic stress induced by treatment of Swiss 3T3 cells with the non-permeant solutes sucrose or sorbitol, rapidly and robustly stimulated endogenous focal adhesion kinase (FAK) phosphorylation at Tyr-397, the major autophosphorylation site, and at Tyr-577, within the kinase activation loop. Hyperosmotic stress-stimulated FAK phosphorylation at Tyr-397 occurred via a Src-independent pathway, whereas Tyr-577 phosphorylation was completely blocked by exposure to the Src family kinase inhibitor PP-2. Inhibition of p38 MAP kinase or phosphatidylinositol 3-kinases did not prevent FAK phosphorylation stimulated by hyperosmotic stress. Overexpression of N17 RhoA did not reduce hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts and treatment with the Rho-associated kinase inhibitor Y-27632 did not prevent FAK translocation and tyrosine phosphorylation in response to hyperosmotic stress. Overexpression of N17 Rac only slightly altered the hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts. In contrast, overexpression of the N17 mutant of Cdc42 disrupted hyperosmotic stress-stimulated FAK Tyr-397 localization to focal contacts. Additionally, treatment of cells with Clostridium difficile toxin B potently inhibited hyperosmotic stress-induced FAK tyrosine phosphorylation. Furthermore, FAK null fibroblasts compared with their FAK containing controls show markedly increased sensitivity, manifest by subsequent apoptosis, to sustained hyperosmotic stress. Our results indicate that FAK plays a fundamental role in protecting cells from hyperosmotic stress, and that the pathway(s) that mediates FAK autophosphorylation at Tyr-397 in response to osmotic stress can be distinguished from the pathways utilized by many other stimuli, including neuropeptides and bioactive lipids (Rho- and Rho-associated kinase-dependent), tyrosine kinase receptor agonists (phosphatidylinositol 3-kinase-dependent), and integrins (Src-dependent).     

Interactions between two cytoskeleton-associated tyrosine kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase

The calcium-dependent tyrosine kinase (CADTK), also known as Pyk2/RAFTK/CAKbeta/FAK2, is a cytoskeleton-associated tyrosine kinase. We compared CADTK regulation with that of the highly homologous focal adhesion tyrosine kinase (FAK). First, we generated site-specific CADTK mutants. Mutation of Tyr402 eliminated autophosphorylation and significantly decreased kinase activity. Mutation of Tyr881, a putative Src kinase phosphorylation site predicted to bind Grb2, had little effect on CADTK regulation. Src family tyrosine kinases resulted in CADTK tyrosine phosphorylation even when co-expressed with the Tyr402/Tyr881 double mutant, suggesting that Src/Fyn etc. phosphorylate additional tyrosine residues. Interestingly, CADTK tyrosine-phosphorylated FAK when both were transiently expressed, but FAK did not phosphorylate CADTK. Biochemical experiments confirmed direct CADTK phosphorylation of FAK. This phosphorylation utilized tyrosine residues other than Tyr397, Tyr925, or Tyr576/Tyr577, suggesting that new SH2-binding sites might be created by CADTK-dependent FAK phosphorylation. Last, expression of the CADTK carboxyl terminus (CRNK) abolished CADTK but not FAK autophosphorylation. In contrast, FAK carboxyl terminus overexpression inhibited both FAK and CADTK autophosphorylation, suggesting that a FAK-dependent cytoskeletal function may be necessary for CADTK activation. Thus, CADTK and FAK, which both bind to some, but not necessarily the same, cytoskeletal elements, may be involved in coordinate regulation of cytoskeletal structure and signaling.     

Dual role of Fyn in the regulation of FAK+6,7 by cannabinoids in hippocampus

In hippocampus endocannabinoids modulate synaptic function and plasticity and increase tyrosine phosphorylation of several proteins, including focal adhesion kinase (FAK). Autophosphorylation of FAK on Tyr-397 is generally a critical step for its activation, allowing the recruitment of Src family kinases, and phosphorylation of FAK and associated proteins. We have examined the mechanisms of the regulation of FAK by cannabinoids in rat and mouse hippocampal slices. Anandamide and 2-arachidonoylglycerol, two endocannabinoids, and Delta9-tetrahydrocannabinol, stimulated tyrosine phosphorylation of FAK+6,7, a neuronal splice isoform of FAK, on several residues including Tyr-397. Cannabinoids increased phosphorylation of p130-Cas, a protein associated with FAK, but had no effect on PYK2, a tyrosine kinase related to FAK and enriched in hippocampus. Pharmacological experiments and the use of knockout mice demonstrated that the effects of cannabinoids were mediated through CB1 receptors. These effects were sensitive to manipulation of cAMP-dependent protein kinase, suggesting that they were mediated by inhibition of a cAMP pathway. PP2, an Src family kinase inhibitor, prevented the effects of cannabinoids on p130-Cas and on FAK+6,7 tyrosines 577 and 925, but not 397, indicating that FAK autophosphorylation was upstream of Src family kinases in response to CB1-R stimulation. Endocannabinoids increased the association of Fyn, but not Src, with FAK+6,7. In hippocampal slices from Fyn -/- mice, the levels of p130-Cas were increased, and the effects of endocannabinoids on tyrosine phosphorylation, including of Tyr-397, were completely abolished. These results demonstrate the specific functional association of Fyn with FAK+6,7 in a pathway regulated by endocannabinoids, in which Fyn may play roles dependent and independent of its catalytic activity.     

Depolarization activates ERK and proline-rich tyrosine kinase 2 (PYK2) independently in different cellular compartments in hippocampal slices

In the hippocampus, extracellular signal-regulated kinase (ERK) and the non-receptor protein proline-rich tyrosine kinase 2 (PYK2) are activated by depolarization and involved in synaptic plasticity. Both are also activated under pathological conditions following ischemia, convulsions, or electroconvulsive shock. Although in non-neuronal cells PYK2 activates ERK through the recruitment of Src-family kinases (SFKs), the link between these pathways in the hippocampus is not known. We addressed this question using K(+)-depolarized rat hippocampal slices. Depolarization increased the phosphorylation of PYK2, SFKs, and ERK. These effects resulted from Ca(2+) influx through voltage-gated Ca(2+) channels and were diminished by GF109203X, a protein kinase C inhibitor. Inhibition of SFKs with PP2 decreased PYK2 tyrosine phosphorylation dramatically, but not its autophosphorylation on Tyr-402. Moreover, PYK2 autophosphorylation and total tyrosine phosphorylation were profoundly altered in fyn-/- mice, revealing an important functional relationship between Fyn and PYK2 in the hippocampus. In contrast, ERK activation was unaltered by PP2, Fyn knock-out, or LY294002, a phosphatidyl-inositol-3-kinase inhibitor. ERK activation was prevented by MEK inhibitors that had no effect on PYK2. Immunofluorescence of hippocampal slices showed that PYK2 and ERK were activated in distinct cellular compartments in somatodendritic regions and nerve terminals, respectively, with virtually no overlap. Activation of ERK was critical for the rephosphorylation of a synaptic vesicle protein, synapsin I, following depolarization, underlining its functional importance in nerve terminals. Thus, in hippocampal slices, in contrast to cell lines, depolarization-induced activation of non-receptor tyrosine kinases and ERK occurs independently in distinct cellular compartments in which they appear to have different functional roles.     

Phosphospecific site tyrosine phosphorylation of p125FAK and proline-rich kinase 2 is differentially regulated by cholecystokinin receptor type A activation in pancreatic acini

The focal adhesion kinases, p125FAK and proline-rich kinase 2 (PYK2), are involved in numerous processes as adhesion, cytoskeletal changes, and growth. These kinases have 45% homology and share three tyrosine phosphorylation (TyrP) sites. Little information exists on the ability of stimulants to cause TyrP of each kinase site and the cellular mechanism involved. We explored the ability of the neurotransmitter/hormone, CCK, to stimulate TyrP at each site. In rat pancreatic acini, CCK stimulated TyrP at each site in both kinases. TyrP was rapid except for pY397FAK. The magnitude of TyrP differed with the different FAK and PYK2 sites. The CCK dose-response curve for TyrP for sites in each kinase was similar. CCK-JMV, an agonist of the high affinity receptor state and antagonist of the low affinity receptor state, was less efficacious than CCK at each FAK/PYK2 site and inhibited CCK maximal stimulation. Thapsigargin decreased CCK-stimulated TyrP of pY402PYK2 and pY925FAK but not the other sites. GF109203X reduced TyrP of only the PYK2 sites, pY402 and pY580. GF109203X with thapsigargin decreased TyrP of pY402PYK2 and the three FAK sites more than either inhibitor alone. Basal TyrP of pY397FAK was greater than other sites. These results demonstrate that CCK stimulates tyrosine phosphorylation of each of the three homologous phosphorylation sites in FAK and PYK2. However, CCK-stimulated TyrP at these sites differs in kinetics, magnitude, and participation of the high/low affinity receptor states and by protein kinase C and [Ca2+]i. These results show that phosphorylation of these different sites is differentially regulated and involves different intracellular mechanisms in the same cell.     

Activation of focal adhesion kinase by protein kinase C epsilon in neonatal rat ventricular myocytes

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase critical for both cardiomyocyte survival and sarcomeric assembly during endothelin (ET)-induced cardiomyocyte hypertrophy. ET-induced FAK activation requires upstream activation of one or more isoenzymes of protein kinase C (PKC). Therefore, with the use of replication-defective adenoviruses (Adv) to overexpress constitutively active (ca) and dominant negative (dn) mutants of PKCs, we examined which PKC isoenzymes are necessary for FAK activation and which downstream signaling components are involved. FAK activation was assessed by Western blot analysis with an antibody specific for FAK autophosphorylated at Y397 (Y397pFAK). ET (10 nmol/l; 2-30 min) resulted in the time-dependent activation of FAK which was inhibited by chelerythrine (5 micromol/l; 1 h pretreatment). Adv-caPKC epsilon, but not Adv-caPKC delta, activated FAK compared with a control Adv encoding beta-galactosidase. Conversely, Adv-dnPKC epsilon inhibited ET-induced FAK activation. Y-27632 (10 micromol/l; 1 h pretreatment), an inhibitor of Rho-associated coiled-coil-containing protein kinases (ROCK), prevented ET- and caPKC epsilon-induced FAK activation as well as cofilin phosphorylation. Pretreatment with cytochalasin D (1 micromol/l, 1 h pretreatment) also inhibited ET-induced Y397pFAK and cofilin phosphorylation and caPKC epsilon-induced Y397pFAK. Neither inhibitor, however, interfered with ET-induced ERK1/2 activation. Finally, PP2 (50 micromol/l; 1 h pretreatment), a highly selective Src inhibitor, did not alter basal or ET-induced Y397pFAK. PP2 did, however, reduce basal and ET-induced phosphorylation of other sites on FAK, namely, Y576, Y577, Y861, and Y925. We conclude that the ET-induced signal transduction pathway resulting in downstream Y397pFAK is partially dependent on PKC epsilon, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src.     

Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK.

A number of cellular processes, such as proliferation, differentiation, and transformation, are regulated by cell-extracellular matrix interactions. Previous studies have identified a novel tyrosine kinase, the focal adhesion kinase p125FAK, as a component of cell adhesion plaques. p125FAK was identified as a 125-kDa tyrosine-phosphorylated protein in cells transformed by the v-src oncogene. p125FAK is an intracellular protein composed of three domains: a central domain with homology to protein tyrosine kinases, flanked by two noncatalytic domains of 400 amino acids which bear no significant homology to previously cloned proteins. p125FAK is believed to play an important regulatory role in cell adhesion because it localizes to cell adhesion plaques and because its phosphorylation on tyrosine residues is regulated by binding of cell surface integrins to the extracellular matrix. Recent studies have shown that Src, through its SH2 domain, stably associates with pp125FAK and that this association prevents dephosphorylation of pp125FAK in vitro by protein tyrosine phosphatases. In this report, we identify Tyr-397 as the primary in vivo and in vitro site of p125FAK tyrosine phosphorylation and association with Src. Substituting phenylalanine for tyrosine at position 397 significantly reduces p125FAK tyrosine phosphorylation and association with Src but does not abolish p125FAK kinase activity. In addition, p125FAK kinase is able to trans-phosphorylate Tyr-397 in vitro in a kinase-deficient p125FAK variant. Phosphorylation of Tyr-397 provides a site [Y(P)AEI] that fits the consensus sequence for the binding of Src.     

Src and focal adhesion kinase mediate mechanical strain-induced proliferation and ERK1/2 phosphorylation in human H441 pulmonary epithelial cells

Pulmonary epithelial cells are exposed to repetitive deformation during physiological breathing and mechanical ventilation. Such deformation may influence pulmonary growth, development, and barotrauma. Although deformation stimulates proliferation and activates extracellular signal-regulated kinases (ERK1/2) in human pulmonary epithelial H441 cells, the upstream mechanosensors that induce ERK activation are poorly understood. We investigated whether c-Src or focal adhesion kinase (FAK) mediates cyclic mechanical strain-induced ERK1/2 activation and proliferation in human pulmonary epithelial (NCI-H441) cells. The H441 and A549 cells were grown on collagen I-precoated membranes and were subjected to an average 10% cyclic mechanical strain at 20 cycles/min. Cyclic strain activated Src within 2 min by increasing phosphorylation at Tyr⁴¹⁸, followed by rapid phosphorylation of FAK at Tyr³⁹⁷ and Tyr⁵⁷⁶ and ERK1/2 at Thr²⁰²/Tyr²⁰⁴ (n = 5, P < 0.05). Twenty-four (A549 cells) and 24–72 h (H441 cells) of cyclic mechanical strain increased cell numbers compared with static culture. Twenty-four hours of cyclic strain also increased H441 FAK, Src, and ERK phosphorylation without affecting total FAK, Src, or ERK protein. The mitogenic effect was blocked by Src (10 µmol/l PP2 or short interfering RNA targeted to Src) or MEK (50 µmol/l PD-98059) inhibition. PP2 also blocked strain-induced phosphorylation of FAK-Tyr⁵⁷⁶ and ERK-Thr²⁰²/Tyr²⁰⁴ but not FAK-Tyr³⁹⁷. Reducing FAK by FAK-targeted short interfering RNA blocked mechanical strain-induced mitogenicity and significantly attenuated strain-induced ERK activation but not strain-induced Src phosphorylation. Together, these results suggest that repetitive mechanical deformation induced by ventilation supports pulmonary epithelial proliferation by a pathway involving Src, FAK, and then ERK signaling. extracellular signal-regulated kinase; mitogenic; signaling     

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.     

Repetitive deformation activates focal adhesion kinase and ERK mitogenic signals in human Caco-2 intestinal epithelial cells through Src and Rac1

Intestinal epithelial cells are subject to repetitive deformation during peristalsis and villous motility, whereas the mucosa atrophies during sepsis or ileus when such stimuli are abnormal. Such repetitive deformation stimulates intestinal epithelial proliferation via focal adhesion kinase (FAK) and extracellular signal-regulated kinases (ERK). However, the upstream mediators of these effects are unknown. We investigated whether Src and Rac1 mediate deformation-induced FAK and ERK phosphorylation and proliferation in human Caco-2 and rat IEC-6 intestinal epithelial cells. Cells cultured on collagen-I were subjected to an average 10% cyclic strain at 10 cycles/min. Cyclic strain activated Rac1 and induced Rac1 translocation to cell membranes. Mechanical strain also induced rapid sustained phosphorylation of c-Src at Tyr(418), Rac1 at Ser(71), FAK at Tyr(397) and Tyr(576), and ERK1/2 at Thr(202)/Tyr(204). The mitogenic effect of cyclic strain was blocked by inhibition of Src (PP2 or short interfering RNA) or Rac1 (NSC23766). Src or Rac1 inhibition also prevented strain-induced FAK phosphorylation at Tyr(576) and ERK phosphorylation but not FAK phosphorylation at Tyr(397). Reducing FAK using short interfering RNA blocked strain-induced mitogenicity and attenuated ERK phosphorylation but not Src or Rac1 phosphorylation. Src inhibition blocked strain-induced Rac1 phosphorylation, but Rac inhibition did not alter Src phosphorylation. Transfection of a two-tyrosine phosphorylation-deficient FAK mutant Y576F/Y577F prevented activation of cotransfected myc-ERK2 by cyclic strain. Repetitive deformation induced by peristalsis or villus motility may support the gut mucosa by a pathway involving Src, Rac1, FAK, and ERK. This pathway may present important targets for interventions to prevent mucosal atrophy during prolonged ileus or fasting.     

Preferential phosphorylation of focal adhesion kinase tyrosine 861 is critical for mediating an anti-apoptotic response to hyperosmotic stress

The results presented here demonstrate that focal adhesion kinase (FAK) Tyr-861 is the predominant tyrosine phosphorylation site stimulated by hyperosmotic stress in a variety of cell types, including epithelial cell lines (ileum-derived IEC-18, colon-derived Caco2, and stomach-derived NCI-N87), FAK null fibroblasts re-expressing FAK, and Src family kinase triple null fibroblasts (SYF cells) in which c-Src has been restored (YF cells). We show that hyperosmotic stress-stimulated FAK phosphorylation in epithelial cells is inhibited by Src family kinase inhibitors PP2 and SU6656 and that it does not occur in SYF cells. Unexpectedly, hyperosmotic stress-induced phosphorylation of FAK at Tyr-397, Tyr-576, and most dramatically at Tyr-861 was completely insensitive to the F-actin-disrupting agents, latrunculin A and cytochalasin D. Finally, we show that in FAK null cells exposed to hyperosmotic stress or growth factor withdrawal, re-expression of wild type FAK restored cell survival, whereas re-expression of FAK mutated from tyrosine to phenylalanine at position 861 (FAKY861F) did not. Our results indicate that FAK Tyr-861 phosphorylation is required for mammalian cell survival of hyperosmotic stress. Furthermore, the results suggest that FAK is an upstream regulator (rather than downstream effector) of F-actin reorganization in response to hyperosmotic stress. We propose that FAK/c-Src bipartite enzyme is a sensor of cytoplasmic shrinkage, and that the phosphorylation on FAK Tyr-861 by Src and subsequent reorganization of F-actin can initiate an anti-apoptotic signaling pathway that protects cells from hyperosmotic stress.     

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.     

Phosphospecific antibodies reveal focal adhesion kinase activation loop phosphorylation in nascent and mature focal adhesions and requirement for the autophosphorylation site.

Focal adhesion kinase (FAK) is a key signaling molecule regulating cellular responses to integrin-mediated adhesion. Integrin engagement promotes FAK phosphorylation at multiple sites to achieve full FAK activation. Phosphorylation of FAK Tyr-397 creates a binding site for Src-family kinases, and phosphorylation of FAK Tyr-576/Tyr-577 in the kinase domain activation loop enhances catalytic activity. Using novel phosphospecific antibody reagents, we show that FAK activation loop phosphorylation is significantly elevated in cells expressing activated Src and is an early event following cell adhesion to fibronectin. In both cases, this regulation is largely dependent on Tyr-397. We also show that the FAK activation loop tyrosines are required for maximal Tyr-397 phosphorylation. Finally, immunostaining analyses revealed that tyrosine-phosphorylated forms of FAK are present in both newly forming and mature focal adhesions. Our findings support a model for reciprocal activation of FAK and Src-family kinases and suggest that FAK/Src signaling may occur during both focal adhesion assembly and turnover.     

Differential regulation of Pyk2 phosphorylation at Tyr-402 and Tyr-580 in intestinal epithelial cells: roles of calcium, Src, Rho kinase, and the cytoskeleton

The calcium-dependent proline-rich tyrosine kinase Pyk2 is activated by tyrosine phosphorylation, associates with focal adhesion proteins, and has been linked to proliferative and migratory responses in a variety of mesenchymal and epithelial cell types. Full Pyk2 activation requires phosphorylation at functionally distinct sites, including autophosphorylation site Tyr-402 and catalytic domain site Tyr-580, though the mechanisms involved are unclear. The pathways mediating Pyk2 phosphorylation at Tyr-402 and Tyr-580 were therefore investigated. Both sites were rapidly and transiently phosphorylated following cell stimulation by Ang II or LPA. However, only Tyr-580 phosphorylation was rapidly enhanced by intracellular Ca(2+) release, or inhibited by Ca(2+) depletion. Conversely, Tyr-402 phosphorylation was highly sensitive to inhibition of actin stress fibers, or of Rho kinase (ROK), an upstream regulator of stress fiber assembly. Ang II also induced a delayed (30-60 min) secondary phosphorylation peak occurring at Tyr-402 alone. Unlike the homologous focal adhesion kinase (FAK), Pyk2 phosphorylation was sensitive neither to the Src inhibitor PP2, nor to truncation of its N-terminal region, which contains a putative autoinhibitory FERM domain. These results better define the mechanisms involved in Pyk2 activation, demonstrating that autophosphorylation is ROK- and stress fiber-dependent, while transphosphorylation within the kinase domain is Ca(2+)-dependent and Src-independent in intestinal epithelial cells. This contrasts with the tight sequential coupling of phosphorylation seen in FAK activation, and further underlines the differences between these closely related kinases.     

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.     

Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397

Plating suspended Swiss 3T3 cells onto fibronectin-coated dishes promoted phosphorylation of endogenous focal adhesion kinase (FAK) at Tyr-397, the major autophosphorylation site, and at Tyr-577, located in the activation loop, as revealed by site-specific antibodies that recognize the phosphorylated form of these residues. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 (PP-2) markedly reduced the phosphorylation of both Tyr-397 and Tyr-577 induced by fibronectin. Furthermore, fibronectin-mediated FAK phosphorylation at Tyr-397 was dramatically reduced in SYF cells (deficient in Src, Yes, and Fyn expression). Stimulation of Swiss 3T3 cells with bombesin also induced a rapid increase in the phosphorylation of endogenous FAK at Tyr-397. In contrast to the results obtained with fibronectin, PP-2 did not prevent FAK Tyr-397 phosphorylation stimulated by bombesin at a concentration (10 micrometer) that suppressed bombesin-induced FAK Tyr-577 phosphorylation. Similarly, PP-2 did not prevent Tyr-397 phosphorylation in Swiss 3T3 cells stimulated with other G protein-coupled receptor agonists including vasopressin, bradykinin, endothelin, and lysophosphatidic acid. Lysophosphatidic acid also induced FAK phosphorylation at Tyr-397 in SYF cells. Our results identify, for first time, the existence of Src-dependent and Src-independent pathways leading to FAK autophosphorylation at Tyr-397 stimulated by adhesion-dependent signals and G protein-coupled receptor agonists in the same cell.     

Transient forebrain ischemia effects FAK-coupled signaling in gerbil hippocampus

Focal adhesion kinase (FAK) is thought to play a major role in transducing extracellular matrix (ECM)-derived survival signals into cells. The function of FAK is linked to its autophosphorylation at Tyr-397 and then recruitment of several effector molecules. Thus, modulation of FAK activity may affect several intracellular signaling pathways and may participate in a variety of pathological settings. In the present study, we investigated the effect of short-term 5 min forebrain ischemia on levels and Tyr-397 phosphorylation of focal adhesion kinase and the interaction of this enzyme with Src protein tyrosine kinase and adapter protein p130Cas, involved in FAK-mediated signaling pathway in gerbil hippocampus. The total amount of focal adhesion kinase as well as its Tyr-397 phosphorylation declined substantially between 24 and 48 h after the insult, particularly in CA1 region of hippocampus. Concomitantly, a decreased amount of FAK/Src kinase complex has been observed. These data indicate that inhibition of FAK/Src-coupled signaling pathway may participate in the ischemia-induced neuronal degeneration in gerbil hippocampus. The temporal profile of FAK down-regulation in CA1 area coincides with metalloproteinases (MMPs) activation. These results suggest that extracellular proteolysis might belong to the mechanisms which govern the FAK-coupled pathway in ischemic hippocampus.     

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.     

An endogenous inhibitor of focal adhesion kinase blocks Rac1/JNK but not Ras/ERK-dependent signaling in vascular smooth muscle cells

Humoral factors and extracellular matrix are critical co-regulators of smooth muscle cell (SMC) migration and proliferation. We reported previously that focal adhesion kinase (FAK)-related non-kinase (FRNK) is expressed selectively in SMC and can inhibit platelet-derived growth factor BB homodimer (PDGF-BB)-induced proliferation and migration of SMC by attenuating FAK activity. The goal of the current studies was to identify the mechanism by which FAK/FRNK regulates SMC growth and migration in response to diverse mitogenic signals. Transient overexpression of FRNK in SMC attenuated autophosphorylation of FAK at Tyr-397, reduced Src family-dependent tyrosine phosphorylation of FAK at Tyr-576, Tyr-577, and Tyr-881, and reduced phosphorylation of the FAK/Src substrates Cas and paxillin. However, FRNK expression did not alter the magnitude or dynamics of ERK activation induced by PDGF-BB or angiotensin II. Instead, FRNK expression markedly attenuated PDGF-BB-, angiotensin II-, and integrin-stimulated Rac1 activity and attenuates downstream signaling to JNK. Importantly, constitutively active Rac1 rescued the proliferation defects in FRNK expressing cells. Based on these observations, we hypothesize that FAK activation is required to integrate integrin signals with those from receptor tyrosine kinases and G protein-coupled receptors through downstream activation of Rac1 and that in SMC, FRNK may control proliferation and migration by buffering FAK-dependent Rac1 activation.