Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.     

Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.     

A small molecule FAK kinase inhibitor,GSK2256098, inhibits growth and survival of pancreatic ductal adenocarcinoma cells

Focal adhesion kinase (FAK) hyperactivation is common in pancreatic ductal adenocarcinoma (PDAC). A small molecule, GSK2256098 (GlaxoSmithKline), has been developed to inhibit FAK activity through targeting the phosphorylation site of FAK, tyrosine (Y) 397. We sought to determine whether GSK2256098 inhibition of FAK Y397 phosphorylation attenuates PDAC-associated cell proliferation, motility and survival. Cultured PDAC cells were used as cellular models of GSK2256098-impaired abnormal growth. Western blot analysis, cell viability analysis, clonogenic survival, soft-agar and wound healing assays were performed. The responses of 6 PDAC cell lines in regards to FAK Y397 phosphorylation or activity to GSK2256098 treatments (0.1–10 μM) ranged from low (less than 20% inhibition) to high (more than 90% inhibition). The least and most sensitive cell lines (PANC-1 and L3.6P1) were selected for further analysis. GSK2256098 inhibition of FAK Y397 phosphorylation correlated with decreased levels of phosphorylated Akt and ERK in L3.6P1 cells. GSK2256098 decreased cell viability, anchorage-independent growth, and motility in a dose dependent manner. Current studies demonstrate that small molecule kinase inhibitors targeting FAK Y397 phosphorylation can inhibit PDAC cell growth. Assessments of FAK Y397 phosphorylation in biopsies may be used as a biomarker to select the subgroup of responsive patients and/or monitor the effects of GSK2256098 on FAK-modulated tumor growth during treatment.     

Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells

Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/min. Phosphorylation of Src-Tyr 418, FAK-Tyr 397-Tyr 576-Tyr 925, and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 micromol/l) or specific short interfering (si)RNA. Src inhibition also prevented strain-induced FAK phosphorylation at Tyr 397 and Tyr 576 but not FAK-Tyr 925 or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of NH2-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F, and Y397F-Y576F-Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK-Tyr 925 phosphorylation under static conditions, it nevertheless exhibited increased FAK-Tyr 925 phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-Tyr 925-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting.     

Stretch-induced cell proliferation is mediated by FAK-MAPK pathway

Previously we reported that a uni-axial cyclic stretch treatment of rat 3Y1 fibroblasts induced focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation (Wang et al., 2001) [Wang, J.G., Miyazu, M., Matsushita, E., Sokabe, M., Naruse, K., 2001. Uni-axial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation. Biochem. Biophys. Res. Comm. 288, 356-361]. In the present study, we investigated whether stretch-induced MAPK activation leads to proliferation of fibroblasts. 3Y1 fibroblasts were subjected to a uni-axial cyclic stretch treatment (1 Hz, 120% in length) and the bromodeoxyuridine (BrdU) incorporation was measured to access cell proliferation. BrdU incorporation increased in a time-dependent manner and became significant within 6 hours. To investigate the involvement of FAK, we transiently expressed FAK mutants that lacked tyrosine phosphorylation site (s) (F397Y, F925Y, F397/925Y). Transient expression of wild-type FAK or mock vector did not inhibit the stretch-induced BrdU incorporation, however, the FAK mutants significantly blocked BrdU incorporation. Treatment of the cells with MAPK inhibitors, PD98059 or SB203580, blocked extracellular signal-regulated kinase (ERK) phosphorylation and p38 MAPK phosphorylation, respectively, and also blocked stretch-induced BrdU incorporation. These results suggest that the stretch-induced FAK activation followed by MAPK activation plays an important role in the stretch-induced proliferation of 3Y1 fibroblasts.     

Focal adhesion kinase signaling controls cyclic tensile strain enhanced collagen I-induced osteogenic differentiation of human mesenchymal stem cells

Focal adhesion kinase (FAK) is a key integrator of integrin-mediated signals from the extracellular matrix to the cytoskeleton and downstream signaling molecules. FAK is activated by phosphorylation at specific tyrosine residues, which then stimulate downstream signaling including the ERK1/2 pathway, leading to a variety of cellular responses. In this study, we examined the effects of FAK point mutations at tyrosine residues (Y397, Y925, Y861, and Y576/7) on osteogenic differentiation of human mesenchymal stem cells exposed to collagen I and cyclic tensile strain. Our results demonstrate that FAK signaling emanating from Y397, Y925, and to a lesser extent Y576/7, but not from Y861, controls osteogenic differentiation through an ERK1/2 pathway, as measured by expression levels of key osteogenesis marker genes and subsequent matrix mineralization. These data indicate that FAK is a critical decision maker in extracellular matrix/strain-enhanced osteogenic differentiation.     

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.     

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.     

Targeting of focal adhesion kinase by small interfering RNAs reduces chondrocyte redifferentiation capacity in alginate beads culture with type II collagen

Type II collagen is a major protein that maintains biological and mechanical characteristics in articular cartilage. Focal adhesion kinase (FAK) is known to play a central role in integrin signaling of cell–extracellular matrix (ECM) interactions, and chondrocyte–type II collagen interactions are very important for cartilage homeostasis. In this study, we focused on phosphorylation of FAK and MAP kinase in chondrocyte–type II collagen interaction and dedifferentiation, and the effects of FAK knockdown on chondrocyte‐specific gene expression and cell proliferation were determined. The addition of exogenous type II collagen to chondrocytes increased levels of tyrosine phosphorylation, p‐FAK_Y397, and p‐ERK1/2. In contrast, expression levels of p‐FAK_Y397 and p‐ERK1/2, but not p‐Smad2/3, were decreased in dedifferentiated chondrocytes with loss of type II collagen expression. Type II collagen expression was significantly increased when dedifferentiated chondrocytes were transferred to alginate beads with TGF‐β1 or type II collagen, but transfected cells with small interfering RNA for FAK (FAK‐siRNA) inhibited mRNA expression of type II collagen and SOX‐6 compared to the control. These FAK‐siRNA‐transfected cells could not recover type II collagen even in the presence of TGF‐β1 or type II collagen in alginate beads culture. We also found that FAK‐siRNA‐transfected cells decreased cell proliferation rate, but there was no effect on glycosaminoglycans (GAGs) secretion. We suggest that FAK is essentially required in chondrocyte communication with type II collagen by regulating type II collagen expression and cell proliferation. J. Cell. Physiol. 218: 623–630, 2009. © 2008 Wiley‐Liss, Inc.     

SRC catalytic but not scaffolding function is needed for integrin-regulated tyrosine phosphorylation, cell migration, and cell spreading

Src family kinases (SFKs) are crucial for signaling through a variety of cell surface receptors, including integrins. There is evidence that integrin activation induces focal adhesion kinase (FAK) autophosphorylation at Y397 and that Src binds to and is activated by FAK to carry out subsequent phosphorylation events. However, it has also been suggested that Src functions as a scaffolding molecule through its SH2 and SH3 domains and that its kinase activity is not necessary. To examine the role of SFKs in integrin signaling, we have expressed various Src molecules in fibroblasts lacking other SFKs. In cells plated on fibronectin, FAK could indeed autophosphorylate at Y397 independently of Src but with lower efficiency than when Src was present. This step was promoted by kinase-inactive Src, but Src kinase activity was required for full rescue. Src kinase activity was also required for phosphorylation of additional sites on FAK and for other integrin-directed functions, including cell migration and spreading on fibronectin. In contrast, Src mutations in the SH2 or SH3 domain greatly reduced binding to FAK, Cas, and paxillin but had little effect on tyrosine phosphorylation or biological assays. Furthermore, our indirect evidence indicates that Src kinase activity does not need to be regulated to promote cell migration and FAK phosphorylation. Although Src clearly plays important roles in integrin signaling, it was not concentrated in focal adhesions. These results indicate that the primary role of Src in integrin signaling is as a kinase. Indirect models for Src function are proposed.     

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.     

Focal adhesion kinase is negatively regulated by phosphorylation at tyrosine 407

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr(407) was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr(407) increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr(397) phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr(397) and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr(407) phosphorylation negatively regulates the enzymatic and biological activities of FAK.     

Src family kinase involvement in muscarinic receptor-induced tyrosine phosphorylation in differentiated SH-SY5Y cells

Muscarinic receptor-mediated changes in protein tyrosine phosphorylation were examined in differentiated human neuroblastoma SH-SY5Y cells. Treatment of differentiated cells with 1 mM carbachol caused rapid increases in the tyrosine phosphorylation of focal adhesion kinase (FAK), Cas, and paxillin. The src family kinase-selective inhibitor PP1 reduced carbachol-stimulated tyrosine phosphorylation of FAK, Cas, and paxillin by 50 to 75%. In contrast, carbachol-stimulated activation of ERK1/2 was unaffected by PP1. Src family kinase activation by carbachol was further demonstrated by increased carbachol-induced tyrosine phosphorylation of the src-substrate, p120, and tyrosine phosphorylation of the src family kinase activation-associated autophosphorylation site. Site-specific FAK phosphotyrosine antibodies were used to determine that the carbachol-stimulated increase in the autophosphorylation of FAK was unaffected by pretreatment with PP1, whereas the carbachol-stimulated increase in the src family kinase-mediated phosphotyrosine of FAK was completely blocked by pretreatment with PP1. In SH-SY5Y cell lines stably overexpressing Fyn, the phosphotyrosine immunoreactivity of FAK was 625% that of control cells. Thus, muscarinic receptors activate protein tyrosine phosphorylation in differentiated cells, and the tyrosine phosphorylation of FAK, Cas, and paxillin, but not ERK1/2, is mediated by a src family tyrosine kinase activated in response to stimulation of muscarinic receptors.     

OipA plays a role in Helicobacter pylori-induced focal adhesion kinase activation and cytoskeletal re-organization

The initial signalling events leading to Helicobacter pylori infection associated changes in motility, cytoskeletal reorganization and elongation of gastric epithelial cells remain poorly understood. Because focal adhesion kinase (FAK) is known to play important roles in regulating actin cytoskeletal organization and cell motility we examined the effect of H. pylori in gastric epithelial cells co-cultured with H. pylori or its isogenic cag pathogenicity island (PAI) or oipA mutants. H. pylori induced FAK phosphorylation at distinct tyrosine residues in a dose- and time-dependent manner. Autophosphorylation of FAK Y397 was followed by phosphorylation of Src Y418 and resulted in phosphorylation of the five remaining FAK tyrosine sites. Phosphorylated FAK and Src activated Erk and induced actin stress fibre formation. FAK knock-down by FAK-siRNA inhibited H. pylori- mediated Erk phosphorylation and abolished stress fibre formation. Infection with oipA mutants reduced phosphorylation of Y397, Y576, Y577, Y861 and Y925, inhibited stress fibre formation and altered cell morphology. cag PAI mutants reduced phosphorylation of only FAK Y407 and had less effect on stress fibre formation than oipA mutants. We propose that activation of FAK and Src are responsible for H. pylori -induced induction of signalling pathways resulting in the changes in cell phenotype important for pathogenesis.     

Novel Role of Src in Priming Pyk2 Phosphorylation

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.     

Activation of extracellular-regulated kinases by normal and mutant EGF receptors

Glioblastoma cells express a mutant EGF receptor (EGFRvIII) that has constitutive tyrosine kinase activity and enhances their tumorigenicity. Here we show that EGFRvIII promotes constitutive phosphorylation of extracellular regulated kinases (ERKs) in glioblastoma cells in the absence of EGF. EGFRvIII also promoted constitutive activation of phosphoinositide 3-kinase in these cells, as assessed by phosphorylation of protein kinase B/akt. As expected, phosphorylation of protein kinase B/akt was blocked by the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. Less expectedly, we found that this treatment also blocked EGFRvIII-induced phosphorylation of ERKs. In contrast, ERK phosphorylation induced by EGF-activated normal EGF receptor in the same cells was largely unaffected by treatment with phosphoinositide 3-kinase inhibitors. This difference in behavior between the normal receptor and EGFRvIII was not due to differences in the levels of activated EGFRvIII and wild-type EGF receptor, as the two types of receptor were tyrosine phosphorylated to a similar extent under the experimental conditions used. EGFRvIII activation of ERKs was also sensitive to the phospholipase C inhibitor U73122, whereas ERK activation by normal EGF receptor was not. These results show that EGFRvIII and wild-type EGF receptor preferentially use different signaling pathways to induce ERK phosphorylation. The different mechanisms of ERK activation used by normal and mutant EGF receptors may be important in understanding the potent tumorigenic activity of EGFRvIII.     

Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

Akt directly regulates focal adhesion kinase through association and serine phosphorylation: implication for pressure-induced colon cancer metastasis

Although focal adhesion kinase (FAK) is typically considered upstream of Akt, extracellular pressure stimulates cancer cell adhesion via Akt-dependent FAK activation. How Akt regulates FAK is unknown. We studied Akt-FAK interaction in colon cancer cells under 15 mmHg increased extracellular pressure. Pressure enhanced Akt-FAK association, blocked by inhibiting FAK or silencing Akt1 but not Akt2, and stimulated FAK serine phosphorylation in Caco-2 and human colon cancer cells from surgical specimens Akt1-dependently. FAK includes three serine (S517/601/695) and one threonine (T600)-containing consensus sequences for Akt phosphorylation. Studying S->A nonphosphorylatable point mutants suggests that these sites coordinately upregulate FAK Y397 tyrosine phosphorylation, which conventionally initiates FAK activation, and mediate pressure-induced cancer cell adhesion. FAK(T600A) mutation did not prevent pressure-induced FAK(Y397) phosphorylation or adhesion. Akt1 appeared to directly bind FAK, and this binding did not depend on the FAK autophosphorylation site (Y397). In addition, our results demonstrated that Akt phosphorylated FAK at three novel serine phosphorylation sites, which were also not required for FAK-Akt binding. This novel interaction suggests that FAK and Akt may be dual kinase targets to prevent cancer cell adhesion and metastasis.     

The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation

We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit beta1 (Y783 and Y795) to phenylalanines markedly reduces the capability of beta1A integrins to mediate directed cell migration. In this study, beta1-dependent cell spreading was found to be delayed in GD25 cells expressing beta1A(Y783/795F) compared to that in wild-type GD25-beta1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to beta1-dependent adhesion in GD25-beta1A(Y783/795F) cells compared to that in wild-type GD25-beta1A or mutants in which only a single tyrosine was altered (beta1A(Y783F) or beta1A(Y795F)). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via beta1A(Y783/795F) lies at the level of the initial autophosphorylation step. Indeed, beta1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the beta1A(Y783/795F) cells, consistent with the impairment in FAK activation. In contrast, p130(CAS) overall tyrosine phosphorylation was unaffected by the beta1 mutations. Despite the defect in beta1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-beta1A(Y783/795F) cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit beta1A are critical mediators of FAK activation and cell spreading in GD25 cells.     

Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 tyrosine sites involved in ERK activation

The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and coimmunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner.