Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration

Directed cell migration requires the coordination of growth factor and cell adhesion signaling and is of fundamental importance during embryonic development, wound repair, and pathological conditions such as tumor metastasis. Herein, we demonstrate that the ArfGAP, paxillin-kinase-linker (PKL/GIT2), is tyrosine phosphorylated in response to platelet-derived growth factor (PDGF) stimulation, in an adhesion dependent manner and is necessary for directed cell migration. Using a combination of pharmacological inhibitors, knockout cells and kinase mutants, FAK, and Src family kinases were shown to mediate PDGF-dependent PKL tyrosine phosphorylation. In fibroblasts, expression of a PKL mutant lacking the principal tyrosine phosphorylation sites resulted in loss of wound-induced cell polarization as well as directional migration. PKL phosphorylation was necessary for PDGF-stimulated PKL binding to the focal adhesion protein paxillin and expression of paxillin or PKL mutants defective in their respective binding motifs recapitulated the polarization defects. RNA interference or expression of phosphorylation mutants of PKL resulted in disregulation of PDGF-stimulated Rac1 and PAK activities, reduction of Cdc42 and Erk signaling, as well as mislocalization of βPIX. Together these studies position PKL as an integral component of growth factor and cell adhesion cross-talk signaling, controlling the development of front–rear cell polarity and directional cell migration.     

Targeting of alpha(v) integrins interferes with FAK activation and smooth muscle cell migration and invasion

Aberrant migration of smooth muscle cells (SMCs) is a key feature of restenosis. Since extracellular matrix proteins and their receptors of the integrin family play a critical role in this process, it is instrumental to understand their contribution to cell migration and invasive motility of SMC on the molecular level. Therefore, we investigated the role of alpha(v)-containing integrins expressed by primary human coronary artery smooth muscle cells (hCASMCs) in vitronectin (VN)-initiated signaling events and cell migration. In hCASMC plated on VN, alpha(v)-containing integrins were localized at focal adhesion sites. Haptotactic stimulation through VN led to a dose-dependent increase in cell migration and concomitantly to enhanced tyrosine phosphorylation of focal adhesion kinase. Both events were completely blocked by a specific inhibitor of integrin alpha(v). Additionally, the integrin alpha(v) inhibitor abolished PDGF-BB-stimulated chemotactic migration. Confocal microscopy confirmed the increased tyrosine phosphorylation at VN-initiated focal contact sites in hCASMC, that was abolished upon alpha(v) inhibition. In vitro invasion of hCASMC was severely compromised in the presence of the integrin alpha(v) inhibitor paralleled by decreased levels of secreted matrix metalloprotease 2 (MMP-2). Together, integrin alpha(v) inhibition abrogates tyrosine phosphorylation at focal adhesion sites and diminishes MMP-2 secretion leading to reduced migration and invasion of hCASMCs.     

Epidermal Growth Factor–induced Contraction Regulates Paxillin Phosphorylation to Temporally Separate Traction Generation from De-adhesion

Directed cell migration is mediated by cycles of protrusion, adhesion, traction generation on the extracellular matrix and retraction. However, how the events after protrusion are timed, and what dictates their temporal order is completely unknown. We used acute epidermal growth factor (EGF) stimulation of epidermal keratinocytes to initiate the cell migration cycle to study the mechanism of the timing of adhesion, traction generation, and de-adhesion. Using microscopic and biochemical assays, we surprisingly found that at ∼2 min after EGF stimulation protrusion, activation of myosin-II, traction generation, adhesion assembly, and paxillin phosphorylation occurred nearly simultaneously, followed by a 10-min delay during which paxillin became dephosphorylated before cell retraction. Inhibition of myosin-II blocked both the EGF-stimulated paxillin phosphorylation and cell retraction, and a paxillin phosphomimic blocked retraction. These results suggest that EGF-mediated activation of myosin-II acts as a mechanical signal to promote a cycle of paxillin phosphorylation/dephosphorylation that mediates a cycle of adhesion strengthening and weakening that delays cell retraction. Thus, we reveal for the first time a mechanism by which cells may temporally segregate protrusion, adhesion, and traction generation from retraction during EGF-stimulated cell migration.     

骨桥蛋白通过NF-κB上调钙蛋白酶小亚基1促进肝癌细胞迁移

骨桥蛋白(osteopontin,OPN)参与调控多种信号途径激活转移相关基因,进而促进细胞迁移.钙蛋白酶小亚基1(calpain small subunit1,Capn4)与肿瘤转移密切相关,在许多肿瘤及其转移组织中高表达.为了探讨OPN促进肝癌细胞迁移的分子机制,应用报告基因检测、RT-PCR、免疫印迹及伤口愈合等方法检测了肝癌细胞中OPN对Capn4的调控作用及其对肝癌细胞迁移的影响.结果显示,在HepG2细胞中过表达OPN后,Capn4的启动子转录活性显著增强,同时mRNA及蛋白质表达水平也明显上调.在HepG2细胞中应用siRNA干扰OPN的表达可导致Capn4启动子转录活性受到明显抑制,同时mRNA及蛋白质表达水平也显著下调.应用核转录因子-κB(NF-κB)的抑制剂PDTC可抑制由过表达OPN导致的HepG2细胞中Capn4的上调.伤口愈合实验显示,OPN可以通过上调Capn4促进肝癌细胞迁移.因此,研究发现,OPN通过NF-κB上调Capn4的表达,进而促进肝癌细胞的迁移,这一发现对进一步阐明肝癌细胞迁移的分子机制具有重要意义.     

Phosphorylation of Ser 21 in Fyn regulates its kinase activity,focal adhesion targeting,and is required for cell migration

The tyrosine kinase Fyn is a member of the Src family kinases which are important in many integrin‐mediated cellular processes including cell adhesion and migration. Fyn has multiple phosphorylation sites which can affect its kinase activity. Among these phosphorylation sites, the serine 21 (S21) residue of Fyn is a protein kinase A (PKA) recognition site within an RxxS motif of the amino terminal SH4 domain of Fyn. In addition, S21 is critical for Fyn kinase‐linked cellular signaling. Mutation of S21A blocks PKA phosphorylation of Fyn and alters its tyrosine kinase activity. Expression of Fyn S21A in cells lacking Src family kinases (SYF cell) led to decreased tyrosine phosphorylation of focal adhesion kinase resulting in reduced focal adhesion targeting, which slowed lamellipodia dynamics and thus cell migration. These changes in cell motility were reflected by the fact that cells expressing Fyn S21A were severely deficient in their ability to assemble and disassemble focal adhesions. Taken together, our findings indicate that phosphorylation of S21 within the pPKA recognition site (RxxS motif) of Fyn regulates its tyrosine kinase activity and controls focal adhesion targeting, and that this residue of Fyn is critical for transduction of signals arising from cell‐extracellular matrix interactions. J. Cell. Physiol. 226: 236–247, 2010. © 2010 Wiley‐Liss, Inc.     

The docking protein FRS2alpha controls a MAP kinase-mediated negative feedback mechanism for signaling by FGF receptors

The docking protein FRS2alpha functions as a major mediator of signaling by FGF and NGF receptors. Here we demonstrate that, in addition to tyrosine phosphorylation, FRS2alpha is phosphorylated by MAP kinase on multiple threonine residues in response to FGF stimulation or by insulin, EGF, and PDGF, extracellular stimuli that do not induce tyrosine phosphorylation of FRS2alpha. Prevention of FRS2alpha threonine phosphorylation results in constitutive tyrosine phosphorylation of FRS2alpha in unstimulated cells and enhanced tyrosine phosphorylation of FRS2alpha, MAPK stimulation, cell migration, and proliferation in FGF-stimulated cells. Expression of an FRS2alpha mutant deficient in MAPK phosphorylation sites induces anchorage-independent cell growth and colony formation in soft agar. These experiments reveal a novel MAPK-mediated, negative feedback mechanism for control of signaling pathways that are dependent on FRS2 and a mechanism for heterologous control of signaling via FGF receptors.     

Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The mu- and m-calpains consist of a mu- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4(-/-) mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4(-/-) cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4(-/-) cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, alpha-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4(+/+) and Capn4(-/-) cells. FAK, alpha-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4(-/-) cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4(-/-) embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.     

The endosomal adaptor protein APPL1 impairs the turnover of leading edge adhesions to regulate cell migration

Cell migration is a complex process that requires the integration of signaling events that occur in distinct locations within the cell. Adaptor proteins, which can localize to different subcellular compartments, where they bring together key signaling proteins, are emerging as attractive candidates for controlling spatially coordinated processes. However, their function in regulating cell migration is not well understood. In this study, we demonstrate a novel role for the adaptor protein containing a pleckstrin-homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1) in regulating cell migration. APPL1 impairs migration by hindering the turnover of adhesions at the leading edge of cells. The mechanism by which APPL1 regulates migration and adhesion dynamics is by inhibiting the activity of the serine/threonine kinase Akt at the cell edge and within adhesions. In addition, APPL1 significantly decreases the tyrosine phosphorylation of Akt by the nonreceptor tyrosine kinase Src, which is critical for Akt-mediated cell migration. Thus, our results demonstrate an important new function for APPL1 in regulating cell migration and adhesion turnover through a mechanism that depends on Src and Akt. Moreover, our data further underscore the importance of adaptor proteins in modulating the flow of information through signaling pathways.     

ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte migration involve intracellular calcium signaling in brain endothelial cell lines

Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells using two rat brain endothelial cell lines (RBE4 and GP8), we report in this paper that ICAM-1 cross-linking induces a sustained tyrosine phosphorylation of the phosphatidylinositol-phospholipase C (PLC)gamma1, with a concomitant increase in both inositol phosphate production and intracellular calcium concentration. Our results suggest that PLC are responsible, via a calcium- and protein kinase C (PKC)-dependent pathway, for p60Src activation and tyrosine phosphorylation of the p60Src substrate, cortactin. PKCs are also required for tyrosine phosphorylation of the cytoskeleton-associated proteins, focal adhesion kinase and paxillin, but not for ICAM-1-coupled p130Cas phosphorylation. PKC's activation is also necessary for stress fiber formation induced by ICAM-1 cross-linking. Finally, cell pretreatment with intracellular calcium chelator or PKC inhibitors significantly diminishes transmonolayer migration of activated T lymphocytes, without affecting their adhesion to brain endothelial cells. In summary, our data demonstrate that ICAM-1 cross-linking induces calcium signaling which, via PKCs, mediates phosphorylation of actin-associated proteins and cytoskeletal rearrangement in brain endothelial cell lines. Our results also indicate that these calcium-mediated intracellular events are essential for lymphocyte migration through the blood-brain barrier.     

Hepatitis B virus X protein drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration

Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). However, the mechanism remains unclear. Recently, we have reported that HBx promotes hepatoma cell migration through the upregulation of calpain small subunit 1 (Capn4). In addition, several reports have revealed that osteopontin (OPN) plays important roles in tumor cell migration. In this study, we investigated the signaling pathways involving the promotion of cell migration mediated by HBx. We report that HBx stimulates several factors in a network manner to promote hepatoma cell migration. We showed that HBx was able to upregulate the expression of osteopontin (OPN) through 5-lipoxygenase (5-LOX) in HepG2-X/H7402-X (stable HBx-transfected cells) cells. Furthermore, we identified that HBx could increase the expression of 5-LOX through nuclear factor-κB (NF-κB). We also found that OPN could upregulate Capn4 through NF-κB. Interestingly, we showed that Capn4 was able to upregulate OPN through NF-κB in a positive feedback manner, suggesting that the OPN and Capn4 proteins involving cell migration affect each other in a network through NF-κB. Importantly, NF-κB plays a crucial role in the regulation of 5-LOX, OPN and Capn4. Thus, we conclude that HBx drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration. This finding provides new insight into the mechanism involving the promotion of cell migration by HBx.     

Spatial and temporal coordination of traction forces in one-dimensional cell migration

ABSTRACT

Migration of a fibroblast along a collagen fiber can be regarded as cell locomotion in one-dimension (1D). In this process, a cell protrudes forward, forms a new adhesion, produces traction forces, and releases its rear adhesion in order to advance itself along a path. However, how a cell coordinates its adhesion formation, traction forces, and rear release in 1D migration is unclear. Here, we studied fibroblasts migrating along a line of microposts. We found that when the front of a cell protruded onto a new micropost, the traction force produced at its front increased steadily, but did so without a temporal correlation in the force at its rear. Instead, the force at the front coordinated with a decrease in force at the micropost behind the front. A similar correlation in traction forces also occurred at the rear of a cell, where a decrease in force due to adhesion detachment corresponded to an increase in force at the micropost ahead of the rear. Analysis with a bio-chemo-mechanical model for traction forces and adhesion dynamics indicated that the observed relationship between traction forces at the front and back of a cell is possible only when cellular elasticity is lower than the elasticity of the cellular environment.     

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration.     

Phosphoproteomic analysis identifies insulin enhancement of discoidin domain receptor 2 phosphorylation

The discoidin domain receptors (DDRs) are collagen binding receptor tyrosine kinases that play important roles in cell migration, invasion and adhesion. Crosstalk between growth factor signaling and components of the extracellular matrix are drivers of cellular function but the integrated signaling networks downstream of such crosstalk events have not been extensively characterized. In this report, we have employed mass spectrometry-based quantitative phosphotyrosine analysis to identify crosstalk between DDR2 and the insulin receptor. Our phosphoproteomic analysis reveals a cluster of phosphorylation sites in which collagen and insulin cooperate to enhance phosphotyrosine levels. Importantly, Y740 on the DDR2 catalytic loop was found in this cluster indicating that insulin acts to promote collagen I signaling by increasing the activity of DDR2. Furthermore, we identify two additional migration associated proteins that are candidate substrates downstream of DDR2 activation. Our data suggests that insulin promotes collagen I signaling through the upregulation of DDR2 phosphorylation which may have important consequences in DDR2 function in health and disease.     

Inhibition of integrin-mediated adhesion and signaling disrupts retinal development

Integrins are the major family of cell adhesion receptors that mediate cell adhesion to the extracellular matrix (ECM). Integrin-mediated adhesion and signaling play essential roles in neural development. In this study, we have used echistatin, an RGD-containing short monomeric disintegrin, to investigate the role of integrin-mediated adhesion and signaling during retinal development in Xenopus. Application of echistatin to Xenopus retinal-derived XR1 glial cells inhibited the three stages of integrin-mediated adhesion: cell attachment, cell spreading, and formation of focal adhesions and stress fibers. XR1 cell attachment and spreading increased tyrosine phosphorylation of paxillin, a focal adhesion associated protein, while echistatin significantly decreased phosphorylation levels of paxillin. Application of echistatin or beta(1) integrin function blocking antibody to the embryonic Xenopus retina disrupted retinal lamination and produced rosette structures with ectopic photoreceptors in the outer retina. These results indicate that integrin-mediated cell-ECM interactions play a critical role in cell adhesion, migration, and morphogenesis during vertebrate retinal development.     

Protein-tyrosine phosphatase PTPD1 regulates focal adhesion kinase autophosphorylation and cell migration

PTPD1 is a cytosolic nonreceptor tyrosine phosphatase and a positive regulator of the Src-epidermal growth factor transduction pathway. We show that PTPD1 localizes along actin filaments and at adhesion plaques. PTPD1 forms a stable complex via distinct molecular modules with actin, Src tyrosine kinase, and focal adhesion kinase (FAK), a scaffold protein kinase enriched at adhesion plaques. Overexpression of PTPD1 promoted cell scattering and migration, short hairpin RNA-mediated silencing of endogenous PTPD1, or expression of PTPD1 mutants lacking either catalytic activity (PTPD1(C1108S)) or the FERM domain (PTPD1(Delta1-325)) significantly reduced cell motility. PTPD1 and Src catalytic activities were both required for epidermal growth factor-induced FAK autophosphorylation at its active site and for downstream propagation of ERK1/2 signaling. Our findings demonstrate that PTPD1 is a component of a multivalent scaffold complex nucleated by FAK at specific intracellular sites. By modulating Src-FAK signaling at adhesion sites, PTPD1 promotes the cytoskeleton events that induce cell adhesion and migration.     

P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.     

Galectin-1 interacts with beta-1 subunit of integrin

Galectin-1, a beta-galactoside-binding dimeric lectin, is involved in adhesion, migration, and proliferation of vascular smooth muscle cells (SMC), the key steps in the development of atherosclerosis and restenosis. Here we investigated the molecular basis of the interactions between galectin-1 and SMCs. Galectin-1 modulated SMC attachment in a dose- and beta-galactoside-dependent manner. Direct binding of galectin-1 to beta1 integrin was detected by the immune precipitation of beta1 integrin after chemical cross-linking of 125I-labelled galectin-1 to the cell surface proteins. Galectin-1 transiently increased availability of beta1 integrins on the cell surface to antibodies against beta1 integrin. Incubation of SMCs with galectin-1 transiently increased the amount of the active form of beta1 integrin and tyrosine phosphorylation of two cytoskeleton-associated proteins; one of them coincided with focal adhesion kinase (FAK). Galectin-1 is likely to affect SMC adhesion by interacting with beta1 integrin on the cell surface of SMCs and inducing outside-in signalling.     

Fer-mediated cortactin phosphorylation is associated with efficient fibroblast migration and is dependent on reactive oxygen species generation during integrin-mediated cell adhesion

The molecular details linking integrin engagement to downstream cortactin (Ctn) tyrosine phosphorylation are largely unknown. In this report, we show for the first time that Fer and Ctn are potently tyrosine phosphorylated in response to hydrogen peroxide (H2O2) in a variety of cell types. Working with catalytically inactive fer and src/yes/fyn-deficient murine embryonic fibroblasts (ferDR/DR and syf MEF, respectively), we observed that H2O2-induced Ctn tyrosine phosphorylation is primarily dependent on Fer but not Src family kinase (SFK) activity. We also demonstrated for the first time that Fer is activated by fibronectin engagement and, in concert with SFKs, mediates Ctn tyrosine phosphorylation in integrin signaling pathways. Reactive oxygen species (ROS) scavengers or the NADPH oxidase inhibitor, diphenylene iodonium, attenuated integrin-induced Fer and Ctn tyrosine phosphorylation. Taken together, these findings provide novel genetic evidence that a ROS-Fer signaling arm contributes to SFK-mediated Ctn tyrosine phosphorylation in integrin signaling. Lastly, a migration defect in ferDR/DR MEF suggests that integrin signaling through the ROS-Fer-Ctn signaling arm may be linked to mechanisms governing cell motility. These data demonstrate for the first time an oxidative link between integrin adhesion and an actin-binding protein involved in actin polymerization.     

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

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