Tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin in glomeruli from diabetic rats

A study of group comprising all road accidents caused by drivers of private cars who were under the influence of alcohol (BAC > = 0.3 g/kg; X = 1.56 +/- 0.62 g/kg) that occurred in a defined area over the span of one calendar year (n = 625) was compared with a randomly selected control group of 718 road accidents in which the drivers had not been under the influence of alcohol. The drivers in the study group were marginally younger than the ones in the control group. However, there was no evidence of an alcohol related increase in the risk of an accident associated with younger age. The sex ratio in the study group corresponded to that, generally found amongst people driving under the influence of alcohol. In the study group there was no evidence of a restricted manner and extent of car use, based on the distances between the sites of the accidents and the offenders' homes. However, the proportion of accidents occurring out of towns was greater in the study group. Alcohol associated accidents occurred more frequently in the evenings and at night, which reflects habitual drinking patterns. Therefore these accidents occurred mainly in darkness and twilight. Surprisingly, unfavorable weather conditions such as rain or ice did not lead to an increase in accidents due to alcohol. In fact, in the study group, proportionally fewer accidents occurred on icy roads. Both injury to persons and damage to property were more severe in the study group. While no relationship between accident severity and blood alcohol concentration could be proven within the study group, the risk of death or severe injury was 3 to 4 times greater in this group than in the control group.     

Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor

The experiments presented here were designed to examine the contribution of p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation to the activation of the mitogen-activated protein kinase cascade induced by bombesin, lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF) in Swiss 3T3 cells. We found that tyrosine phosphorylation of p125FAK in response to these growth factors is completely abolished in cells treated with cytochalasin D or in cells that were suspended in serum-free medium for 30 min. In marked contrast, the activation of p42mapk by these factors was independent of the integrity of the actin cytoskeleton and of the interaction of the cells with the extracellular matrix. The protein kinase C inhibitor GF 109203X and down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol esters blocked bombesin-stimulated activation of p42mapk, p90rsk, and MAPK kinase-1 but did not prevent bombesin-induced tyrosine phosphorylation of p125FAK. Furthermore, LPA-induced p42mapk activation involved a pertussis toxin-sensitive guanylate nucleotide-binding protein, whereas tyrosine phosphorylation of p125FAK in response to LPA was not prevented by pretreatment with pertussis toxin. Finally, PDGF induced maximum p42mapk activation at concentrations (30 ng/ml) that failed to induce tyrosine phosphorylation of p125FAK. Thus, our results demonstrate that p42mapk activation in response to bombesin, LPA, and PDGF can be dissociated from p125FAK tyrosine phosphorylation in Swiss 3T3 cells.     

Epidermal growth factor modulates tyrosine phosphorylation of p130Cas. Involvement of phosphatidylinositol 3'-kinase and actin cytoskeleton

Epidermal growth factor (EGF) treatment of Rat-1 cells expressing human EGF receptor results in the modification of the tyrosine phosphorylation of the p130 Crk-associated substrate (Cas), a novel signaling molecule residing in focal adhesions. At low, mitogenic concentrations (<10 ng/ml), EGF treatment induced a rapid and transient tyrosine phosphorylation of Cas and promoted the formation of a Cas-adapter protein Crk complex in intact cells. The increase in tyrosine phosphorylation of Cas paralleled an increase in the cellular content of actin stress fibers and occurred via a pathway that depended on the integrity of the cytoskeleton. Further, phosphatidylinositol 3'-kinase activity was found to be required for the EGF-stimulated Cas phosphorylation and actin polymerization. At high concentrations (>30 ng/ml), EGF treatment resulted in the tyrosine dephosphorylation of Cas in a time-dependent manner with a concomitant decrease in the length and number of actin stress fibers. Thus, Cas exhibits an unusual bell-shaped dose-response curve in response to EGF stimulation. These results demonstrate a novel signaling role for EGF in inducing changes in tyrosine phosphorylation of Cas and Cas-Crk complex formation and suggest that Cas could be a signaling component in EGF-mediated signal transduction.     

p125Fak focal adhesion kinase is a substrate for the insulin and insulin-like growth factor-I tyrosine kinase receptors

The focal adhesion kinase p125(Fak) is a widely expressed cytosolic tyrosine kinase, which is involved in integrin signaling and in signal transduction of a number of growth factors. In contrast to tyrosine kinase receptors such as the platelet-derived growth factor and the hepatocyte growth factor receptors, which induce p125(Fak) phosphorylation, insulin has been shown to promote its dephosphorylation. In this study, we compared p125(Fak) phosphorylation in insulin-stimulated cells maintained in suspension or in an adhesion state. We found that, in nonattached cells, insulin promotes p125(Fak) phosphorylation, whereas dephosphorylation occurred in attached cells. This was observed in Rat-1 fibroblasts overexpressing the insulin receptor, as well as in Hep G2 hepatocytes and in 3T3-L1 adipocytes expressing more natural levels of insulin receptors. Insulin-induced p125(Fak) phosphorylation correlated with an increase in paxillin phosphorylation, indicating that p125(Fak) kinase activity may be stimulated by insulin. Mixing of purified insulin or insulin-like growth factor-I (IGF-I) receptors with p125(Fak) resulted in an increase in p125(Fak) phosphorylation. Using a kinase-deficient p125(Fak) mutant, we found that this protein is a direct substrate of the insulin and IGF-I receptor tyrosine kinases. This view is supported by two additional findings. (i) A peptide corresponding to p125(Fak) sequence comprising amino acids 568-582, which contains tyrosines 576 and 577 of the kinase domain regulatory loop, is phosphorylated by the insulin receptor; and (ii) p125(Fak) phosphorylation by the insulin receptor is prevented by addition of this peptide. Finally, we observed that p125(Fak) phosphorylation by the receptor results in its activation. Our results show that the nature of the cross-talk between the insulin/IGF-I receptors and p125(Fak) is dependent on the cell architecture, and hence the interaction of the insulin/IGF-I signaling system with the integrin system will vary accordingly.     

Galpha12 and Galpha13 stimulate Rho-dependent tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130 Crk-associated substrate

We examined whether constitutively active mutants of the Galpha proteins Galpha12 and Galpha13, which together comprise the G12 subfamily of Galpha proteins, induce Rho-dependent tyrosine phosphorylation of the focal adhesion proteins p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate. We report that transient expression of the constitutively active mutants of Galpha12 or of Galpha13 in human embryonic kidney 293 cells stimulates tyrosine phosphorylation of a set of proteins of Mr of 110,000-130,000, 97,000, and 60,000-70,000. We identified p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate as prominent tyrosine-phosphorylated proteins in human embryonic kidney 293 cells expressing constitutively active Galpha12 and Galpha13. In common with the increased tyrosine phosphorylation of these proteins mediated by mitogens acting through heptahelical receptors, the Galpha12- and Galpha13-mediated increase in tyrosine phosphorylation is blocked by cytochalasin D, which specifically disrupts the actin cytoskeleton, and by the Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho. Our results support the hypothesis that Galpha12 and Galpha13 activate Rho and suggest that Galpha12 and Galpha13 may mediate the tyrosine phosphorylation of p125 focal adhesion kinase, paxillin, and p130 Crk-associated substrate.     

EGF stimulates tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin in rat pancreatic acini by a phospholipase C-independent process that depends on phosphatidylinositol 3-kinase, the small GTP-binding protein, p21rho, and the integrity of the actin cytoskeleton

Epidermal growth factor (EGF) is a potent mitogen in many cell types including pancreatic cells. Recent studies show that the effects of some growth factors on growth and cell migration are mediated by tyrosine phosphorylation of the cytosolic tyrosine kinase p125 focal adhesion kinase (p125FAK) and the cytoskeletal protein, paxillin. The aim of the present study was to determine whether EGF activates this pathway in rat pancreatic acini and causes tyrosine phosphorylation of each of these proteins, and to examine the intracellular pathways involved. Treatment of pancreatic acini with EGF induced a rapid, concentration-dependent increase in p125FAK and paxillin tyrosine phosphorylation. Depletion of the intracellular calcium pool or inhibition of PKC activation had no effect on the response to EGF. However, inhibition of the phosphatidylinositol 3-kinase (PI3-kinase) or inactivation of p21rho inhibited EGF-stimulated phosphorylation of p125FAK and paxillin by more than 70%. Finally, cytochalasin D, a selective disrupter of the actin filament network, completely inhibited EGF-stimulated tyrosine phosphorylation of both proteins. All these treatments did not modify EGF receptor autophosphorylation in response to EGF. These results identify p125FAK and paxillin as components of the intracellular pathways stimulated after EGF receptor occupation in rat pancreatic acini. Activation of this cascade requires activation of PI3-kinase and participation of p21rho, but not PKC activation and calcium mobilization.     

Differential regulation of focal adhesion kinase and mitogen-activated protein kinase tyrosine phosphorylation during insulin-like growth factor-I-mediated cytoskeletal reorganization

In SH-SY5Y human neuroblastoma cells, insulin-like growth factor (IGF)-I mediates membrane ruffling and growth cone extension. We have previously shown that IGF-I activates the tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) 2. In the current study, we examined which signaling pathway underlies IGF-I-mediated FAK phosphorylation and cytoskeletal changes and determined if an intact cytoskeleton was required for IGF-I signaling. Treatment of SH-SY5Y cells with cytochalasin D disrupted the actin cytoskeleton and prevented any morphological changes induced by IGF-I. Inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked IGF-I-mediated changes in the actin cytoskeleton as measured by membrane ruffling. In contrast, PD98059, a selective inhibitor of ERK kinase, had no effect on IGF-I-induced membrane ruffling. In parallel with effects on the actin cytoskeleton, cytochalasin D and PI 3-K inhibitors blocked IGF-I-induced FAK tyrosine phosphorylation, whereas PD98059 had no effect. It is interesting that cytochalasin D did not block IGF-I-induced ERK2 tyrosine phosphorylation. Therefore, it is likely that FAK and ERK2 tyrosine phosphorylations are regulated by separate pathways during IGF-I signaling. Our study suggests that integrity as well as dynamic motility of the actin cytoskeleton mediated by PI 3-K is required for IGF-I-induced FAK tyrosine phosphorylation, but not for ERK2 activation.     

Cytotoxic necrotizing factor 1 from Escherichia coli and dermonecrotic toxin from Bordetella bronchiseptica induce p21(rho)-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 cells

Treatment of Swiss 3T3 cells with cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli and dermonecrotic toxin (DNT) from Bordetella bronchiseptica, which directly target and activate p21(rho), stimulated tyrosine phosphorylation of focal adhesion kinase (p125(fak)) and paxillin. Tyrosine phosphorylation induced by CNF1 and DNT occurred after a pronounced lag period (2 h), and was blocked by either lysosomotrophic agents or incubation at 22 degrees C. CNF1 and DNT stimulated tyrosine phosphorylation of p125(fak) and paxillin, actin stress fiber formation, and focal adhesion assembly with similar kinetics. Cytochalasin D and high concentrations of platelet-derived growth factor disrupted the actin cytoskeleton and completely inhibited CNF1 and DNT induced tyrosine phosphorylation. Microinjection of Clostridium botulinum C3 exoenzyme which ADP-ribosylates and inactivates p21(rho) function, prevented tyrosine phosphorylation of focal adhesion proteins in response to either CNF1 or DNT. In addition, our results demonstrated that CNF1 and DNT do not induce protein kinase C activation, inositol phosphate formation, and Ca2+ mobilization. Moreover, CNF1 and DNT stimulated DNA synthesis without activation of p42(mapk) and p44(mapk) providing additional evidence for a novel p21(rho)-dependent signaling pathway that leads to entry into the S phase of the cell cycle in Swiss 3T3.     

NCAM140 interacts with the focal adhesion kinase p125(fak) and the SRC-related tyrosine kinase p59(fyn)

Axonal growth cones respond to adhesion molecules and extracellular matrix components by rapid morphological changes and growth rate modification. Neurite outgrowth mediated by the neural cell adhesion molecule (NCAM) requires the src family tyrosine kinase p59(fyn) in nerve growth cones, but the molecular basis for this interaction has not been defined. The NCAM140 isoform, which is found in migrating growth cones, selectively co-immunoprecipitated with p59(fyn) from nonionic detergent (Brij 96) extracts of early postnatal mouse cerebellum and transfected rat B35 neuroblastoma and COS-7 cells. p59(fyn) did not associate significantly with the NCAM180 isoform, which is found at sites of stable neural cell contacts, or with the glycophosphatidylinositol-linked NCAM120 isoform. pp60(c-)src, a tyrosine kinase that promotes neurite growth on the neuronal cell adhesion molecule L1, did not interact with any NCAM isoform. Whereas p59(fyn) was constitutively associated with NCAM140, the focal adhesion kinase p125(fak), a nonreceptor tyrosine kinase known to mediate integrin-dependent signaling, became recruited to the NCAM140-p59(fyn) complex when cells were reacted with antibodies against the extracellular region of NCAM. Treatment of cells with a soluble NCAM fusion protein or with NCAM antibodies caused a rapid and transient increase in tyrosine phosphorylation of p125(fak) and p59(fyn). These results suggest that NCAM140 binding interactions at the cell surface induce the assembly of a molecular complex of NCAM140, p125(fak), and p59(fyn) and activate the catalytic function of these tyrosine kinases, initiating a signaling cascade that may modulate growth cone migration.     

Serine and threonine phosphorylation of the paxillin LIM domains regulates paxillin focal adhesion localization and cell adhesion to fibronectin

We have previously shown that the LIM domains of paxillin operate as the focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to serve as binding sites for, and substrates of serine/threonine kinases. The activities of the LIM2- and LIM3-associated kinases were stimulated after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localization of paxillin after adhesion to fibronectin was investigated. An avian paxillin-CHO.K1 model system was used to explore the role of paxillin phosphorylation in paxillin localization to FAs. We found that mutations of paxillin that mimicked LIM domain phosphorylation accelerated fibronectin-induced localization of paxillin to focal contacts. Further, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation significantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was specific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in response to fibronectin adhesion had no effect on the rate of FA localization or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Additionally, these results provide the first evidence that paxillin contributes to "inside-out" integrin-mediated signal transduction.     

Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells

This study aimed at evaluating the influence of submaximal isometric contraction on pressure pain thresholds (PPTs) in 14 fibromyalgia (FM) patients and 14 healthy volunteers, before and after skin hypoesthesia. PPTs were determined with pressure algometry over m. quadriceps femoris before, during and following an isometric contraction. Maximum voluntary contraction (MVC) was assessed using a computerized dynamometer. A contraction of 22% MVC on average was held until exhaustion (max. 5 min) and PPTs were assessed every 30 sec. A local anesthetic cream and a control cream were applied following a double-blind design and PPTs were reassessed. In healthy volunteers PPTs increased during contraction (P < 0.001), then decreased after the end of contraction (P < 0.001) but remained above precontraction values during the 5 min of post-contraction assessments (P < 0.001). In FM patients PPTs decreased in the middle of the contraction period (P < 0.05) and remained below precontraction levels during the rest of the contraction period (P < 0.05) and during the 5 min of post-contraction assessment (immediately post-contraction NS; 2.5 min post-contraction P < 0.01; 5 min post-contraction P < 0.05). The normalized PPTs were significantly lower in patients than in controls during contraction (start P < 0.01; middle P < 0.001; end P < 0.001) and at all times during post-contraction assessments (P < 0.001). Anesthetic cream raised PPTs at rest in controls (P < 0.01) but not in FM patients, and did not influence contraction or post-contraction PPTs in either group. Therefore, the increased pressure pain sensibility in FM patients is more pronounced deep to the skin. The observed decrease of PPTs during isometric contraction in FM patients could be due to sensitization of mechanonociceptors caused by muscle ischemia and/or dysfunction in pain modulation during muscle contraction.     

Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase

The focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To gain insight into FAK function, we examined the potential interaction of FAK with intracellular signaling molecules containing the Src homology 2 domains. We report here the stable association of FAK with phosphatidylinositol 3-kinase (PI3-kinase; EC 2.7.1.137) in NIH 3T3 mouse fibroblasts. This interaction was stimulated by cell adhesion concomitant with FAK activation. We also found that recombinant FAK bound to the p85 subunit of PI 3-kinase directly in vitro and that autophosphorylation of recombinant FAK in vitro increased its binding to PI 3-kinase. We detected increased tyrosine phosphorylation of the p85 subunit of PI 3-kinase during cell adhesion and observed direct phosphorylation of p85 by FAK in vitro. Together, these results suggest that PI 3-kinase may be a FAK substrate in vivo and serve as an effector of FAK.     

Activation of human endothelial cells via S-endo-1 antigen (CD146) stimulates the tyrosine phosphorylation of focal adhesion kinase p125(FAK)

S-Endo-1 antigen (CD146), a transmembrane receptor also known as MUC18/MCAM, is a member of the immunoglobulin superfamily and belongs to a group of cell adhesion molecules. CD146 is highly expressed on the whole vascular tree. We demonstrate here that engagement of CD146 on human endothelial cells isolated from cord blood results in tyrosine phosphorylation of a large panel of cellular proteins, although no tyrosine phosphorylation of CD146 was detected. In particular, CD146 cross-linking induces the tyrosine phosphorylation of the protein tyrosine kinase p125(FAK) as well as p125(FAK) association with paxillin, both events being inhibited by cytochalasin D. No direct association of CD146 with p125(FAK) was observed. Consistent with these data, CD146 associates with p59(fyn), a Src family kinase known to phosphorylate p125(FAK). The identification of a signaling pathway initiated by CD146 engagement and which includes p59(fyn), p125(FAK), and paxillin indicates that CD146 participates in outside-in signaling in endothelial cells.     

Mechanism of down-regulation of c-kit receptor. Roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase, and protein kinase C

The receptor tyrosine kinase Kit and Kit ligand (KL), encoded at the murine white spotting (W) and steel (Sl) loci, respectively, function in hematopoiesis, melanogenesis, and gametogenesis. To understand the mechanism of turnover of Kit in mast cells, mutant receptors generated in vitro were heterologously expressed in Wsb/Wsh mast cells lacking endogenous c-kit expression, and the effects of mutations on KL-induced internalization and ubiquitination/degradation of Kit were studied. Upon binding of KL, KL.Kit receptor complexes were rapidly internalized, and the turnover was accelerated by ubiquitin-mediated degradation. Inactivation of the Kit kinase resulted in a reduced rate of internalization of KL.Kit complexes, degradation of kinase-inactive receptor complexes was relatively slow, and receptor ubiquitination was absent. But abolishment of KL-induced receptor association and activation of phosphatidylinositol 3'-kinase and of tyrosine 821 autophosphorylation did not affect KL-induced internalization and ubiquitination/degradation of Kit. Furthermore, Kit receptors can be down-regulated by proteolytic cleavage induced by either activation of protein kinase C or by isopropyl alcohol. In summary, KL-induced internalization of KL.Kit complexes and ubiquitination/degradation require an active kinase. By contrast, proteolytic cleavage of Kit mediated by protein kinase C activation is independent of kinase activity.     

Tyrosine phosphorylation of Crk-associated substrates by focal adhesion kinase. A putative mechanism for the integrin-mediated tyrosine phosphorylation of Crk-associated substrates

Integrin-ligand binding induces the tyrosine phosphorylation of various proteins including focal adhesion kinase (pp125(FAK)) and Crk-associated substrate (Cas). FAK is activated and autophosphorylated by the ligation of integrins, although the substrate of FAK has not been revealed. We show here that p130(Cas) and Cas-L are FAK substrates. FAK directly phosphorylates Cas proteins primarily at the YDYVHL sequence that is conserved among all Cas proteins. Furthermore, the phosphorylated YDYVHL sequence is a binding site for Src family protein-tyrosine kinases, and the recruited Src family kinase phosphorylates the other tyrosine residues within Cas. The Cas-L YDYVHL sequence is phosphorylated upon integrin-ligand binding, and this integrin-mediated tyrosine phosphorylation is inhibited by the cotransfection of the FAK COOH-terminal domain that does not contain a kinase domain. These findings strongly suggest that FAK initiates integrin-mediated tyrosine phosphorylation of Cas proteins; then, Src family tyrosine kinases, which are recruited to phosphorylated Cas and FAK, further phosphorylate Cas proteins.     

Activation of protein kinase C-zeta and phosphatidylinositol 3'-kinase and promotion of macrophage differentiation by insulin-like growth factor-I

Phosphoinositides that are phosphorylated at the D3 position have been reported to activate an atypical, Ca2-independent protein kinase C (PKC) isoform designated PKC-zeta, and overexpression of this enzyme leads to monocytic differentiation. In this study, we cultured human HL-60 promyeloid cells with vitamin D3 and insulin-like growth factor-I (IGF-I), a 70-amino-acid peptide that activates phosphatidylinositol 3'-kinase (PI 3-kinase) in murine promyeloid cells. Two days later, the proportion of cells differentiating into macrophages in serum-free medium, as assessed by expression of the alpha-subunit of the beta2 integrin CD11b, increased from 5 +/- 1% to 25 +/- 3%. Addition of IGF-I increased the proportion of cells differentiating into CD11b-positive macrophages to 78 +/- 5%. In the absence of vitamin D3, IGF-I did not induce expression of CD11b (6 +/- 1%). The IGF-I-promoted macrophage differentiation was blocked specifically by preincubation of HL-60 cells with a mAb (alphaIR3) directed against the IGF type I receptor. Similarly, pretreatment of cells with either alphaIR3 or an IGF-binding protein, IGFBP-3, led to a 75% inhibition of CD11b expression when cells were cultured with vitamin D3 in serum-containing medium. IGF-I, but not vitamin D3, caused a sevenfold increase in the enzymatic activity of both PI 3-kinase and atypical PKC-zeta. Inhibition of IGF-I-inducible PI 3-kinase with either wortmannin or LY294002 abrogated the IGF-I-induced activation of PKC-zeta and totally blocked the enhancement in macrophage differentiation caused by IGF-I. These data establish that PKC-zeta is a putative downstream target of PI 3-kinase that is activated during IGF-I-promoted macrophage differentiation.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

A transmembrane-anchored chimeric focal adhesion kinase is constitutively activated and phosphorylated at tyrosine residues identical to pp125FAK

Focal adhesion kinase, pp125FAK, is a nonmyristylated cytosolic tyrosine kinase unrelated to protein-tyrosine kinase families categorized to date. The kinase activity and tyrosine phosphorylation of pp125FAK are induced by beta 1 and beta 3 integrin-mediated cell adherence or aggregation. pp125FAK is also a tyrosine phosphorylation substrate in v-src-transformed cells and is localized to focal adhesion contracts of adherent fibroblasts and carcinoma cells. In this report, we have transiently expressed in COS cells a transmembrane-anchored chimeric receptor kinase, CD2FAK, consisting of CD2 and pp125FAK. We analyzed its kinase activity and tyrosine phosphorylation and compared to those of pp125FAK. We found that CD2FAK exhibited constitutive kinase activity and a high basal tyrosine phosphorylation level when COS transfectants were suspended in serum-free media. The kinase activity of CD2FAK was similarly up-regulated upon beta 1 integrin-mediated cell adherence as the endogenous pp125FAK. Both CD2FAK and pp125FAK appeared to be active as autophosphorylating kinases as shown by mutation of the ATP binding site. We determined the major tyrosine phosphorylation site, Tyr397, identical for both the constitutively activated CD2FAK and pp125FAK in response to beta 1 integrin-mediated cell adherence by site-directed mutagenesis. Deletions of the NH2- or the COOH-terminal noncatalytic domain of FAK, including Tyr397 did not lead to abolition of the kinase activity of pp125FAK or CD2FAK. Taken together, CD2FAK exhibits properties of an activated pp125FAK and the kinase activity does not appear to require tyrosine phosphorylation in vitro or in vivo.