Clostridium difficile toxin A binds colonocyte Src causing dephosphorylation of focal adhesion kinase and paxillin

Clostridium difficile toxin A impairs tight junction function of colonocytes by glucosylation of Rho family proteins causing actin filament disaggregation and cell rounding. We investigated the effect of toxin A on focal contact formation by assessing its action on focal adhesion kinase (FAK) and the adapter protein paxillin. Exposure of NCM460 human colonocytes to toxin A for 1 h resulted in complete dephosphorylation of FAK and paxillin, while protein tyrosine phosphatase activity was reduced. Blockage of toxin A-associated glucosyltransferase activity by co-incubation with UDP-2′3′ dialdehyde did not reduce toxin A-induced FAK and paxillin dephosphorylation. GST-pull down and in vitro kinase activity experiments demonstrated toxin A binding directly to the catalytic domain of Src with suppression of its kinase activity. Direct binding of toxin A to Src, independent of any effect on protein tyrosine phosphatase or Rho glucosylation, inhibits Src kinase activity followed by FAK/paxillin inactivation. These mechanisms may contribute to toxin A inhibition of colonocyte focal adhesion that occurs in human colonic epithelium exposed to toxin A.     

Mechanosensitive tyrosine phosphorylation of paxillin and focal adhesion kinase in tracheal smooth muscle

We investigatedthe role of the integrin-associated proteins focal adhesion kinase(FAK) and paxillin as mediators of mechanosensitive signal transductionin tracheal smooth muscle. In muscle strips contracted isometricallywith ACh, we observed higher levels of tyrosine phosphorylation of FAKand paxillin at the optimal muscle length(L_o) than atshorter muscle lengths of 0.5 or 0.75 L_o. Paxillinphosphorylation was also length sensitive in muscles activated byK⁺ depolarization and adjustedrapidly to changes in muscle length imposed after contractileactivation by either ACh or K⁺depolarization. Ca²⁺ depletion didnot affect the length sensitivity of paxillin and FAK phosphorylationin muscles activated with ACh, indicating that the mechanotransductionprocess can be mediated by aCa²⁺-independent pathway. SinceCa²⁺-depleted muscles do notgenerate significant active tension, this suggests that themechanotransduction mechanism is sensitive to muscle length rather thantension. We conclude that FAK and paxillin participate in anintegrin-mediated mechanotransduction process in tracheal smoothmuscle. We propose that this pathway may initiate alterations in smoothmuscle cell structure and contractility via the remodeling of actinfilaments and/or via the mechanosensitive regulation ofsignaling molecules involved in contractile protein activation.

     

Protein tyrosine phosphatase-dependent proteolysis of focal adhesion complexes in endothelial cell apoptosis

Adenosine and/or homocysteine causes endothelial cell apoptosis, a mechanism requiring protein tyrosine phosphatase (PTPase) activity. We investigated the role of focal adhesion contact disruption in adenosine-homocysteine endothelial cell apoptosis. Analysis of focal adhesion kinase (FAK), paxillin, and vinculin demonstrated disruption of focal adhesion complexes after 4 h of treatment with adenosine-homocysteine followed by caspase-induced proteolysis of FAK, paxillin, and p130(CAS). No significant changes were noted in tyrosine phosphorylation of FAK or paxillin. Pretreatment with the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone prevented adenosine-homocysteine-induced DNA fragmentation and FAK, paxillin, and p130(CAS) proteolysis. Asp-Glu-Val-Asp-ase activity was detectable in endothelial cells after 4 h of treatment with adenosine-homocysteine. The PTPase inhibitor sodium orthovanadate did not prevent endothelial cell retraction or FAK, paxillin, or vinculin redistribution. Sodium orthovanadate did block adenosine-homocysteine-induced FAK, paxillin, and p130(CAS) proteolysis and Asp-Glu-Val-Asp-ase activity. Thus disruption of focal adhesion contacts and caspase-induced degradation of focal adhesion contact proteins occurs in adenosine-homocysteine endothelial cell apoptosis. Focal adhesion contact disruption induced by adenosine-homocysteine is independent of PTPase or caspase activation. These studies demonstrate that disruption of focal adhesion contacts is an early, but not an irrevocable, event in endothelial cell apoptosis.     

PKC-regulated myogenesis is associated with increased tyrosine phosphorylation of FAK,Cas, and paxillin,formation of Cas-CRK complex,and JNK activation

Previous reports suggest that PKC plays an important role in regulating myogenesis. However, the regulatory signaling pathways are not fully understood. We examined the effects of PKC downregulation on signaling events during skeletal muscle differentiation. We found that downregulation of PKC results in increased myogenesis in C2C12 cells as measured by creatine kinase activity and myogenin expression. We showed that, during differentiation, downregulation of PKC expression results in increased tyrosine phosphorylation of FAK, Cas, and paxillin, concomitant with enhanced Cas-CrkII complex formation, which leads to activation of JNK2. But in proliferated muscle cells, PKC inhibition results in FAK and Cas tyrosine dephosphorylation. Further, disruption of actin cytoskeleton by cytochalasin D prevents the activation of FAK and Cas as well as the formation of Cas-CrkII complex stimulated by PKC downregulation during muscle cell differentiation. Finally, we observed that PKC downregulation increases the tyrosine phosphorylation of focal adhesion associated proteins. Based on the above data, we propose that PKC downregulation results in enhanced tyrosine phosphorylation of FAK, Cas, and paxillin, thus promoting the establishment of Cas-CrkII complex, leading to activation of JNK and that these interactions are dependent upon the integrity of actin cytoskeleton during muscle cell differentiation. Data presented here significantly contribute to elucidating the regulatory role of PKC in myogenesis possibly through integrin signaling pathway.     

Hsp72 interacts with paxillin and facilitates the reassembly of focal adhesions during recovery from ATP depletion

The cytoprotective effect of heat stress proteins on epithelial cell detachment, an important cause of acute, ischemic renal failure, was examined after ATP depletion by evaluating focal adhesion complex (FAC) integrity. The intracellular distribution of FAC proteins (paxillin, talin, and vinculin) was assessed by immunohistochemistry before, during, and after exposure of renal epithelial cells to metabolic inhibitors. The resulting ATP depletion caused reversible re-distribution of all three proteins from focal adhesions to the cytosol. Paxillin, a key adaptor protein, was selected as a surrogate marker for FAC integrity in subsequent studies. Prior heat stress increased hsp72, a molecular chaperone, in both the Triton X-100-soluble and -insoluble protein fractions. Compared with ATP depleted control, heat stress significantly decreased paxillin and hsp72 shift from the Triton X-100 soluble to the insoluble protein fraction (an established marker of denaturation and aggregation); increased paxillin-hsp72 interaction detected by co-immunoprecipitation; enhanced paxillin extractability from Triton X-100-insoluble precipitates, increased the reformation of focal adhesions, and improved cell attachment (p < 0.05). To determine whether hsp72 mediates protection afforded by heat stress, cells were infected with adenovirus containing human hsp72 or empty vector. Hsp72 overexpression increased its interaction with paxillin and improved focal adhesion reformation during recovery, mimicking the protective effects of heat stress. These data suggest that hsp72 facilitates the reassembly of focal adhesions and improves cell attachment by reducing paxillin denaturation and increasing its re-solubilization after ATP depletion.     

Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane

Cytoskeletal reorganization of the smooth muscle cell in response to contractile stimulation may be an important fundamental process in regulation of tension development. We used confocal microscopy to analyze the effects of cholinergic stimulation on localization of the cytoskeletal proteins vinculin, paxillin, talin and focal adhesion kinase (FAK) in freshly dissociated tracheal smooth muscle cells. All four proteins were localized at the membrane and throughout the cytoplasm of unstimulated cells, but their concentration at the membrane was greater in acetylcholine (ACh)-stimulated cells. Antisense oligonucleotides were introduced into tracheal smooth muscle tissues to deplete paxillin protein, which also inhibited contraction in response to ACh. In cells dissociated from paxillin-depleted muscle tissues, redistribution of vinculin to the membrane in response to ACh was prevented, but redistribution of FAK and talin was not inhibited. Muscle tissues were transfected with plasmids encoding a paxillin mutant containing a deletion of the LIM3 domain (paxillin LIM3 dl 444–494), the primary determinant for targeting paxillin to focal adhesions. Expression of paxillin LIM3 dl in muscle tissues also inhibited contractile force and prevented cellular redistribution of paxillin and vinculin to the membrane in response to ACh, but paxillin LIM3 dl did not inhibit increases in intracellular Ca²⁺ or myosin light chain phosphorylation. Our results demonstrate that recruitment of paxillin and vinculin to smooth muscle membrane is necessary for tension development and that recruitment of vinculin to the membrane is regulated by paxillin. Vinculin and paxillin may participate in regulating the formation of linkages between the cytoskeleton and integrin proteins that mediate tension transmission between the contractile apparatus and the extracellular matrix during smooth muscle contraction. tissue transfection; plasmids; cytoskeleton; talin; immunofluorescence     

Y-27632, an Inhibitor of Rho-Associated Kinases, Prevents Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin Induced by Bombesin: Dissociation from Tyrosine Phosphorylation of p130

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.     

Y-27632, an inhibitor of Rho-associated kinases, prevents tyrosine phosphorylation of focal adhesion kinase and paxillin induced by bombesin: dissociation from tyrosine phosphorylation of p130(CAS)

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.     

Paxillin phosphorylation and complexing with Erk and FAK are regulated by PLD activity in MDA-MB-231 cells

MDA-MB-231 cells are highly aggressive human breast adenocarcinoma cells that depend on PLD activity for survival. In response to the stress of serum withdrawal, there is increased motility and invasiveness of these cells that is associated with a rapid increase in PLD activity. In addition, PLD activity is elevated in response to most mitogenic signals. Similar to PLD, paxillin, a focal adhesion adaptor protein, and Erk, mitogen-activated protein kinase, play vital roles in cell motility through regulation of focal adhesion dynamics. Here, we addressed whether there is a functional correlation between paxillin and PLD that may influence cancer cell motility. We investigated the role of PLD activity on paxillin regulation, Erk activation and formation of a paxillin-Erk and paxillin-FAK association. Inhibition of PLD activity led to an increase in paxillin tyrosine phosphorylation, a decrease in Erk activation, as measured by phosphorylation, and enhanced association of paxillin with Erk. In addition, we found that paxillin tyrosine phosphorylation depends upon Erk activity and may be a consequence of an increased association with FAK. Taken together, these results suggest that Erk activity is governed by PLD activity and regulates the tyrosine phosphorylation of paxillin, potentially explaining its role in cell motility. This study indicated that PLD, Erk, paxillin and FAK participate in the same signaling pathway in this breast cancer cell line.     

Signaling transduction pathway of angiotensin II in human mesangial cells: mediation of focal adhesion and GTPase activating proteins

Human mesangial cells (HMCs) respond to angiotensin II stimulation, which modulates their physiological activities, i.e., contraction and proliferation. It has been revealed that focal adhesion kinase (FAK) and paxillin participate in the angiotensin II-mediated signaling and cytoskeletal rearrangements at focal adhesion. We investigated the influences of cell adhesion upon angiotensin II effects in HMCs. In adherent cells, both FAK and paxillin were tyrosine phosphorylated by angiotensin II, while the cell detachment completely inhibited the tyrosine phosphorylation of paxillin. Activation of p44/42 mitogen-activated protein (MAP) kinase by angiotensin II was accentuated in suspended cells. Moreover, p190, a member of Rho GTPase activating protein (GAP), and RasGAP were coprecipitated with paxillin in adherent cells and angiotensin II stimulation reduced the formation of paxillin-p190 and paxillin-RasGAP complexes. These results suggest that the formation of focal adhesion complexes accelerated by accumulation of mesangial matrices may inhibit the proliferation of HMCs by modulating MAP kinase activity and be related to mesangial cell depletion.     

Cross-correlated fluctuation analysis reveals phosphorylation-regulated paxillin-FAK complexes in nascent adhesions

We used correlation methods to detect and quantify interactions between paxillin and focal adhesion kinase (FAK) in migrating cells. Cross-correlation raster-scan image correlation spectroscopy revealed that wild-type paxillin and the phosphorylation-inhibiting paxillin mutant Y31F-Y118F do not interact with FAK in the cytosol but a phosphomimetic mutant of paxillin, Y31E-Y118E, does. By extending cross-correlation number and brightness analysis to the total internal reflection fluorescence modality, we were able to show that tetramers of paxillin and FAK form complexes in nascent adhesions with a 1:1 stoichiometry ratio. The phosphomimetic mutations on paxillin increase the size of the complex and the assembly rate of nascent adhesions, suggesting that the physical molecular aggregation of paxillin and FAK regulates adhesion formation. In contrast, when phosphorylation is inhibited, the interaction decreases and the adhesions tend to elongate rather than turn over. These direct in vivo data show that the phosphorylation of paxillin is specific to adhesions and leads to localized complex formation with FAK to regulate the dynamics of nascent adhesions.     

Cyclic strain promotes shuttling of PYK2/Hic-5 complex from focal contacts in osteoblast-like cells

We showed that cyclic strain (CS) of osteoblastic cells induced tyrosine phosphorylation of two homologous tyrosine kinases FAK and PYK2, and of two homologous adaptor proteins paxillin and Hic5, with similar kinetics. Immunostaining showed that all four proteins were localized to focal contacts in controls. In contrast, the dynamics of their subcellular localization observed after CS differed. While FAK and paxillin remained at the focal contact, Hic-5 and PYK2 translocated outside ventral focal contacts as early as 30 min after CS and were sequestered by the cytoskeleton. Co-immunoprecipitation showed that the association of PYK2/Hic-5 and PYK2/FAK increased with time after strain while that of paxillin and Hic-5 decreased. Altogether these results suggested that CS regulates focal contact activity in osteoblasts by modulating PYK2-containing complexes in particular by shuttling out of the focal contact the adaptor Hic-5 and favoring the anchorage of FAK within contacts.     

Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells

The relationship between focal adhesion protein (FAK) activity and loss of cell-matrix contact during apoptosis is not entirely clear nor has the role of FAK in chemically induced apoptosis been studied. We investigated the status of FAK phosphorylation and cleavage in renal epithelial cells during apoptosis caused by the nephrotoxicant dichlorovinylcysteine (DCVC). DCVC treatment caused a loss of cell-matrix contact which was preceded by a dissociation of FAK from the focal adhesions and tyrosine dephosphorylation of FAK. Paxillin was also dephosphorylated at tyrosine. DCVC treatment activated caspase-3 which was associated with cleavage of FAK. However, FAK cleavage occurred after cells had already lost focal adhesions indicating that cleavage of FAK by caspases is not responsible for loss of FAK from focal adhesions. Accordingly, although inhibition of caspase activity with zVAD-fmk blocked activation of caspase-3, FAK cleavage, and apoptosis, it neither affected dephosphorylation nor translocation of FAK or paxillin. However, zVAD-fmk completely blocked the cell detachment caused by DCVC treatment. Orthovanadate prevented DCVC-induced tyrosine dephosphorylation of both FAK and paxillin; however, it did not inhibit DCVC-induced apoptosis and actually potentiated focal adhesion disorganization and cell detachment. Thus, FAK dephosphorylation and loss of focal adhesions are not due to caspase activation; however, caspases are required for FAK proteolysis and cell detachment.     

Expression of fibroblast growth factor family during postnatal skeletal muscle hypertrophy

The potential role of the fibroblast growth factor (FGF) familyduring stretch-induced postnatal skeletal muscle hypertrophy wasanalyzed by using an avian wing-weighting model. After 2 or 11 days ofweighted stretch, anterior latissimus dorsi (ALD) muscles were, onaverage, 34 (P < 0.01) and 85%(P < 0.01) larger, respectively, than unweighted ALD control muscles. By using quantitative RT-PCR, FGF-1 mRNA expression was found to be significantly decreased in ALDmuscles stretched for 2 or 11 days. In contrast, FGF-4 and FGF-10 mRNAexpression was significantly increased 2 days after initiation ofstretch. FGF-2, FGF-10, fibroblast growth factor receptor 1, andFREK mRNA expression was significantly increased at 11 days poststretch. Increases in FGF-2 and FGF-4 protein could bedetected throughout the myofiber periphery after 11 days of stretch. Ona cellular level, FGF-2 and FGF-4 proteins were differentiallylocalized. This differential expression pattern and proteinlocalization of the FGF family in response to stretch-induced hypertrophy suggest distinct roles for individual FGFs during thepostnatal hypertrophy process.

     

Insulin stimulates spreading of skeletal muscle cells involving the activation of focal adhesion kinase,phosphatidylinositol 3-kinase and extracellular signal regulated kinases

Insulin plays an important role in muscle cell survival and proliferation. However, there is no report showing the role of insulin in spreading of muscle cells. In the present report, we showed that insulin enhances muscle cell spreading concomitant with enhanced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Moreover, insulin can stimulate the cell spreading even in presence of integrin alpha5 blockers although to a lesser extent as compared to control. Cell adhesion was not dependent on insulin and serum, and decreased in presence of integrin blockers. We found direct association of FAK with affinity purified insulin receptors using in vitro kinase assay. The increase in FAK tyrosine phosphorylation was associated with increase in its kinase activity and further supported by increased phosphotyrosine accumulation on focal adhesions and increased membrane localization of FAK after stimulation by insulin. Moreover, insulin-mediated muscle cell spreading was dependent upon phosphatidylinositol 3-kinase (PI 3-kinase) activity. PI 3-kinase activity was found to be associated with FAK and the FAK associated PI 3-kinase activity enhanced when cells were plated in presence of insulin. We also observed activation of MAP kinases, i.e., ERK-1/-2 during insulin mediated muscle cell spreading. In conclusion, FAK, PI 3-kinase, and MAP kinase are important components of pathway(s) that regulate insulin stimulated muscle cell spreading.     

Subcellular responses of p53 and Id2 in fast and slow skeletal muscle in response to stretch-induced overload.

Tumor suppressor p53 and inhibitor of DNA-binding/differentiation Id2 were examined after 7 or 21 days of wing weighting in fast patagialis (PAT) and slow anterior latissimus dorsi (ALD) wing muscles of young adult and old Japanese quails. The contralateral wing served as the intra-animal control. Seven days of loading increased PAT and ALD muscle weight by 28 and 96%, respectively, in young birds. PAT and ALD muscle weight was 49 and 179% greater, respectively, than control muscles after 21 days of loading in young birds. In aged birds, no PAT or ALD hypertrophy was found after 7 days of loading; however, PAT and ALD muscle weight increased by 29 and 102%, respectively, after 21 days of loading. Id2 protein in the nuclear muscle fraction increased in both PAT and ALD muscles from young adult and old birds that were loaded for 7 days and in ALD muscles after 21 days of loading relative to contralateral control muscles. Nuclear p53 protein was greater in 7- or 21-day loaded PAT and ALD muscles relative to control muscles in both age groups. Cytosolic Id2 and p53 protein contents were not changed in loaded PAT or ALD muscles relative to control muscles at any time point. These data suggest that nuclear, but not cytosolic, Id2 and p53 are responsive to stretch-induced muscle overload. Moreover, the attenuated ability of the aged skeletal muscle to achieve hypertrophy does not appear to be explained by the subcellular changes in Id2 and p53 content with overload.     

Paxillin modulates squamous cancer cell adhesion and is important in pressure-augmented adhesion

Paxillin is an adapter protein regulating signaling and focal adhesion assembly that has been linked to malignant potential in many malignancies. Overexpression of paxillin has been noted in aggressive tumors. Integrin-mediated binding through the focal adhesion complex is important in metastatic adhesion and is upregulated by extracellular pressure in malignant colonocytes through FAK and Src activation. Neither head and neck cancers nor paxillin have been studied in this regard. We hypothesized that paxillin would play a role in modulating squamous cancer adhesion both at baseline and under conditions of increased extracellular pressure. Using SCC25 tongue squamous cancer cells stably transfected with either an empty selection vector or paxillin expression and selection vectors, we studied adhesion to collagen, paxillin, FAK, and Src expression and phosphorylation in cells maintained for 30 min under ambient or 15 mmHg increased pressure conditions. Paxillin-overexpressing cells exhibited adhesion 121 +/- 2.9% of that observed in vector-only cells (n = 6, P < 0.001) under ambient pressure. Paxillin-overexpression reduced FAK phosphorylation. Pressure stimulated adhesion to 118 +/- 2.3% (n = 6, P < 0.001) of baseline in vector-only cells, similar to its effect in the parental line, and induced paxillin, FAK, and Src phosphorylation. However, increased pressure did not stimulate adhesion or phosphorylate paxillin, FAK, or Src further in paxillin-overexpressing cells. Metastasizing squamous cancer cell adhesiveness may be increased by paxillin-overexpression or by paxillin activation by extracellular pressure during surgical manipulation or growth within a constraining compartment. Targeting paxillin in patients with malignancy and minimal tumor manipulation during surgical resection may be important therapeutic adjuncts.     

Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding

Paxillin is a 68-kD focal adhesion phosphoprotein that interacts with several proteins including members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal proteins vinculin and the tyrosine kinase, focal adhesion kinase (FAK). This suggests a function for paxillin as a molecular adaptor, responsible for the recruitment of structural and signaling molecules to focal adhesions. The current study defines the vinculin- and FAK-interaction domains on paxillin and identifies the principal paxillin focal adhesion targeting motif. Using truncation and deletion mutagenesis, we have localized the vinculin-binding site on paxillin to a contiguous stretch of 21 amino acids spanning residues 143-164. In contrast, maximal binding of FAK to paxillin requires, in addition to the region of paxillin spanning amino acids 143-164, a carboxyl-terminal domain encompassing residues 265-313. These data demonstrate the presence of a single binding site for vinculin, and at least two binding sites for FAK that are separated by an intervening stretch of 100 amino acids. Vinculin- and FAK-binding activities within amino acids 143-164 were separable since mutation of amino acid 151 from a negatively charged glutamic acid to the uncharged polar residue glutamine (E151Q) reduced binding of vinculin to paxillin by >90%, with no reduction in the binding capacity for FAK. The requirement for focal adhesion targeting of the vinculin- and FAK-binding regions within paxillin was determined by transfection into CHO.K1 fibroblasts. Significantly and surprisingly, paxillin constructs containing both deletion and point mutations that abrogate binding of FAK and/or vinculin were found to target effectively to focal adhesions. Additionally, expression of the amino-terminal 313 amino acids of paxillin containing intact vinculin- and FAK-binding domains failed to target to focal adhesions. This indicated other regions of paxillin were functioning as focal adhesion localization motifs. The carboxyl-terminal half of paxillin (amino acids 313-559) contains four contiguous double zinc finger LIM domains. Transfection analyses of sequential carboxyl-terminal truncations of the four individual LIM motifs and site-directed mutagenesis of LIM domains 1, 2, and 3, as well as deletion mutagenesis, revealed that the principal mechanism of targeting paxillin to focal adhesions is through LIM3. These data demonstrate that paxillin localizes to focal adhesions independent of interactions with vinculin and/or FAK, and represents the first definitive demonstration of LIM domains functioning as a primary determinant of protein subcellular localization to focal adhesions.     

Dissociation of focal adhesion kinase and paxillin tyrosine phosphorylation induced by bombesin and lysophosphatidic acid from epidermal growth factor receptor transactivation in Swiss 3T3 cells

Tyrosine phosphorylation of the nonreceptor tyrosine kinase p125 focal adhesion kinase (FAK) and the adapter protein paxillin is rapidly increased by multiple agonists, including bombesin (BOM) and lysophosphatidic acid (LPA), through heptahelical G protein-coupled receptors (GPCRs). The pathways involved remain incompletely understood. The experiments presented here were designed to test the role of epidermal growth factor receptor (EGFR) transactivation in the rapid increase of tyrosine phosphorylation of FAK and paxillin induced by GPCR agonists. Our results show that treatment with the selective EGFR tyrosine kinase inhibitor AG 1478, at concentrations that completely blocked the increase in tyrosine phosphorylation of these proteins induced by EGF, did not affect the stimulation of tyrosine phosphorylation of either FAK or paxillin induced by multiple GPCR agonists including LPA, BOM, vasopressin, bradykinin, and endothelin. Similar results were obtained when Swiss 3T3 cells were treated with another highly specific inhibitor of the EGF receptor kinase activity, PD-158780. Collectively, our results clearly dissociate EGFR transactivation from the tyrosine phosphorylation of FAK and paxillin induced by multiple GPCR agonists.