Leupaxin is similar to paxillin in focal adhesion targeting and tyrosine phosphorylation but has distinct roles in cell adhesion and spreading

Focal adhesion (FA) formation is induced by extracellular matrix-stimulated integrin clustering and activation of receptors for diffusible factors. Leupaxin (LPXN) is a member of the paxillin family of FA proteins expressed in many cancer cell lines. We found activation of gastrin-releasing peptide receptor (GRPr) by bombesin (BN) stimulated LPXN translocation from cytoplasm to FAs. Using mutagenesis, we identified LIM3 as the primary FA targeting domain for LPXN and showed BN-induced LPXN tyrosine phosphorylation on residues 22, 62 and 72. A LIM3 point mutant of LPXN failed to target to FAs and had no BN-stimulated tyrosine phosphorylation. Conversely, a non-phosphorylatable mutant (Y22/62/72F) translocated to FAs after BN addition. Stimulation of FA formation using vinblastine also induced LPXN translocation and tyrosine phosphorylation. Therefore, dynamic LPXN tyrosine phosphorylation requires translocation to FAs. LPXN and paxillin had opposite roles in adhesion to collagen I (CNI) in MDA-MB-231 breast cancer cells. LPXN siRNA stimulated whereas paxillin siRNA inhibited cell adhesion. Knockdown of both LPXN and paxillin behaved similarly to paxillin knockdown alone, suggesting LPXN''s function in adhesion might depend on paxillin. Additionally, LPXN regulated cell spreading on CNI but not on fibronectin whereas paxillin knockdown suppressed spreading on both substrates. These results demonstrate that although LPXN and paxillin''s FA targeting and tyrosine phosphorylation are similar, each protein has distinct functions.Key words: focal adhesion, tyrosine phosphorylation, bombesin, adhesion, spreading     

Roles for the tubulin- and PTP-PEST-binding paxillin LIM domains in cell adhesion and motility

Cell dynamics mediated through cell-extracellular matrix contacts, such as adhesion and motility involve the precise regulation of large complexes of structural and signaling molecules called focal adhesions (FAs). Paxillin is a multi-domain FA adaptor protein containing five amino-terminal paxillin leucine-aspartate repeat (LD) motifs and four carboxyl-terminal Lin-11 Isl-1 and Mec-3 (LIM) domains. The LD motifs support paxillin binding to actopaxin, integrin linked kinase (ILK), FA kinase (FAK), paxillin kinase linker (PKL) and vinculin. Of the LIM domains, LIM2 and 3 comprise the paxillin FA-targeting motif, with phosphorylation of these domains modulating paxillin targeting and cell adhesion to fibronectin (Fn). The identity of the paxillin FA targeting partner remains to be determined; however, the LIM domains mediate interactions with tubulin and the protein-tyrosine phosphatase (PTP)-PEST. PTP-PEST binding requires both LIM3 and 4, whereas, the precise LIM target of tubulin binding is not known. In this report, we demonstrate that the individual paxillin LIM2 and 3 domains support specific binding to tubulin and suggest a potential role for this interaction in the regulation of paxillin sub-cellular compartmentalization. In addition, expression of paxillin molecules with mutations in the tubulin- and PTP-PEST-binding LIM domains differentially impaired Chinese hamster ovary K1 (CHO.K1) cell adhesion and migration to Fn. Perturbation of LIM3 or 4 inhibited adhesion while mutation of LIM2 or 4 decreased cell motility. Interestingly, expression of tandem LIM2-3 inhibited cell adhesion and spreading while LIM3-4 stimulated a well-spread polarized phenotype. These data offer further support for a critical role for paxillin in cell adhesion and motility.     

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.     

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.     

FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion

 Cell migration is a highly complex process that requires the coordinated formation of membrane protrusion and focal adhesions (FAs). Focal adhesion kinase (FAK), a major signaling component of FAs, is involved in the disassembly process of FAs through phosphorylation and dephosphorylation of its tyrosine residues, but the role of such phosphorylations in nascent FA formation and turnover near the cell front and in cell protrusion is less well understood. In the present study, we demonstrate that, depending on the phosphorylation status of Tyr-925 residue, FAK modulates cell migration via two specific mechanisms. FAK^−/− mouse embryonic fibroblasts (MEFs) expressing nonphosphorylatable Y925F-FAK show increased interactions between FAK and unphosphorylated paxillin, which lead to FA stabilization and thus decreased FA turnover and reduced cell migration. Conversely, MEFs expressing phosphomimetic Y925E-FAK display unchanged FA disassembly rates, show increase in phosphorylated paxillin in FAs, and exhibit increased formation of nascent FAs at the cell leading edges. Moreover, Y925E-FAK cells present enhanced cell protrusion together with activation of the p130^CAS/Dock180/Rac1 signaling pathway. Together, our results demonstrate that phosphorylation of FAK at Tyr-925 is required for FAK-mediated cell migration and cell protrusion.     

Paxillin enables attachment-independent tyrosine phosphorylation of focal adhesion kinase and transformation by RAS

Paxillin and HIC5 are closely related adapter proteins that regulate cell migration and are tyrosine-phosphorylated by focal adhesion kinase (FAK). Paxillin, HIC5, and FAK tyrosine phosphorylation increase upon cell attachment and decrease upon detachment from extracellular matrix. Unexpectedly, we found that although FAK tyrosine phosphorylation in attached cells did not require paxillin, in detached fibroblasts there was remaining FAK tyrosine phosphorylation that required expression of paxillin and was not supported by HIC5. The support of attachment-independent FAK tyrosine phosphorylation required the paxillin LIM domains and suggested that paxillin might facilitate oncogenic transformation. Paxillin but not HIC5 augmented anchorage-independent cell proliferation induced by RAS. Both anchorage-independent FAK tyrosine phosphorylation and RAS-induced colony formation required multiple docking sites on paxillin, including LD4 (docking sites for FAK-Src and GIT1/2-PIX-NCK-PAK complex), LD5, and all four carboxyl-terminal LIM domains (that bind tubulin and PTP-PEST). Analysis using paxillin mutants dissociated domains of paxillin that are required for regulation of cell migration from domains that are required for anchorage-independent cell proliferation and demonstrated essential functions of the paxillin LIM domains that are not found in HIC5 LIM domains. These results highlight the role of paxillin in facilitating attachment-independent signal transduction implicated in cancer.     

Kindlin Binds Migfilin Tandem LIM Domains and Regulates Migfilin Focal Adhesion Localization and Recruitment Dynamics

Focal adhesions (FAs), sites of tight adhesion to the extracellular matrix, are composed of clusters of transmembrane integrin adhesion receptors and intracellular proteins that link integrins to the actin cytoskeleton and signaling pathways. Two integrin-binding proteins present in FAs, kindlin-1 and kindlin-2, are important for integrin activation, FA formation, and signaling. Migfilin, originally identified in a yeast two-hybrid screen for kindlin-2-interacting proteins, is a LIM domain-containing adaptor protein found in FAs and implicated in control of cell adhesion, spreading, and migration. By binding filamin, migfilin provides a link between kindlin and the actin cytoskeleton. Here, using a combination of kindlin knockdown, biochemical pulldown assays, fluorescence microscopy, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP), we have established that the C-terminal LIM domains of migfilin dictate its FA localization, shown that these domains mediate an interaction with kindlin in vitro and in cells, and demonstrated that kindlin is important for normal migfilin dynamics in cells. We also show that when the C-terminal LIM domain region is deleted, then the N-terminal filamin-binding region of the protein, which is capable of targeting migfilin to actin-rich stress fibers, is the predominant driver of migfilin localization. Our work details a correlation between migfilin domains that drive kindlin binding and those that drive FA localization as well as a kindlin dependence on migfilin FA recruitment and mobility. We therefore suggest that the kindlin interaction with migfilin LIM domains drives migfilin FA recruitment, localization, and mobility.     

Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation

Focal adhesions (FAs) are mechanosensitive adhesion and signaling complexes that grow and change composition in response to myosin II–mediated cytoskeletal tension in a process known as FA maturation. To understand tension-mediated FA maturation, we sought to identify proteins that are recruited to FAs in a myosin II–dependent manner and to examine the mechanism for their myosin II–sensitive FA association. We find that FA recruitment of both the cytoskeletal adapter protein vinculin and the tyrosine kinase FA kinase (FAK) are myosin II and extracellular matrix (ECM) stiffness dependent. Myosin II activity promotes FAK/Src-mediated phosphorylation of paxillin on tyrosines 31 and 118 and vinculin association with paxillin. We show that phosphomimic mutations of paxillin can specifically induce the recruitment of vinculin to adhesions independent of myosin II activity. These results reveal an important role for paxillin in adhesion mechanosensing via myosin II–mediated FAK phosphorylation of paxillin that promotes vinculin FA recruitment to reinforce the cytoskeletal ECM linkage and drive FA maturation.     

Rsu1 contributes to regulation of cell adhesion and spreading by PINCH1-dependent and - independent mechanisms

Cell adhesion and migration are complex processes that require integrin activation, the formation and dissolution of focal adhesion (FAs), and linkage of actin cytoskeleton to the FAs. The IPP (ILK, PINCH, Parvin) complex regulates FA formation via binding of the adaptor protein ILK to β1 integrin, PINCH and parvin. The signaling protein Rsu1 is linked to the complex via binding PINCH1. The role of Rsu1 and PINCH1 in adhesion and migration was examined in non-transformed mammary epithelial cells. Confocal microscopy revealed that the depletion of either Rsu1 or PINCH1 by siRNA in MCF10A cells decreased the number of focal adhesions and altered the distribution and localization of β1 integrin, vinculin, talin and paxillin without affecting the levels of FA protein expression. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. In addition, constitutive phosphorylation of actin regulatory proteins occurred in the absence of PINCH1. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function by regulating levels of PINCH1. However, while both Rsu1- or PINCH1-depleted cells retained the ability to activate adhesion signaling in response to EGF stimulation, only Rsu1 was required for EGF-induced p38 Map Kinase phosphorylation and ATF2 activation, suggesting an Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with an Rsu1 mutant that does not bind to PINCH1 failed to restore FAs or migration but did promote spreading and constitutive p38 activation. These data show that Rsu1-PINCH1 association with ILK and the IPP complex is required for regulation of adhesion and migration but that Rsu1 has a critical role in linking integrin-induced adhesion to activation of p38 Map kinase signaling and cell spreading. Moreover, it suggests that Rsu1 may regulate p38 signaling from the IPP complex affecting other functions including survival.     

Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness

The ArfGAP paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT)2 has been implicated in regulating cell spreading and motility through its transient recruitment of the p21-activated kinase (PAK) to focal adhesions. The Nck-PAK-PIX-PKL protein complex is recruited to focal adhesions by paxillin upon integrin engagement and Rac activation. In this report, we identify tyrosine-phosphorylated PKL as a protein that associates with the SH3-SH2 adaptor Nck, in a Src-dependent manner, after cell adhesion to fibronectin. Both cell adhesion and Rac activation stimulated PKL tyrosine phosphorylation. PKL is phosphorylated on tyrosine residues 286/392/592 by Src and/or FAK and these sites are required for PKL localization to focal adhesions and for paxillin binding. The absence of either FAK or Src-family kinases prevents PKL phosphorylation and suppresses localization of PKL but not GIT1 to focal adhesions after Rac activation. Expression of an activated FAK mutant in the absence of Src-family kinases partially restores PKL localization, suggesting that Src activation of FAK is required for PKL phosphorylation and localization. Overexpression of the nonphosphorylated GFP-PKL Triple YF mutant stimulates cell spreading and protrusiveness, similar to overexpression of a paxillin mutant that does not bind PKL, suggesting that failure to recruit PKL to focal adhesions interferes with normal cell spreading and motility.     

Paxillin-beta-catenin interactions are involved in Rac/Cdc42-mediated endothelial barrier-protective response to oxidized phospholipids

Oxidized phospholipids may appear in the pulmonary circulation as a result of acute lung injury or inflammation. We have previously described barrier-protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on human pulmonary endothelial cells (EC) mediated by small GTPases Rac and Cdc42. This work examined OxPAPC-induced focal adhesion (FA) and adherens junction (AJ) remodeling and potential interactions between FA and AJ protein complexes involved in OxPAPC-induced EC barrier enhancement. Immunofluorescence analysis, subcellular fractionation, and coimmunoprecipitation assays have shown that OxPAPC induced translocation and peripheral accumulation of FA complexes containing paxillin, focal adhesion kinase, vinculin, GIT1, and GIT2, increased association of AJ proteins vascular endothelial-cadherin, p120-catenin, alpha-, beta-, and gamma-catenins, and dramatically enhanced cell junction areas covered by AJ. Coimmunoprecipitation, pulldown assays, and confocal microscopy studies have demonstrated that OxPAPC promoted novel interactions between FA and AJ complexes via paxillin and beta-catenin association, which was critically dependent on Rac and Cdc42 activities and was abolished by pharmacological or small interfering RNA (siRNA)-mediated inhibition of Rac and Cdc42. Depletion of beta-catenin using the siRNA approach attenuated OxPAPC-induced paxillin translocation to the cell periphery, but also significantly decreased interaction of paxillin with AJ protein complex. In turn, paxillin knockdown by specific siRNA attenuated AJ enhancement in response to OxPAPC. These results show for the first time the novel interactions between FA and AJ protein complexes critical for EC barrier regulation by OxPAPC.     

Hepatocyte growth factor induces ERK-dependent paxillin phosphorylation and regulates paxillin-focal adhesion kinase association.

Hepatocyte growth factor (HGF) modulates cell adhesion, migration, and branching morphogenesis in cultured epithelial cells, events that require regulation of cell-matrix interactions. Using mIMCD-3 epithelial cells, we studied the effect of HGF on the focal adhesion proteins, focal adhesion kinase (FAK) and paxillin and their association. HGF was found to increase the tyrosine phosphorylation of paxillin and to a lesser degree FAK. In addition, HGF induced association of paxillin and activated ERK, correlating with a gel retardation of paxillin that was prevented with the ERK inhibitor U0126. The ability of activated ERK to phosphorylate and induce gel retardation of paxillin was confirmed in vitro in both full-length and amino-terminal paxillin. Several potential ERK phosphorylation sites in paxillin flank the paxillin-FAK association domains, so the ability of HGF to regulate paxillin-FAK association was examined. HGF induced an increase in paxillin-FAK association that was inhibited by pretreatment with U0126 and reproduced by in vitro phosphorylation of paxillin with ERK. The prevention of the FAK-paxillin association with U0126 correlated with an inhibition of the HGF-mediated FAK tyrosine phosphorylation and inhibition of HGF-dependent cell spreading and adhesion. An examination of cellular localization of FAK and paxillin demonstrated that HGF caused a condensation of focal adhesion complexes at the leading edges of cell processes and FAK-paxillin co-localization in these large complexes. Thus, these data suggest that HGF can induce serine/threonine phosphorylation of paxillin most probably mediated directly by ERK, resulting in the recruitment and activation of FAK and subsequent enhancement of cell spreading and adhesion.     

Roles of microfilaments and microtubules in paxillin dynamics

We investigated the roles of microfilaments and microtubules in the localization and tyrosine phosphorylation of paxillin, a focal adhesion-associated signaling molecule, in bovine aortic endothelial cells (BAECs). Paxillin tyrosine phosphorylation is inhibited by cytochalasin D (CD), but slightly increased by colchicine and paclitaxol (taxol). CD also caused an overall disassembly of paxillin-containing focal adhesions (paxillin-FAs) and translocation of paxillin to the cytoplasm and perinuclear region with a diffuse distribution. Meanwhile, colchicine and taxol caused a disassembly of paxillin-FAs from cell periphery and lamellipodia, and their assembly in cell center. These results indicate that actin filaments are important in paxillin assembly in the FAs of the whole ECs and that microtubules are critical in paxillin assembly in cell periphery and lamellipodia; thus the microfilaments and microtubules play differential roles in the dynamics of paxillin assembly/disassembly. Our findings also suggest that tyrosine phosphorylation is an important element in paxillin dynamics at FAs.     

p130cas but not paxillin is essential for Caco-2 intestinal epithelial cell spreading and migration on collagen IV

We have previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src kinase and stimulates Src-dependent tyrosine phosphorylation of p130cas. We observed that collagen IV also stimulated Src-dependent phosphorylation of both paxillin Tyr31 and paxillin Tyr118. Caco-2 transfection with paxillin or p130cas siRNAs inhibited expression of these proteins by more than 90% for at least 5 days after transfection. Although p130cas siRNA inhibited cell spreading on collagen IV by 33%, three different paxillin siRNAs did not inhibit cell spreading. p130cas siRNA did not affect Src Tyr416 or Src Tyr527 phosphorylation, FAK Tyr397 phosphorylation, or Src-dependent phosphorylation of FAK Tyr925, suggesting that p130cas did not inhibit cell spreading by altering FAK or Src activity. Rat p130cas expression after siRNA knock-out of endogenous human p130cas in Caco-2 cells reduced cell spreading inhibition by 71%. In contrast, expression of rat p130cas from which the Src-phosphorylated substrate domain was deleted did not rescue siRNA inhibition of cell spreading. Combined treatment with siRNAs to Crk and CrkL, which bind to the p130cas substrate domain, inhibited cell spreading by 54%. Both p130cas siRNA and the combined Crk/CrkL siRNAs strongly inhibited (52 and 46% inhibition, respectively) Caco-2 sheet migration on collagen IV and noticeably inhibited lamellipodial extension, whereas paxillin siRNA only inhibited migration by 18% and did not noticeably affect lamellipodial extension. These results suggest that Src may regulate Caco-2 migration on collagen IV via both p130cas and paxillin but that Src phosphorylation of p130cas is more important for this process.     

Paxillin binding is not the sole determinant of focal adhesion localization or dominant-negative activity of focal adhesion kinase/focal adhesion kinase-related nonkinase

The carboxy-terminal 150 residues of the focal adhesion kinase (FAK) comprise the focal adhesion-targeting sequence, which is responsible for its subcellular localization. The mechanism of focal adhesion targeting has not been fully elucidated. We describe a mutational analysis of the focal adhesion-targeting sequence of FAK to further examine the mechanism of focal adhesion targeting and explore additional functions encoded by the carboxy-terminus of FAK. The results demonstrate that paxillin binding is dispensable for focal adhesion targeting of FAK. Cell adhesion-dependent tyrosine phosphorylation strictly correlated with the ability of mutants to target to focal adhesions. Focal adhesion targeting was also a requirement for maximal FAK-dependent tyrosine phosphorylation of paxillin and FAK-related nonkinase (FRNK)-dependent inhibition of endogenous FAK function. However, there were additional requirements for these latter functions because we identified mutants that target to focal adhesions, yet are defective for the induction of paxillin phosphorylation or the dominant-negative function of FRNK. Furthermore, the paxillin-binding activity of FRNK mutants did not correlate with their ability to inhibit FAK, suggesting that FRNK has other targets in addition to paxillin.     

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.     

Calcium rises locally trigger focal adhesion disassembly and enhance residency of focal adhesion kinase at focal adhesions

Focal adhesion kinase (FAK) activity and Ca(2+) signaling led to a turnover of focal adhesions (FAs) required for cell spreading and migration. We used yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca(2+) sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca(2+) variations at FA sites and FA dynamics. Discrete subcellular Ca(2+) oscillators initiate both propagating and abortive Ca(2+) waves in migrating U87 astrocytoma cells. Ca(2+)-dependent FA disassembly occurs when the Ca(2+) wave reaches individual FAs, indicating that local but not global Ca(2+) increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. The FAK-Ycam recovery half-time (17 s) at FAs was slowed (to 29 s) by Ca(2+) elevation. FAK-related non-kinase domain-Ycam had a faster, Ca(2+)-insensitive recovery half-time (11 s), which is consistent with the effect of Ca(2+) on FAK-Ycam dynamics not being due to a general modification of the dynamics of FA components. Because FAK association at FAs was prolonged by Ca(2+) and FAK autophosphorylation was correlated to intracellular Ca(2+) levels, we propose that local Ca(2+) elevations increase the residency of FAK at FAs, possibly by means of tyrosine phosphorylation of FAK, thereby leading to increased activation of its effectors involved in FA disassembly.     

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.     

Endothelial contraction and monolayer hyperpermeability are regulated by Src kinase

Endothelial monolayer hyperpermeability is regulated by a myosin light chain phosphorylation (MLCP)-dependent contractile mechanism. In this study, we tested the role of Src-dependent tyrosine phosphorylation to modulate endothelial contraction and monolayer barrier function with the use of the myosin phosphatase inhibitor calyculin A (CalA) to directly elevate MLCP with the Src family tyrosine kinase inhibitor herbimycin A (HA) in bovine pulmonary artery endothelial cells (EC). CalA stimulated an increase in MLCP, Src kinase activity, an increase in the tyrosine phosphorylation of paxillin and focal adhesion (FA) kinase (p125(FAK)), and monolayer hyperpermeability. Microscopic examination of CalA-treated EC revealed a contractile morphology characterized by peripheral contractile bands of actomyosin filaments and stress fibers linked to phosphotyrosine-containing FAs. These CalA-dependent events were HA sensitive. HA alone stimulated an improvement in monolayer barrier formation by reducing the levels of MLCP and phosphotyrosine-containing proteins and the number of large paracellular holes. These data show that Src kinase plays an important role in regulating monolayer hyperpermeability through adjustments in tyrosine phosphorylation, MLCP, and EC contraction.     

Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase

Vascular endothelial growth factor (VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury. VEGF-induced endothelial cell migration is a key step in the angiogenic response and is mediated by an accelerated rate of focal adhesion complex assembly and disassembly. In this study, we identified the signaling mechanisms by which VEGF regulates human brain microvascular endothelial cell (HBMEC) integrity and assembly of focal adhesions, complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. We found that VEGF treatment of HBMECs plated on laminin or fibronectin stimulated cytoskeletal organization and increased focal adhesion sites. Pretreating cells with VEGF antibodies or with the specific inhibitor SU-1498, which inhibits Flk-1/KDR receptor phosphorylation, blocked the ability of VEGF to stimulate focal adhesion assembly. VEGF induced the coupling of focal adhesion kinase (FAK) to integrin alphavbeta5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130cas. Additionally, FAK and related adhesion focal tyrosine kinase (RAFTK)/Pyk2 kinases were tyrosine-phosphorylated by VEGF and found to be important for focal adhesion sites. Overexpression of wild type RAFTK/Pyk2 increased cell spreading and the migration of HBMECs, whereas overexpression of catalytically inactive mutant RAFTK/Pyk2 markedly suppressed HBMEC spreading ( approximately 70%), adhesion ( approximately 82%), and migration ( approximately 65%). Furthermore, blocking of FAK by the dominant-interfering mutant FRNK (FAK-related non-kinase) significantly inhibited HBMEC spreading and migration and also disrupted focal adhesions. Thus, these studies define a mechanism for the regulatory role of VEGF in focal adhesion complex assembly in HBMECs via activation of FAK and RAFTK/Pyk2.