High-molecular-weight serum protein complexes differentially promote cell migration and the focal adhesion localization of the urokinase receptor in human glioma cells

The distribution of the urokinase-type plasminogen activator receptor (uPAR) on human glioma cells was examined as a function of culture conditions, using immunofluorescence and immunophotoelectron microscopy. Both uPAR colocalization with focal adhesion proteins and glioma cell motility were maximal in medium containing whole serum or a serum fraction retained by a 500,000 mol wt cutoff centrifugal concentration filter. High motility also took place in medium containing a serum fraction passed by the 500,000 cutoff filter but retained by a 100,000 cutoff filter and in minimal medium containing added vitronectin; however, under these conditions only a small percentage of the otherwise abundant focal adhesions contained colocalized uPAR. Glioma cells in minimal medium with added laminin migrated with a highly elongated morphology but without either classical focal adhesions or well-defined uPAR labeling. In contrast, glioma cells in minimal medium with no additions did not migrate, nor did they adhere well or display defined labeling patterns for focal adhesion proteins or uPAR. The results indicate that high-molecular-weight serum protein complexes promote both uPAR-focal adhesion colocalization and cell migration in glioma cells. However, conditions can be selected in which migration takes place with minimal uPAR-focal adhesion localization, as well as in the absence of apparent focal adhesions.     

Tyrosine phosphorylation and cytoskeletal tension regulate the release of fibroblast adhesions

We have investigated the mechanisms by which fibroblasts release their adhesions to the extracellular matrix substrata using a permeabilized cell system in which the adhesions remain relatively stable. A large number of different molecules were assayed for their effect on focal adhesion stability using immunofluorescence with antibodies against different focal adhesion constituents. ATP uniquely stimulates a rapid breakdown of focal adhesions, and at high ATP concentrations (> 5 mM), many cells are released from the dish. The remaining cells appear contracted with talin, alpha-actinin, and vinculin localized diffusely throughout the cell. Integrin containing tracks of variable intensity outline the regions where cells had resided before they detached from the substratum. At lower ATP concentrations (0.5-5 mM) the cells remain spread; however the focal adhesion components, including integrin, show an array of phenotypes ranging from diffusely localized throughout the cell to a localization in small, thin focal adhesions. Okadaic acid, a serine, threonine phosphatase inhibitor, enhances the contracted phenotype, even at low concentrations (0.5 mM) of ATP. The localization of focal adhesion components is different in okadaic acid-treated cells. In highly contracted cells, integrin is present in tracks where the cells resided before the contraction; however focal adhesions are no longer apparent. Talin, vinculin, and alpha-actinin localize in trabecular networks toward the periphery of the cell. Interestingly, phosphotyrosine staining as well as nascent, intracellular integrin precedes the recruitment of focal adhesion constituents into the trabecular network. The ATP-stimulated focal adhesion breakdown appears to operate through two mechanisms. First, ATP stimulates the tyrosine phosphorylation of several cytoskeletally associated proteins. These tyrosine phosphorylations correlated well with focal adhesion breakdown. Furthermore, addition of a recombinant, constitutively active tyrosine phosphatase inhibits both the tyrosine phosphorylations and the breakdown of the focal adhesions. None of the major tyrosine phosphoproteins are FAK, integrin, tensin, paxillin, or other phosphoproteins implicated in focal adhesion assembly. The second mechanism is cell contraction. High ATP concentrations, or lower ATP concentrations in the presence of okadaic acid induce cell contraction. Inhibiting the contraction by addition of a heptapeptide IRICRKG, which blocks the actin-myosin interaction, also inhibits focal adhesion breakdown. Neither the peptide nor the phosphatase inhibits focal adhesion breakdown under all conditions suggesting that both tension and tyrosine phosphorylations mediate the release of adhesions.     

Variation in cell-substratum adhesion in relation to cell cycle phases

The quantification of focal adhesion sites offers an assessable method of measuring cell-substrate adhesion. Such measurement can be hindered by intra-sample variation that may be cell cycle derived. A combination of autoradiography and immunolabelling techniques, for scanning electron microscopy (SEM), were utilised simultaneously to identify both S-phase cells and their focal adhesion sites. Electron-energy 'sectioning' of the sample, by varying the accelerating voltage of the electron beam, combined with backscattered electron (BSE) imaging, allowed for S-phase cell identification in one energy 'plane' image and quantitation of immunogold label in another. As a result, it was possible simultaneously to identify S-phase cells and their immunogold-labelled focal adhesions sites on the same cell. The focal adhesion densities were calculated both for identified S-phase cells and the remaining non-S-phase cells present. The results indicated that the cell cycle phase was a significant factor in determining the density of focal adhesions, with non-S-phase cells showing a larger adhesion density than S-phase cells. Focal adhesion morphology was also seen to correspond to cell cycle phase; with 'dot' adhesions being more prevalent on smaller non-S-phase and the mature 'dash' type on larger S-phase cells. This study demonstrated that when quantitation of focal adhesion sites is required, it is necessary to consider the influence of cell cycle phases on any data collected.     

Tetraspanin CD9 in cell migration

Tetraspanin CD9 is associated with integrin adhesion receptors and it was reported that CD9 regulates integrin-dependent cell migration and invasion. Pro- and anti-migratory effects of CD9 have been linked to adhesion-dependent signalling pathways, including phosphorylation of FAK (focal adhesion kinase) and activation of phosphoinositide 3-kinase, p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase). In the present paper, we describe a novel mechanism whereby CD9 specifically controls localization of talin1, one of the critical regulators of integrin activation, to focal adhesions: CD9-deficiency leads to impaired localization of talin1 to focal adhesions and correlates with increased motility of breast cancer cells.     

Preferential Localization of Tyrosine-Phosphorylated Paxillin in Focal Adhesions

Focal adhesions are sites for integrin-mediated attachment of cultured cells to the extracellular matrix. Localization studies have shown that focal adhesions can be stained by antiphosphotyrosine antibodies, but the role of tyrosine-phosphorylated proteins in focal adhesions is not known. By using ventral plasma membranes prepared from chicken embryo fibroblasts spread on the substrate, we present evidence for the preferential localization of a minor pool of tyrosine-phosphorylated paxillin in focal adhesions. Ventral plasma membranes showed an enrichment in β1-integrins, and in several tyrosine-phosphorylated polypeptides, while focal adhesion proteins like vinculin and paxillin, although localized to focal adhesions in ventral plasma membranes, were not particularly enriched in these preparations compared to whole cell lysates. Biochemical and morphological analysis of ventral plasma membranes showed a dramatic increase in the level of tyrosine-phosphorylation of the pool of paxillin localized to the adhesive sites, when compared to the paxillin present in whole cell lysates. The observed preferential localization of tyrosine-phosphorylated paxillin to focal adhesions may represent a general mechanism to compartmentalize focal adhesion components from large non-phosphorylated, cytosolic pools.     

Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions.     

Preferential Localization of Tyrosine-Phosphorylated Paxillin in Focal Adhesions

Focal adhesions are sites for integrin-mediated attachment of cultured cells to the extracellular matrix. Localization studies have shown that focal adhesions can be stained by antiphosphotyrosine antibodies, but the role of tyrosine-phosphorylated proteins in focal adhesions is not known. By using ventral plasma membranes prepared from chicken embryo fibroblasts spread on the substrate, we present evidence for the preferential localization of a minor pool of tyrosine-phosphorylated paxillin in focal adhesions. Ventral plasma membranes showed an enrichment in β1-integrins, and in several tyrosine-phosphorylated polypeptides, while focal adhesion proteins like vinculin and paxillin, although localized to focal adhesions in ventral plasma membranes, were not particularly enriched in these preparations compared to whole cell lysates. Biochemical and morphological analysis of ventral plasma membranes showed a dramatic increase in the level of tyrosine-phosphorylation of the pool of paxillin localized to the adhesive sites, when compared to the paxillin present in whole cell lysates. The observed preferential localization of tyrosine-phosphorylated paxillin to focal adhesions may represent a general mechanism to compartmentalize focal adhesion components from large non-phosphorylated, cytosolic pools.     

Mechanisms of cellular adhesion : II. The interplay between adhesion, the cytoskeleton and morphology in substrate-attached cells

cAMP/theophylline exaggerates cell shape—whether the fibroblastic morphology of controls or the epithelioid shape of colchicine-treated cells. The ultrastructural basis is that cAMP/theophylline increases the number and linearity of microtubules and microfilament bundles, although where also treated with colchicine, the cells adopt a well-spread shape maintained by microfilament bundles alone. Since interference reflection microscopy shows that colchicine promotes the marked alignment of focal contacts (which terminate microfilament bundles) it is concluded that microtubules encourage angular cell form and modify the pattern of adhesions by influencing the directionality of microfilament bundle formation although they are inessential for the maintenance of the spread form or adhesion per se.     

Protein Phosphatase 1 δ is Associated with Focal Adhesions

In all mammalian cells protein phosphatase-1 (PP1) exists in three isoforms, defined as α, γ1 and δ. Immunofluorescence studies with isoform-specific antibodies indicated that δ, but not α or γ1, is enriched at focal adhesions in HeLa cells, fibroblasts, endothelial cells and keratinocytes. This was confirmed also by interference reflection microscopy, which indicated that PP1δ was in areas of tight adhesion of the membrane to the extracellular matrix at sites where the microfilament cytoskeleton is organized. In all the cell types so far considered the PP1δ in focal adhesions represented only a small aliquot of the total PP1δ, which was predominantly localized to the nucleus. The association of PP1δ to focal adhesions was confirmed by the co-immunoprecipitation of PP1δ with the focal adhesion kinase pp125^FAK and with the αv integrin. Comparison between the amount of PP1δ associated with focal adhesion proteins and that of PP1δ recovered in an anti-PP1δ immunoprecipitate confirmed that only a minor amount of the enzyme was associated with the focal adhesions. Since some focal adhesion proteins are phosphorylated on Ser/Thr, it is likely that PP1δ may be involved in the regulation of focal adhesion functions and particularly in the signaling pathway generated by cell-substratum adhesion.     

Calpain mediates integrin-induced signaling at a point upstream of Rho family members.

Integrin-induced adhesion leads to cytoskeletal reorganizations, cell migration, spreading, proliferation, and differentiation. The details of the signaling events that induce these changes in cell behavior are not well understood but they appear to involve activation of Rho family members which activate signaling molecules such as tyrosine kinases, serine/threonine kinases, and lipid kinases. The result is the formation of focal complexes, focal adhesions, and bundles and networks of actin filaments that allow the cell to spread. The present study shows that mu-calpain is active in adherent cells, that it cleaves proteins known to be present in focal complexes and focal adhesions, and that overexpression of mu-calpain increased the cleavage of these proteins, induced an overspread morphology and induced an increased number of stress fibers and focal adhesions. Inhibition of calpain with membrane permeable inhibitors or by expression of a dominant negative form of mu-calpain resulted in an inability of cells to spread or to form focal adhesions, actin filament networks, or stress fibers. Cells expressing constitutively active Rac1 could still form focal complexes and actin filament networks (but not focal adhesions or stress fibers) in the presence of calpain inhibitors; cells expressing constitutively active RhoA could form focal adhesions and stress fibers. Taken together, these data indicate that calpain plays an important role in regulating the formation of focal adhesions and Rac- and Rho-induced cytoskeletal reorganizations and that it does so by acting at sites upstream of both Rac1 and RhoA.     

Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy

Background  

In anchorage dependent cells, myosin generated contractile forces affect events closely associated with adhesion such as the formation of stress fibers and focal adhesions, and temporally distal events such as entry of the cell into S-phase. As occurs in many signaling pathways, a phosphorylation reaction (in this case, phosphorylation of myosin light chain) is directly responsible for cell response. Western blotting has been useful in measuring intracellular phosphorylation events, but cells are lysed in the process of sample preparation for western blotting, and spatial information such as morphology, localization of the phosphorylated species, and the distribution of individual cell responses across the population is lost. We report here a reliable automated microscopy method for quantitative measurement of myosin light chain phosphorylation in adherent cells. This method allows us to concurrently examine cell morphology, cell-cell contact, and myosin light chain diphosphorylation in vascular smooth muscle cells.     

A Highlights from MBoC Selection: Induction of focal adhesions and motility in Drosophila S2 cells

Focal adhesions are dynamic structures that interact with the extracellular matrix on the cell exterior and actin filaments on the cell interior, enabling cells to adhere and crawl along surfaces. We describe a system for inducing the formation of focal adhesions in normally non–ECM-adherent, nonmotile Drosophila S2 cells. These focal adhesions contain the expected molecular markers such as talin, vinculin, and p130Cas, and they require talin for their formation. The S2 cells with induced focal adhesions also display a nonpolarized form of motility on vitronectin-coated substrates. Consistent with findings in mammalian cells, the degree of motility can be tuned by changing the stiffness of the substrate and was increased after the depletion of PAK3, a p21-activated kinase. A subset of nonmotile, nonpolarized cells also exhibited focal adhesions that rapidly assembled and disassembled around the cell perimeter. Such cooperative and dynamic fluctuations of focal adhesions were decreased by RNA interference (RNAi) depletion of myosin II and focal adhesion kinase, suggesting that this behavior requires force and focal adhesion maturation. These results demonstrate that S2 cells, a cell line that is well studied for cytoskeletal dynamics and readily amenable to protein manipulation by RNAi, can be used to study the assembly and dynamics of focal adhesions and mechanosensitive cell motility.     

Skeletal muscle LIM protein 1 regulates integrin-mediated myoblast adhesion,spreading, and migration

The skeletal muscle LIM protein 1 (SLIM1) is highly expressed in skeletal and cardiac muscle, and itsexpression is downregulated significantly in dilated humancardiomyopathy. However, the function of SLIM1 is unknown. In thisstudy, we investigated the intracellular localization of SLIM1.Endogenous and recombinant SLIM1 localized to the nucleus, stressfibers, and focal adhesions in skeletal myoblasts plated onfibronectin, collagen, or laminin. However, after inhibition ofintegrin signaling either by plating on poly-L-lysine or bysoluble RGD peptide, SLIM1 localized diffusely in the cytosol, withdecreased nuclear expression. Disruption of the actin cytoskeleton bycytochalasin D did not inhibit nuclear localization of SLIM1 inintegrin-activated cells. Green fluorescent protein-tagged SLIM1shuttled in the nucleus of untransfected NIH 3T3 cells, in aheterokaryon fusion assay. Overexpression of SLIM1 in Sol8 myoblastsinhibited cell adhesion and promoted cell spreading and migration.These studies show SLIM1 localizes in an integrin-dependent manner tothe nucleus and focal adhesions where it functions downstream ofintegrin activation to promote cell spreading and migration.

     

Upregulation of paxillin and focal adhesion signaling follows Dystroglycan Complex deletions and promotes a hypertensive state of differentiation

Anchorage to matrix is mediated for many cells not only by integrin-based focal adhesions but also by a parallel assembly of integral and peripheral membrane proteins known as the Dystroglycan Complex. Deficiencies in either dystrophin (mdx mice) or γ-sarcoglycan (γSG^?/? mice) components of the Dystroglycan Complex lead to upregulation of numerous focal adhesion proteins, and the phosphoprotein paxillin proves to be among the most prominent. In mdx muscle, paxillin-Y31 and Y118 are both hyper-phosphorylated as are key sites in focal adhesion kinase (FAK) and the stretch-stimulatable pro-survival MAPK pathway, whereas γSG^?/? muscle exhibits more erratic hyper-phosphorylation. In cultured myotubes, cell tension generated by myosin-II appears required for localization of paxillin to adhesions while vinculin appears more stably integrated. Overexpression of wild-type (WT) paxillin has no obvious effect on focal adhesion density or the physical strength of adhesion, but WT and a Y118F mutant promote contractile sarcomere formation whereas a Y31F mutant shows no effect, implicating Y31 in striation. Self-peeling of cells as well as Atomic Force Microscopy (AFM) probing of cells with or without myosin-II inhibition indicate an increase in cell tension within paxillin-overexpressing cells. However, prednisolone, a first-line glucocorticoid for muscular dystrophies, decreases cell tension without affecting paxillin at adhesions, suggesting a non-linear relationship between paxillin and cell tension. Hypertension that results from upregulation of integrin adhesions is thus a natural and treatable outcome of Dystroglycan Complex down-regulation.     

Paxillin-dependent paxillin kinase linker and p21-activated kinase localization to focal adhesions involves a multistep activation pathway

The precise temporal-spatial regulation of the p21-activated serine-threonine kinase PAK at the plasma membrane is required for proper cytoskeletal reorganization and cell motility. However, the mechanism by which PAK localizes to focal adhesions has not yet been elucidated. Indirect binding of PAK to the focal adhesion protein paxillin via the Arf-GAP protein paxillin kinase linker (PKL) and PIX/Cool suggested a mechanism. In this report, we demonstrate an essential role for a paxillin-PKL interaction in the recruitment of activated PAK to focal adhesions. Similar to PAK, expression of activated Cdc42 and Rac1, but not RhoA, stimulated the translocation of PKL from a generally diffuse localization to focal adhesions. Expression of the PAK regulatory domain (PAK1-329) or the autoinhibitory domain (AID 83-149) induced PKL, PIX, and PAK localization to focal adhesions, indicating a role for PAK scaffold activation. We show PIX, but not NCK, binding to PAK is necessary for efficient focal adhesion localization of PAK and PKL, consistent with a PAK-PIX-PKL linkage. Although PAK activation is required, it is not sufficient for localization. The PKL amino terminus, containing the PIX-binding site, but lacking paxillin-binding subdomain 2 (PBS2), was unable to localize to focal adhesions and also abrogated PAK localization. An identical result was obtained after PKLDeltaPBS2 expression. Finally, neither PAK nor PKL was capable of localizing to focal adhesions in cells overexpressing paxillinDeltaLD4, confirming a requirement for this motif in recruitment of the PAK-PIX-PKL complex to focal adhesions. These results suggest a GTP-Cdc42/GTP-Rac triggered multistep activation cascade leading to the stimulation of the adaptor function of PAK, which through interaction with PIX provokes a functional PKL PBS2-paxillin LD4 association and consequent recruitment to focal adhesions. This mechanism is probably critical for the correct subcellular positioning of PAK, thereby influencing the ability of PAK to coordinate cytoskeletal reorganization associated with changes in cell shape and motility.     

Phosphorylation of SHP-2 regulates interactions between the endoplasmic reticulum and focal adhesions to restrict interleukin-1-induced Ca2+ signaling

Interleukin-1 (IL-1)-induced Ca2+ signaling in fibroblasts is constrained by focal adhesions. This process involves the proteintyrosine phosphatase SHP-2, which is critical for IL-1-induced phosphorylation of phospholipase Cgamma1, thereby enhancing IL-1-induced Ca2+ release and ERK activation. Currently, the mechanisms by which SHP-2 modulates Ca2+ release from the endoplasmic reticulum are not defined. We used immunoprecipitation and fluorescence protein-tagged SHP-2 or endoplasmic reticulum (ER)-protein expression vectors, and an ER-specific calcium indicator, to examine the functional relationships between SHP-2, focal adhesions, and IL-1-induced Ca2+ release from the ER. By total internal reflection fluorescence microscopy to image subplasma membrane compartments, SHP-2 co-localized with the ER-associated proteins calnexin and calreticulin at sites of focal adhesion formation in fibroblasts. IL-1beta promoted time-dependent recruitment of SHP-2 and ER proteins to focal adhesions; this process was blocked in cells treated with small interfering RNA for SHP-2 and in cells expressing a Y542F SHP-2 mutant. IL-1 stimulated inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the ER subjacent to the plasma membrane that was tightly localized around fibronectin-coated beads and was reduced 4-fold in cells expressing Tyr-542 SHP-2 mutant. In subcellular fractions enriched for ER proteins, immunoprecipitation demonstrated that IL-1-enhanced association of SHP-2 with the type 1 inositol 1,4,5-trisphosphate receptor was dependent on Tyr-542 of SHP-2. We conclude that Tyr-542 of SHP-2 modulates IL-1-induced Ca2+ signals and association of the ER with focal adhesions.     

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.     

Calpain-mediated proteolysis of paxillin negatively regulates focal adhesion dynamics and cell migration

The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using time-lapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.     

FAK potentiates Rac1 activation and localization to matrix adhesion sites: a role for betaPIX

FAK, a cytoplasmic protein tyrosine kinase, is activated and localized to focal adhesions upon cell attachment to extracellular matrix. FAK null cells spread poorly and exhibit altered focal adhesion turnover. Rac1 is a member of the Rho-family GTPases that promotes membrane ruffling, leading edge extension, and cell spreading. We investigated the activation and subcellular location of Rac1 in FAK null and FAK reexpressing fibroblasts. FAK reexpressers had a more robust pattern of Rac1 activation after cell adhesion to fibronectin than the FAK null cells. Translocation of Rac1 to focal adhesions was observed in FAK reexpressers, but seldom in FAK null cells. Experiments with constitutively active L61Rac1 and dominant negative N17Rac1 indicated that the activation state of Rac1 regulated its localization to focal adhesions. We demonstrated that FAK tyrosine-phosphorylated betaPIX and thereby increased its binding to Rac1. In addition, betaPIX facilitated the targeting of activated Rac1 to focal adhesions and the efficiency of cell spreading. These data indicate that FAK has a role in the activation and focal adhesion translocation of Rac1 through the tyrosine phosphorylation of betaPIX.     

Targeting membrane-localized focal adhesion kinase to focal adhesions: roles of tyrosine phosphorylation and SRC family kinases

In the present study, we examined regulation of activated focal adhesion kinase localization in focal adhesions. By using focal adhesion kinase fused to an inert transmembrane anchor, we found that the focal contact targeting region within focal adhesion kinase was preserved in the membrane-targeted fusion protein. However, upon tyrosine phosphorylation, full-length focal adhesion kinase became excluded from focal adhesions. This negative regulation of localization could be abolished by mutating key amino acid residues of focal adhesion kinase shown previously to be involved in adhesion-mediated signal transduction. Hyper-phosphorylation of endogenous focal adhesion kinase induced by pervanadate resulted in a similar reduction of localization at focal adhesions. We also show here that Src family kinases are essential for the phosphorylation-dependent exclusion of focal adhesion kinase from focal adhesions. We propose here a molecular model for the tyrosine phosphorylation-dependent regulation of focal adhesion kinase organization involving Src kinases and an inhibitory phosphorylation of the C-terminal (Tyr-925) tyrosine residue.