A conformational switch in vinculin drives formation and dynamics of a talin-vinculin complex at focal adhesions

Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin-cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or alpha-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to Kd approximately 10(-7) is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.     

Focal adhesion kinase mediates defects in the force-dependent reinforcement of initial integrin-cytoskeleton linkages in metastatic colon cancer cell lines

Micro-environmental clues, including the biophysical interpretation of the extracellular matrix, are critical to proliferation, apoptosis and migration. Here, we show that metastatic human colon cancer cell lines display altered matrix interaction. Interaction of colon cancer cells with collagen I depends on integrins (mainly alpha(1)/beta(1)) but metastatic cells display delayed spreading and reduced extension of lamellipodia. In addition, cells show defective strengthening of integrin-cytoskeleton linkages upon mechanical stimulation, as determined by laser trapping experiments and binding of large beads to the cell surface. However, adhesion to pliable surfaces is ameliorated in metastatic variants. These changes are caused by constitutive activation of focal adhesion kinase (FAK) and can be modulated by changing expression and/or activity of FAK via RNA-interference or expression of inhibitory constructs, respectively. In addition, consistent with defective strengthening of integrin-cytoskeleton linkages, metastatic cell lines show reduced random motility. Taken together these data suggest that constitutive activation of FAK causes defects in spreading, reinforcement of integrin-cytoskeleton linkages and migration and at the same time could ameliorate the adhesion of metastatic cells to suboptimal surfaces.     

SRC-mediated phosphorylation of focal adhesion kinase couples actin and adhesion dynamics to survival signaling

Integrin-associated focal adhesions not only provide adhesive links between cellular actin and extracellular matrix but also are sites of signal transmission into the cell interior. Many cell responses signal through focal adhesion kinase (FAK), often by integrin-induced autophosphorylation of FAK or phosphorylation by Src family kinases. Here, we used an interfering FAK mutant (4-9F-FAK) to show that Src-dependent FAK phosphorylation is required for focal adhesion turnover and cell migration, by controlling assembly of a calpain 2/FAK/Src/p42ERK complex, calpain activation, and proteolysis of FAK. Expression of 4-9F-FAK in FAK-deficient fibroblasts also disrupts F-actin assembly associated with normal adhesion and spreading. In addition, we found that FAK's ability to regulate both assembly and disassembly of the actin and adhesion networks may be linked to regulation of the protease calpain. Surprisingly, we also found that the same interfering 4-9F-FAK mutant protein causes apoptosis of serum-deprived, transformed cells and suppresses anchorage-independent growth. These data show that Src-mediated phosphorylation of FAK acts as a pivotal regulator of both actin and adhesion dynamics and survival signaling, which, in turn, control apparently distinct processes such as cell migration and anchorage-independent growth. This also highlights that dynamic regulation of actin and adhesions (which include the integrin matrix receptors) is critical to signaling output and biological responses.     

Deoxycholic acid differentially regulates focal adhesion kinase phosphorylation: role of tyrosine phosphatase ShP2

Environmental factors, including dietary fats, are implicated in colonic carcinogenesis. Dietary fats modulate secondary bile acids including deoxycholic acid (DCA) concentrations in the colon, which are thought to contribute to the nutritional-related component of colon cancer risk. Here we demonstrate, for the first time, that DCA differentially regulated the site-specific phosphorylation of focal adhesion kinase (FAK). DCA decreased adhesion of HCA-7 cells to the substratum and induced dephosphorylation of FAK at tyrosine-576/577 (Tyr-576/577) and Tyr-925. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by DCA stimulation. Interestingly, we found that c-Src was constitutively associated with FAK and DCA actually activated Src, despite no change in FAK-397 and an inhibition of FAK-576 phosphorylation. DCA concomitantly and significantly increased association of tyrosine phosphatase ShP2 with FAK. Incubation of immunoprecipitated FAK, in vitro, with glutathione-S-transferase-ShP2 fusion protein resulted in tyrosine dephosphorylation of FAK in a concentration-dependent manner. Antisense oligodeoxynucleotides directed against ShP2 decreased ShP2 protein levels and attenuated DCA-induced FAK dephosphorylation. Inhibition of FAK by adenoviral-mediated overexpression of FAK-related nonkinase and gene silencing of Shp2 both abolished DCA's effect on cell adhesion, thus providing a possible mechanism for inside-out signaling by DCA in colon cancer cells. Our results suggest that DCA differentially regulates focal adhesion complexes and that tyrosine phosphatase ShP2 has a role in DCA signaling.     

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.     

Talin1 is critical for force-dependent reinforcement of initial integrin-cytoskeleton bonds but not tyrosine kinase activation

Cells rapidly transduce forces exerted on extracellular matrix contacts into tyrosine kinase activation and recruitment of cytoskeletal proteins to reinforce integrin-cytoskeleton connections and initiate adhesion site formation. The relationship between these two processes has not been defined, particularly at the submicrometer level. Using talin1-deficient cells, it appears that talin1 is critical for building early mechanical linkages. Deletion of talin1 blocked laser tweezers, force-dependent reinforcement of submicrometer fibronectin-coated beads and early formation of adhesion sites in response to force, even though Src family kinases, focal adhesion kinase, and spreading were activated normally. Recruitment of vinculin and paxillin to sites of force application also required talin1. FilaminA had a secondary role in strengthening fibronectin-integrin-cytoskeleton connections and no role in stretch-dependent adhesion site assembly. Thus, force-dependent activation of tyrosine kinases is independent of early force-dependent structural changes that require talin1 as part of a critical scaffold.     

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

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

LKB1 Represses Focal Adhesion Kinase (FAK) Signaling via a FAK-LKB1 Complex to Regulate FAK Site Maturation and Directional Persistence

Liver kinase β1 (LKB1, also known as STK11) is a serine/threonine kinase that has multiple cellular functions including the regulation of cell polarity and motility. Murine proteomic studies show that LKB1 loss causes aberrant adhesion signaling; however, the mechanistic underpinnings of this relationship are unknown. We show that cells stably depleted of LKB1 or its co-activator STRADα have increased phosphorylation of focal adhesion kinase (FAK) at Tyr³⁹⁷/Tyr⁸⁶¹ and enhanced adhesion to fibronectin. LKB1 associates in a complex with FAK and LKB1 accumulation at the cellular leading edge is mutually excluded from regions of activated Tyr³⁹⁷-FAK. LKB1-compromised cells lack directional persistence compared with wild-type cells, but this is restored through subsequent pharmacological FAK inhibition or depletion, showing that cell directionality is mediated through LKB1-FAK signaling. Live cell confocal imaging reveals that LKB1-compromised cells lack normal FAK site maturation and turnover, suggesting that defects in adhesion and directional persistence are caused by aberrant adhesion dynamics. Furthermore, re-expression of full-length wild-type or the LKB1 N-terminal domain repressed FAK activity, whereas the kinase domain or C-terminal domain alone did not, indicating that FAK suppression is potentially regulated through the LKB1 N-terminal domain. Based upon these results, we conclude that LKB1 serves as a FAK repressor to stabilize focal adhesion sites, and when LKB1 function is compromised, aberrant FAK signaling ensues, resulting in rapid FAK site maturation and poor directional persistence.     

Phosphorylated alpha-actinin and protein-tyrosine phosphatase 1B coregulate the disassembly of the focal adhesion kinase x Src complex and promote cell migration

The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein alpha-actinin at Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) is an alpha-actinin phosphatase. PTP 1B-dependent dephosphorylation of alpha-actinin was seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type alpha-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the alpha-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered by PTP 1B. In addition, we found that phosphorylated alpha-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation at Tyr-397 in FAK triggered by wild-type alpha-actinin and PTP 1B caused a significant increase in cell migration. We propose that phosphorylated alpha-actinin disrupts the FAK x Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel feedback loop involving phosphorylated alpha-actinin and PTP 1B that regulates FAK x Src interaction and cell migration.     

Regulation of Matrix Assembly through Rigidity-dependent Fibronectin Conformational Changes

Cells sense and respond to the mechanical properties of their microenvironment. We investigated whether these properties affect the ability of cells to assemble a fibrillar fibronectin (FN) matrix. Analysis of matrix assembled by cells grown on FN-coated polyacrylamide gels of varying stiffnesses showed that rigid substrates stimulate FN matrix assembly and activation of focal adhesion kinase (FAK) compared with the level of assembly and FAK signaling on softer substrates. Stimulating integrins with Mn²⁺ treatment increased FN assembly on softer gels, suggesting that integrin binding is deficient on soft substrates. Guanidine hydrochloride-induced extension of the substrate-bound FN rescued assembly on soft substrates to a degree similar to that of Mn²⁺ treatment and increased activation of FAK along with the initiation of assembly at FN matrix assembly sites. In contrast, increasing actin-mediated cell contractility did not rescue FN matrix assembly on soft substrates. Thus, rigidity-dependent FN matrix assembly is determined by extracellular events, namely the engagement of FN by cells and the induction of FN conformational changes. Extensibility of FN in response to substrate stiffness may serve as a mechanosensing mechanism whereby cells use pericellular FN to probe the stiffness of their environment.     

FAK blunts adenosine-homocysteine-induced endothelial cell apoptosis: requirement for PI 3-kinase

Treatment of cultured bovine pulmonary endothelial cells (BPAEC) with adenosine (Ado) alone or in combination with homocysteine (Hc) leads to disruption of focal adhesion complexes, caspase-dependent degradation of components of focal adhesion complexes, and subsequent apoptosis. Endothelial cells transiently overexpressing paxillin or p130(Cas) cDNAs underwent Ado-Hc-induced apoptosis to an extent similar to that of cells transfected with vector alone. However, overexpression of focal adhesion kinase (FAK) cDNA blunted Ado-Hc-induced apoptosis. FAK constructs lacking the central catalytic domain or containing a point mutation, rendering the catalytic domain enzymatically inactive, did not provide protection from apoptosis. Constructs containing a mutation in the major autophosphorylation site (tyrosine-397) similarly did not prevent cell death. A FAK mutant in amino acid 395, deficient in phosphatidylinositol 3-kinase (PI 3-kinase) binding, was not able to blunt apoptosis. Finally, overexpression of FAK did not provide protection from apoptosis in the presence of LY-294002, a PI 3-kinase inhibitor. Taken together, these data suggest that the survival signals mediated by overexpression of FAK in response to Ado-Hc-induced apoptosis require a PI 3-kinase-dependent pathway.     

Activation-enhanced alpha(IIb)beta(3)-integrin-cytoskeleton interactions outside of focal contacts require the alpha-subunit

Integrins link the cell's cytoskeleton to the extracellular matrix, as well as to receptors on other cells. These links occur not only at focal contacts but also at smaller integrin-containing protein complexes outside of focal contacts. We previously demonstrated the importance of focal contact-independent integrin-cytoskeleton interactions of beta(2) integrins: activation of adhesion resulted from a release of integrins from cytoskeletal constraints. To determine whether changes in integrin-cytoskeleton interactions were related to activation of the integrin, we used single particle tracking to examine focal contact-independent cytoskeletal associations of alpha(IIb)beta(3)-integrin, in which activation results in a large conformational change. Direct activation of alpha(IIb)beta(3) by mutation did not mimic activation of lymphocytes with phorbol ester, because it enhanced integrin-cytoskeleton interactions, whereas activation of lymphocytes decreased them. Using additional integrin mutants, we found that both alpha- and beta-cytoplasmic domains were required for these links. This suggests that 1) both beta(2)- and beta(3)-integrins interact with the cytoskeleton outside of focal contacts; 2) activation of a cell and activation of an integrin are distinct processes, and both can affect integrin-cytoskeleton interactions; and 3) the role of the alpha-subunit in integrin-cytoskeleton interactions in at least some circumstances is more direct than generally supposed.     

PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration

We investigated the molecular and cellular actions of receptor protein tyrosine phosphatase (PTP) alpha in integrin signaling using immortalized fibroblasts derived from wild-type and PTP alpha-deficient mouse embryos. Defects in PTP alpha-/- migration in a wound healing assay were associated with altered cell shape and focal adhesion kinase (FAK) phosphorylation. The reduced haptotaxis to fibronectin (FN) of PTP alpha-/- cells was increased by expression of active (but not inactive) PTP alpha. Integrin-mediated formation of src-FAK and fyn-FAK complexes was reduced or abolished in PTP alpha-/- cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397. Reintroduction of active (but not inactive) PTP alpha restored FAK Tyr-397 phosphorylation. FN-induced cytoskeletal rearrangement was retarded in PTP alpha-/- cells, with delayed filamentous actin stress fiber assembly and focal adhesion formation. This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2. These results, together with the reduced src/fyn tyrosine kinase activity in PTP alpha-/- fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTP alpha functions in integrin signaling and cell migration as an Src-PTK activator. Our paper establishes that PTP alpha is required for early integrin-proximal events, acting upstream of FAK to affect the timely and efficient phosphorylation of FAK Tyr-397.     

Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin‐containing focal adhesions

Actin–myosin contractility modulates focal adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast adhesion strengthening using a hydrodynamic adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in adhesion strengthening to levels 30–40% higher than serum‐free cultures. Inhibition of myosin light chain kinase or Rho‐kinase blocked phosphorylation of myosin light chain to similar extents and eliminated the serum‐induced enhancements in strengthening. Blebbistatin‐induced inhibition of myosin II reduced serum‐induced adhesion strength to similar levels as those obtained by blocking myosin light chain phosphorylation. Reductions in adhesion strengthening by inhibitors of contractility correlated with loss of vinculin and talin from focal adhesions without changes in integrin binding. In vinculin‐null cells, inhibition of contractility did not alter adhesive force, whereas controls displayed a 20% reduction in adhesion strength, indicating that the effects of contractility on adhesive force are vinculin‐dependent. Furthermore, in cells expressing FAK, inhibitors of contractility reduced serum‐induced adhesion strengthening as well as eliminated focal adhesion assembly. In contrast, in the absence of FAK, these inhibitors did not alter adhesion strength or focal adhesion assembly. These results indicate that contractility modulates adhesion strengthening via FAK‐dependent, vinculin‐containing focal adhesion assembly. J. Cell. Physiol. 223:746–756, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Invasion of host cells by Salmonella typhimurium requires focal adhesion kinase and p130Cas

Salmonella typhimurium colonizes the intestinal epithelium by injecting an array of effector proteins into host cells that induces phagocytic uptake of attached bacteria. However, the host molecules targeted by these effectors remain poorly defined. Here, we demonstrate that S. typhimurium induces formation of focal adhesion-like complexes at sites of bacterial attachment and that both focal adhesion kinase (FAK) and the scaffolding protein p130Cas are required for Salmonella uptake. Entry of Salmonella into FAK(-/-) cells is dramatically impaired and can be restored to control levels by expression of wild-type FAK. Surprisingly, reconstitution of bacterial internalization requires neither the kinase domain of FAK nor activation of c-Src, but does require a C-terminal PXXP motif through which FAK interacts with Cas. Infection of Cas(-/-) cells is also impaired, and reconstitution of invasiveness requires the central Cas YXXP repeat domain. The invasion defect in Cas(-/-) cells can be suppressed by overexpression of FAK, suggesting a functional link between FAK and Cas in the regulation of Salmonella invasion. Together, these findings reveal a novel role for focal adhesion proteins in the invasion of host cells by Salmonella.     

Caspase-mediated cleavage of p130cas in etoposide-induced apoptotic Rat-1 cells

Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell-cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD(416)G and DSPD(748)G) in vitro, and point mutations substituting the Asp of DVPD(416)G and DSPD(748)G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD(416)G generated a 74-kDa fragment, which was in turn cleaved at DSPD(748)G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas-paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.     

Hic-5-Reduced Cell Spreading on Fibronectin: Competitive Effects between Paxillin and Hic-5 through Interaction with Focal Adhesion Kinase

Hic-5 is a paxillin homologue that is localized to focal adhesion complexes. Hic-5 and paxillin share structural homology and interacting factors such as focal adhesion kinase (FAK), Pyk2/CAKbeta/RAFTK, and PTP-PEST. Here, we showed that Hic-5 inhibits integrin-mediated cell spreading on fibronectin in a competitive manner with paxillin in NIH 3T3 cells. The overexpression of Hic-5 sequestered FAK from paxillin, reduced tyrosine phosphorylation of paxillin and FAK, and prevented paxillin-Crk complex formation. In addition, Hic-5-mediated inhibition of spreading was not observed in mouse embryo fibroblasts (MEFs) derived from FAK(-/-) mice. The activity of c-Src following fibronectin stimulation was decreased by about 30% in Hic-5-expressing cells, and the effect of Hic-5 was restored by the overexpression of FAK and the constitutively active forms of Rho-family GTPases, Rac1 V12 and Cdc42 V12, but not RhoA V14. These observations suggested that Hic-5 inhibits cell spreading through competition with paxillin for FAK and subsequent prevention of downstream signal transduction. Moreover, expression of antisense Hic-5 increased spreading in primary MEFs. These results suggested that the counterbalance of paxillin and Hic-5 expression may be a novel mechanism regulating integrin-mediated signal transduction.     

Alpha-actinin-1 phosphorylation modulates pressure-induced colon cancer cell adhesion through regulation of focal adhesion kinase-Src interaction

Physical forces including pressure, strain, and shear can be converted into intracellular signals that regulate diverse aspects of cell biology. Exposure to increased extracellular pressure stimulates colon cancer cell adhesion by a beta(1)-integrin-dependent mechanism that requires an intact cytoskeleton and activation of focal adhesion kinase (FAK) and Src. alpha-Actinin facilitates focal adhesion formation and physically links integrin-associated focal adhesion complexes with the cytoskeleton. We therefore hypothesized that alpha-actinin may be necessary for the mechanical response pathway that mediates pressure-stimulated cell adhesion. We reduced alpha-actinin-1 and alpha-actinin-4 expression with isoform-specific small interfering (si)RNA. Silencing of alpha-actinin-1, but not alpha-actinin-4, blocked pressure-stimulated cell adhesion in human SW620, HT-29, and Caco-2 colon cancer cell lines. Cell exposure to increased extracellular pressure stimulated alpha-actinin-1 tyrosine phosphorylation and alpha-actinin-1 interaction with FAK and/or Src, and enhanced FAK phosphorylation at residues Y397 and Y576. The requirement for alpha-actinin-1 phosphorylation in the pressure response was investigated by expressing the alpha-actinin-1 tyrosine phosphorylation mutant Y12F in the colon cancer cells. Expression of Y12F blocked pressure-mediated adhesion and inhibited the pressure-induced association of alpha-actinin-1 with FAK and Src, as well as FAK activation. Furthermore, siRNA-mediated reduction of alpha-actinin-1 eliminated the pressure-induced association of alpha-actinin-1 and Src with beta(1)-integrin receptor, as well as FAK-Src complex formation. These results suggest that alpha-actinin-1 phosphorylation at Y12 plays a crucial role in pressure-activated cell adhesion and mechanotransduction by facilitating Src recruitment to beta(1)-integrin, and consequently the association of FAK with Src, to enhance FAK phosphorylation.