Vinculin Phosphorylation Research Articles

Dysfunctional mechanotransduction regulates the progression of PIK3CA-driven vascular malformations.

Somatic activating mutations in PIK3CA are common drivers of vascular and lymphatic malformations. Despite common biophysical signatures of tissues susceptible to lesion formation, including compliant extracellular matrix and low rates of perfusion, lesions vary in clinical presentation from localized cystic dilatation to diffuse and infiltrative vascular dysplasia. The mechanisms driving the differences in disease severity and variability in clinical presentation and the role of the biophysical microenvironment in potentiating progression are poorly understood. Here, we investigate the role of hemodynamic forces and the biophysical microenvironment in the pathophysiology of vascular malformations, and we identify hemodynamic shear stress and defective endothelial cell mechanotransduction as key regulators of lesion progression. We found that constitutive PI3K activation impaired flow-mediated endothelial cell alignment and barrier function. We show that defective shear stress sensing in PIK3CA E542K endothelial cells is associated with reduced myosin light chain phosphorylation, junctional instability, and defective recruitment of vinculin to cell-cell junctions. Using 3D microfluidic models of the vasculature, we demonstrate that PIK3CA E542K microvessels apply reduced traction forces and are unaffected by flow interruption. We further found that draining transmural flow resulted in increased sprouting and invasion responses in PIK3CA E542K microvessels. Mechanistically, constitutive PI3K activation decreased cellular and nuclear elasticity resulting in defective cellular tensional homeostasis in endothelial cells which may underlie vascular dilation, tissue hyperplasia, and hypersprouting in PIK3CA-driven venous and lymphatic malformations. Together, these results suggest that defective nuclear mechanics, impaired cellular mechanotransduction, and maladaptive hemodynamic responses contribute to the development and progression of PIK3CA-driven vascular malformations.

Abstract P2074: Site-specific Phosphorylation Of Vinculin Regulates N-cadherin Junction Assembly

Introduction: During physiological and pathological growth, cardiomyocytes adapt and remodel in response to changes in mechanical load. The cytoskeleton plays a central role in tissue remodeling as it both senses mechanical stress and mediates structural remodeling which affects the functional response within the cardiomyocyte. Abl kinase-dependent vinculin (VCL) phosphorylation at tyrosine (Y) residue 822 (pVCL-Y822) has been implicated in force-induced cytoskeletal remodeling and adhesion strengthening at cadherin junctions. However, the functional significance of VCL phosphorylation in the working myocardium remains unknown. Objective: To determine the role of vinculin-mediated mechanotransduction in cardiac development, homeostasis, and disease. Methods and Results: Using a phospho-specific antibody, we found that pVCL-Y822 correlates with dynamic junction remodeling in embryonic cardiomyocytes which declines in the postnatal heart when N-cadherin junctions assemble at the intercalated disc. Notably, pVCL-Y822 is upregulated in border zone cardiomyocytes following myocardial infarction. To investigate VCL-mediated mechanotransduction in the heart, CRISPR-mediated genome editing was used to mutate the tyrosine at position 822 to a non-phosphorylatable phenylalanine (F) in the mouse Vcl gene. Vcl Y822F mice were born in the expected Mendelian frequency and are viable. Blocking VCL phosphorylation causes a preferential reduction of mutant VCL protein at the myocyte termini together with the N-cadherin-catenin adhesion complex. Whereas, α5/β1 integrin and fibronectin are both increased along the lateral border of the Vcl Y822F postnatal cardiomyocytes. Vcl Y822F mice exhibit reduced cardiac function with age; however, cardiac remodeling is attenuated in the mutant hearts. Conclusion: This novel animal model demonstrates how blocking vinculin Y822 phosphorylation affects the balance between cadherin adhesions and integrin adhesions and thereby highlights the importance of understanding the crosstalk between the two adhesion systems and its implications for disease pathogenesis.

Stiff Substrates Increase Inflammation-Induced Endothelial Monolayer Tension and Permeability

Arterial stiffness and inflammation are associated with atherosclerosis, and each have individually been shown to increase endothelial monolayer tension and permeability. The objective of this study was to determine if substrate stiffness enhanced endothelial monolayer tension and permeability in response to inflammatory cytokines. Porcine aortic endothelial cells were cultured at confluence on polyacrylamide gels of varying stiffness and treated with either tumor necrosis factor-α (TNFα) or thrombin. Monolayer tension was measured through vinculin localization at the cell membrane, traction force microscopy, and phosphorylated myosin light chain quantity and actin fiber colocalization. Cell permeability was measured by cell-cell junction confocal microscopy and a dextran permeability assay. When treated with TNFα or thrombin, endothelial monolayers on stiffer substrates showed increased traction forces, vinculin at the cell membrane, and vinculin phosphorylation, suggesting elevated monolayer tension. Interestingly, VE-cadherin shifted toward a smaller molecular weight in endothelial monolayers on softer substrates, which may relate to increased VE-cadherin endocytosis and degradation. Phosphorylated myosin light chain colocalization with actin stress fibers increased in endothelial monolayers treated with TNFα or thrombin on stiffer substrates, indicating elevated cell monolayer contractility. Endothelial monolayers also developed focal adherens intercellular junctions and became more permeable when cultured on stiffer substrates in the presence of the inflammatory cytokines. Whereas each of these effects was likely mitigated by Rho/ROCK, Rho/ROCK pathway inhibition via Y27632 disrupted cell-cell junction morphology, showing that cell contractility is required to maintain adherens junction integrity. These data suggest that stiff substrates change intercellular junction protein localization and degradation, which may counteract the inflammation-induced increase in endothelial monolayer tension and thereby moderate inflammation-induced junction loss and associated endothelial monolayer permeability on stiffer substrates.

Clonorchis sinensis excretory-secretory products promote the migration and invasion of cholangiocarcinoma cells by activating the integrin β4–FAK/Src signaling pathway

Cholangiocarcinoma (CCA) is a slow-growing but highly metastatic cancer. Its metastatic potential largely explains its high mortality rate. A recognized risk factor for CCA development is infection with the liver flukes Opisthorchis viverrini and Clonorchis sinensis. We previously reported that the excretory-secretory products (ESPs) of C. sinensis promoted the three-dimensional aggregation and invasion of CCA cells. In the present study, a quantitative real-time PCR array of extracellular matrix (ECM) and adhesion molecules was used to examine the regulatory mechanism of ESP-mediated CCA cell migration and invasion. In particular, the expression levels of integrin α isoforms and β4 were upregulated in response to ESPs. Increased expression of integrin β4 was probably correlated with activation of focal adhesion kinase (FAK) and the steroid receptor coactivator (Src) family kinase and the subsequent activation of two downstream focal adhesion molecules, paxillin and vinculin. Moreover, inhibition of FAK/Src activation reduced paxillin and vinculin phosphorylation and attenuated ESP-induced CCA cell migration and invasion. These findings suggest that the integrin β4–FAK/Src signaling axis may play a crucial role in clonorchiasis-associated CCA metastasis during tumor progression.

Vinculin phosphorylation at residues Y100 and Y1065 is required for cellular force transmission.

The focal adhesion protein vinculin connects the actin cytoskeleton, through talin and integrins, with the extracellular matrix. Vinculin consists of a globular head and tail domain, which undergo conformational changes from a closed auto-inhibited conformation in the cytoplasm to an open conformation in focal adhesions. Src-mediated phosphorylation has been suggested to regulate this conformational switch. To explore the role of phosphorylation in vinculin activation, we used knock-out mouse embryonic fibroblasts re-expressing different vinculin mutants in traction microscopy, magnetic tweezer microrheology, FRAP and actin-binding assays. Compared to cells expressing wild-type or constitutively active vinculin, we found reduced tractions, cytoskeletal stiffness, adhesion strength, and increased vinculin dynamics in cells expressing constitutively inactive vinculin or vinculin where Src-mediated phosphorylation was blocked by replacing tyrosine at position 100 and/or 1065 with a non-phosphorylatable phenylalanine residue. Replacing tyrosine residues with phospho-mimicking glutamic acid residues restored cellular tractions, stiffness and adhesion strength, as well as vinculin dynamics, and facilitated vinculin-actin binding. These data demonstrate that Src-mediated phosphorylation is necessary for vinculin activation, and that phosphorylation controls cytoskeletal mechanics by regulating force transmission between the actin cytoskeleton and focal adhesion proteins.

Serine phosphorylation on position 1033 of vinculin impacts cellular mechanics

This study evaluates the influence of S1033 vinculin phosphorylation on the mechanical properties of cells. We demonstrate that MEFvcl KO cells transfected with the non-phosphorylatable eGFP-vinculin mutant S1033A are of lower stiffness compared to MEFvcl Rescue and phospho-mimicking mutant S1033D cells, which were of similar stiffness. Analogous, 2D traction microscopy indicates that MEFvcl Rescue and MEF mutant S1033D cells generate similar strain energy, but mutant S1033A cells display ∼50% less strain energy. Fluorescence recovery after photobleaching demonstrates that the recovery time for mutant S1033A was significantly lower compared to MEFvcl Rescue and mutant S1033D and that the mobile fraction was smaller for MEFvcl Rescue and mutant S1033D than for mutant S1033A cells. This indicates that serine phosphorylation is required for the activation of vinculin and force transmission in focal adhesions.

Vinculin phosphorylation differentially regulates mechanotransduction at cell-cell and cell-matrix adhesions.

Cells experience mechanical forces throughout their lifetimes. Vinculin is critical for transmitting these forces, yet how it achieves its distinct functions at cell-cell and cell-matrix adhesions remains unanswered. Here, we show vinculin is phosphorylated at Y822 in cell-cell, but not cell-matrix, adhesions. Phosphorylation at Y822 was elevated when forces were applied to E-cadherin and was required for vinculin to integrate into the cadherin complex. The mutation Y822F ablated these activities and prevented cells from stiffening in response to forces on E-cadherin. In contrast, Y822 phosphorylation was not required for vinculin functions in cell-matrix adhesions, including integrin-induced cell stiffening. Finally, forces applied to E-cadherin activated Abelson (Abl) tyrosine kinase to phosphorylate vinculin; Abl inhibition mimicked the loss of vinculin phosphorylation. These data reveal an unexpected regulatory mechanism in which vinculin Y822 phosphorylation determines whether cadherins transmit force and provides a paradigm for how a shared component of adhesions can produce biologically distinct functions.

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation, in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine stimulation of tracheal smooth muscle tissues induces the recruitment of vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr(1065) on its C terminus increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of vinculin phosphorylation at Tyr(1065) in regulating the conformation and function of vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr(1065) (vinculin Y1065F and vinculin Y1065E) and vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr(1065) phosphorylation affects smooth muscle contraction and the conformation and cellular functions of vinculin. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr(1065) phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. We conclude that the phosphorylation of vinculin at tyrosine 1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to contractile stimulation.

Vinculin phosphorylation differentially regulates mechanotransduction at cell–cell and cell–matrix adhesions

Vinculin phosphorylation differentially regulates mechanotransduction at cell–cell and cell–matrix adhesions

Conformational Changes in Vinculin Measured by Fluorescence Resonance Energy Transfer (FRET) during Airway Smooth Muscle (ASM) Contraction Depend on Vinculin Phosphorylation at Tyrosine 1065

Vinculin links integrins to actin filaments by binding to talin and actin. Vinculin can exist in a folded conformation with these binding sites masked, thus preventing its binding to actin and talin, or in an open ligand‐binding state with actin and talin binding sites exposed. ACh (acetylcholine) stimulation of ASM tissues increases the association of vinculin with actin and talin and stimulates vinculin phosphorylation at Tyr 1065 (Huang et al. JBC, 2011). We expressed non‐phosphorylatable vinculin Y1065F in ASM tissues to inhibit endogenous vinculin phosphorylation; this depressed contraction and inhibited the interaction of vinculin with talin. We constructed vinculin Wt and vinculin Y1065F FRET probes such that a FRET signal was generated when vinculin was in the closed conformation, but not in an open conformation. Probes were expressed in ASM tissues and FRET signals were analyzed in dissociated cells. Wt vinculin maintained a folded conformation in the cytoplasm of unstimulated ASM cells. ACh stimulation increased the open conformation of Wt vinculin at the membrane, but vinculin Y1065F maintained a closed conformation with or without ACh stimulation. We conclude that ACh stimulation of ASM causes a conformational change in vinculin that allows it to bind to talin and actin, and that phosphorylation at Tyr 1065 is required for vinculin to maintain an open ligand‐binding conformation. Support: NIH HL‐29289; HL074099

Role of Molecular Determinants of Store-operated Ca2+ Entry (Orai1, Phospholipase A2 Group 6, and STIM1) in Focal Adhesion Formation and Cell Migration

Store-operated Ca(2+) entry is important for cell migration. This study presents characterization of localization and roles of Orai1, STIM1, and PLA2g6 in adhesion dynamics during cell migration. Orai1 and PLA2g6 are involved in adhesion formation at the front, whereas STIM1 participates in both adhesion formation and disassembly. Results uncovered new parameters of Orai1, STIM1, and PLA2g6 involvement in cell migration. Store-operated Ca(2+) entry and its major determinants are known to be important for cell migration, but the mechanism of their involvement in this complex process is unknown. This study presents a detailed characterization of distinct roles of Orai1, STIM1, and PLA2g6 in focal adhesion (FA) formation and migration. Using HEK293 cells, we discovered that although molecular knockdown of Orai1, STIM1, or PLA2g6 resulted in a similar reduction in migration velocity, there were profound differences in their effects on number, localization, and lifetime of FAs. Knockdown of STIM1 caused an increase in lifetime and number of FAs, their redistribution toward lamellae region, and an increase in cell tail length. In contrast, the number of FAs in Orai1- or PLA2g6-deficient cells was significantly reduced, and FAs accumulated closer to the leading edge. Assembly rate and Vinculin phosphorylation of FAs was similarly reduced in Orai1, PLA2g6, or STIM1-deficient cells. Although Orai1 and PLA2g6 accumulated and co-localized at the leading edge, STIM1 distribution was more complex. We found STIM1 protrusions in lamellipodia, which co-localized with FAs, whereas major accumulation could be seen in central and retracting parts of the cell. Interestingly, knockdown of Orai1 and PLA2g6 produced similar and non-additive effect on migration, whereas knockdown of STIM1 simultaneously with either Orai1 or PLA2g6 produced additional inhibition. Together these data suggest that although Orai1, PLA2g6, and STIM1 play major roles in formation of new FAs at the leading edge, STIM1 may also be involved in Orai1- and PLA2g6-independent disassembly of FAs in the back of cells.

Phosphorylation Primes Vinculin for Activation

Vinculin phosphorylation has been implicated as a potential mechanism for focal adhesion growth and maturation. Four vinculin residues—Y100, S1033, S1045, and Y1065—are phosphorylated by kinases during focal adhesion maturation. In this study, phosphorylation at each of these residues is simulated using molecular dynamics models. The simulations demonstrate that once each phosphorylated vinculin structure is at equilibrium, significant local conformational changes result that may impact either vinculin activation or vinculin binding to actin and PIP2. Simulation of vinculin activation after phosphorylation shows that the added phosphoryl groups can prime vinculin for activation. It remains to be seen if vinculin can be phosphorylated at S1033 in vivo, but these simulations highlight that in the event of a S1033 phophorylation vinculin will likely be primed for activation.

Serum‐ and glucocorticoid‐dependent kinase‐1‐induced cell migration is dependent on vinculin and regulated by the membrane androgen receptor

The serum- and glucocorticoid-dependent kinases 1-3 (SGK1-3) are downstream effectors of phosphatidylinositol 3-kinases, implicated in various cell responses including colon cancer tumorigenesis in mice. Here, we investigated the role of SGK1 in the regulation of cell motility. Using Caco-2 colon tumor and HEK293 embryonic kidney cells, we report that transfection with the constitutively active SGK1 mutant (SGK1-SD) significantly enhanced cell motility. The cell-adhesion protein vinculin was effectively dephosphorylated in SGK1-SD-transfected cells. Treatment of the cells with phosphatase inhibitors restored vinculin phosphorylation and inhibited cell migration, indicating a significant role for vinculin phosphorylation in SGK1-induced motility. SGK1-SD-enhanced cell motility was inhibited by activation of membrane androgen-binding sites (mAR) via testosterone-conjugates in both cell lines, whereas intracellular androgen receptor (iAR)-silencing and flutamide treatment revealed that these effects were clearly independent of the interaction of SGK1 with the classical androgen receptors (iAR). More importantly, mAR activation restored vinculin phosphorylation in SGK1-SD-transfected cells, whereas silencing of vinculin fully reversed the mAR-induced inhibition of the migratory capacity, implying that this protein is directly involved in cell motility regulation by SGK1 and mAR. This study indicates for the first time that SGK1 regulates cell migration via vinculin dephosphorylation, a mechanism that is controlled by mAR function.

SGK1 Sensitivity of Platelet Migration

Recent observations pointed to the ability of platelets to migrate and thus to invade the inflamed vascular wall. Platelet migration could be stimulated by stromal cell-derived factor-1 (SDF-1), an effect dependent on phosphatidylinositide-3-kinase (PI3K) and paralleled by activation and phosphorylation of Wiskott-Aldrich syndrome protein (WASP). Migration is inhibited by vinculin, which is similarly regulated by phosphorylation. PI3K-sensitive kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored whether SGK1 modifies WASP and vinculin phosphorylation in murine platelets and participates in the regulation of platelet migration. Platelets were isolated from gene-targeted mice lacking SGK1 (sgk1^-/-) and from their wild type littermates (sgk1^+/+). Platelet migration stimulated with SDF-1 was significantly less pronounced in sgk1^-/-platelets than in sgk1^+/+ platelets. Moreover, SDF-1 significantly induced WASP phosphorylation, an effect again reduced in platelets lacking SGK1. Phosphorylation of vinculin was significantly enhanced in sgk1^-/-platelets and was significantly reduced following treatment of platelets with Ca²⁺ chelator BAPTA. Immunohistochemical analysis of in vivo experiments in intestinal vessels after vascular inflammation revealed that transmigration of platelets into inflamed vessel walls was significantly less pronounced in sgk1^-/-than in sgk1^+/+ mice. In conclusion, SGK1 is a powerful regulator of platelet migration.

Ethanol suppresses phagosomal adhesion maturation, Rac activation, and subsequent actin polymerization during FcγR-mediated phagocytosis

Clinical and laboratory investigations have provided evidence that ethanol suppresses normal lung immunity. Our initial studies revealed that acute ethanol exposure results in transient suppression of phagocytosis of Pseudomonas aeruginosa by macrophages as early as 3h after initial exposure. Focusing on mechanisms by which ethanol decreases macrophage Fcγ-receptor (FcγR) phagocytosis we targeted the study on the focal adhesion and cytoskeletal elements that are necessary for phagosome progression. Ethanol inhibited macrophage phagocytosis of IgG-coated bead recruitment of actin to the site of the phagosome, dampened the phosphorylation of vinculin, but had no effect on paxillin phosphorylation suggesting a loss in “phagosomal adhesion” maturation. Moreover, our observations revealed that FcγR-phagocytosis induced Rac activation, which was increased by only 50% in ethanol exposed cells, compared to 175% in the absence of ethanol. This work is the first to show evidence of the cellular mechanisms involved in the ethanol-induced suppression of FcγR-mediated phagocytosis.

Quantitative mapping of averaged focal adhesion dynamics in migrating cells by shape normalization

The spatially ordered formation and disassembly of focal adhesions is a basic requirement for effective cell locomotion. Because focal adhesions couple the contractile actin-myosin network to the substrate, their distribution determines the pattern of traction forces propelling the cell in a certain direction. In the present study, we quantitatively analyzed the spatial patterning of cell-substrate adhesion in migrating cells by mapping averaged focal adhesion growth dynamics to a standardized cell coordinate system. These maps revealed distinct zones of focal adhesion assembly, disassembly and stability and were strongly interrelated with corresponding actin flow and traction force patterns. Moreover, the mapping technique enables precise detection of even minute responses of adhesion dynamics upon targeted signaling perturbations. For example, the partial inhibition of vinculin phosphorylation was followed by the reduced number of newly formed adhesions, whereas growth dynamics of existing adhesions remained unaffected.

Activation of Vinculin Induced by Cholinergic Stimulation Regulates Contraction of Tracheal Smooth Muscle Tissue

Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.

Becoming stable and strong: The interplay between vinculin exchange dynamics and adhesion strength during adhesion site maturation

The coordinated formation and release of focal adhesions is necessary for cell attachment and migration. According to current models, these processes are caused by temporal variations in protein composition. Protein incorporation into focal adhesions is believed to be controlled by phosphorylation. Here, we tested the exchange dynamics of GFP-vinculin as marker protein of focal adhesions using the method of Fluorescence Recovery After Photobleaching. The relevance of the phosphorylation state of the protein, the age of focal adhesions and the acting force were investigated. For stable focal adhesions of stationary keratinocytes, we determined an exchangeable vinculin fraction of 52% and a recovery halftime of 57 s. Nascent focal adhesions of moving cells contained a fraction of exchanging vinculin of 70% with a recovery halftime of 36 s. Upon maturation, mean saturation values and recovery halftimes decreased to levels of 49% and 42 s, respectively. Additionally, the fraction of stably incorporated vinculin increased with cell forces and decreased with vinculin phosphorylation within these sites. Experiments on a nonphosphorylatable vinculin mutant construct at phosphorylation site tyr1065 confirmed the direct interplay between phosphorylation and exchange dynamics of adhesion proteins during adhesion site maturation.

Activation of Vinculin Induced by Cholinergic Stimulation Regulates Tension Development in Tracheal Smooth Muscle (TSM)

The adhesion protein vinculin functions in focal adhesions to link actin filaments to membrane adhesion junctions by binding to the integrin‐binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). In the inactive (closed) state, Vh and Vt bind to each other, masking the binding sites for actin and talin; in the active (open) state, vinculin binding sites for talin and actin are exposed. We hypothesized that the contractile activation of TSM might regulate conformation of vinculin and thereby contribute to the regulation of tension development. Stimulation of TSM tissues with acetylcholine (ACh) increased the co‐immunoprecipitation of vinculin and talin from tissue extracts, and increased vinculin phosphorylation at tyr 1065, an indicator of vinculin activation. Recombinant Vh (EGFP‐Vh) expressed in TSM tissues competed with endogenous vinculin for binding to talin and inhibited vinculin tyr 1065 phosphorylation. Expression of EGFP‐Vh depressed TSM contraction to ACh by 60% and inhibited ACh‐induced actin polymerization. EGFP‐Vh did not affect myosin light chain phosphorylation. We conclude that vinculin is activated in TSM by ACh, and that vinculin regulates tension by promoting connections between actin filaments and talin‐integrin adhesion complexes and by facilitating the initiation of actin polymerization.Support: AHA 0820074Z, HL 29289, HL 074099

Paxillin-Y118 phosphorylation contributes to the control of Src-induced anchorage-independent growth by FAK and adhesion

BackgroundFocal adhesion kinase (FAK) and Src are protein tyrosine kinases that physically and functionally interact to facilitate cancer progression by regulating oncogenic processes such as cell motility, survival, proliferation, invasiveness, and angiogenesis.MethodTo understand how FAK affects oncogenesis through the phosphorylation of cellular substrates of Src, we analyzed the phosphorylation profile of a panel of Src substrates in parental and v-Src-expressing FAK+/+ and FAK-/- mouse embryo fibroblasts, under conditions of anchorage-dependent (adherent) and -independent (suspension) growth.ResultsTotal Src-induced cellular tyrosine phosphorylation as well as the number of phosphotyrosyl substrates was higher in suspension versus adherent cultures. Although the total level of Src-induced cellular phosphorylation was similar in FAK+/+ and FAK-/- backgrounds, the phosphorylation of some substrates was influenced by FAK depending on adherence state. Specifically, in the absence of FAK, Src induced higher phosphorylation of p190RhoGAP, paxillin (poY118) and Crk irrespective of adhesion state, PKC-δ (poY311), connexin-43 (poY265) and Sam68 only under adherent conditions, and p56Dok-2 (poY351) and p120catenin (poY228) only under suspension conditions. In contrast, FAK enhanced the Src-induced phosphorylation of vinculin (poY100 and poY1065) and p130CAS (poY410) irrespective of adherence state, p56Dok-2 (poY351) and p120catenin (poY228) only under adherent conditions, and connexin-43 (poY265), cortactin (poY421) and paxillin (poY31) only under suspension conditions. The Src-induced phosphorylation of Eps8, PLC-γ1 and Shc (poY239/poY240) were not affected by either FAK or adherence status. The enhanced anchorage-independent growth of FAK-/-[v-Src] cells was selectively decreased by expression of paxillinY118F, but not by WT-paxillin, p120cateninY228F or ShcY239/240F, identifying for the first time a role for paxillinpoY118 in Src-induced anchorage-independent growth. Knockdown of FAK by siRNA in the human colon cancer lines HT-25 and RKO, resulted in increased paxillinpoY118 levels under suspension conditions as well as increased anchorage-independent growth, supporting the notion that FAK attenuates anchorage-independent growth by suppressing adhesion-dependent phosphorylation of paxillinY118.ConclusionThese data suggest that phosphorylation of Src substrates is a dynamic process, influenced temporally and spatially by factors such as FAK and adhesion.