Protein Paxillin Research Articles

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

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

Dependence of EGF-Induced Increases in Corneal Epithelial Proliferation and Migration on GSK-3 Inactivation

This study was designed to determine in human corneal epithelial cells (HCEC) whether the balance between epidermal growth factor (EGF)-induced increases in proliferation and migration is dependent on the duration and magnitude of extracellular signal-regulated kinase (Erk)1/2 activation. Western blot analysis evaluated the phosphorylation status of Erk1/2 and phosphoinositide 3-kinase (PI3-K) along with cell cycle kinases, paxillin, and mitogen kinase protein phosphatase (MKP)-1. Proliferation and migration rates were determined by [(3)H]-thymidine incorporation and scratch wound healing assay, respectively. EGF induced increases in paxillin Ser-126 phosphorylation and cyclin D1 expression through transient Erk1/2 phosphorylation. However, preinhibition of glycogen synthase kinase (GSK)-3 activation with 20 microM SB415286 prolonged and augmented this Erk1/2 response to EGF but decreased cyclin D1 expression, whereas p27Kip1 levels rose. In turn, the mitogenic response fell, whereas paxillin phosphorylation occurred 45 minutes sooner than without SB415286. In contrast, blocking PI3-K activation with LY294002 (50 microM) eliminated EGF-induced GSK-3 inhibition and Erk1/2 phosphorylation as well as increases in proliferation and migration. SB415286 or U0126 (10 microM) suppression of Erk1/2 phosphorylation blocked EGF-induced MKP-1 phosphorylation. Inhibition of EGF-induced increases in proliferation and migration by LY294002 was associated with sustained MKP-1 phosphorylation induced by GSK-3. Prolonging MKP-1 phosphorylation by LY294002 increased p27Kip1, whereas cyclin D1 levels fell. GSK-3-induced MKP-1 phosphorylation mediates negative feedback control between EGF receptor-linked PI3-K and ERK signaling pathways. Inhibition of such control prolongs Erk1/2 activation and alters the balance between EGF-induced increases in proliferation and migration. Therefore, these responses to EGF can be modulated through altering the feedback between these two pathways.

Role of c-Met in Cancer: Emphasis on Lung Cancer

Lung cancer remains the leading cause of cancer death. It is often diagnosed at late stages and is treated systemically with cytotoxic chemotherapy, which is generally ineffective. Research efforts have focused on developing therapies targeted to growth factor receptor pathways, such as epidermal growth factor receptor (EGFR), but the results from clinical trials overall show very small improvements in survival. Research on signaling pathways dysregulated in lung cancer is ongoing, including investigation of the hepatocyte growth factor receptor (HGFR) or c-Met. Protein tyrosine kinases, such as EGFR and c-Met, are a family of oncogenes that regulate important cellular processes, such as differentiation, proliferation, cell cycle, motility, and apoptosis. Hepatocyte growth factor (HGF), a ligand for c-Met, is secreted by mesodermal cells during development. It produces multiple effects upon binding to its receptor (HGFR/c-Met) and regulates proliferation, motility, mitogenesis, and morphogenesis. Studies in cell lines isolated from various tumors show that several intracellular pathways participate in c-Met signaling, including growth factor receptor-bound protein 2 (Grb2), mitogen-activated protein (MAP) kinase, phosphoinositol 3-kinase (PI3K), and phospholipase C-gamma (PLC-gamma). c-Met is overexpressed in many tumors. However, overexpression may not be sufficient to cause increased activity; the receptor needs to be activated. In some cases, the kinases are constitutively active due to mutations in the gene. The cytoskeletal protein paxillin also appears to be activated along with c-Met. Correlative studies from patient tissue samples and cell lines have rendered the same information, indicating that the signaling pathways dysregulated are complex and interdependent. Mutations in human c-Met have been exogenously expressed in Caenorhabditis elegans, which can serve as a model for determining the role of gene mutations in a whole organism. Several inhibitors of c-Met/HGF binding are in development, including some in phase I trials. Their effectiveness in improving cancer outcomes will be determined in the near future.

Roles of focal adhesion kinase and paxillin in focal adhesion dynamics of living endothelial cells

Focal adhesion kinase (FAK) is a signaling protein that can activate the adaptor protein paxillin, which is also localized at focal adhesions (FAs) and associated with integrins, to influence the cytoskeleton and membrane protrusions, thereby regulating FA dynamics and cell motility. In living ECs transfected with GFP‐FAK and DsRed‐paxillin, concurrent monitoring of the intracellular dynamics of FAK and paxillin by time‐lapse video recording and dual‐color fluorescence imaging showed the co‐localization of FAK and paxillin at FAs. The ratio of the fluorescence intensities (FI) of FAK/paxillin was determined during assembly/disassembly of FAs. The FAK/paxillin FI ratio of FA sites was higher in the cytoplasm than in the perinuclear region. This ratio in the cytoplasm increased as the dynamics of FA sites became active, especially at the leading edge of the migrating EC. Our findings show the roles of FAK and paxillin in the assembly/disassembly of FAs and in cell adhesion. We conclude that both molecules play important roles in modulating the FA dynamics and cellular signaling during cell migration. This work was supported by NHLBI Research Grants HL‐080518 and HL‐085159 (S.C.).

Distribution of cytoskeletal and adhesion proteins in adult zebrafish skeletal muscle.

The organization of cytoskeletal and adhesion proteins in skeletal muscle is critical for its contractile function. Zebrafish has become a paramount model for studies of vertebrate biology, including muscle. However, only a few studies have been published using immunolabeling to specifically localize proteins in adult zebrafish muscle. To fully appreciate the distribution of cytoskeletal and adhesion proteins, and therefore to better correlate the adult muscle with its myogenesis, we used indirect immunofluorescence microscopy of frozen adult zebrafish skeletal muscle sections. Here we describe the fish muscle cytoskeletal architecture and location of the major myofibrillar proteins desmin, alpha-actinin, myosin, titin, troponin, tropomyosin and nebulin, the adhesion proteins vinculin and paxillin, and the extracellular matrix proteins laminin and fibronectin. Electron microscopical analysis in ultra-thin sections of adult zebrafish skeletal muscle showed bundles of collagen fibers and fibroblastic cells in the extracellular space of the myosepta.

A Role for the Juxtamembrane Cytoplasm in the Molecular Dynamics of Focal Adhesions

Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in live cells, with in silico simulations and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplasmic pool. The juxtamembrane region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentration and dynamics. Based on these findings, we propose a new model for the regulation of FA dynamics in which this juxtamembrane domain acts as an intermediary layer, enabling an efficient regulation of FA formation and reorganization.

Activation of c-Src and Fyn Kinases by Protein-tyrosine Phosphatase RPTPα Is Substrate-specific and Compatible with Lipid Raft Localization

Src family tyrosine kinases (SFKs) function in multiple signaling pathways, raising the question of how appropriate regulation and substrate choice are achieved. SFK activity is modulated by several protein-tyrosine phosphatases, among which RPTPalpha and SHP2 are the best established. We studied how RPTPalpha affects substrate specificity and regulation of c-Src and Fyn in response to epidermal growth factor and platelet-derived growth factor. We find that RPTPalpha, in a growth factor-specific manner, directs the specificity of these kinases toward a specific subset of SFK substrates, particularly the focal adhesion protein Paxillin and the lipid raft scaffolding protein Cbp/PAG. A significant fraction of RPTPalpha is present in lipid rafts, where its targets Fyn and Cbp/PAG reside, and growth factor-mediated SFK activation within this compartment is strictly dependent on RPTPalpha. Forced concentration of RPTPalpha into lipid rafts is compatible with activation of Fyn. Finally, RPTPalpha-induced phosphorylation of Paxillin and Cbp/PAG induces recruitment of the SFK inhibitory kinase Csk, indicative of negative feedback loops limiting SFK activation by RPTPalpha. Our findings indicate that individual SFK-controlling PTPs play important and specific roles in dictating SFK substrate specificity and regulatory mechanism.

Ultrastructural localization of actin and actin-binding proteins in the nucleus

Nuclear actin plays an important role in such processes as chromatin remodeling, transcriptional regulation, RNA processing, and nuclear export. Recent research has demonstrated that actin in the nucleus probably exists in dynamic equilibrium between monomeric and polymeric forms, and some of the actin-binding proteins, known to regulate actin dynamics in cytoplasm, have been also shown to be present in the nucleus. In this paper, we present ultrastructural data on distribution of actin and various actin-binding proteins (alpha-actinin, filamin, p190RhoGAP, paxillin, spectrin, and tropomyosin) in nuclei of HeLa cells and resting human lymphocytes. Probing extracts of HeLa cells for the presence of actin-binding proteins also confirmed their presence in nuclei. We report for the first time the presence of tropomyosin and p190RhoGAP in the cell nucleus, and the spatial colocalization of actin with spectrin, paxillin, and alpha-actinin in the nucleolus.

Migration Potential of Human Mesenchymal Stromal Cells Is Determined by Origin of Tissue Rather Than Their Developmental Stage

It is thought that adult mesenchymal stromal cells (MSC) are important for tissue repair and maintenance. Crucial in these processes is the presence of MSC at the site of injury, however the recruitment and migration of MSC towards their destiny is poorly understood. With respect to future cell therapy, we are studying the process of migration of various human mesenchymal stem cell sources, and hypothesize that only a subpopulation of ex vivo expanded mesenchymal stem cells is capable of specific homing. For this purpose, MSC from different sources i.e. fetal lung (FL), fetal bone marrow (FBM), adult bone marrow (ABM) and adult adipose tissue (AT) were derived by plastic adherence and subsequently expanded. All MSC sources were characterized as CD73+, CD90+, CD105+, CD34− and CD45−. MSC (P4-9) were allowed to migrate for 4h towards SDF-1a, PDGF-BB, HGF, bFGF or FCS over fibronectin-coated 12 mm pore size transwell plates. FL-MSC migrated significantly better towards SDF-1a as compared to ABM-MSC or AT-MSC. This enhanced migration capacity towards SDF-1a is specific for FL-MSC since AT-MSC migrated better towards FCS as compared to FL-MSC. Even ABM-MSC responded better to FCS than FL-MSC. This suggests that MSC originating from all sources are able to migrate but require different triggers to induce migration. In order to elucidate whether the observed differences in migration potential were due to developmental stage, cultured MSC derived from fetal bone marrow were tested as well. No significant differences in migration capacity were observed between adult and fetal BM- MSC for any of the (chemotactic) stimuli evaluated. Interestingly, FL-MSC had a significant increased migration capacity as compared to FBM-MSC towards SDF-1a, PDGF-BB and HGF, suggesting that the origin of tissue may determine migration capacity of ex vivo expanded MSC. Since it was observed that only a small percentage of the cultured MSC were able to migrate towards the various stimuli, checkerboard migration was performed to elucidate whether a synergistic effect could be observed. No synergistic effect was observed between SDF and PDGF, SDF and FCS or PDGF and FCS in FL-MSC, suggesting that there may be one subpopulation of MSC that possesses migratory capacities. When studying the SDF-1-induced migratory subpopulation in more detail, it was observed that, after migration, migratory MSC originating from all tissue sources maintain their proliferation and differentiation capacity and express CXCR4 at a higher level than MSC that did not migrate. To be able to migrate, cells have to rearrange their actin cytoskeleton and focal adhesions. These processes can be initiated by various chemokines and growth factors. In response to SDF or FCS, morphological changes were observed in ABM-MSC by confocal microscopy. Cells became smaller and membrane protrusions appeared, whereas this was absent in the control. Furthermore, upon stimulation with SDF, PDGF and FCS, tyrosine-phosphorylation of the adapter protein paxillin that links the actin cytoskeleton to focal adhesions was increased. In conclusion, our results suggest that migration potential of ex vivo expanded MSC derived from various adult and fetal tissues have different migratory capacity towards growth factor and chemokine stimuli and may involve paxillin phosphorylation. Our data indicate that further studies on the migratory subpopulation(s) within the heterogeneous population of culture expanded MSC will contribute to unravel how and which MSC will be of interest for future cellular therapies.

Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays

Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-Fc epsilonRI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-Fc epsilonRI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-Fc epsilonRI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-Fc epsilonRI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (alpha5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-Fc epsilonRI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-Fc epsilonRI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the Fc epsilonRI beta subunit but enhances stimulated Ca(2+) release from intracellular stores. The results suggest that paxillin associated with clustered IgE-Fc epsilonRI has a net positive effect on Fc epsilonRI signaling.

Structural Analysis of the Interactions Between Paxillin LD Motifs and α-Parvin

SummaryThe adaptor protein paxillin contains five conserved leucine-rich (LD) motifs that interact with a variety of focal adhesion proteins, such as α-parvin. Here, we report the first crystal structure of the C-terminal calponin homology domain (CHC) of α-parvin at 1.05 Å resolution and show that it is able to bind all the LD motifs, with some selectivity for LD1, LD2, and LD4. Cocrystal structures with these LD motifs reveal the molecular details of their interactions with a common binding site on α-parvin-CHC, which is located at the rim of the canonical fold and includes part of the inter-CH domain linker. Surprisingly, this binding site can accommodate LD motifs in two antiparallel orientations. Taken together, these results reveal an unusual degree of binding degeneracy in the paxillin/α-parvin system that may facilitate the assembly of dynamic signaling complexes in the cell.

Breast Tumor Kinase Phosphorylates p190RhoGAP to Regulate Rho and Ras and Promote Breast Carcinoma Growth, Migration, and Invasion

Breast tumor kinase (Brk), an Src-like nonreceptor tyrosine kinase, is overexpressed in breast cancer and several other cancer types. Our previous study indicates that Brk promotes cell migration and tumor invasion by phosphorylating the focal adhesion protein paxillin. Here, we report the identification of p190RhoGAP-A (p190) as a Brk substrate. Brk phosphorylates p190 at the Y(1105) residue both in vitro and in vivo, thereby promoting the association of p190 with p120RasGAP (p120). As a consequence, Brk stimulates p190 and attenuates p120 functions, leading to RhoA inactivation and Ras activation, respectively. In carcinoma cells expressing high levels of Brk, endogenous Brk functions as a key contributor to epidermal growth factor-induced p190 tyrosine phosphorylation. We present evidence showing that p190 phosphorylation plays essential roles in both migratory and proliferative effects of Brk. Furthermore, disruption of p190 phosphorylation-induced p190/p120 complex in breast cancer cells abolishes not only the abilities of Brk to regulate RhoA and Ras but also the stimulatory effects of Brk on proliferation, migration, invasion, transformation, and tumorigenicity. Together, our findings reveal a previously unknown function of Brk in regulating both RhoA and Ras by phosphorylating p190 and provide evidence for the crucial roles of this Brk-elicited signaling pathway in promoting breast malignancy.

The signaling hubs at the crossroad of longevity and age-related disease networks

The established human age-related disease proteins (ARDPs) and longevity-associated proteins (LAPs) together with their first-order interacting partners form scale-free networks which significantly overlap. About half of the common proteins are involved in signal transduction. These proteins are strongly interconnected and in turn form a common signaling network which comprises over 40% of all hubs (proteins with multiple interactions) in the human interactome. Along with the insulin pathway, the common signaling network is remarkably enriched with the focal adhesion and adherens junction proteins whose relation to the control of lifespan is yet to be fully addressed. The examples of such candidate proteins include several hubs, focal adhesion kinase PTK2 and the extracellular proteins fibronectin FN1, paxillin PXN, and vinculin VCL. The results of the network-based analysis highlight the potential importance of these pathways, especially hubs, in linking the human longevity and age-related diseases.

Cell adhesion in zebrafish myogenesis: Distribution of intermediate filaments, microfilaments, intracellular adhesion structures and extracellular matrix

To overcome the limitations of in vitro studies, we have been studying myogenesis in situ in zebrafish embryos, at a sub-cellular level. While in previous works we focused on myofibrillogenesis and some aspects of adhesion structures, here we describe in more detail cell adhesion structures and interactions among cytoskeletal components, membrane and extracellular matrix during zebrafish muscle development. We studied the intermediate filaments, and we describe the full range of desmin distribution in zebrafish development, from perinuclear to striated, until its deposition around the intersomite septa of older somites. This adhesion structure, positive for desmin and actin, has not been previously observed in myogenesis in vitro. We also show that actin is initially located in the intersomite septum region whereas it is confined to the myofibrils later on. While actin localization changes during development, the adhesion complex proteins vinculin, paxillin, talin, dystrophin, laminin and fibronectin always appear exclusively at the intersomite septa, and appear to be co-distributed, even though the extracellular proteins accumulates before the intracellular ones. Contrary to the adhesion proteins, that are continuously distributed, desmin and sarcomeric actin form triangular aggregates among the septa and the cytoskeleton. We studied the cytoskeletal linker plectin as well, and we show that it has a distribution similar to desmin and not to actin. We conclude that the in situ adhesion structures differ from their in vitro counterparts, and that the actual zebrafish embryo myogenesis is quite different than that which occurs in in vitro systems.

Cross talk between paxillin and Rac is critical for mediation of barrier-protective effects by oxidized phospholipids

We previously reported that the barrier-protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cells (ECs) delineate the role of Rac- and Cdc42-dependent mechanisms and described the involvement of the focal adhesion (FA) protein paxillin in enhancement of the EC barrier upon OxPAPC challenge. This study examined a potential role of paxillin in the feedback mechanism of Rac regulation by FAs in OxPAPC-stimulated ECs. Our results demonstrate that OxPAPC induced Rac-dependent, Rho-independent peripheral accumulation of paxillin-containing FAs and time-dependent paxillin phosphorylation. Molecular inhibition of Rac decreased association of paxillin with the Rac-specific guanine nucleotide exchange factor beta-PIX. Molecular inhibition of paxillin also attenuated OxPAPC-induced enhancement of adherens junctions critical for the EC barrier-protective response, accumulation of vascular endothelial cadherin in the membrane fractions, and decreased activation of Rac and its effector p21-activated kinase (PAK1). Expression of paxillin with a mutated PAK1-dependent phosphorylation site (S273A) attenuated OxPAPC-induced PAK1 activation and the EC barrier-protective response. These results suggest that PAK1-specific paxillin phosphorylation at Ser(273) is critically involved in the positive-feedback regulation of the Rac-PAK1 pathway and may contribute to sustained enhancement of the EC barrier caused by oxidized phospholipids.

Myometrial mechanoadaptation during pregnancy: implications for smooth muscle plasticity and remodelling

The smooth muscle of the uterus during pregnancy presents a unique circumstance of physiological mechanotransduction as the tissue remodels in response to stretches imposed by the growing foetus(es), yet the nature of the molecular and functional adaptations remain unresolved. We studied, in myometrium isolated from non-pregnant (NP) and pregnant mice, the active and passive length–tension curves by myography and the expression and activation by immunoblotting of focal adhesion-related proteins known in other systems to participate in mechanosensing and mechanotransduction. In situ uterine mass correlated with pup number and weight throughout pregnancy. In vitro myometrial active, and passive, length-tension curves shifted significantly to the right during pregnancy indicative of altered mechanosensitivity; at term, maximum active tension was generated following 3.94 ± 0.33-fold stretch beyond slack length compared to 1.91 ± 0.12-fold for NP mice. Moreover, mechanotransduction was altered during pregnancy as evidenced by the progressive increase in absolute force production at each optimal stretch. Pregnancy was concomitantly associated with an increased expression of the dense plaque-associated proteins FAK and paxillin, and elevated activation of FAK, paxillin, c-Src and extracellular signal-regulated kinase (ERK1/2) which reversed 1 day post-partum. Electron microscopy revealed close appositioning of neighbouring myometrial cells across a narrow extracellular cleft adjoining plasmalemmal dense plaques. Collectively, these results suggest a physiological basis of myometrial length adaptation, long known to be a property of many smooth muscles, whereupon plasmalemmal dense plaque proteins serve as molecular signalling and structural platforms contributing to functional (contractile) remodelling in response to chronic stretch.

JNK regulates cell migration through promotion of tyrosine phosphorylation of paxillin

The adaptor protein paxillin plays an important role in cell migration. Although the c-Jun amino-terminal kinase (JNK) phosphorylation of paxillin on Ser 178 has been found to be critical for cell migration, the precise mechanism by which JNK regulates cell migration is still not very clear. Here, the migration of human corneal epithelial (HCE) cells was used to determine which signaling pathways are involved in EGF-induced paxillin phosphorylation. Paxillin was phosphorylated on Tyr 31 and Tyr 118 after induction of migration by EGF in HCE cells. Specific inhibition of JNK activation by inhibitor SP600125 or overexpression of a dominant-negative JNK mutant not only blocked EGF-induced cell migration, but also eliminated tyrosine phosphorylation of paxillin on Tyr 31 and Tyr 118. HCE cells overexpressing paxillin-S178A mutant also exhibited lower mobility, and reduced phosphorylation of Tyr 31 and Tyr 118. However, paxillin-S178A-inhibited cell migration can be rescued by overexpression of paxillin-Y31E/Y118E mutant. Importantly, inhibition of JNK by SP600125 or overexpression of paxillin-S178A mutant prevented the association of FAK with paxillin. Taken together, these results suggest that phosphorylation of paxillin on Ser 178 by JNK is required for the association of paxillin with FAK, and subsequent tyrosine phosphorylation of paxillin.

Paxillin Is Involved in the Differential Regulation of Endothelial Barrier by HGF and VEGF

Circulating levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) are increased during acute lung injury; however, combined effects of HGF and VEGF on pulmonary endothelial cell (EC) permeability remain to be elucidated. We have previously shown differential remodeling of focal adhesions (FA) caused by barrier-protective and barrier-disruptive mechanical and chemical stimuli. This study examined a role of FA protein paxillin in the pulmonary EC barrier responses induced by HGF and VEGF. VEGF increased, but HGF decreased, pulmonary EC permeability. These effects were accompanied by differential patterns of site-specific phosphorylation of focal adhesion kinase (FAK) and paxillin and FA redistribution. HGF antagonized random FA formation caused by VEGF challenge and promoted FA accumulation at the cell periphery. HGF attenuated VEGF-induced paxillin redistribution, FA remodeling, and endothelial permeability. SiRNA-based paxillin knockdown attenuated VEGF-induced EC permeability, myosin light chain phosphorylation, and stress fiber and paracellular gap formation. Paxillin knockdown also decreased HGF-induced EC barrier enhancement and suppressed activation of Rac and its effector PAK1. Expression of paxillin-S(273) deficient on PAK1 phosphorylation site prevented HGF-induced cytoskeletal remodeling. These data show a dual role of paxillin in the HGF- and VEGF-mediated endothelial barrier regulation and suggest essential paxillin role in the modulation of Rac-Rho crosstalk. Our results also support a model of pulmonary EC barrier recovery during resolution of ALI via switch from VEGF to HGF signaling.

A Rictor-Myo1c Complex Participates in Dynamic Cortical Actin Events in 3T3-L1 Adipocytes

Insulin signaling through phosphatidylinositol 3-kinase (PI 3-kinase) activates the protein kinase Akt through phosphorylation of its threonine 308 and serine 473 residues by the PDK1 protein kinase and the Rictor-mammalian target of rapamycin complex (mTORC2), respectively. Remarkably, we show here that the Rictor protein is also present in cultured adipocytes in complexes containing Myo1c, a molecular motor that promotes cortical actin remodeling. Interestingly, the Rictor-Myo1c complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Furthermore, while RNA interference-directed silencing of Rictor results in the expected attenuation of Akt phosphorylation at serine 473, depletion of Myo1c is without effect. In contrast, loss of either Rictor or Myo1c inhibits phosphorylation of the actin filament regulatory protein paxillin at tyrosine 118. Furthermore, Myo1c-induced membrane ruffling of 3T3-L1 adipocytes is also compromised following Rictor knockdown. Interestingly, neither the mTORC2 inhibitor rapamycin nor the PI 3-kinase inhibitor wortmannin affects paxillin tyrosine 118 phosphorylation. Taken together, our findings suggest that the Rictor-Myo1c complex is distinct from mTORC2 and that Myo1c, in conjunction with Rictor, participates in cortical actin remodeling events.

Combined expression of the non-receptor protein tyrosine kinases FAK and Src in primary colorectal cancer is associated with tumor recurrence and metastasis formation

Purpose The protein tyrosine kinase focal adhesion kinase (FAK) and Src in association with phosphorylation of the adapter protein paxillin are essential in tumor metastasis formation. Elevated levels of FAK, Src and paxillin may increase the metastatic potential of colorectal tumor cells. The aim of the current study was to examine the expression of FAK, Src, and paxillin using immunohistochemistry in the context of disease progression and to evaluate its clinical significance as a prognostic factor. Experimental design The relationship between FAK, Src and paxillin levels and colorectal cancer progression was evaluated by immunohistochemistry in 104 colorectal cancer specimens with clinical follow up. In addition, FAK, Src and paxillin expression levels were quantified in 68 colorectal tumors and corresponding liver metastases. Results FAK and paxillin expression individually did not significantly impact time to recurrence ( p = 0.09, and p = 0.89 respectively). Src expression was associated with tumor recurrence p = 0.03. However, tumors that expressed both high FAK and Src levels had a significant shorter time to recurrence ( p = 0.004, hazard ratio: 2.98, 95% CI 1.14–6.31). FAK, Src and paxillin showed equivalent levels in corresponding liver metastases compared to the primary tumors ( p = 0.67, p = 0.28 and p = 0.34 respectively). Conclusions These findings show that high levels of FAK and Src combined were predictive for recurrence of colorectal cancer. In addition, expression of FAK, Src and paxillin in colorectal cancer were maintained in corresponding distant metastases.