Association Of Paxillin Research Articles

Hic-5 interacts with GIT1 with a different binding mode from paxillin.

Hic-5, a member of the paxillin family of adaptor molecules, is localized at focal adhesion and implicated in integrin-mediated signaling. Hic-5 and paxillin exhibit structural homology and share interacting factors, however, diverse functions are suggested for them. In this study, we carried out yeast two-hybrid screening to identify Hic-5 interacting factors using its LD3-4 region, which includes the Hic-5-specific amino acid sequence, as a bait. Through the screening, we identified GIT1, an Arf GTPase-activating protein, as a Hic-5 binding protein. The interaction of these two proteins was mediated by the LD3 motif of Hic-5 and the C-terminal region, which includes a paxillin-binding subdomain, of GIT1. Although GIT1 is known as a paxillin-binding protein, we only observed weak association of paxillin with GIT1 in the overexpression system. In contrast, Hic-5 firmly bound to GIT1 under the same conditions. In addition, the paxillin/GIT1 complex contained PIX, a guanine nucleotide exchange factor, whereas the Hic-5/GIT1 complex contained a smaller amount of PIX. These results suggested that paxillin and Hic-5 associate with GIT1 with different binding modes, and that the Hic-5 complex possesses static features compared with the paxillin complex, which contains both positive and negative regulators of GTPases involved in actin dynamics. Moreover, Hic-5-mediated inhibition of cell spreading was restored by co-expression of the C-terminal fragment of GIT1, which perturbs the interaction of Hic-5 with endogenous GIT1. Thus, it was demonstrated that Hic-5 and GIT1 interact functionally in addition to showing a physical association.

Hepatocyte Growth Factor Induces ERK-dependent Paxillin Phosphorylation and Regulates Paxillin-Focal Adhesion Kinase Association

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

Binding of Paxillin to the α9 Integrin Cytoplasmic Domain Inhibits Cell Spreading

alpha(9)beta(1) integrin is a member of the beta(1) integrin family, plays an important role in extravasation of neutrophils at sites of acute inflammation, and is required for the normal development of the lymphatic system. The alpha(9) and alpha(4) integrin subunits are most closely related and form a subfamily of integrin alpha subunits. Previously, we have reported that the alpha(4) cytoplasmic domain directly and tightly binds paxillin, an intracellular signaling adaptor molecule. This interaction accounts for some of the unusual functional responses to alpha(4) integrin-mediated cell adhesion, including stimulation of cell migration and inhibition of cell spreading and focal adhesion formation. In the current studies, we have examined the interaction between the alpha(9) cytoplasmic domain and paxillin. Here we report that the alpha(9) cytoplasmic domain binds paxillin directly and tightly and that the alpha(9)-paxillin association inhibits cell spreading. We have identified amino acid residues in the alpha(9) cytoplasmic domain, Trp(999) and Trp(1001), that are critical for paxillin binding, and alanine substitution of either Trp(999) or Trp(1001) blocks paxillin binding. Furthermore, these mutations also reverse the effect of the alpha(9) cytoplasmic domain on cell spreading. Thus, the alpha(9) and alpha(4) integrin subunits form a paxillin-binding subfamily of integrin alpha subunits, and direct binding of paxillin to the alpha(9) cytoplasmic domain mediates some of the biological activities of the alpha(9)beta(1) integrin.

The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinΔLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin–PKL interaction. In cells overexpressing paxillinΔLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLΔPBS2) induced phenotypic changes reminiscent of paxillinΔLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

Involvement of Focal Adhesion Kinase in Escherichia coli Invasion of Human Brain Microvascular Endothelial Cells

Escherichia coli K1 traversal across the blood-brain barrier is an essential step in the pathogenesis of neonatal meningitis. We have previously shown that invasive E. coli promotes the actin rearrangement of brain microvascular endothelial cells (BMEC), which constitute a lining of the blood-brain barrier, for invasion. However, signal transduction mechanisms involved in E. coli invasion are not defined. In this report we show that tyrosine kinases play a major role in E. coli invasion of human BMEC (HBMEC). E. coli induced tyrosine phosphorylation of HBMEC cytoskeletal proteins, focal adhesion kinase (FAK), and paxillin, with a concomitant increase in the association of paxillin with FAK. Overexpression of a dominant interfering form of the FAK C-terminal domain, FRNK (FAK-related nonkinase), significantly inhibited E. coli invasion of HBMEC. Furthermore, we found that FAK kinase activity and the autophosphorylation site (Tyr397) are important in E. coli invasion of HBMEC, whereas the Grb2 binding site (Tyr925) is not required. Immunocytochemical studies demonstrated that FAK is recruited to focal plaques at the site of bacterial entry. Consistent with the invasion results, overexpression of FRNK, a kinase-negative mutant (Arg454 FAK), and a Src binding mutant (Phe397 FAK) inhibited the accumulation of FAK at the bacterial entry site. The overexpression of FAK mutants in HBMEC also blocked the E. coli-induced tyrosine phosphorylation of FAK and its association with paxillin. These observations provide evidence that FAK tyrosine phosphorylation and its recruitment to the cytoskeleton play a key role in E. coli invasion of HBMEC.

Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells.

Identification of signaling molecules that regulate cell migration is important for understanding fundamental processes in development and the origin of various pathological conditions. The migration of Nara Bladder Tumor II (NBT-II) cells was used to determine which signaling molecules are specifically involved in the collagen-mediated locomotion. We show here that paxillin is tyrosine phosphorylated after induction of motility on collagen. Overexpression of paxillin mutants in which tyrosine 31 and/or tyrosine 118 were replaced by phenylalanine effectively impaired cell motility. Moreover, stimulation of motility by collagen preferentially enhanced the association of paxillin with the SH2 domain of the adaptor protein CrkII. Mutations in both tyrosine 31 and 118 diminished the phosphotyrosine content of paxillin and prevented the formation of the paxillin-Crk complex, suggesting that this association is necessary for collagen-mediated NBT-II cell migration. Other responses to collagen, such as cell adhesion and spreading, were not affected by these mutations. Overexpression of wild-type paxillin or Crk could bypass the migration-deficient phenotype. Both the SH2 and the SH3 domains of CrkII are shown to play a critical role in this collagen-mediated migration. These results demonstrate the important role of the paxillin-Crk complex in the collagen-induced cell motility.

Binding of paxillin to α4 integrins modifies integrin-dependent biological responses

The alpha4 integrins are indispensable for embryogenesis, haematopoiesis and immune responses, possibly because alpha4 regulates cellular functions differently from other integrins through its cytoplasmic tail. We used novel mimics of the alpha4 tail to identify molecules that could account for alpha4-specific signalling. Here we report that the alpha4 tail, but not several other alpha-subunit tails, binds tightly to the signalling adaptor paxillin. Paxillin physically associated with alpha4 integrins in Jurkat T cells at high stoichiometry, and joining the alpha4 tail to alphaIIb resulted in a complex of integrin alphaIIbbeta3 with paxillin. This association markedly enhanced the rates of alphaIIbbeta3-dependent phosphorylation of focal adhesion kinase and cell migration. It also reduced cell spreading, focal adhesion and stress fibre formation. A point mutation within the alpha4 tail that disrupts paxillin binding reversed all of these effects. Furthermore, alpha4beta1-dependent adhesion to VCAM-1 led to spreading of mouse embryonic fibroblasts derived from paxillin-null but not from wild-type mice. Thus, the tight association of paxillin with the alpha4 tail leads to distinct biochemical and biological responses to integrin-mediated cell adhesion.

Activation of protein tyrosine kinase PYK2 by the m1 muscarinic acetylcholine receptor.

Several G protein-coupled receptors are known to direct the tyrosine phosphorylation, and in some cases the activation, of diverse tyrosine kinases. Using a stable cell line approach, we characterize the activation of PYK2, a tyrosine kinase structurally related to focal adhesion kinase, by the G protein-coupled m1 muscarinic acetylcholine receptor. We find that PYK2 tyrosine kinase activity is critical for the m1 receptor-stimulated tyrosine phosphorylation of PYK2. Furthermore, we identify two tyrosine residues that are subject to phosphorylation in response to muscarinic signaling and show that this phosphorylation induces two cytosolic proteins, c-Src and Grb2, to bind to PYK2. This is the first demonstration of the significance played by distinct PYK2 tyrosine residues in G protein-coupled signaling to this kinase. By comparison, though m1 receptors induce the tyrosine phosphorylation of the cytoskeletal protein paxillin, the association of paxillin with PYK2 is unaffected by muscarinic signaling. We also provide evidence that PYK2 specifically phosphorylates the carboxyl-terminal cytosolic portion of the potassium channel Kv1.2 in a manner regulated by the m1 receptor. These results delineate molecular events attending the m1 muscarinic receptor stimulation of this tyrosine kinase and establish PYK2 as an effector of the m1 muscarinic receptor in the regulation of multiple cell functions.

Paxillin Phosphorylation and Association with Lck and Pyk2 in Anti-CD3- or Anti-CD45-stimulated T Cells

Antibodies to either CD3 or CD45 have been shown to induce dramatic changes in cell morphology, increased tyrosine phosphorylation of cellular proteins, and the association of a subset of these proteins with the tyrosine kinase Lck. The current study was initiated to determine the identity of the tyrosine-phosphorylated 70-80 kDa protein that becomes Lck-associated after stimulation with anti-CD45 or anti-CD3. We demonstrate that the cytoskeletal protein paxillin becomes tyrosine-phosphorylated when cells are plated on immobilized antibodies specific for CD45 or CD3. Only tyrosine-phosphorylated paxillin is associated with Lck, suggesting that the association is through the SH2 domain of Lck. Consistent with this we demonstrate that the SH2 domain of Lck binds tyrosine-phosphorylated paxillin. In contrast, the association of paxillin with the FAK-related kinase Pyk2 was found to be constitutive and not altered by the phosphorylation of either protein. Finally, we establish that the phosphorylation of paxillin is dependent on the expression of Lck. Taken together, these results demonstrate that paxillin is physically associated with kinases from two different families in T cells and suggest that paxillin may function as an adaptor protein linking cellular signals with cytoskeletal changes during T cell activation.

Entamoeba histolytica: collagen-induced AP-1 DNA binding activity

The interaction of Entamoeba histolytica trophozoites with collagen induces the synthesis and release of electron-dense granules containing a collagenase activity that is an important factor in the pathogenicity of the parasite. The binding is thought to be mediated by an `integrin-like' collagen receptor. In the signal transduction mechanisms activated by collagen, pp125 FAK and p42 MAPK are involved. Using immunoprecipitation assays coupled to Western blot analysis, we demonstrate here the collagen-dependent association of paxillin and Src with pp125 FAK. Furthermore, collagen induces a time-dependent increase in the DNA binding activity of the activator protein 1, which is well correlated with an increase in Fos expression. Our results suggest that a stimulus-transcription coupling triggered by collagen in E. histolytica trophozoites might activate or repress genes involved in tissue invasiveness.

Entamoeba histolytica: collagen-induced AP-1 DNA binding activity.

The interaction of Entamoeba histolytica trophozoites with collagen induces the synthesis and release of electron-dense granules containing a collagenase activity that is an important factor in the pathogenicity of the parasite. The binding is thought to be mediated by an 'integrin-like' collagen receptor. In the signal transduction mechanisms activated by collagen, pp125FAK and p42MAPK are involved. Using immunoprecipitation assays coupled to Western blot analysis, we demonstrate here the collagen-dependent association of paxillin and Src with pp125FAK. Furthermore, collagen induces a time-dependent increase in the DNA binding activity of the activator protein 1, which is well correlated with an increase in Fos expression. Our results suggest that a stimulus-transcription coupling triggered by collagen in E. histolytica trophozoites might activate or repress genes involved in tissue invasiveness.

Association of Bovine Papillomavirus Type 1 E6 oncoprotein with the focal adhesion protein paxillin through a conserved protein interaction motif.

We have found that the E6 oncoprotein of Bovine Papillomavirus Type 1 (BE6) as well as the E6 protein of the cancer associated HPV-16 (16E6) interact with the focal adhesion protein paxillin. Mutational analysis of paxillin revealed that BE6 binds paxillin through small protein interaction motifs called LD motifs that have been previously identified as important in regulating association of paxillin with vinculin and focal adhesion kinase (FAK), and that BE6 can interact with at least two separate binding sites on paxillin. The LD motifs of paxillin that bind BE6 share homology with the E6 binding site of E6-AP, a ubiquitin ligase that together with 16E6 targets the degradation of the p53 tumor suppressor. Paxillin binding to BE6 excludes simultaneous binding to E6-AP. Mutational analysis of BE6 can distinguish the interaction of BE6 with E6-AP compared to paxillin and revealed that the interaction of BE6 with paxillin may be necessary for the induction of anchorage independent growth of cells by BE6.

Signal Transduction in Human Hematopoietic Cells by Vascular Endothelial Growth Factor Related Protein, a Novel Ligand for the FLT4 Receptor

AbstractWe have recently identified a novel ligand of the vascular endothelial growth factor (VEGF) family termed VEGF-related protein (VRP), which specifically binds to the FLT4 receptor. To characterize the signaling events after VRP engagement of its cognate receptor in hematopoietic cells, a population of human erythroleukemia (HEL) cells, termed HEL-JW, expressing high levels of FLT4 receptor was isolated. Stimulation of HEL-JW cells with VRP alone and in combination with the c-kit ligand/stem cell factor increased cell growth. VRP induced tyrosine phosphorylation of various proteins, including the FLT4 receptor. Further characterization of these tyrosine phosphorylated molecules revealed that Shc, Grb2, and SOS form a complex with the activated FLT4 receptor. HEL-JW cells also expressed RAFTK, a recently identified member of the focal adhesion kinase family. RAFTK was phosphorylated and activated upon VRP treatment, and there was an enhanced association of this kinase with the adaptor protein Grb2. Furthermore, the c-Jun NH2-terminal kinase (JNK), involved in growth activation and shown to mediate RAFTK signaling in other cell types, was activated by VRP stimulation. We also observed that VRP treatment of HEL-JW cells resulted in the phosphorylation of the cytoskeletal protein paxillin. This treatment resulted in an increased association of paxillin with RAFTK, which was mediated by the C-terminal region of RAFTK. These studies indicate that VRP stimulation induced the formation of a signaling complex at its activated receptor as well as activation of RAFTK. VRP-mediated activation of RAFTK may facilitate signal transduction to the cytoskeleton and downstream to the JNK pathway in FLT4-expressing blood cells.

Integrin-Mediated Tyrosine Phosphorylation and Redistribution of Paxillin during Neuronal Adhesion

Integrins are important receptors for neuronal adhesion to laminin, which is one of the best promoters of neurite outgrowth. The present study was carried out to understand some of the intracellular mechanisms which allow integrin-mediated neurite extension on laminin. In chicken retinal neurons, integrin-mediated adhesion to laminin and antibody-induced integrin clustering caused an increase in tyrosine phosphorylation of paxillin and focal adhesion kinase. The kinetics of phosphorylation and dephosphorylation of these proteins were different in neurons plated on laminin, compared to neurons in which the receptors were clustered with anti-integrin antibodies. Analysis of sucrose velocity gradients could not show any association of paxillin and focal adhesion kinase with the integrin receptors. On the other hand, by using digitonin and milder extraction conditions, we found an enrichment of the tyrosine-phosphorylated polypeptides in the cytoskeletal, digitonin-insoluble fraction. Furthermore, neuronal adhesion induced a dramatic increase in the fraction of tyrosine-phosphorylated paxillin recovered with the digitonin-insoluble fraction, suggesting redistribution of this protein following adhesion of neurons to laminin. Localization studies on the detergent-insoluble fraction showed codistribution of both paxillin and focal adhesion kinase with integrins. We also found that paxillin tyrosine phosphorylation, but not paxillin expression, is developmentally regulated in the retina. Our results show that integrin-mediated neuronal adhesion leads to the accumulation of a pool of highly phosphorylated proteins at adhesion sites. There they may be responsible for the reorganization of the cytoskeleton, which underlies the process of neurite extension.

Paxillin association in vitro with integrin cytoplasmic domain peptides

Short cytoplasmic domains of integrin heterodimers are crucial for transduction of signals generated by adhesion of cells to the extracellular matrix. Here, we describe the use of peptides mimicking the intracellular tails of integrin α 5 β 1 to assay in vitro associations with cytoskeletal proteins. Our results suggest that the focal adhesion protein, paxillin, may interact directly with the intracellular region of the integrin β 1 subunit. Paxillin is known to form stable complexes with several signaling molecules, including focal adhesion kinase. Physical interaction between paxillin and the β 1 cytoplasmic domain suggests a model in which paxillin may function as a key intermediary in integrinmediated signal transduction.