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Christopher E Turner - One of the best experts on this subject based on the ideXlab platform.

  • Evolution and Expression of Paxillin Genes in Teleost Fish.
    PloS one, 2016
    Co-Authors: Andrew E. Jacob, Christopher E Turner, Jeffrey D. Amack
    Abstract:

    Background Paxillin family proteins regulate intracellular signaling downstream of extracellular matrix adhesion. Tissue expression patterns and cellular functions of Paxillin proteins during embryo development remain poorly understood. Additionally, the evolution of this gene family has not been thoroughly investigated. Results This report characterizes the evolution and expression of a novel Paxillin gene, called Paxillin-b, in Teleosts. Alignments indicate that Teleost Paxillin-a and Paxillin-b proteins are highly homologous to each other and to human Paxillin. Phylogenetic and synteny analyses suggest that these genes originated from the duplication of an ancestral Paxillin gene that was in a common ancestor of Teleosts and Tetrapods. Analysis of the spatiotemporal expression profiles of Paxillin-a and Paxillin-b using zebrafish revealed both overlapping and distinct domains for Paxillin-a and Paxillin-b during embryo development. Localization of zebrafish Paxillin orthologs expressed in mammalian cells demonstrated that both proteins localize to focal adhesions, similar to mammalian Paxillin. This suggests these proteins regulate adhesion-dependent processes in their endogenous tissues. Conclusion Paxillin-a and Paxillin-b were generated by duplication in Teleosts. These genes likely play similar roles as Paxillin genes in other organisms. This work provides a framework for functional investigation of Paxillin family members during development using the zebrafish as an in vivo model system.

  • Paxillin is essential for PTP-PEST-dependent regulation of cell spreading and motility: a role for Paxillin kinase linker.
    Journal of Cell Science, 2005
    Co-Authors: Jennifer S. Jamieson, Michael C. Brown, David A. Tumbarello, Maxime Hallé, Michel L. Tremblay, Christopher E Turner
    Abstract:

    The tyrosine phosphatase PTP-PEST has been implicated in the regulation of cell spreading and migration through dephosphorylation of focal adhesion proteins and inhibition of Rac GTPase activity. The focal adhesion adaptor protein Paxillin is also necessary for normal cell migration and binds directly to PTP-PEST. In this study, we have utilized PTP-PEST(-/-) and Paxillin(-/-) fibroblasts to demonstrate that Paxillin is essential for PTP-PEST inhibition of cell spreading and membrane protrusion as well as inhibition of adhesion-induced Rac activation. Furthermore, we show that Paxillin-binding is necessary for PTP-PEST stimulation of cell migration. Mutation analysis indicates that PTP-PEST function involves binding to the Paxillin C-terminal LIM domains, and signaling through the tyrosine 31 and 118 phosphorylation sites, as well as the LD4 motif of the Paxillin N-terminus. Using 'substrate trapping' approaches and immunoprecipitation, we show that the ARF GAP Paxillin kinase linker PKL/GIT2, a Paxillin LD4 binding partner, is a substrate for PTP-PEST. Additionally, the PKL-Paxillin interaction was necessary for PTP-PEST inhibition of cell spreading. These data provide mechanistic insight into how the Paxillin-PTP-PEST interaction contributes to integrin signaling events associated with the spatiotemporal regulation of key modulators of the cytoskeleton and cell motility machinery.

  • Regulation of Paxillin family members during epithelial-mesenchymal transformation: a putative role for Paxillin δ
    Journal of Cell Science, 2005
    Co-Authors: David A. Tumbarello, Michael C. Brown, Sara E. Hetey, Christopher E Turner
    Abstract:

    Epithelial-mesenchymal transformation (EMT) and the resulting induction of cell motility are essential components of tissue remodeling during embryonic development and wound repair, as well as tumor progression to an invasive metastatic phenotype. Paxillin, a multi-domain adaptor and phosphoprotein has previously been implicated in integrin signaling and cell motility. In this report we characterize a novel Paxillin gene product, Paxillin delta, generated from an evolutionarily conserved internal translation initiation site within the full-length Paxillin mRNA. Paxillin delta, which lacks the key phosphorylation sites Y31 and Y118 as well as the ILK and actopaxin binding LD1 motif, exhibits a restricted distribution to epithelial cell types and is downregulated during TGF-beta1-induced EMT of normal murine mammary gland (NMuMG) epithelial cells. Interestingly, Hic-5, a Paxillin superfamily member, exhibits a reciprocal protein expression profile to Paxillin delta. In addition, Paxillin delta expression is maintained following NMuMG differentiation in a 3D collagen I gel while other focal adhesion components are downregulated. Paxillin delta protein expression coincided with reduced Paxillin tyrosine phosphorylation in NMuMG cells and Paxillin delta overexpression in CHO.K1 cells inhibited adhesion-mediated tyrosine phosphorylation of Paxillin. Forced expression of Paxillin delta in NMuMG cells suppressed cell migration whereas Hic-5 overexpression stimulated motility. Together our data support a role for Paxillin delta as a naturally occurring functional antagonist of Paxillin signaling potentially through suppression of a Crk-mediated pathway during processes associated with cell migration.

  • Regulation of Paxillin family members during epithelial-mesenchymal transformation: a putative role for Paxillin delta.
    Journal of cell science, 2005
    Co-Authors: David A. Tumbarello, Michael C. Brown, Sara E. Hetey, Christopher E Turner
    Abstract:

    Epithelial-mesenchymal transformation (EMT) and the resulting induction of cell motility are essential components of tissue remodeling during embryonic development and wound repair, as well as tumor progression to an invasive metastatic phenotype. Paxillin, a multi-domain adaptor and phosphoprotein has previously been implicated in integrin signaling and cell motility. In this report we characterize a novel Paxillin gene product, Paxillin delta, generated from an evolutionarily conserved internal translation initiation site within the full-length Paxillin mRNA. Paxillin delta, which lacks the key phosphorylation sites Y31 and Y118 as well as the ILK and actopaxin binding LD1 motif, exhibits a restricted distribution to epithelial cell types and is downregulated during TGF-beta1-induced EMT of normal murine mammary gland (NMuMG) epithelial cells. Interestingly, Hic-5, a Paxillin superfamily member, exhibits a reciprocal protein expression profile to Paxillin delta. In addition, Paxillin delta expression is maintained following NMuMG differentiation in a 3D collagen I gel while other focal adhesion components are downregulated. Paxillin delta protein expression coincided with reduced Paxillin tyrosine phosphorylation in NMuMG cells and Paxillin delta overexpression in CHO.K1 cells inhibited adhesion-mediated tyrosine phosphorylation of Paxillin. Forced expression of Paxillin delta in NMuMG cells suppressed cell migration whereas Hic-5 overexpression stimulated motility. Together our data support a role for Paxillin delta as a naturally occurring functional antagonist of Paxillin signaling potentially through suppression of a Crk-mediated pathway during processes associated with cell migration.

  • rnf5 a ring finger protein that regulates cell motility by targeting Paxillin ubiquitination and altered localization
    Molecular and Cellular Biology, 2003
    Co-Authors: Christine Didier, Christopher E Turner, Sheila M. Thomas, Limor Broday, Anindita Bhoumik, Sharon Israeli, Shoichi Takahashi, Koh Nakayama, Scott C Henderson, Hisataka Sabe
    Abstract:

    RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans. The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C. elegans which shows homology with human Paxillin. Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of Paxillin, resulting in its ubiquitination. RNF5 requires intact RING and C-terminal domains to mediate Paxillin ubiquitination. Whereas RNF5 mediates efficient ubiquitination of Paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of Paxillin ubiquitination. Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology. Expression of RNF5 increases the cytoplasmic distribution of Paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth. Concomitantly, RNF5 expression results in inhibition of cell motility. Via targeting of Paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.

Francesco Michelangeli - One of the best experts on this subject based on the ideXlab platform.

  • the mechanism of inhibition of the sarco endoplasmic reticulum ca2 atpase by Paxilline
    Archives of Biochemistry and Biophysics, 2002
    Co-Authors: Jonathan G Bilmen, Laura L Wootton, Francesco Michelangeli
    Abstract:

    Abstract Paxilline, an indole alkaloid mycotoxin from Penicillium paxilli, is an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). Paxilline inhibited differing isoforms of SERCA with IC50s between 5 and 50 μM . It inhibited more potently the purified Ca2+ ATPase activity from skeletal muscle with an IC50 of 5 μM . Detailed effects of this inhibitor on the Ca2+ and ATP dependence upon activity indicate that it affects the high-affinity Ca2+-binding (E1) form of the ATPase. In addition, Paxilline is a “competitive” inhibitor with respect to high concentrations of ATP, increasing the regulatory binding site Km, without affecting the catalytic binding site Km. At higher concentrations, Paxilline inhibits phosphoenzyme formation from ATP and inorganic phosphate, without affecting nucleotide binding. We therefore suggest that Paxilline has two effects on the Ca2+ ATPase. At lower concentrations ( 5–10 μM ), Paxilline inhibits the ATP-dependent acceleration of Ca2+ release from the phosphoenzyme and/or phosphoenzyme decay. At higher concentrations, Paxilline inhibits phosphoenzyme formation.

  • The mechanism of inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase by Paxilline.
    Archives of Biochemistry and Biophysics, 2002
    Co-Authors: Jonathan G Bilmen, Laura L Wootton, Francesco Michelangeli
    Abstract:

    Abstract Paxilline, an indole alkaloid mycotoxin from Penicillium paxilli, is an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). Paxilline inhibited differing isoforms of SERCA with IC50s between 5 and 50 μM . It inhibited more potently the purified Ca2+ ATPase activity from skeletal muscle with an IC50 of 5 μM . Detailed effects of this inhibitor on the Ca2+ and ATP dependence upon activity indicate that it affects the high-affinity Ca2+-binding (E1) form of the ATPase. In addition, Paxilline is a “competitive” inhibitor with respect to high concentrations of ATP, increasing the regulatory binding site Km, without affecting the catalytic binding site Km. At higher concentrations, Paxilline inhibits phosphoenzyme formation from ATP and inorganic phosphate, without affecting nucleotide binding. We therefore suggest that Paxilline has two effects on the Ca2+ ATPase. At lower concentrations ( 5–10 μM ), Paxilline inhibits the ATP-dependent acceleration of Ca2+ release from the phosphoenzyme and/or phosphoenzyme decay. At higher concentrations, Paxilline inhibits phosphoenzyme formation.

Jonathan G Bilmen - One of the best experts on this subject based on the ideXlab platform.

  • the mechanism of inhibition of the sarco endoplasmic reticulum ca2 atpase by Paxilline
    Archives of Biochemistry and Biophysics, 2002
    Co-Authors: Jonathan G Bilmen, Laura L Wootton, Francesco Michelangeli
    Abstract:

    Abstract Paxilline, an indole alkaloid mycotoxin from Penicillium paxilli, is an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). Paxilline inhibited differing isoforms of SERCA with IC50s between 5 and 50 μM . It inhibited more potently the purified Ca2+ ATPase activity from skeletal muscle with an IC50 of 5 μM . Detailed effects of this inhibitor on the Ca2+ and ATP dependence upon activity indicate that it affects the high-affinity Ca2+-binding (E1) form of the ATPase. In addition, Paxilline is a “competitive” inhibitor with respect to high concentrations of ATP, increasing the regulatory binding site Km, without affecting the catalytic binding site Km. At higher concentrations, Paxilline inhibits phosphoenzyme formation from ATP and inorganic phosphate, without affecting nucleotide binding. We therefore suggest that Paxilline has two effects on the Ca2+ ATPase. At lower concentrations ( 5–10 μM ), Paxilline inhibits the ATP-dependent acceleration of Ca2+ release from the phosphoenzyme and/or phosphoenzyme decay. At higher concentrations, Paxilline inhibits phosphoenzyme formation.

  • The mechanism of inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase by Paxilline.
    Archives of Biochemistry and Biophysics, 2002
    Co-Authors: Jonathan G Bilmen, Laura L Wootton, Francesco Michelangeli
    Abstract:

    Abstract Paxilline, an indole alkaloid mycotoxin from Penicillium paxilli, is an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). Paxilline inhibited differing isoforms of SERCA with IC50s between 5 and 50 μM . It inhibited more potently the purified Ca2+ ATPase activity from skeletal muscle with an IC50 of 5 μM . Detailed effects of this inhibitor on the Ca2+ and ATP dependence upon activity indicate that it affects the high-affinity Ca2+-binding (E1) form of the ATPase. In addition, Paxilline is a “competitive” inhibitor with respect to high concentrations of ATP, increasing the regulatory binding site Km, without affecting the catalytic binding site Km. At higher concentrations, Paxilline inhibits phosphoenzyme formation from ATP and inorganic phosphate, without affecting nucleotide binding. We therefore suggest that Paxilline has two effects on the Ca2+ ATPase. At lower concentrations ( 5–10 μM ), Paxilline inhibits the ATP-dependent acceleration of Ca2+ release from the phosphoenzyme and/or phosphoenzyme decay. At higher concentrations, Paxilline inhibits phosphoenzyme formation.

Ravi Salgia - One of the best experts on this subject based on the ideXlab platform.

  • role played by Paxillin and Paxillin tyrosine phosphorylation in hepatocyte growth factor sphingosine 1 phosphate mediated reactive oxygen species generation lamellipodia formation and endothelial barrier function
    Pulmonary circulation, 2015
    Co-Authors: Peter V. Usatyuk, Ravi Salgia, Jeffrey R Jacobson, Anne E Cress, Joe G N Garcia, Viswanathan Natarajan
    Abstract:

    Paxillin is a multifunctional and multidomain focal adhesion adaptor protein. It serves as an important scaffolding protein at focal adhesions by recruiting and binding to structural and signaling molecules. Paxillin tyrosine phosphorylation at Y31 and Y118 is important for Paxillin redistribution to focal adhesions and angiogenesis. Hepatocyte growth factor (HGF) and sphingosine-1-phosphate (S1P) are potent stimulators of lamellipodia formation, a prerequisite for endothelial cell migration. The role played by Paxillin and its tyrosine phosphorylated forms in HGF- or S1P-induced lamellipodia formation and barrier function is unclear. HGF or S1P stimulated lamellipodia formation, tyrosine phosphorylation of Paxillin at Y31 and Y118, and c-Abl in human lung microvascular endothelial cells (HLMVECs). Knockdown of Paxillin with small interfering RNA (siRNA) or transfection with Paxillin mutants (Y31F or Y118F) mitigated HGF- or S1P-induced lamellipodia formation, translocation of p47 (phox) to lamellipodia, and reactive oxygen species (ROS) generation in HLMVECs. Furthermore, exposure of HLMVECs to HGF or S1P stimulated c-Abl-mediated tyrosine phosphorylation of Paxillin at Y31 and Y118 in a time-dependent fashion, and down-regulation of c-Abl with siRNA attenuated HGF- or S1P-mediated lamellipodia formation, translocation of p47 (phox) to lamellipodia, and endothelial barrier enhancement. In vivo, knockdown of Paxillin with siRNA in mouse lungs attenuated ventilator-induced lung injury. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of Paxillin at Y31 and Y118 regulates HGF- or S1P-mediated lamellipodia formation, ROS generation in lamellipodia, and endothelial permeability.

  • Role played by Paxillin and Paxillin tyrosine phosphorylation in hepatocyte growth factor/sphingosine-1-phosphate-mediated reactive oxygen species generation, lamellipodia formation, and endothelial barrier function.
    Pulmonary circulation, 2015
    Co-Authors: Peter V. Usatyuk, Ravi Salgia, Jeffrey R Jacobson, Anne E Cress, Joe G N Garcia, Viswanathan Natarajan
    Abstract:

    Paxillin is a multifunctional and multidomain focal adhesion adaptor protein. It serves as an important scaffolding protein at focal adhesions by recruiting and binding to structural and signaling molecules. Paxillin tyrosine phosphorylation at Y31 and Y118 is important for Paxillin redistribution to focal adhesions and angiogenesis. Hepatocyte growth factor (HGF) and sphingosine-1-phosphate (S1P) are potent stimulators of lamellipodia formation, a prerequisite for endothelial cell migration. The role played by Paxillin and its tyrosine phosphorylated forms in HGF- or S1P-induced lamellipodia formation and barrier function is unclear. HGF or S1P stimulated lamellipodia formation, tyrosine phosphorylation of Paxillin at Y31 and Y118, and c-Abl in human lung microvascular endothelial cells (HLMVECs). Knockdown of Paxillin with small interfering RNA (siRNA) or transfection with Paxillin mutants (Y31F or Y118F) mitigated HGF- or S1P-induced lamellipodia formation, translocation of p47 (phox) to lamellipodia, and reactive oxygen species (ROS) generation in HLMVECs. Furthermore, exposure of HLMVECs to HGF or S1P stimulated c-Abl-mediated tyrosine phosphorylation of Paxillin at Y31 and Y118 in a time-dependent fashion, and down-regulation of c-Abl with siRNA attenuated HGF- or S1P-mediated lamellipodia formation, translocation of p47 (phox) to lamellipodia, and endothelial barrier enhancement. In vivo, knockdown of Paxillin with siRNA in mouse lungs attenuated ventilator-induced lung injury. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of Paxillin at Y31 and Y118 regulates HGF- or S1P-mediated lamellipodia formation, ROS generation in lamellipodia, and endothelial permeability.

  • The Noncatalytic Domain of Protein-tyrosine Phosphatase-PEST Targets Paxillin for Dephosphorylation in Vivo
    The Journal of biological chemistry, 2000
    Co-Authors: Yu Shen, Ravi Salgia, James D Griffin, Patrick D. Lyons, Marion Anne Cooley, Dominique Davidson, André Veillette, Michael D Schaller
    Abstract:

    Abstract The noncatalytic domain of protein-tyrosine phosphatase (PTP)-PEST contains a binding site for the focal adhesion-associated protein Paxillin. This binding site has been narrowed to a 52-residue sequence that is composed of two nonoverlapping, weak Paxillin binding sites. The PTP-PEST binding site on Paxillin has been mapped to the two carboxyl-terminal LIM (lin11, isl-1, and mec-3) domains. Transient expression of PTP-PEST reduced tyrosine phosphorylation of p130cas, as anticipated. A PTP-PEST mutant defective for binding p130cas does not cause a reduction in its tyrosine phosphorylation in vivo. Expression of PTP-PEST also caused a reduction of phosphotyrosine on Paxillin. Expression of mutants of PTP-PEST with deletions in the Paxillin-binding site did not associate with Paxillin in vivo and failed to cause a reduction in the phosphotyrosine content of Paxillin. These results demonstrate that Paxillin can serve as a PTP-PEST substrate in vivo and support the model that a noncatalytic domain interaction recruits Paxillin to PTP-PEST to facilitate its dephosphorylation.

  • expression of the focal adhesion protein Paxillin in lung cancer and its relation to cell motility
    Oncogene, 1999
    Co-Authors: Ravi Salgia, Darren S Ewaniuk, Youbin Wang, Martin Sattler, Wenche Chen, William G Richards, Evan Pisick, Geoffrey I Shapiro, Barrett J Rollins, Lan Bo Chen
    Abstract:

    Lung cancer can lead to abnormalities of the actin cytoskeleton structure which may be important in transformation. In this study, we have investigated the expression of the cytoskeletal associated protein Paxillin in lung cancer. Paxillin is a 68 kDa focal adhesion protein, with four tandem LIM domains at the C-terminus, involved in growth factor receptor, integrin and oncogenic signaling such as v-src, BCR/ABL, and E6 of the papilloma virus. In non-small cell lung cancer (NSCLC) cell lines, Paxillin localized to the focal adhesions. The possible role of Paxillin in lung cancer cells was assessed by overexpressing green fluorescence protein (GFP)-Paxillin construct in two separate NSCLC cell lines (Calu-1 and H661). Over the course of 48 h, GFP-Paxillin consistently caused the cells to become round and to decrease cell motility as compared to normal controls, GFP-N-terminus Paxillin, or GFP-LIM transfected cells. Because some lung cancers may be quite aggressive and metastasize quickly, which may be related to the cytoskeleton, we determined the expression of Paxillin in NSCLC and small cell lung cancer (SCLC) cell lines and patient tumor tissues. Expression of Paxillin in NSCLC and SCLC cell lines were determined by Northern blot and Western blot analysis. The expression of Paxillin was consistently low in SCLC cell lines, whereas there was Paxillin expression in NSCLC cell lines. There was a variability of expression of Paxillin in NSCLC tumor tissue as compared to normal lung tissue. In contrast, by immunohistochemistry, we show that there was no detectable expression of Paxillin in 5/5 SCLC patients. This data suggests that absence or low level of Paxillin protein expression may cause certain lung cancers, such as SCLC, to be more motile and possibly more aggressive.

  • the bovine papillomavirus e6 protein binds to the ld motif repeats of Paxillin and blocks its interaction with vinculin and the focal adhesion kinase
    Journal of Biological Chemistry, 1997
    Co-Authors: Xiao Tong, Ravi Salgia, James D Griffin, Jianliang Li, Peter M Howley
    Abstract:

    Abstract The bovine papillomavirus type 1 (BPV-1) E6 oncoprotein can transform fibroblasts and induce anchorage-independent growth and disassembly of the actin stress fibers. We have previously shown that the E6 protein interacts with the focal adhesion protein, Paxillin, suggesting a direct role of E6 in the disruption of the actin cytoskeleton. We have now mapped the E6 binding sites on Paxillin to the LD motif repeats region, which has been implicated in mediating Paxillin binding to two other focal adhesion proteins, vinculin and the focal adhesion kinase. The five LD motif repeats identified in Paxillin do not contribute equally to its interaction with E6. The first LD repeat is most critical for Paxillin binding to E6 both in vitro and in vivo. Furthermore, the binding of recombinant wild-type E6 protein to Paxillin blocked the interaction of several cellular proteins with Paxillin, including vinculin and the focal adhesion kinase. A mutant E6 protein (H105) which does not bind to Paxillin had no effect on the binding of these cellular proteins to Paxillin. These data suggest that E6 disruption of the actin stress fibers occurs through blocking the interaction of Paxillin with its cellular effectors such as vinculin and the focal adhesion kinase.

Michael C. Brown - One of the best experts on this subject based on the ideXlab platform.

  • Paxillin is essential for PTP-PEST-dependent regulation of cell spreading and motility: a role for Paxillin kinase linker.
    Journal of Cell Science, 2005
    Co-Authors: Jennifer S. Jamieson, Michael C. Brown, David A. Tumbarello, Maxime Hallé, Michel L. Tremblay, Christopher E Turner
    Abstract:

    The tyrosine phosphatase PTP-PEST has been implicated in the regulation of cell spreading and migration through dephosphorylation of focal adhesion proteins and inhibition of Rac GTPase activity. The focal adhesion adaptor protein Paxillin is also necessary for normal cell migration and binds directly to PTP-PEST. In this study, we have utilized PTP-PEST(-/-) and Paxillin(-/-) fibroblasts to demonstrate that Paxillin is essential for PTP-PEST inhibition of cell spreading and membrane protrusion as well as inhibition of adhesion-induced Rac activation. Furthermore, we show that Paxillin-binding is necessary for PTP-PEST stimulation of cell migration. Mutation analysis indicates that PTP-PEST function involves binding to the Paxillin C-terminal LIM domains, and signaling through the tyrosine 31 and 118 phosphorylation sites, as well as the LD4 motif of the Paxillin N-terminus. Using 'substrate trapping' approaches and immunoprecipitation, we show that the ARF GAP Paxillin kinase linker PKL/GIT2, a Paxillin LD4 binding partner, is a substrate for PTP-PEST. Additionally, the PKL-Paxillin interaction was necessary for PTP-PEST inhibition of cell spreading. These data provide mechanistic insight into how the Paxillin-PTP-PEST interaction contributes to integrin signaling events associated with the spatiotemporal regulation of key modulators of the cytoskeleton and cell motility machinery.

  • Regulation of Paxillin family members during epithelial-mesenchymal transformation: a putative role for Paxillin δ
    Journal of Cell Science, 2005
    Co-Authors: David A. Tumbarello, Michael C. Brown, Sara E. Hetey, Christopher E Turner
    Abstract:

    Epithelial-mesenchymal transformation (EMT) and the resulting induction of cell motility are essential components of tissue remodeling during embryonic development and wound repair, as well as tumor progression to an invasive metastatic phenotype. Paxillin, a multi-domain adaptor and phosphoprotein has previously been implicated in integrin signaling and cell motility. In this report we characterize a novel Paxillin gene product, Paxillin delta, generated from an evolutionarily conserved internal translation initiation site within the full-length Paxillin mRNA. Paxillin delta, which lacks the key phosphorylation sites Y31 and Y118 as well as the ILK and actopaxin binding LD1 motif, exhibits a restricted distribution to epithelial cell types and is downregulated during TGF-beta1-induced EMT of normal murine mammary gland (NMuMG) epithelial cells. Interestingly, Hic-5, a Paxillin superfamily member, exhibits a reciprocal protein expression profile to Paxillin delta. In addition, Paxillin delta expression is maintained following NMuMG differentiation in a 3D collagen I gel while other focal adhesion components are downregulated. Paxillin delta protein expression coincided with reduced Paxillin tyrosine phosphorylation in NMuMG cells and Paxillin delta overexpression in CHO.K1 cells inhibited adhesion-mediated tyrosine phosphorylation of Paxillin. Forced expression of Paxillin delta in NMuMG cells suppressed cell migration whereas Hic-5 overexpression stimulated motility. Together our data support a role for Paxillin delta as a naturally occurring functional antagonist of Paxillin signaling potentially through suppression of a Crk-mediated pathway during processes associated with cell migration.

  • Regulation of Paxillin family members during epithelial-mesenchymal transformation: a putative role for Paxillin delta.
    Journal of cell science, 2005
    Co-Authors: David A. Tumbarello, Michael C. Brown, Sara E. Hetey, Christopher E Turner
    Abstract:

    Epithelial-mesenchymal transformation (EMT) and the resulting induction of cell motility are essential components of tissue remodeling during embryonic development and wound repair, as well as tumor progression to an invasive metastatic phenotype. Paxillin, a multi-domain adaptor and phosphoprotein has previously been implicated in integrin signaling and cell motility. In this report we characterize a novel Paxillin gene product, Paxillin delta, generated from an evolutionarily conserved internal translation initiation site within the full-length Paxillin mRNA. Paxillin delta, which lacks the key phosphorylation sites Y31 and Y118 as well as the ILK and actopaxin binding LD1 motif, exhibits a restricted distribution to epithelial cell types and is downregulated during TGF-beta1-induced EMT of normal murine mammary gland (NMuMG) epithelial cells. Interestingly, Hic-5, a Paxillin superfamily member, exhibits a reciprocal protein expression profile to Paxillin delta. In addition, Paxillin delta expression is maintained following NMuMG differentiation in a 3D collagen I gel while other focal adhesion components are downregulated. Paxillin delta protein expression coincided with reduced Paxillin tyrosine phosphorylation in NMuMG cells and Paxillin delta overexpression in CHO.K1 cells inhibited adhesion-mediated tyrosine phosphorylation of Paxillin. Forced expression of Paxillin delta in NMuMG cells suppressed cell migration whereas Hic-5 overexpression stimulated motility. Together our data support a role for Paxillin delta as a naturally occurring functional antagonist of Paxillin signaling potentially through suppression of a Crk-mediated pathway during processes associated with cell migration.

  • Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton.
    The Journal of biological chemistry, 2000
    Co-Authors: Lourdes Herreros, Christopher E Turner, Michael C. Brown, José Luis Rodríguez-fernández, José Luis Alonso-lebrero, Carlos Cabañas, Francisco Sánchez-madrid, Natividad Longo, Paloma Sánchez-mateos
    Abstract:

    Next Section Abstract Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter protein, binding several structural and signaling molecules. α-Tubulin was identified as an interacting protein in a two-hybrid screen using the Paxillin C-terminal LIM domain as a bait. In vitro binding assays with glutathione S-transferase-Paxillin demonstrated an interaction of α-tubulin with the C terminus of Paxillin. Another member of the tubulin family, γ-tubulin, bound to both the N and the C terminus of Paxillin. The interaction between Paxillin and both α- and γ-tubulin in vivo was confirmed by co-immunoprecipitation from human T lymphoblasts. Immunofluorescence studies revealed that, in adherent T cells, Paxillin localized to sites of cell-matrix interaction as well as to a large perinuclear region. Confocal microscopy revealed that this region corresponds to the lymphocyte microtubule organizing center, where Paxillin colocalizes with α- and γ-tubulin. The localization of Paxillin to this area was observed in cells in suspension as well as during adhesion to integrin ligands. These data constitute the first characterization of the interaction of Paxillin with the microtubule cytoskeleton, and suggest that Paxillin, in addition to its well established role at focal adhesions, could also be associated with the lymphocyte microtubule network.

  • Paxillin ld4 motif binds pak and pix through a novel 95 kd ankyrin repeat arf gap protein a role in cytoskeletal remodeling
    Journal of Cell Biology, 1999
    Co-Authors: Christopher E Turner, Michael C. Brown, Sheila M. Thomas, Joseph A. Perrotta, M C Riedy, Sotiris N Nikolopoulos, Rosa A Mcdonald, Shubha Bagrodia, Phillip S Leventhal
    Abstract:

    Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named Paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677–678) have been implicated in Paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual Paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for Paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase–activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with Paxillin is mediated by a new 95-kD protein, p95PKL (Paxillin-kinase linker), which binds directly to Paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the Paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF–GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two Paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a Paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST–LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate Paxillin as a mediator of p21 GTPase–regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.