title: Thrombospondin induces RhoA inactivation through FAK-dependent signaling to stimulate focal adhesion disassembly.
abstract: Cells utilize dynamic interactions with the extracellular matrix to adapt to changing environmental conditions. Thrombospondin 1 (TSP1) induces focal adhesion disassembly and cell migration through a sequence (hep I) in its heparin-binding domain signaling through the calreticulin-low density lipoprotein receptor-related protein receptor complex. This involves the Galphai-dependent activation of ERK and phosphoinositide (PI) 3-kinase, both of which are required for focal adhesion disassembly. Focal adhesion kinase (FAK) regulates adhesion dynamics, acting in part by modulating RhoA activity, and FAK is implicated in ERK and PI 3-kinase activation. In this work, we sought to determine the role of FAK in TSP1-induced focal adhesion disassembly. TSP1/hep I does not stimulate focal adhesion disassembly in FAK knockout fibroblasts, whereas re-expressing FAK rescues responsiveness. Inhibiting FAK signaling through FRNK or FAK Y397F expression in endothelial cells also abrogates this response. TSP1/hep I stimulates a transient increase in FAK phosphorylation that requires calreticulin and Galphai, but not ERK or PI 3-kinase. Hep I does not activate ERK or PI 3-kinase in FAK knockout fibroblasts, suggesting activation occurs downstream of FAK. TSP1/hep I stimulates RhoA inactivation with kinetics corresponding to focal adhesion disassembly in a FAK, ERK, and PI 3-kinase-dependent manner. Furthermore, hep I does not stimulate focal adhesion disassembly in cells expressing constitutively active RhoA, suggesting that RhoA inactivation is required for this response. This is the first work to illustrate a connection between FAK phosphorylation in response to a soluble factor and RhoA inactivation, as well as the first report of PI 3-kinase and ERK in FAK regulation of RhoA activity.
authors.affiliation: Department of Pathology, Division of Molecular and Cellular Pathology and the Cell Adhesion and Matrix Research Center, University of Alabama, Birmingham, Alabama 35294-9340, USA.
authors.forename: Anthony Wayne
authors.initials: AW
authors.lastname: Orr
authors.forename: Manuel Antonio
authors.initials: MA
authors.lastname: Pallero
authors.forename: Wen-Cheng
authors.initials: WC
authors.lastname: Xiong
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Murphy-Ullrich
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D037282:Calreticulin / Q000378:metabolism; D002417:Cattle; D002465:Cell Movement; D002478:Cells, Cultured; D019281:Dimerization; D004730:Endothelium, Vascular / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007700:Kinetics; D008856:Microscopy, Fluorescence; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D015398:Signal Transduction; D019699:Thrombospondins / Q000502:physiology*; D013997:Time Factors; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D037282:Calreticulin; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D019699:Thrombospondins; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D048052:Mitogen-Activated Protein Kinase 3; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020742:rhoA GTP-Binding Protein