title: Cell surface transglutaminase promotes RhoA activation via integrin clustering and suppression of the Src-p190RhoGAP signaling pathway.
abstract: Tissue transglutaminase (tTG) is a multifunctional protein that serves as cross-linking enzyme and integrin-binding adhesion coreceptor for fibronectin on the cell surface. Previous work showed activation of small GTPase RhoA via enzymatic transamidation by cytoplasmic tTG. Here, we report an alternative nonenzymatic mechanism of RhoA activation by cell surface tTG. Direct engagement of surface tTG with specific antibody or the fibronectin fragment containing modules I(6)II(1,2)I(7-9) increases RhoA-GTP levels. Integrin-dependent signaling to RhoA and its downstream target Rho-associated coiled-coil containing serine/threonine protein kinase (ROCK) is amplified by surface tTG. tTG expression on the cell surface elevates RhoA-GTP levels in nonadherent and adherent cells, delays maximal RhoA activation upon cell adhesion to fibronectin and accelerates a rise in RhoA activity after binding soluble integrin ligands. These data indicate that surface tTG induces integrin clustering regardless of integrin-ligand interactions. This notion is supported by visualization of integrin clusters, increased susceptibility of integrins to chemical cross-linking, and biochemical detection of large integrin complexes in cells expressing tTG. In turn, integrin aggregation by surface tTG inhibits Src kinase activity and decreases activation of the Src substrate p190RhoGAP. Moreover, pharmacological inhibition of Src kinase reveals inactivation of Src signaling as the primary cause of elevated RhoA activity in cells expressing tTG. Together, these findings show that surface tTG amplifies integrin-mediated signaling to RhoA/ROCK via integrin clustering and down-regulation of the Src-p190RhoGAP regulatory pathway.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Janiak
authors.forename: Evgeny A
authors.initials: EA
authors.lastname: Zemskov
authors.forename: Alexey M
authors.initials: AM
authors.lastname: Belkin
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002448:Cell Adhesion; D002462:Cell Membrane / Q000201:enzymology; D016764:Cell Polarity; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D004789:Enzyme Activation; D005353:Fibronectins / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D020690:GTPase-Activating Proteins; D019012:Integrin beta1 / Q000276:immunology / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D012097:Repressor Proteins; D015398:Signal Transduction; D011503:Transglutaminases / Q000235:genetics / Q000502:physiology*; D015854:Up-Regulation; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; C513041:Arhgap35 protein, mouse; C513042:Arhgap5 protein, mouse; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D005353:Fibronectins; D020690:GTPase-Activating Proteins; D019012:Integrin beta1; D008024:Ligands; D012097:Repressor Proteins; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D016392:Proto-Oncogene Proteins pp60(c-src); D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein