title: The IL-1 receptor and Rho directly associate to drive cell activation in inflammation.
abstract: IL-1-stimulated mesenchymal cells model molecular mechanisms of inflammation. Binding of IL-1 to the type I IL-1 receptor (IL-1R) clusters a multi-subunit signaling complex at focal adhesion complexes. Since Rho family GTPases coordinately organize actin cytoskeleton and signaling to regulate cell phenotype, we hypothesized that the IL-1R signaling complex contained these G proteins. IL-1 stimulated actin stress fiber formation in serum-starved HeLa cells in a Rho-dependent manner and rapidly activated nucleotide exchange on RhoA. Glutathione S-transferase (GST) fusion proteins, containing either the full-length IL-1R cytosolic domain (GST-IL-1Rcd) or the terminal 68 amino acids of IL-1R required for IL-1-dependent signal transduction, specifically coprecipitated both RhoA and Rac-1, but not p21(ras), from Triton-soluble HeLa cell extracts. In whole cells, a small-molecular-weight G protein coimmunoprecipitated by anti-IL-1R antibody was a substrate for C3 transferase, which specifically ADP-ribosylates Rho GTPases. Constitutively activated RhoA, loaded with [gamma-32P]GTP, directly interacted with GST-IL-1Rcd in a filter-binding assay. The IL-1Rcd-RhoA interaction was functionally important, since a dominant inhibitory mutant of RhoA prevented IL-1Rcd-directed transcriptional activation of the IL-6 gene. Consistent with our previous data demonstrating that IL-1R-associated myelin basic protein (MBP) kinases are necessary for IL-1-directed gene expression, cellular incorporation of C3 transferase inhibited IL-1R-associated MBP kinase activity both in solution and in gel kinase assays. In summary, IL-1 activated RhoA, which was physically associated with IL-1Rcd and necessary for activation of cytosolic nuclear signaling pathways. These findings suggest that IL-1-stimulated, Rho-dependent cytoskeletal reorganization may cluster signaling molecules in specific architectures that are necessary for persistent cell activation in chronic inflammatory disease.
authors.affiliation: Department of Medicine and Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: R
authors.initials: R
authors.lastname: Singh
authors.forename: B
authors.initials: B
authors.lastname: Wang
authors.forename: A
authors.initials: A
authors.lastname: Shirvaikar
authors.forename: S
authors.initials: S
authors.lastname: Khan
authors.forename: S
authors.initials: S
authors.lastname: Kamat
authors.forename: J R
authors.initials: JR
authors.lastname: Schelling
authors.forename: M
authors.initials: M
authors.lastname: Konieczkowski
authors.forename: J R
authors.initials: JR
authors.lastname: Sedor
mesh_terms: D036002:ADP Ribose Transferases / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D001905:Botulinum Toxins; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002457:Cell Extracts; D002460:Cell Line; D011232:Chemical Precipitation; D003599:Cytoskeleton / Q000502:physiology; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase; D038362:Glycogen Synthase Kinase 3; D006367:HeLa Cells; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D007375:Interleukin-1 / Q000378:metabolism / Q000494:pharmacology; D017472:Receptors, Interleukin-1 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D020744:rac GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein
chemical_list: D000199:Actins; D002457:Cell Extracts; D007375:Interleukin-1; D017472:Receptors, Interleukin-1; D011993:Recombinant Fusion Proteins; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D005982:Glutathione Transferase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D001905:Botulinum Toxins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein