The IL-1 receptor and Rho directly associate to drive cell activation in inflammation
R. Singh, … , M. Konieczkowski, J.R. Sedor
R. Singh, … , M. Konieczkowski, J.R. Sedor
Published June 1, 1999
Citation Information: J Clin Invest. 1999;103(11):1561-1570. https://doi.org/10.1172/JCI5754.
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Article

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 p21ras, 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 [γ-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

R. Singh, B. Wang, A. Shirvaikar, S. Khan, S. Kamat, J.R. Schelling, M. Konieczkowski, J.R. Sedor

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Figure 5

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A substrate for C3 transferase coprecipitates with native IL-1R. Quiesce...
A substrate for C3 transferase coprecipitates with native IL-1R. Quiescent HeLa cells were stimulated with IL-1α (20 ng/mL) for 10 minutes, and HeLa membranes were prepared as described in Methods. Membrane-associated Rho proteins in HeLa membranes were ADP-ribosylated in the presence (+) or absence (–) of C3 transferase and [32P]NAD. Ribosylated membrane extracts were used directly (Total lysate), or were subsequently analyzed by immunoprecipitation using an anti–IL-1R antibody (IL-1RAb) or specific small GTPase antibodies directed against H-Ras (H-Ras-Ab), Rac-1 (Rac-1-Ab), or RhoA (RhoA-Ab). Immunoprecipitated proteins were separated by SDS-PAGE, and [32P]NAD-ribosylated RhoA GTPases were viewed by autoradiography. The [32P]NAD-ribosylated Rho proteins are indicated by the arrow at right, and molecular mass markers (in kilodaltons) are shown at left. These autoradiographs are representative of 3 independent experiments.