Singh R, Wang B, Shirvaikar A, Khan S, Kamat S, Schelling J R, Konieczkowski M, Sedor J R
Department of Medicine and Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Clin Invest. 1999 Jun;103(11):1561-70. doi: 10.1172/JCI5754.
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.
白细胞介素-1（IL-1）刺激的间充质细胞可模拟炎症的分子机制。IL-1与I型IL-1受体（IL-1R）结合，在粘着斑复合物处聚集一个多亚基信号复合物。由于Rho家族小GTP酶协同组织肌动蛋白细胞骨架和信号传导以调节细胞表型，我们推测IL-1R信号复合物包含这些G蛋白。IL-1以Rho依赖的方式刺激血清饥饿的HeLa细胞中肌动蛋白应激纤维的形成，并迅速激活RhoA上的核苷酸交换。谷胱甘肽S-转移酶（GST）融合蛋白，包含全长IL-1R胞质结构域（GST-IL-1Rcd）或IL-1依赖信号转导所需的IL-1R的末端68个氨基酸，能从Triton可溶的HeLa细胞提取物中特异性共沉淀RhoA和Rac-1，但不能沉淀p21（ras）。在全细胞中，抗IL-1R抗体共免疫沉淀的小分子G蛋白是C3转移酶的底物，C3转移酶可特异性地对Rho GTP酶进行ADP核糖基化。持续激活的、负载[γ-32P]GTP的RhoA在滤膜结合试验中直接与GST-IL-1Rcd相互作用。IL-1Rcd-RhoA相互作用在功能上很重要，因为RhoA的显性抑制突变体可阻止IL-1Rcd指导的IL-6基因转录激活。与我们之前的数据一致，即IL-1R相关髓鞘碱性蛋白（MBP）激酶对IL-1指导的基因表达是必需的，在溶液和凝胶激酶试验中，C3转移酶的细胞内掺入均抑制了IL-1R相关MBP激酶活性。总之，IL-1激活了RhoA，RhoA与IL-1Rcd物理相关，且对胞质-核信号通路的激活是必需的。这些发现表明，IL-1刺激的、Rho依赖的细胞骨架重组可能会在特定结构中聚集信号分子，这些结构对于慢性炎症性疾病中细胞的持续激活是必需的。
