Centre de Recherche du CHUQ, Département de Microbiologie-Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, Québec G1V 4G2, Canada.
J Biol Chem. 2011 Apr 29;286(17):15073-84. doi: 10.1074/jbc.M110.213660. Epub 2011 Mar 3.
We previously described a non-classical mechanism that arrests FcγRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of FcγRIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated FcγRIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the FcγRIIa degradation process because the cholesterol-depleting agent methyl-β-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented FcγRIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of FcγRIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate FcγRIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated FcγRIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of FcγRIIa signaling.
我们之前描述了一种非经典机制，该机制一旦被免疫复合物或调理病原体结合，就会阻止人类中性粒细胞中的 FcγRIIa 信号转导。FcγRIIa 的结合导致其被泛素连接酶 c-Cbl 泛素化，并被蛋白酶体降解。在此，我们进一步研究了一些调节该新途径的事件。衔接蛋白 CIN85 在其他系统中被描述为参与调节 c-Cbl 依赖性途径。我们发现 CIN85 在人中性粒细胞中表达，并且在受体交联后像 c-Cbl 一样从细胞质易位到质膜。CIN85 还被募集到与刺激的 FcγRIIa 与 c-Cbl 一起分配的相同高密度去污剂抗性膜部分。这些微区的完整性对于 FcγRIIa 降解过程至关重要，因为胆固醇耗竭剂甲基-β-环糊精抑制了该事件。用 siRNA 沉默二丁酰环 AMP 分化的 PLB 985 细胞中的 CIN85 表达可防止 FcγRIIa 降解并增加 IgG 介导的吞噬作用。共聚焦显微镜显示，CIN85 的存在对于 FcγRIIa 在内吞作用过程中的正确分拣是必不可少的。我们还提供了直接证据表明 CIN85 是丝氨酸/苏氨酸激酶 PKC 的底物。经典 PKC 正向调节 FcγRIIa 的泛素化和降解，因为这些事件被经典 PKC 抑制剂 Gö6976 抑制。我们得出结论，c-Cbl 介导的刺激的 FcγRIIa 的泛素化和降解被衔接蛋白 CIN85 以 PKC 依赖性方式正向调节，并且这些事件有助于 FcγRIIa 信号的终止。
