促炎分子盘状结构域受体2在泛素化修饰过程中与泛素分子的偶联方式

The attachment manner of ubiquitin to proinflammatory molecule discoidin domain receptor 2

目的研究泛素分子中哪些位点的赖氨酸(Lys)残基参与了具有促炎效应的膜受体盘状结构域受体2(DDR2)的多聚泛素化修饰。方法将DDR2、Cbl-b及泛素分子不同位点Lys的突变体共转染HEK293T细胞,以胶原蛋白刺激细胞诱导DDR2发生活化。利用免疫沉淀方法分离细胞中的DDR2,Western blot法检测DDR2与不同泛素分子的偶联情况。结果仅保留27位Lys的泛素分子偶联到DDR2分子上的能力与野生型泛素分子相接近,保留33位Lys的泛素分子有较微弱与DDR2偶联的能力。27位Lys突变可使泛素分子彻底丧失与DDR2相偶联的能力,而33位Lys突变使得泛素分子偶联DDR2的能力部分丧失。结论 Cbl-b介导的DDR2的多泛素化修饰主要是通过泛素分子的Lys27偶联的,Lys33可能也部分参与了DDR2的泛素化修饰。

Objective To understand which lysine（K） residue in ubiquitin（Ub） is used to form a poly-Ubs chain on discoidin domain receptor 2（DDR2）. Methods The constructs encoding human DDR2 and Cbl-b were transiently transfected into HEK293 T cells together with the Ub mutants with lysine mutation at different sites,and the transfected cells were stimulated with collagens in order to induce the activation of DDR2. Immunoprecipitation was performed to isolate DDR2,and the immunoprecipitates were then subjected to immunoblot analysis for the conjugation of DDR2 with Ub. Results Ub K27-only mutant displayed the strongest poly-Ub chain formation on DDR2,while Ub K33-only mutant exhibited weak ability to be conjugated with DDR2. These findings were further confirmed by Ub K27 R and K33 R mutants,which reduced DDR2 polyubiquitination. Conclusion DDR2 is linked to a poly Ub chain predominantly through K27 conjugation and slightly through K33.