Ubc9融合表达时SUMO化修饰影响蛋白激酶Cθ在免疫突触的聚集

SUMOylation Affects Aggregation of PKCθ at the Immunological Synapse When Ubc9 Is Fusion Expressed

SUMO化修饰具有广泛的功能,包括调控神经突触的形成和传递。神经突触和免疫突触共享某些特性,而蛋白激酶Cθ(protein kinase Cθ,PKCθ)是蛋白激酶C(protein kinase C,PKC)家族中唯一能在抗原刺激后定位于免疫突触中央超分子活化簇的一员。为了探讨SUMO化修饰对免疫突触的影响,该文采用"Ubc9融合介导的SUMO化修饰系统(Ubc9 fusion-directed SUMOylation system,UFDS)"方法检测Ubc9-PKCθ与SUMO的相互作用;应用体外定点突变技术构建多个SUMO化修饰位点突变的Ubc9-PKCθ;通过荧光显微镜观察Raji B-Jurkat T细胞之间的接触面。结果显示,Ubc9-PKCθ能被SUMO化修饰,且抗原刺激后能在免疫突触聚集;而当多个SUMO化修饰位点突变后,Ubc9-PKCθ的SUMO化水平显著下调,抗原刺激后虽然还是被招募到免疫突触上,但呈现弥散状态。综上所述,Ubc9融合表达时SUMO化修饰影响PKCθ在免疫突触的聚集。

SUMOylation has diverse functions, including regulating the neural synapse formation and transmission. The neural and immunological synapses share common features, while protein kinase Cθ（PKCθ） is the only PKC（protein kinase C） isoform which is localized in the c SMAC of the immunological synapse following antigen stimulation. To unravel the effects of sumoylation on the immunological synapse, this study measured the interaction between Ubc9-PKCθ and SUMO by Ubc9 fusion-directed SUMOylation system（UFDS）, constructed multipoint KR mutant of Ubc9-PKCθ by PCR site-directed mutagenesis, observed the contact surface between Raji B and Jurkat T cells by fluorescence microscope. The results showed that Ubc9-PKCθ could be modified by SUMO, and accumulated at the immunological synapse after antigen stimulation. Furthermore, after multiple SUMOylation sites were mutated, the sumoylated Ubc9-PKCθ had a significant reduction, and was still targeted to the immunological synapse but displayed a diffuse pattern. In conclusion, SUMOylation can affect aggregation of PKCθ at the immunological synapse when Ubc9 is fusion expressed.