PKA磷酸化p21^(WAF)后对其泛素化修饰和稳定性的影响

Phosphorylation of p21^(WAF) by PKA affects its ubiquitination and stability

目的:探索p21被蛋白激酶A(PKA)磷酸化修饰后对其泛素化修饰和稳定性的影响。方法:构建p21可能磷酸化位点的突变体p21 T145A,观察PKA体外磷酸化修饰p21;W estern印迹分析PKA特异性抑制剂H89对p21稳定性的影响;通过免疫沉淀分析H89对p21泛素化修饰的影响以及p21 Thr 145突变前后和Skp2结合能力的变化。结果:PKA体外磷酸化修饰p21的Thr 145;PKA特异性抑制剂H89能够提高p21的稳定性并抑制p21的泛素化修饰;野生型(w ild)p21比p21 T145A结合Skp2的能力强。结论:PKA磷酸化修饰p21的Thr145后导致其结合E3连接酶结合亚基Skp2的能力增强,从而促进p21的泛素化修饰,导致p21稳定性的降低。

Objective：To investigate whether the phosphorylation of p21 by PKA affects its ubiquitination and stability. Methods： Site-directed mutagenesis was used to engineer p21 construct defective in potential acceptor site for PKA-dependent phosphorylation. Western blotting was used to analyze the stability of p21 after PKA specific inhibitor H89 treatment and the immunoprecipitation analysis was used to investigate whether H89 inhibits the ubiquitination of p21 and whether p21 phosphorylation at Thr 145 affects its Skp2 binding ability. Results： It was confirmed that PKA specifically phosphorylated p21 at Thr 145 by in vitro kinase analysis. PKA specific inhibitor H89 treatment significantly inhibited the ubiquitination and increased protein stability of p21 in HeLa cells. The p21 T145A construct displayed decrease in Skp2 binding compared withwild p21. Conclusion： PKA phosphorylates p21 at Thr 145 in vitro. The phosphorylation of p21 might increase p21 binding to Skp2 and accelerate the ubiquitination of p21 and its degradation.