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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 explore the effect of p21 phosphorylation modification on the ubiquitination and stability of p21 phosphorylated by protein kinase A (PKA). Methods: The p21 T145A mutant with p21 phosphorylation site was constructed and the phosphorylation of p21 was examined by in vitro phosphorylation of p21. The effect of PKA-specific inhibitor H89 on p21 stability was analyzed by Western blotting. The effect of H89 on p21 ubiquitination Modification and changes in the binding ability of Skp2 before and after p21 Thr 145 mutation. Results: Phosphorylation of p21 by PKA in vitro modified Thr 145; PKA-specific inhibitor H89 enhanced the stability of p21 and inhibited ubiquitination of p21; wild type (w ild) p21 had stronger ability of binding to Skp2 than p21 T145A. CONCLUSION: Phosphorylation of Thr145 by p21 phosphorylation of p21 leads to its enhanced ability to bind E3 ligase to subunit Skp2, which promotes the ubiquitination of p21 and leads to the decrease of p21 stability.