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目的:探讨P73蛋白上存在的能够被polo样激酶3(polo-like kinases 3,Plk3)磷酸化的结构域或位点,并分析Plk3对P73介导肿瘤细胞凋亡的影响。方法:免疫共沉淀法检测COS-7细胞中Plk3与P73蛋白之间的相互作用,荧光免疫染色法检测Plk3与P73蛋白在细胞中的定位。制备不同的P73缺失突变体GST融合蛋白,体外点突变法构建GST-P73(1～130)点突变的P73(T86A)(第86位苏氨酸点突变为丙氨酸)质粒,体外磷酸化实验分析P73中被Plk3磷酸化的结构域或位点。通过检测PARP蛋白的裂解分析Plk3对P73介导人宫颈癌HeLa细胞凋亡的影响。结果:Plk3与P73蛋白之间存在相互作用,Plk3与P73蛋白共定位于COS-7细胞核中。制备获得不同的P73缺失突变体GST融合蛋白,Plk3在P73蛋白N端第63～113位氨基酸残基之间磷酸化P73蛋白。GST-P73(1～130)融合蛋白第86位苏氨酸点突变为丙氨酸(T86A)之后,不影响GST-P73(1～130)蛋白的磷酸化状态。Plk3可抑制P73介导的HeLa细胞凋亡。结论:Plk3通过与P73蛋白结合,诱导P73蛋白N端第63～113位氨基酸磷酸化,但第86位苏氨酸并非Plk3的特异作用位点;此外Plk3抑制P73介导的HeLa细胞凋亡。
OBJECTIVE: To investigate the phosphorylation of Plk3 in P73 protein, and analyze the effect of Plk3 on P73-mediated apoptosis of tumor cells. METHODS: The interaction between Plk3 and P73 in COS-7 cells was detected by co-immunoprecipitation. The localization of Plk3 and P73 in cells was detected by immunofluorescence staining. P73 (T86A) (86th site of threonine mutation to alanine) with GST-P73 (1 ~ 130) point mutation was constructed by in vitro mutagenesis to construct different P73 deletion mutants GST fusion protein, in vitro phosphorylation The Plk3 phosphorylated domains or sites in P73 were experimentally analyzed. The effect of Plk3 on P73-mediated apoptosis of human cervical carcinoma HeLa cells was examined by detecting the cleavage of PARP protein. Results: There was interaction between Plk3 and P73 protein, Plk3 and P73 protein co-localized in COS-7 cell nucleus. Different P73 deletion mutant GST fusion proteins were obtained, and Plk3 phosphorylated P73 protein between amino acid residues 63- 113 of the N-terminal of P73 protein. The point mutation of threonine at position 86 of GST-P73 (1 ~ 130) fusion protein to alanine (T86A) did not affect the phosphorylation of GST-P73 (1 ~ 130). Plk3 can inhibit P73-mediated HeLa cell apoptosis. CONCLUSION: Plk3 induces the phosphorylation of amino acids 63- 113 of N-terminal of P73 protein by binding with P73 protein, but the 86th threonine is not the specific site of Plk3. Plk3 also inhibits P73-mediated HeLa cell apoptosis.