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 structural domains and sites of P73 which can be phosphorylated by polo like kinases 3 (Plk3), and to analyze the effect of Plk3 on P73mediated apoptosis. Methods: Coimmunoprecipitation experiment was used to examine the interaction between Plk3 and P73. Immunofluorescence was used to examine the localization of Plk3 and P73 in cells. Different deletion mutants of GSTP73 fusion protein were prepared. A sitemutation plasmid of GSTP73 (1130) was constructed by converting threonine86 to alanine86 (T86A) and was named GSTP73 (1130) (T86A). The phosphorylated domains and sites of P73 by Plk3 were determined by in vitro phosphorylation assay. The effect of Plk3 on P73mediated apoptosis of HeLa cells was examined by cleaved PARP detection. Results: Plk3 could interact with P73; Plk3 and P73 colocated in the cell nuclei. Different deletion mutants of GSTP73 fusion protein were successfully prepared, and Plk3 phosphorylated P73 at Nterminal 63113 amino residues. Point mutation (T86A) of GSTP73 (1130) fusion protein could not influence the phosphorylation status of P73 by Plk3. Furthermore, Plk3 inhibited P73mediated apoptosis in HeLa cells. Conclusion: Plk3 can interact with and phosphorylate P73 at Nterminal 63113 amino residues (but not at the 86 threonine), thereby inhibiting P73mediated apoptosis of HeLa cells.

河北省卫生厅科研基金资助项目（No. 20090155）