酵母PAP1与PHO85激酶复合物对PHO4的磷酸化  

Phosphorylation of PHO4 Protein by the PHO85PAP1 Kinase Complex

作　　者：吴健生[1] 夏赞贤[1] 曹铮 敖世洲[1] 

机构地区：[1]中国科学院上海生物化学研究所分子生物学国家重点实验室

出　　处：《生物化学与生物物理学报》1998年第1期63-68,共6页

摘　　要：酵母ＰＨＯ８５结合蛋白ＰＡＰ１与其它的ＰＨＯ８５结合蛋白ＰＨＯ８０、ＰＣＬ１及ＰＣＬ２有较高的同源性。我们构建了ＰＡＰ１与ＨＡ抗原的融合基因，利用抗－ＨＡ抗体进行免疫沉淀。体外翻译的融合ＨＡ标记肽的ＰＡＰ１与体外翻译的ＰＨＯ８５的免疫共沉淀证明了ＰＡＰ１与ＰＨＯ８５的结合。同时纯化了大肠杆菌表达的ＰＨＯ４蛋白，并构建了ＰＨＯ８５∷ＴＲＰ１缺失株。ＰＡＰ１与ＨＡ的融合基因被置于酵母ＡＤＨ１启动子的控制之下，转入酵母ＹＰＨ４９９和ＰＨＯ８５∷ＴＲＰ１菌株中，抽提总蛋白，利用ＨＡ抗体制备免疫沉淀复合物。ＰＡＰ１的免疫沉淀复合物能够在体外磷酸化ＰＨＯ４蛋白，并且这种磷酸化作用是ＰＨＯ８５依赖的，而与酵母的ＣＤＣ２８蛋白激酶无关。这表明ＰＡＰ１能与ＰＨＯ８５形成细胞周期蛋白－ＣＤＫ复合物，ＰＡＰ１可能是一种细胞周期蛋白。Ｎｏｒｔｈｅｒｎ杂交显示ＰＡＰ１基因的转录在细胞周期的各时相中没有明显的差异。构建了ＰＡＰ１基因的缺失株，酸性磷酸酯酶活力分析表明，ＰＡＰ１在ＰＨＯ系统中没有作用。Homology comparison of the novel PAP1 protein indicated that PAP1 protein is highly homologous to PHO85associated protein PHO80, PCL1 and PCL2. We constructed the fused HAPAP1 gene in frame for immunoprecipitation with antiHA monoclonal antibody. Coimmunoprecipitation of fused HAPAP1 and PHO85 protein, which were translated in vitro, verified the association of PAP1 with PHO85 protein. Simultaneously, we purified PHO4 protein expressed in E. coli and constructed a PHO85∷TRP1 strain. After arranging the fused gene under the control of yeast ADH1 promoter, we transformed the resulting plasmids into yeast YPH499 and PHO85∷TRP1 respectively, then prepared the yeast lysates for immunoprecipitation with antiHA. We found that the immunoprecipitant complex of PAP1 can phosphorylate PHO4 protein in vitro, and the kinase activity is PHO85associated, but is not related to CDC28 protein kinase. These data suggest that PAP1 is a putative cyclin and can form cyclinCDK complex with PHO85 protein. Northern blot with PAP1 gene as probe indicated there was no obvious difference during the different phase of the cellcycle in the transcription of PAP1 gene. We constructed the PAP1∷TRP1 strain, and the rAPase activity analysis showed PAP1 had no function in the PHO system.

关 键 词：酵母 激酶 复合物 PHO4 磷酸化 

分 类 号：Q556[生物学—生物化学]