酵母蛋白质YJL084c的磷酸化分析  

Analysis of Phosphorylation of YJL084c, a Yeast Protein

作　　者：石晓钟[1] 敖世洲[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所分子生物学国家重点实验室,上海200031

出　　处：《生物化学与生物物理学报》2002年第4期433-438,共6页

基　　金：国家自然科学基金重大项目 (No .3 9893 3 2 0 )~~

摘　　要：PCL6、PCL7(PAP1)和PHO80为酵母蛋白激酶PHO85的细胞周期蛋白因子 ,同属于PHO80亚类 ,它们在蛋白质序列上有较高的同源性 ,彼此在功能上有一定程度的重叠。YLR190w为先前鉴定的PCL7 PHO85的一个底物 ,根据BLAST比较 ,YJL0 84c与YJLR190w在一段区域内有一定的同源性 ,并且根据已公布公共数据库 ,YJL0 84c是PCL6的一个结合蛋白。利用双杂交系统分析了YJL0 84c与这三个细胞周期蛋白因子之间的相互作用 ,表明PCL7也可以结合YJL0 84c。利用免疫共沉淀证实它们在体内的相互作用 ;通过体外GST沉降分析 ,验证它们的体外相互作用。YJL0 84c的体外翻译产物可以被PCL7 PHO85复合物磷酸化 ,进一步分别表达纯化YJL0 84c的氨基末端、中间和羧端三部分的GST融合蛋白片段 ,PCL7 PHO85复合物可以磷酸化其中间部分 ,而对N末端和C末端则未表现出磷酸化。以该中间片段蛋白质为底物 ,进一步研究高低磷条件对其磷酸化的影响。PCL7 PHO85对YJL0 84c的磷酸化受到无机磷条件的明显影响 :高磷条件下 ,磷酸化程度高 ;而低磷条件下磷酸化程度低。有意思的是 ,PCL6 PHO85免疫沉淀复合物也表现出同样的磷酸激酶特征。于是分析PHO81与这三个细胞周期因子之间相互作用。此外 ,还构建了Yjl0 84c的缺失株 ,分析了缺失的可能影响。PCL6, PCL7(PAP1), and PHO80 belong to the PHO80 subfamily of PCLs (PHO85 cyclins), share high homology in protein sequences, and function with some similarity. YLR190w, the substrate of PCL7 PHO85, shares homology with YJL084c in a 140 amino acid region. In addition, YJL084c was reported as a PCL6 binding protein. Here, it was found that there was association between YJL084c and PCL7, and their interaction was confirmed by co immunoprecipitation assay and GST pull down assay. The in vitro translational product of YJL084c could be phosphorylated by PCL7 PHO85 complex. Also, the GST fusion protein of the middle region expressed in E.coli could be phosphorylated, while the amino terminal or the carboxyl terminal could not. Interestingly, PCL6 PHO85 complex had the same characters; effect of phosphate condition on the phosphorylation was shown in both PCL6 PHO85 and PCL7 PHO85. YPH499: Yjl084c ∷ LEU 2 was constructed by homologous recombination. PUT4 was reported as a YJL084c binding protein, but no difference was observed between wild strain and the Yjl084c null mutant on MP medium. In addition, the interaction between PHO81 and all of the three cyclins was analyzed.

关 键 词：酵母蛋白质 YJL084c 磷酸化分析 酵母双杂交系统 

分 类 号：Q516[生物学—生物化学]