p62~(dok)蛋白氨基酸和cDNA序列测定和分析

目的 :分析p6 2 dok氨基酸和cDNA序列 ,探讨p6 2 dok酪氨酸磷酸化及其与p2 1ras鸟苷三磷酸酶激活蛋白(p2 1rasGAP)的关系。方法 :从过渡表达人类胰岛素受体的中国仓鼠卵巢细胞 (CHO/IR细胞 )中提取、纯化p6 2 dok,进行微肽序列分析 ,设计相应引物PCR、克隆cDNA并测序。利用免疫沉淀和蛋白免疫印迹检测p6 2 dok是否存在酪氨酸磷酸化及与p2 1rasGAP的关系。结果 :p6 2 dokcDNA由 186 3bp组成 ,编码 481个氨基酸 ,含 15个酪氨酸残基 ,富PXXP基序 ,并有一个pleckstrin同源区。当p6 2 dok酪氨酸磷酸化后 ,可与p2 1rasGAP相结合。结论 :p6 2 dok可能是一种新的信息分子 ,并通过p2 1rasGAP参与胰岛素信号转导系统中RAS/MAPK径路的调节。

Sequence analysis of p62~(dok) amino acid and cDNA clone

AIM:To analyse sequences of p62 dok amino acid and cDNA and to investigate p62 dok tyrosine phosphorylation and its relation with p21 ras GAP. METHODS:The purified p62 dok was extracted from CHO/IR cells. The peptide sequence of p62 dok was carried out on a high performance analyzer. PCR was performed with the primers designed from the sequence of p62 dok amino acid. Western blot and immunoprecipitation were used to identify tyrosine phosphorylation of p62 dok and the binding of p62 dok with p21 ras GAP. RESULTS:The p62 dok cDNA is a 1863 bp sequence and code 481 amino acid with 15 tyrosine residues and a putative pleckstrin homology domain. The p62 dok protein is rich in PxxP motif. The tyrosine-phosphorylated p62 dok can bind p21 ras GAP. CONCLUSION:Perhaps p62 dok is a new signaling molecule and play an important role in insulin signaling networks through RAS/MAPK pathway.