Coccetti P, Mauri I, Alberghina L, Martegani E, Parmeggiani A
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Biochem Biophys Res Commun. 1995 Jan 5;206(1):253-9. doi: 10.1006/bbrc.1995.1035.
The minimal active domain of the mouse CDC25Mm, a GDP/GTP exchange factor (GEF) active on H-ras protein, was determined by constructing several deletion mutants of the C-terminal domain of the protein. The functional activity of these fragments was analyzed for the ability to complement the yeast temperature sensitive mutation cdc25-1 and to catalyze the GDP/GTP exchange on Ras proteins in vitro. A C-terminal domain of 256 residues (CDC25Mm 1005-1260) was sufficient for full biological activity in vivo. Deletion of 27 C-terminal amino acids (CDC25Mm 1005-1233) abolished the complementing activity while deletion of 25 N-terminal residues (CDC25Mm 1030-1260 corresponding to the most conserved domain) led to a complete loss of expression. The results in vivo were supported by experiments in vitro. Highly purified CDC25Mm 1005-1260, expressed in E. coli using the pMAL system, enhanced the GDP release from both H-ras p21 and S. cerevisiae Ras2p and its activity was nearly as high as that of CDC25Mm 974-1260. Comparison with the Cdc25p protein yielded further evidence that the minimal active domain of CDC25Mm is shorter than the yeast one.
小鼠CDC25Mm是一种对H-ras蛋白具有活性的GDP/GTP交换因子（GEF），通过构建该蛋白C末端结构域的多个缺失突变体，确定了其最小活性结构域。分析了这些片段的功能活性，以检测其互补酵母温度敏感突变体cdc25-1的能力以及在体外催化Ras蛋白上GDP/GTP交换的能力。一个包含256个残基的C末端结构域（CDC25Mm 1005-1260）在体内具有充分的生物学活性。缺失27个C末端氨基酸（CDC25Mm 1005-1233）消除了互补活性，而缺失25个N末端残基（CDC25Mm 1030-1260，对应于最保守结构域）导致表达完全丧失。体外实验支持了体内实验结果。使用pMAL系统在大肠杆菌中表达的高度纯化的CDC25Mm 1005-1260，增强了H-ras p21和酿酒酵母Ras2p的GDP释放，其活性几乎与CDC25Mm 974-1260一样高。与Cdc25p蛋白的比较进一步证明，CDC25Mm的最小活性结构域比酵母的更短。
