ISOPRENOID ADDITION TO RAS PROTEIN IS THE CRITICAL MODIFICATION FOR ITS MEMBRANE ASSOCIATION AND TRANSFORMING ACTIVITY
ISOPRENOID ADDITION TO RAS PROTEIN IS THE CRITICAL MODIFICATION FOR ITS MEMBRANE ASSOCIATION AND TRANSFORMING ACTIVITY
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DOI：
10.1073/pnas.89.14.6403
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发表时间：
1992-07-15
影响因子：
11.1
通讯作者：
DER, CJ
中科院分区：
文献类型：
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作者：
KATO, K;COX, AD;DER, CJ
我们已经将多种氨基酸取代引入致癌[Val 12] Ki-Ras 4 B蛋白的羧基末端CA 1A 2X序列（C =半胱氨酸; A =脂肪族; X =任何氨基酸），以鉴定允许Ras加工（异戊二烯化、蛋白水解和羧基甲基化）、膜结合和在培养的哺乳动物细胞中转化的氨基酸。虽然在A1位置的所有取代都是耐受的，但在A2和X位置的取代降低了转化活性。A2残基对异戊二烯化和AAX蛋白水解都很重要，而X残基仅决定类异戊二烯修饰的程度和特异性。在活细胞中的Ras处理和无细胞系统中的法尼基化效率之间观察到差异。最后，一个法尼基化的突变体没有经历蛋白水解或羧基甲基化，但仍然显示出有效的膜缔合（几乎等于50%）和转化活性，表明单独的法尼基化可以支持Ras转化活性。由于法尼基化和羧基甲基化对于酵母a因子生物活性都是至关重要的，因此三种CAAX信号修饰可能对不同含CAAX蛋白的功能具有不同的贡献。
We have introduced a variety of amino acid substitutions into carboxyl-terminal CA1A2X sequence (C = cysteine; A = aliphatic; X = any amino acid) of the oncogenic [Val12]Ki-Ras4B protein to identify the amino acids that permit Ras processing (isoprenylation, proteolysis, and carboxyl methylation), membrane association, and transformation in cultured mammalian cells. While all substitutions were tolerated at the A1 position, substitutions at A2 and X reduced transforming activity. The A2 residue was important for both isoprenylation and AAX proteolysis, whereas the X residue dictated the extent and specificity of isoprenoid modification only. Differences were observed between Ras processing in living cells and farnesylation efficiency in a cell-free system. Finally, one farnesylated mutant did not undergo either proteolysis or carboxyl methylation but still displayed efficient membrane association (almost-equal-to 50%) and transforming activity, indicating that farnesylation alone can support Ras transforming activity. Since both farnesylation and carboxyl methylation are critical for yeast a-factor biological activity, the three CAAX-signaled modifications may have different contributions to the function of different CAAX-containing proteins.