Clarke S, Vogel J P, Deschenes R J, Stock J
Department of Molecular Biology, Princeton University, NJ 08544.
Proc Natl Acad Sci U S A. 1988 Jul;85(13):4643-7. doi: 10.1073/pnas.85.13.4643.
The ras oncogene products require membrane localization for their function, and this is thought to be accomplished by the addition of a palmitoyl group to a cysteine residue near the carboxyl terminus of the nascent chain. A lipidated carboxyl-terminal cysteine residue is also found in sequence-related yeast sex factors, and in at least two cases, the alpha-carboxyl group is also methyl esterified. To determine if ras proteins are themselves modified by a similar type of methylation reaction, we incubated rat embryo fibroblasts transformed with p53 and activated Ha-ras oncogenes with L-[methyl-3H]methionine under conditions in which the isotope was converted to the methyl donor S-adenosyl-L-[methyl-3H]methionine. By using an assay that detects methyl ester linkages, we found that immunoprecipitated ras proteins are in fact esterified and that the stability of these esters is consistent with a carboxyl-terminal localization. This methylation reaction may be important in regulating the interaction of ras proteins with plasma membrane components. The presence of analogous carboxyl-terminal tetrapeptide sequences in other proteins may provide a general recognition sequence for lipidation and methylation modification reactions.
原癌基因ras产物的功能需要膜定位，据认为这是通过在新生链羧基末端附近的半胱氨酸残基上添加一个棕榈酰基团来实现的。在序列相关的酵母性因子中也发现了脂酰化的羧基末端半胱氨酸残基，并且在至少两种情况下，α-羧基也被甲酯化。为了确定ras蛋白本身是否通过类似类型的甲基化反应进行修饰，我们在将同位素转化为甲基供体S-腺苷-L-[甲基-³H]蛋氨酸的条件下，用L-[甲基-³H]蛋氨酸孵育用p53和活化的Ha-ras原癌基因转化的大鼠胚胎成纤维细胞。通过使用检测甲酯键的测定方法，我们发现免疫沉淀的ras蛋白实际上被酯化，并且这些酯的稳定性与羧基末端定位一致。这种甲基化反应可能在调节ras蛋白与质膜成分的相互作用中起重要作用。其他蛋白质中类似的羧基末端四肽序列的存在可能为脂酰化和甲基化修饰反应提供一个通用的识别序列。
