Maltese W A, Sheridan K M, Repko E M, Erdman R A
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
J Biol Chem. 1990 Feb 5;265(4):2148-55.
Several proteins in mammalian cells are modified post-translationally by the isoprenoid, farnesol. Incubation of cultured cells with [3H]mevalonate, an isoprenoid precursor, results in the labeling of multiple polypeptides, the most prominent of which migrate in the range of 21-26 kDa on sodium dodecyl sulfate-polyacrylamide gels. In Rat-6 fibroblasts transformed by H-ras, one of the farnesylated proteins was identified as p21ras by two-dimensional immunoblotting. However, this protein accounted for only a small proportion of the [3H]mevalonate-derived radioactivity incorporated into 21-26-kDa proteins. Murine erythroleukemia cells, which did not express immunodetectable quantities of p21ras, contained several 21-26-kDa farnesylated proteins distributed in both the cytosolic and particulate fractions. At least eight of these proteins were capable of binding [alpha-32P]GTP on nitrocellulose membranes. Pulse-chase studies showed that the isoprenoid modification did not necessarily result in the translocation of the cytosolic proteins to the cell membrane. A prominent group of carboxyl-methylated proteins in murine erythroleukemia cells overlapped with the 21-26-kDa farnesylated proteins on one-dimensional sodium dodecyl sulfate gels. Methylation of this group of proteins was selectively abolished when cells were treated with lovastatin, an inhibitor of isoprenoid synthesis. Addition of exogenous mevalonate to the lovastatin-treated cells fully restored carboxyl methylation. These studies suggest that the 21-26-kDa farnesylated proteins in mammalian cells are members of a recently discovered family of low molecular mass GTP-binding proteins which, although ras-related, appear to be distinct structurally and possibly functionally from the products of the ras genes. The observed isoprenoid-dependent carboxyl methylation of a group of 21-26-kDa proteins suggests that the low molecular mass GTP-binding proteins may undergo a series of post-translational C-terminal cysteine modifications (i.e. farnesylation, carboxyl methylation) analogous to those recently elucidated for p21ras.
哺乳动物细胞中的几种蛋白质在翻译后会被类异戊二烯法尼醇修饰。用类异戊二烯前体[3H]甲羟戊酸孵育培养的细胞，会导致多种多肽被标记，其中最突出的多肽在十二烷基硫酸钠-聚丙烯酰胺凝胶上的迁移范围为21 - 26 kDa。在由H-ras转化的大鼠6成纤维细胞中，通过二维免疫印迹法鉴定出一种法尼基化蛋白为p21ras。然而，该蛋白仅占掺入21 - 26 kDa蛋白中的[3H]甲羟戊酸衍生放射性的一小部分。不表达可免疫检测量p21ras的小鼠红白血病细胞含有几种21 - 26 kDa的法尼基化蛋白，分布于胞质和颗粒部分。这些蛋白中至少有八种能够在硝酸纤维素膜上结合[α-32P]GTP。脉冲追踪研究表明，类异戊二烯修饰不一定会导致胞质蛋白转运到细胞膜。在一维十二烷基硫酸钠凝胶上，小鼠红白血病细胞中一组突出的羧基甲基化蛋白与21 - 26 kDa的法尼基化蛋白重叠。当细胞用类异戊二烯合成抑制剂洛伐他汀处理时，这组蛋白的甲基化被选择性消除。向用洛伐他汀处理的细胞中添加外源性甲羟戊酸可完全恢复羧基甲基化。这些研究表明，哺乳动物细胞中21 - 26 kDa的法尼基化蛋白是最近发现的低分子量GTP结合蛋白家族的成员，这些蛋白虽然与ras相关，但在结构上似乎与ras基因产物不同，在功能上可能也不同。观察到的一组21 - 26 kDa蛋白的类异戊二烯依赖性羧基甲基化表明，低分子量GTP结合蛋白可能会经历一系列翻译后C末端半胱氨酸修饰（即法尼基化、羧基甲基化），类似于最近阐明的p21ras的修饰。
