IDENTIFICATION AND PRELIMINARY CHARACTERIZATION OF PROTEIN-CYSTEINE FARNESYLTRANSFERASE
IDENTIFICATION AND PRELIMINARY CHARACTERIZATION OF PROTEIN-CYSTEINE FARNESYLTRANSFERASE
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DOI：
10.1073/pnas.87.19.7541
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发表时间：
1990-10-01
影响因子：
11.1
通讯作者：
BARBACID, M
中科院分区：
文献类型：
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作者：
MANNE, V;ROBERTS, D;BARBACID, M
Ras蛋白必须在靠近羧基端（哺乳动物Ras p21蛋白中的Cys-186）的保守半胱氨酸残基上异丙烯化，才能发挥其生物活性。先前的研究表明甲羟戊酸途径中的一种中间体，很可能是焦磷酸法尼酯，是这种异戊烯基的供体。抑制甲羟戊酸合成可以恢复酿酒酵母突变体RAS2Val-19基因诱导的异常表型，并阻断人类来源的致癌Ras p21蛋白诱导的非洲爪蟾卵母细胞成熟。这些结果提高了使用甲羟戊酸途径抑制剂来阻断ras癌基因转化特性的可能性。不幸的是，甲羟戊酸盐是哺乳动物细胞必需的各种最终产物的前体，如多酚、泛素、血红素a和胆固醇。在这项研究中，我们描述了一种酶活性（ies）能够催化未加工的Ras p21蛋白在体外正确的（Cys-186）残基上的法尼化。这种法酰化活性是热不稳定的，需要Mg2+或Mn2+离子，与时间和酶浓度呈线性关系，并且存在于所有哺乳动物细胞系和测试组织中。凝胶过滤分析显示，部分纯化的法尼基转移酶在250-350 kDa和80-130 kDa处有两个活性峰。体外蛋白法尼基转移酶试验的可用性应该有助于筛选ras癌基因功能的潜在抑制剂，这些抑制剂不会干扰甲羟戊酸途径的其他方面。
Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus (Cys-186 in mammalian Ras p21 proteins) in order to exert their biological activity. Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophophate, is the donor of this isoprenyl group. Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2Val-19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an oncogenic Ras p21 protein of human origin. These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, we describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue. This farnesylating activity is heat-labile, requires Mg2+ or Mn2+ ions, is linear with time and with enzyme concentration, and is present in all mammalian cell lines and tissues tested. Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa. Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway.