Bergo Martin O, Ambroziak Patricia, Gregory Cria, George Amanda, Otto James C, Kim Edward, Nagase Hiroki, Casey Patrick J, Balmain Allan, Young Stephen G
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California 94141-9100, USA.
Mol Cell Biol. 2002 Jan;22(1):171-81. doi: 10.1128/MCB.22.1.171-181.2002.
After isoprenylation, the Ras proteins and other CAAX proteins undergo two additional enzymatic modifications-endoproteolytic release of the last three amino acids of the protein by the protease Rce1 and methylation of the carboxyl-terminal isoprenylcysteine by the methyltransferase Icmt. This postisoprenylation processing is thought to be important for the association of Ras proteins with membranes. Blocking postisoprenylation processing, by inhibiting Rce1, has been suggested as a potential approach for retarding cell growth and blocking cellular transformation. The objective of this study was to develop a cell culture system for addressing these issues. We generated mice with a conditional Rce1 allele (Rce1(flox)) and produced Rce1(flox/flox) fibroblasts. Cre-mediated excision of Rce1 (thereby producing Rce1(Delta/Delta) fibroblasts) eliminated Ras endoproteolytic processing and methylation and caused a partial mislocalization of truncated K-Ras and H-Ras fusion proteins within cells. Rce1(Delta/Delta) fibroblasts grew more slowly than Rce1(flox/flox) fibroblasts. The excision of Rce1 also reduced Ras-induced transformation, as judged by the growth of colonies in soft agar. The excision of Rce1 from a Rce1(flox/flox) skin carcinoma cell line also significantly retarded the growth of cells, and this effect was exaggerated by cotreatment of the cells with a farnesyltransferase inhibitor. These studies support the idea that interference with postisoprenylation processing retards cell growth, limits Ras-induced transformation, and sensitizes tumor cells to a farnesyltransferase inhibitor.
异戊二烯化后，Ras蛋白和其他CAAX蛋白会经历另外两种酶促修饰——蛋白酶Rce1对蛋白质的最后三个氨基酸进行蛋白水解切割，以及甲基转移酶Icmt对羧基末端异戊二烯化半胱氨酸进行甲基化。这种异戊二烯化后加工被认为对Ras蛋白与膜的结合很重要。通过抑制Rce1来阻断异戊二烯化后加工，已被提议作为延缓细胞生长和阻断细胞转化的一种潜在方法。本研究的目的是开发一种细胞培养系统来解决这些问题。我们构建了带有条件性Rce1等位基因（Rce1(flox)）的小鼠，并培养出Rce1(flox/flox)成纤维细胞。Cre介导的Rce1切除（从而产生Rce1(Delta/Delta)成纤维细胞）消除了Ras的蛋白水解加工和甲基化，并导致截短的K-Ras和H-Ras融合蛋白在细胞内出现部分定位错误。Rce1(Delta/Delta)成纤维细胞的生长比Rce1(flox/flox)成纤维细胞更慢。通过软琼脂中集落的生长情况判断，Rce1的切除也降低了Ras诱导的转化。从Rce1(flox/flox)皮肤癌细胞系中切除Rce1也显著延缓了细胞生长，并且用法尼基转移酶抑制剂联合处理细胞会增强这种效果。这些研究支持了这样一种观点，即干扰异戊二烯化后加工会延缓细胞生长、限制Ras诱导的转化，并使肿瘤细胞对法尼基转移酶抑制剂敏感。
