Protein isoprenylation is a lipid posttranslational modification required for the function of many proteins that share a carboxyl-terminal CAAX motif. The X residue determines which isoprenoid will be added to the cysteine. When X is a methionine or serine, the farnesyl-transferase transfers a farnesyl, and when X is a leucine or isoleucine, the geranygeranyl-transferase I, a geranylgeranyl group. But despite its CKVL motif, RhoB was reported to be both geranylgeranylated and farnesylated. Thus, the determinants of RhoB prenylation appear more complex than initially thought. To determine the role of RhoB CAAX motif, we designed RhoB mutants with modified CAAX sequence expressed in baculovirus-infected insect cells. We demonstrated that RhoB was prenylated as a function of the three terminal amino acids, i.e., RhoB bearing the CAIM motif of lamin B or CLLL motif of Rap1A was farnesylated or geranylgeranylated, respectively. Next, we produced a specific polyclonal antibody against farnesyl cysteine methyl ester allowing prenylation analysis avoiding the metabolic labeling restrictions. We confirmed that the unique modification of the RhoB CAAX box was sufficient to direct the RhoB distinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chimera could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB from cells with the anti-farnesyl cysteine antibody suggested that wild-type RhoB is farnesylated in vivo. Taken together, our results demonstrated that the three last carboxyl amino acids are the main determinants for RhoB prenylation and described an anti-farnesyl cysteine antibody as a useful tool for understanding the cellular control of protein farnesylation.

译文

蛋白质异戊二烯化是一种脂质翻译后修饰,是许多具有羧基末端CAAX基序的蛋白质的功能所需的。X残基决定了哪些类异戊二烯将被添加到半胱氨酸中。当X是蛋氨酸或丝氨酸时,法尼基转移酶转移法尼基,而当X是亮氨酸或异亮氨酸时,香叶香叶基转移酶I,香叶香叶基。但是,尽管有CKVL基序,但据报道RhoB既被香叶基化又被法尼基化。因此,RhoB异戊烯化的决定因素似乎比最初认为的更复杂。为了确定RhoB CAAX基序的作用,我们设计了具有在杆状病毒感染的昆虫细胞中表达的修饰CAAX序列的RhoB突变体。我们证明了RhoB作为三个末端氨基酸的函数被异戊二烯化,即带有lamin B的CAIM基序或Rap1A的CLLL基序的RhoB分别被法呢基化或香叶基化。接下来,我们产生了针对法呢基半胱氨酸甲酯的特异性多克隆抗体,从而可以进行异戊烯化分析,从而避免了代谢标记的限制。我们证实,RhoB CAAX盒的独特修饰足以在哺乳动物细胞中引导RhoB独特的异戊烯化,并且相反,RhoA-CKVL嵌合体可以替代异戊烯化。此外,用抗法尼基半胱氨酸抗体从细胞中免疫沉淀内源性RhoB表明,野生型RhoB在体内被法尼基化。合在一起,我们的结果表明,最后三个羧基氨基酸是RhoB异戊烯化的主要决定因素,并描述了抗法尼基半胱氨酸抗体作为了解蛋白质法尼基化的细胞控制的有用工具。

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