Desrosiers R R, Nguyen Q T, Béliveau R
Laboratoire de Médecine Moléculaire, Centre de Cancérologie Charles Bruneau, Université du Québec à Montréal, Succursale Centre-ville, Montréal, Québec, H3C 3P8, Canada.
Biochem Biophys Res Commun. 1999 Aug 11;261(3):790-7. doi: 10.1006/bbrc.1999.0936.
The prenylated protein carboxyl methyltransferase (PPMT) catalyzes the posttranslational methylation of isoprenylated C-terminal cysteine residues found in many signaling proteins such as the small monomeric G proteins and the gamma subunits of heterotrimeric G proteins. Here we report that both membrane-bound PPMT from rat kidney and the recombinant bacterially expressed form of the enzyme required divalent cations for catalytic activity. Unlike EDTA and EGTA, the metal chelator 1,10-phenanthroline strongly inhibited the PPMT activity of kidney intracellular membranes in a dose- and time-dependent manner. 1,10-Phenanthroline was found to inhibit the methylation of the prenylcysteine analog N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, a synthetic substrate for PPMT, with an IC(50) of 2.2 mM. Gel electrophoretic analysis demonstrated that 1,10-phenanthroline almost totally abolished the labeling of methylated proteins in kidney intracellular membranes. Immunoblotting analysis showed that one of the two major peaks of (3)H-methylated proteins in intracellular membranes comigrated with the small G proteins Ras, Cdc42, RhoA, and Rab1. In addition, the methylation of immunoprecipitated Ras and RhoA from kidney intracellular membranes was strongly inhibited when 1,10-phenanthroline was present. Treatment of kidney intracellular membranes with 1,10-phenanthroline increased the proteolytic degradation of PPMT by exogenous trypsin, compared to untreated membranes. We conclude from these data that metal ions are essential for the activity and the stabilization of PPMT. The finding that PPMT is a metalloenzyme may provide new insights into the functions played by this methyltransferase in signal transduction processes.
异戊二烯化蛋白羧基甲基转移酶（PPMT）催化许多信号蛋白（如小单体G蛋白和异三聚体G蛋白的γ亚基）中异戊二烯化的C末端半胱氨酸残基的翻译后甲基化。在此我们报告，大鼠肾脏中的膜结合PPMT和该酶的重组细菌表达形式都需要二价阳离子来发挥催化活性。与EDTA和EGTA不同，金属螯合剂1,10 - 菲咯啉以剂量和时间依赖性方式强烈抑制肾脏细胞内膜的PPMT活性。发现1,10 - 菲咯啉抑制异戊烯基半胱氨酸类似物N - 乙酰 - S - 全反式香叶基香叶基 - L - 半胱氨酸（PPMT的一种合成底物）的甲基化，IC(50)为2.2 mM。凝胶电泳分析表明，1,10 - 菲咯啉几乎完全消除了肾脏细胞内膜中甲基化蛋白的标记。免疫印迹分析显示，细胞内膜中(3)H - 甲基化蛋白的两个主要峰之一与小G蛋白Ras、Cdc42、RhoA和Rab1共迁移。此外，当存在1,10 - 菲咯啉时，肾脏细胞内膜中免疫沉淀的Ras和RhoA的甲基化受到强烈抑制。与未处理的膜相比，用1,10 - 菲咯啉处理肾脏细胞内膜会增加外源性胰蛋白酶对PPMT蛋白的降解。从这些数据我们得出结论，金属离子对于PPMT的活性和稳定性至关重要。PPMT是一种金属酶这一发现可能为该甲基转移酶在信号转导过程中所起的作用提供新的见解。
