Characterization of the prenylated protein methyltransferase in human endometrial carcinoma

The processing of ras and of other GTP-binding proteins includes a final reversible step in which the carboxy terminal prenylated cysteine is methylated by the enzyme prenylated protein methyltransferase (PPMTase). The significance of this modification and of the role of PPMTase in human tumors has yet to be fully elucidated. Here we characterize the PPMTase of human endometrial carcinomas (tumors in which the frequency of ras gene mutations is relatively high) and compare it to the PPMTase of the normal endometrium. Our results show that in both types of tissues the enzyme is bound to the membranes. It can utilize synthetic substrates such as N-acetyl-S-farnesyl-L-cysteine (Km = 18-20 microM) and is blocked by the PPMTase inhibitor S-farnesylthioacetic acid (Ki = 2 microM). In vitro methylation assays and [alpha-32P]GTP blot-overlay assays showed that the major endogenous PPMTase substrates are small GTP-binding proteins. Methylation of these proteins in vitro is blocked by farnesylthioacetic acid. The kinetic properties of PPMTase from the carcinomas and the normal tissues are very similar. However, levels of PPMTase activity (but not of its endogenous substrates) are higher in the carcinomatous endometrium than in the normal one. The elevated enzyme activity is restricted to the crude mitochondrial fraction (8.0 +/- 0.4 vs. 5.4 +/- 0.1 pmol N-acetyl farnesylcysteine methyl ester formed/min/mg protein by the carcinoma and by the normal endometrial preparations, respectively). As this fraction is enriched in plasma membranes, it appears that the elevated enzyme activity could be related to ras protein methylation; if so, selective methylation blockers might inhibit the growth of endometrial carcinomas.