PostprenylationCAAXProcessing Is Required for Proper Localization of Ras but Not Rho GTPases

Abstract

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal-AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the -AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1-and Icmtdeficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.

Control of RhoA Methylation by Carboxylesterase I

“…Methylation has been shown to play an important role in interactions of RhoA with other proteins. It has been demonstrated by us and others (10,(31)(32)(33)(34) that the methylation status of RhoA directly impacts the RhoA-RhoGDI interaction and that RhoGDI plays an important role in the regulation of Rho activity. We hypothesize that, by controlling the methylation of RhoA, CES1 may be involved in the down-regulation of RhoA activity.…”

Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer

“…However, it is possible that ICMTmediated methylation may be critical for the function of only farnesylated proteins, thus greatly reducing the number of targets for ICMT inhibitors (Michaelson et al, 2005). Finally, ICMT-mediated methylation is also needed for the function of a subset of Rab family small GTPases, regulators of vesicular trafficking (Leung et al, 2006), thus expanding the candidate targets for ICMT inhibitors beyond CAAX-terminating proteins.…”

Therapeutic intervention based on protein prenylation and associated modifications

“…The well-known chemotherapeutic compound methotrexate (an antifolate that blocks thymidine synthesis, which is necessary for tumor growth) causes accumulation of S-adenosylhomocysteine, a potent inhibitor of nearly all cellular methyltransferases; it has been suggested that at least some of methotrexate's antitumor activity may stem from its ability to inhibit Icmt, though specificity for Icmt is lacking 48 . It is worth noting that there were initial reservations that specific Icmt inhibitors might be toxic to cells because they would affect all prenylated proteins; these concerns have been significantly mitigated by recent work showing that although the localization of farnesylated proteins is strongly affected when methylation is blocked, the localization of geranylgeranylated proteins (the main class of prenylated proteins) is relatively unaffected 49 . Efforts to identify specific Icmt inhibitors are currently underway.…”