Role of Isoprenylcysteine Carboxylmethyltransferase-catalyzed Methylation in Rho Function and Migration

Abstract

A number of proteins that play key roles in biological regulatory events undergo a process of post-translational modifications termed prenylation. The prenylation pathway consists of three enzymatic steps; the final processed protein is isoprenoidmodified and methylated on the C-terminal cysteine. This protein modification pathway plays a significant role in cancer biology because many oncogenic proteins undergo prenylation. Methylation of the C terminus by isoprenylcysteine carboxylmethyltransferase (Icmt) is the final step in the prenylation pathway. Cysmethynil, a specific Icmt inhibitor discovered in our laboratory, is able to inhibit Ras-mediated signaling, cell growth, and oncogenesis. We sought to examine the role of Icmt-mediated methylation on the behaviors of cancer cells associated with metastatic potential. Our results indicate that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading are also significantly impacted by cysmethynil. To examine the mechanism of Icmt-dependent migration we focused on RhoA and Rac1, prenylated proteins that are important mediators of cell migration through their control of the actin cytoskeleton. Inhibition of Icmt significantly decreases the activation of both RhoA and Rac1; an increase in Rho GDP-dissociation inhibitor (RhoGDI) binding in the absence of methylation appears to contribute to this effect. Furthermore, in the absence of Icmt activity the addition of exogenous RhoA or Rac1 is able to partially rescue directed and random migration, respectively. These findings establish a role for Icmt-mediated methylation in cell migration and advance our understanding of the biological consequences of Rho methylation.Post-translational modifications of proteins play vital roles in many aspects of cell biology. Hence, identifying and understanding the biological impact of these processes is crucial to furthering our basic understanding of how cells function. Numerous proteins that control important biological regulatory events undergo a complex series of post-translational modifications that are directed by the presence of a so-called CaaX motif at their C terminus. This post-translational pathway, termed protein prenylation, is initiated by the attachment of an isoprenoid lipid to an invariant cysteine residue, the C of the CaaX motif (1, 2). Either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid is covalently attached to this cysteine by protein farnesyltransferase (FTase) 2 or protein geranylgeranyltransferase-I (GGTase-I), respectively (3). The prenylation step is followed by cleavage of the three C-terminal amino acids (the -AAX) by an endoplasmic reticulum (ER)-bound protease termed Rce1. Finally, the prenylated cysteine, which is now located at the C terminus, is methylated by isoprenylcysteine carboxylmethyltransferase (Icmt), another integral ER membrane protein (4, 5). The final result of these modifications is a protein that contains a prenylated and meth...

Rho GTPases RhoA and Rac1 Mediate Effects of Dietary Folate on Metastatic Potential of A549 Cancer Cells through the Control of Cofilin Phosphorylation

“…1D), indicating a strong decline in the carboxyl methylation. The loss of carboxyl methylation should prevent the interaction of Rho GTPases with RhoGDI and the extraction from ER into cytoplasm resulting in redistribution of the proteins between the two compartments (32). In agreement with this notion, we observed a strong shift in the subcellular distribution of Rac1 in cells kept on folate-free media compared with cells kept on standard folate-proficient media with a much higher presence of the protein in ER of the former cells (Fig.…”

Control of RhoA Methylation by Carboxylesterase I

“…Icmt-mediated methylation of CaaX proteins plays a role in oncogenesis, autophagy, and in the metastatic behavior of cells (10,11,21,22). In addition, Icmt-mediated methylation is necessary for development and involved in many other areas of biology, including insulin secretion and endothelial monolayer permeability (23)(24)(25)(26).…”

“…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.…”

“…Recently, we demonstrated a link between Icmt activity and Rho-mediated metastatic behavior in breast cancer cells (10). When Icmt activity was suppressed by either genetic or pharmacologic methods, RhoA and Rac1 activation was reduced, resulting in impaired actin cytoskeletal organization and inhibition of migration.…”

Prenylated C17orf37 Induces Filopodia Formation to Promote Cell Migration and Metastasis

“…After the isoprenyl group is added, the modified protein undergoes two additional postprenylation processing steps, which include cleavage of the last three C-terminal amino acids by an endoprotease enzyme named Rce1 (Ras-converting enzyme 1) and finally methylation of the prenylated-cysteine by Icmt (isoprenylcysteine-O-carboxyl methyltransferase) enzyme. These final modifications are also thought to be important for correct subcellular localization and biological activities of these proteins (9,10). The addition of the isoprenyl tail facilitates binding to the cellular membranes and protein-protein interaction and increases the stability of the protein, thereby regulating the overall functional property of these proteins (7).…”