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 isoprenoid-modified 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.
Highlights
The prenylation pathway has been targeted for potential anticancer therapy because most members of the Ras superfamily, which contains many known oncogenes, undergo CAAX processing
While protein prenyltransferase inhibitors still show some promise as anticancer agents, the emerging view that global attenuation of CaaX protein function may be advantageous in blocking cancer cell growth has increased interest in studying the two downstream enzymes involved in CaaX processing
To ensure that only live cells were assayed we chose time points and concentrations of cysmethynil that resulted in minimal cell death and trypan blue dye exclusion was a criteria for counting
Summary
Materials—Recombinant human thrombin was from Enzyme Research Laboratories (South Bend, IN). Cells were washed with PBS, harvested in MLB buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10 mM MgCl2, 1 mM EDTA, 2% glycerol) or GST-FISH buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 10 mM MgCl2) respectively, incubated for 5 min to ensure complete lysis. Rho-GTP in cell lysates was precipitated using the GST fusion of the activated Rho-binding domain Rhotekin and detected by separation on SDS-PAGE followed by immunoblot analysis with a monoclonal Rho-specific antibody (Santa Cruz Biotechnology). Cells were washed with PBS, harvested in lysis buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10 mM MgCl2, 1 mM EDTA, 2% glycerol), incubated for 5 min. Immunoblot analysis and quantitation was performed using the Odyssey System (LICOR, Lincoln, Nebraska) according to the manufacturer’s instructions
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