Abstract
Wrch-1 is a Rho family GTPase that shares strong sequence and functional similarity with Cdc42. Like Cdc42, Wrch-1 can promote anchorage-independent growth transformation. We determined that activated Wrch-1 also promoted anchorage-dependent growth transformation of NIH 3T3 fibroblasts. Wrch-1 contains a distinct carboxyl-terminal extension not found in Cdc42, suggesting potential differences in subcellular location and function. Consistent with this, we found that Wrch-1 associated extensively with plasma membrane and endosomes, rather than with cytosol and perinuclear membranes like Cdc42. Like Cdc42, Wrch-1 terminates in a CAAX tetrapeptide (where C is cysteine, A is aliphatic amino acid, and X is any amino acid) motif (CCFV), suggesting that Wrch-1 may be prenylated similarly to Cdc42. Most surprisingly, unlike Cdc42, Wrch-1 did not incorporate isoprenoid moieties, and Wrch-1 membrane localization was not altered by inhibitors of protein prenylation. Instead, we showed that Wrch-1 is modified by the fatty acid palmitate, and pharmacologic inhibition of protein palmitoylation caused mislocalization of Wrch-1. Most interestingly, mutation of the second cysteine of the CCFV motif (CCFV > CSFV), but not the first, abrogated both Wrch-1 membrane localization and transformation. These results suggest that Wrch-1 membrane association, subcellular localization, and biological activity are mediated by a novel membrane-targeting mechanism distinct from that of Cdc42 and other isoprenylated Rho family GTPases.
Highlights
The Rho family of Ras-related small GTPases is a functionally diverse group of proteins that are best known for their roles in regulation of actin cytoskeleton organization, cell polarity, cell adhesion, vesicular trafficking, transcriptional regulation, and cell cycle progression [1, 2]
The correct subcellular localization and function of Ras and Rho family members are dictated by post-translational modification of the carboxyl-terminal hypervariable domain, including the last four amino acids known as the CAAX motif [17, 18]
Wrch-1 terminates in an atypical CAAX tetrapeptide motif, and its hypervariable domain possesses an additional 21 amino acid residues not found in Cdc42
Summary
Molecular Constructs—pcDNA3 expression constructs encoding wild type (WT) and GTPase-deficient (Q107L) human Wrch-1 was obtained from Dr A. NIH 3T3 cells stably expressing HA epitopetagged pCGN constructs of either empty vector, activated Wrch1(107L)-CCFV, -SCFV, -CSFV, or -SSFV were suspended in DMEM containing 10% calf serum, 1% penicillin/streptomycin, and 0.4% agar (BD Biosciences) at 5 ϫ 104 cells per 35-mm dish. Metabolic Labeling—NIH 3T3 cells were seeded at 2 ϫ 105 cells per 60-mm dish and transiently transfected with HA epitope-tagged pCGN constructs containing Wrch-1(107L) CCFV, -SCFV, -CSFV, -SSFV, H-Ras(61L), K-Ras(12V), or empty vector with FuGENE 6. Human embryonic kidney 293 cells transiently expressing GFP-tagged Wrch-1 proteins were lysed in BMCC lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 0.02% NaN3, and 2% Triton X-100) containing Complete protease inhibitor tablet (Roche Applied Science). Membranes were blocked in 5% nonfat dry milk and probed for HA-tagged Wrch-1 proteins using mouse anti-HA antibody, for -actin as a loading control using mouse anti--actin (Sigma), for endogenous phosphorylated PAK using rabbit anti-phospho-PAK1 (Ser-144)/PAK2 (Ser-141), for total endogenous PAK using rabbit antiPAK1/2/3, or for GFP-tagged proteins using mouse anti-GFP antibody (Clontech), followed by anti-mouse HRP-conjugated antibody or antirabbit HRP-conjugated antibody and SuperSignal West Dura extended duration substrate as above
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