Regulation of the Rho family small GTPase Wrch-1/RhoU by C-terminal tyrosine phosphorylation requires Src.

Wrch-1 is an atypical Rho family small GTPase with roles in migration, epithelial cell morphogenesis, osteoclastogenesis, and oncogenic transformation. Here, we observed rapid relocalization of Wrch-1 from the plasma membrane upon serum stimulation. Studies revealed a requirement for serum-stimulated tyrosine phosphorylation of Wrch-1 at residue Y254 within its C-terminal membrane targeting domain, mediated by the nonreceptor tyrosine kinase Src. Genetic or pharmacological loss of Src kinase activity blocked both phosphorylation and relocalization of Wrch-1. Functionally, Y254 was required for proper Wrch-1 modulation of cystogenesis in three-dimensional culture, and the phospho-deficient mutant, Y254F, was enhanced in Wrch-1-mediated anchorage-independent growth. Mechanistically, C-terminal tyrosine phosphorylation and subsequent relocalization of Wrch-1 downregulated its ability to interact with and activate its effectors by decreasing active Wrch-1-GTP, perhaps by altering proximity to a GEF or GAP. Phospho-deficient Wrch-1(Y254F) remained at the plasma membrane and GTP bound and continued to recruit and activate its effector PAK, even upon serum stimulation. In contrast, a phospho-mimetic mutant, Y254E, was constitutively endosomally localized and GDP bound and failed to recruit PAK unless mutated to be constitutively active/GAP insensitive. C-terminal tyrosine phosphorylation thus represents a new paradigm in posttranslational control of small GTPase localization, activation, and biological function.

Using inhibitors of prenylation to block localization and transforming activity.

The proper subcellular localization and biological activity of most Ras and Rho family small GTPases are dependent on their posttranslational modification by isoprenylation. Farnesyltransferase (FTase) and geranylgeranyl transferase I (GGTase I) are the prenyltransferases that catalyze the irreversible attachment of C15 farnesyl (Ras, Rnd) or C20 (R-Ras, Ral, Rap, Rho, Rac, Cdc42) isoprenoid lipid moieties to these small GTPases and other proteins. Therefore, pharmacological inhibitors of FTase (FTIs) and GGTase I (GGTIs) have been developed to prevent these modifications and thereby to block the lipid-mediated association of Ras and Rho proteins with cellular membranes and the consequent signaling and transforming activities. In addition, other small molecule inhibitors such as farnesyl thiosalicylic acid (FTS) can compete with the isoprenoid moiety of small GTPases for membrane binding sites. Finally, endogenous regulatory proteins such as RhoGDIs can bind to and mask the prenyl groups of small GTPases, leading to their sequestration from membranes. We describe here methods to use each of these categories of prenylation inhibitors to manipulate and investigate the subcellular localization patterns and transforming potential of these Ras and Rho family GTPases.

Biochemical analyses of the Wrch atypical Rho family GTPases.

The Rho family of GTPases comprises a major branch of the Ras superfamily of small GTPases. To date, at least 22 human members have been identified. However, most of our knowledge of Rho GTPase function comes from the study of the three classical Rho GTPases, RhoA, Rac1, and Cdc42. These Rho GTPases function as GDP/GTP-related binary switches that are activated by diverse extracellular signal-mediated stimuli. The activated GTPases then interact with downstream effectors to regulate cytoplasmic signaling networks that in turn regulate actin organization, cell cycle progression, and gene expression. Recently, studies have begun to explore the regulation and function of some of the lesser-known members of the Rho GTPase family. Wrch-1 (Wnt-regulated Cdc42 homolog-1) and the closely related Chp (Cdc42 homologous protein)/Wrch-2 protein comprise a distinct branch of the mammalian Rho GTPase family. Although both share significant sequence and functional similarities with Cdc42, Wrch proteins possess additional N- and C-terminal sequences that distinguish them from the classical Rho GTPases (Cdc42, RhoA, and Rac1). We have determined that Wrch-1 and Wrch2 exhibit unusual GDP/GTP binding properties and undergo posttranslational lipid modifications distinct from those of the classical Rho GTPases. In this chapter, we summarize our experimental approaches used to characterize the biochemical properties of these atypical Rho GTPases.

Visual monitoring of post-translational lipid modifications using EGFP-GTPase probes in live cells.

Modification of small GTPases by lipids is required for their proper subcellular localization and biological activity. Lipids added post-translationally include both farnesyl and geranylgeranyl isoprenoids and the fatty acid palmitate. Thus, specific small molecule inhibitors of these processes cause mislocalization of small GTPases and impair their biological activity. Common biochemical methods of determining the lipid modification status or inhibitor sensitivity of small GTPases, such as in vitro prenylation assays, SDS-PAGE mobility shifts or metabolic labeling, although highly useful in their own right, cannot distinguish differences among specific subpopulations of cells, link lipid modification status with other properties of interest, or provide spatio-temporal information. An alternative method takes advantage of the tight link between small GTPase lipid modification and subcellular localization. The innate localization pattern of the enhanced green fluorescent protein, a common epitope tag frequently used in live cell imaging, is altered by fusion to modified but not unmodified small GTPases. We describe here a technique that takes advantage of these properties to monitor post-translational modifications of these proteins in a rapid, visual manner in live cells.

Transforming activity of the Rho family GTPase, Wrch-1, a Wnt-regulated Cdc42 homolog, is dependent on a novel carboxyl-terminal palmitoylation motif.

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.

Atypical mechanism of regulation of the Wrch-1 Rho family small GTPase.

Rho family GTPases are GDP/GTP-regulated molecular switches that regulate signaling pathways controlling diverse cellular processes. Wrch-1 was identified as a Wnt-1 regulated Cdc42 homolog, upregulated by Wnt1 signaling in Wnt1-transformed mouse mammary cells, and was able to promote formation of filopodia and activate the PAK serine/threonine kinase. Wrch-1 shares significant sequence and functional similarity with the Cdc42 small GTPase. However, Wrch-1 possesses a unique N-terminal 46 amino acid sequence extension that contains putative Src homology 3 (SH3) domain-interacting motifs. We determined the contribution of the N terminus to Wrch-1 regulation and activity. We observed that Wrch-1 possesses properties that distinguish it from Cdc42 and other Rho family GTPases. Unlike Cdc42, Wrch-1 possesses an extremely rapid, intrinsic guanine nucleotide exchange activity. Although the N terminus did not influence GTPase or GDP/GTP cycling activity in vitro, N-terminal truncation of Wrch-1 enhanced its ability to interact with and activate PAK and to cause growth transformation. The N terminus associated with the Grb2 SH3 domain-containing adaptor protein, and this association increased the levels of active Wrch-1 in cells. We propose that Grb2 overcomes N-terminal negative regulation to promote Wrch-1 effector interaction. Thus, Wrch-1 exhibits an atypical model of regulation not seen in other Rho family GTPases.