Posttranslational modifications as regulators of membrane localization and biological activity of the Rho family small GTPase, Wrch-1

Rho proteins are members of the Ras superfamily of small GTPases. They are most well known for their functions in regulating the actin cytoskeleton, but also have normal roles in nearly all aspects of cellular physiology. Aberrant regulation of Rho signaling pathways leads to transformation including uncontrolled growth, invasion and metastasis. As such, mediators of Rho protein activity are subject to intense investigation as potential targets for pharmacological inhibitors. In addition to GTP/GDP cycling, membrane localization of these GTPases is a critical determinant of their transforming ability through spatial regulation of their signaling interaction partners and downstream signaling pathways. In this dissertation, I describe my investigations into regulation of the localization and function of Wrch-1 (Wntregulated Cdc42 homolog-1), a novel member of the Cdc42 subfamily of Rho proteins. Nearly all Rho proteins rely on posttranslational modifications of specific residues within their carboxyl termini for proper delivery to cellular membranes. For example, a required geranylgeranyl or farnesyl isoprenoid lipid moiety is attached by the respective prenyltransferase to a conserved cysteine residue within the carboxyterminal CAAX motif of Rho proteins. Farnesyltransferase and geranylgeranyltransferase inhibitors (FTIs,GGTIs) are under investigation as iv potential anticancer drugs. I sought to determine whether Wrch-1 is a target for FTIs or GGTIs. In addition, Rho family proteins are also modified by phosphorylation and ubiquitylation that can direct protein localization and stability, but the role of these modifications in regulating Rho biological activity is much less well understood. I also investigated how posttranslational modifications might regulate the localization and transforming activity of Wrch-1. I found that Wrch-1 is an atypical Rho protein that requires the addition of palmitoyl fatty acids rather than isoprenyl groups for correct sorting to membranes and for its transforming ability. I defined three distinct membrane targeting motifs in the carboxy-terminal hypervariable domain of Wrch-1 that regulate its interaction with plasma membrane, endomembrane and nuclear locations. Finally, I uncovered a possible role for monoubiquitylation of Wrch-1 in regulating its subcellular location and trafficking. Thus, Wrch-1 biological activity is regulated by its subcellular distribution due to modification by palmitoylation, phosphorylation and ubiquitylation

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

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