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 (Wnt-regulated 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 carboxy-terminal CAAX motif of Rho proteins. Farnesyltransferase and geranylgeranyltransferase inhibitors (FTIs,GGTIs) are under investigation as 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.