A Non-farnesylated Ha-Ras Protein Can Be Palmitoylated and Trigger Potent Differentiation and Transformation

Ha-Ras undergoes post-translational modifications (including attachment of farnesyl and palmitate) that culminate in localization of the protein to the plasma membrane. Because palmitate is not attached without prior farnesyl addition, the distinct contributions of the two lipid modifications to membrane attachment or biological activity have been difficult to examine. To test if palmitate is able to support these crucial functions on its own, novel C-terminal mutants of Ha-Ras were constructed, retaining the natural sites for palmitoylation, but replacing the C-terminal residue of the CAAX signal for prenylation with six lysines. Both the Ext61L and ExtWT proteins were modified in a dynamic fashion by palmitate, without being farnesylated; bound to membranes modestly (40% as well as native Ha-Ras); and retained appropriate GTP binding properties. Ext61L caused potent transformation of NIH 3T3 cells and, unexpectedly, an exaggerated differentiation of PC12 cells. Ext61L with the six lysines but lacking palmitates was inactive. Thus, farnesyl is not needed as a signal for palmitate attachment or removal, and a combination of transient palmitate modification and basic residues can support Ha-Ras membrane binding and two quite different biological functions. The roles of palmitate can therefore be independent of and distinct from those of farnesyl. Reciprocally, if membrane association can be sustained largely through palmitates, farnesyl is freed to interact with other proteins

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes

Role of Ras signaling in hematological malignancies and the potential role of inhibitors of the Ras signaling cascade as anti-cancer agents

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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