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 th e plasma membrane. Because palmitate is not attached without prior farnesyl addition, the distinct contributions of the two lipid modifications to mem brane attachment or biological activity have been difficult to examine. To test if palmitate is able to support these crucial functions on its own, no vel C terminal mutants of Ha-Ras were constructed, retaining the natural si tes for palmitoylation, but replacing the C-terminal residue of the CAAX si gnal for prenylation with six lysines, Both the Ext61L and ExtWT proteins w ere modified in a dynamic fashion by palmitate, without being farnesylated; bound to membranes modestly (40% as well as native Ha-Ras); and retained a ppropriate GTP binding properties. Ext61L caused potent transformation of N IH 3T3 cells and, unexpectedly, an exaggerated differentiation of PC12 cell s, Ext61L with the six lysines but lacking palmitates was inactive. Thus, f arnesyl is not needed as a signal for palmitate attachment or removal, and a combination of transient palmitate modification and basic residues can su pport Ha-Ras membrane binding and two quite different biological functions. The roles of palmitate can therefore be independent of and distinct from t hose of farnesyl. Reciprocally, if membrane association can be sustained la rgely through palmitates, farnesyl is freed to interact with other proteins .