Abstract
Ha-Ras is modified by isoprenoid on Cys(186) and by reversibly attached palmitates at Cys(181) and Cys(184). Ha-Ras loses 90% of its transforming activity if Cys(181) and Cys(184) are changed to serines, implying that palmitates make important contributions to oncogenicity. However, study of dynamic acylation is hampered by an absence of methods for acutely manipulating Ha-Ras palmitoylation in living cells. S-nitrosocysteine (SNC) and, to a more modest extent, S-nitrosoglutathione were found to rapidly increase [(3)H]palmitate incorporation into cellular or oncogenic Ha-Ras in NIH 3T3 cells. In contrast, SNC decreased [(3)H]palmitate labeling of the transferrin receptor and caveolin. SNC accelerated loss of [(3)H]palmitate from Ha-Ras, implying that SNC stimulated deacylation and permitted subsequent reacylation of Ha-Ras. SNC also decreased Ha-Ras GTP binding and inhibited phosphorylation of the kinases ERK1 and ERK2 in NIH 3T3 cells. Thus, SNC altered two important properties of Ha-Ras activation state and lipidation. These results identify SNC as a new tool for manipulating palmitate turnover on Ha-Ras and for studying requirements of repalmitoylation and the relationship between palmitate cycling, membrane localization, and signaling by Ha-Ras.
Highlights
Ha-Ras is a monomeric GTPase that has two types of lipid modifications, both of which must occur in order for the protein to bind efficiently to the plasma membrane [1,2,3]
S-Nitrosocysteine Treatment Leads to both Increases and Decreases in Protein Palmitoylation—Previous results had shown that palmitate incorporation into SNAP-25 and GTPase-activating proteins (GAPs)-43 decreased when the neuronal cell line, PC-12, or primary cultures of rat dorsal root ganglion cells were labeled with [3H]palmitate in the presence of S-nitrosocysteine [49]
To determine if SNC could alter protein palmitoylation in another cell type (NIH 3T3 fibroblasts), three different classes of palmitoylated fibroblast proteins were examined: the transferrin receptor, which is localized in endocytotic vesicles and clathrin-coated pits in the plasma membrane [50]; caveolin, a second transmembrane protein, localized in caveolae [51]; and cellular Ha-Ras (Ha-RasWT), which is plasma membrane-associated through hydrophobic lipid modifications [4, 7]
Summary
Ha-Ras is a monomeric GTPase that has two types of lipid modifications, both of which must occur in order for the protein to bind efficiently to the plasma membrane [1,2,3]. Ha-Ras requires a second lipid to stabilize its membrane interaction This second lipid is the fatty acid palmitate [10], attached through thioester bonds to cysteine 181 or 184 [11, 12]. Despite the transience of a thioacyl group, the presence of cysteines that can be palmitoylated dramatically increases the extent of farnesylated Ha-Ras membrane binding from ϳ10% to Ͼ95% [7, 20]. For Ha-Ras, palmitoylation and plasma membrane targeting are clearly necessary for biological activity, because Ha-Ras mutants that lack both palmitates are poorly transforming in NIH 3T3 cells [7, 8, 20]. The relationships between palmitoylation, submembrane location, and signaling by any of these proteins [26] are difficult to study, since there are few techniques through which the acylation state of such signaling proteins can be varied
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