S-nitrosocysteine increases palmitate turnover on Ha-Ras in NIH 3T3 cells

Ha-Res is modified by isoprenoid on Cys(186) and by reversibly attached pal mitates at Cys(181) and Cys(184). Ha-Ras loses 90% of its transforming acti vity if Cys(181) and Cys(184) changed to serines, implying that palmitates make important contributions to oncogenicity, However, study of dynamic acy lation is hampered by an absence of methods for acutely manipulating Ha-Ras palmitoylation in living cells. S-nitrosocysteine (SNC) and, to a more mod est extent, S-nitrosoglutathione were found to rapidly increase [H-3]palmit ate incorporation into cellular or oncogenic Ha-Ras in NIH 3T3 cells. In co ntrast, SNC decreased [H-3]palmitate labeling of the transferrin receptor a nd caveolin. SNC accelerated loss of [H-3]palmitate from Ha-Ras, implying t hat SNC stimulated deacylation and permitted subsequent reacylation of Ha-R as. 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-Res activation state and lipidation. These results identi fy SNC as a new tool for manipulating palmitate turnover on Ha-Ras and for studying requirements of repalmitoylation and the relationship be tween pal mitate cycling, membrane localization, and signaling by Ha-Ras.