POSTTRANSLATIONAL PROCESSING OF RHOA - CARBOXYL METHYLATION OF THE CARBOXYL-TERMINAL PRENYLCYSTEINE INCREASES THE HALF-LIFE OF RHOA

RhoA and related GTP-binding proteins are modified post-translationall y at their carboxyl terminus to form a prenylcysteine methyl ester. Th e synthesis and posttranslational modification of RhoA and Cdc42 were examined in the RAW264 macrophage cell line, and the effect of carboxy l methylation on protein turnover was determined. Cells were labeled w ith [S-35]cysteine, and RhoA or Cdc42 was immunoprecipitated with spec ific antibodies. Both RhoA and Cdc42 were methylated rapidly in contro l cells, with little accumulation of unmethylated protein. Carboxyl me thylation of RhoA was inhibited by incubation of cells with a carbocyc lic adenosine analog, 3-deazaaristeromycin, resulting in the accumulat ion of unmethylated RhoA. Under these conditions, Cdc42 methylation wa s inhibited only partially, When methylation was inhibited, the RhoA h alf-life decreased from 31 to 12 h, and the Cdc42 half-life decreased from 15 to 11 h. The increased degradation of unmethylated RhoA demons trates a novel function for carboxyl-terminal prenylcysteine carboxyl methylation in protecting RhoA and related proteins from degradation.