GUANINE NUCLEOTIDE-SPECIFIC PHOSPHATE TRANSFER BY GUANINE-NUCLEOTIDE-BINDING REGULATORY PROTEIN BETA-SUBUNITS - CHARACTERIZATION OF THE PHOSPHORYLATED AMINO-ACID

One major substrate protein was phosphorylated with [gamma-P-32]GTP in membranes of human leukemia (HL-60) cells. The phosphoprotein comigra ted with beta-subunits of heterotrimeric GTP-binding proteins (G prote ins) in different gel systems. Upon solubilization of the phosphorylat ed membranes, the phosphoprotein could be immunoprecipitated by a G pr otein beta-subunit-specific antiserum. The beta-subunit phosphorylatio n was transient and was found to be specific for GTP and its analog, g uanosine 5'-O-(gamma-thio)triphosphate. When phosphorylated membranes were incubated with various nucleotides, the bound phosphate was speci fically removed by GDP, suggesting that the phosphate can be retransfe rred onto GDP. Divalent cations, preferentially Mg2+ and Mn2+, were re quired for both phosphorylation and dephosphorylation. The phosphoryla tion was stable against treatment with NaOH but sensitive to treatment with heat, HCl, and hydroxylamine. Moreover, treatment of the membran es with the histidine-modifying agent, diethyl pyrocarbonate, resulted in a loss in phosphate incorporation. The data suggest that G protein beta-subunits are involved in a guanine nucleotide-specific enzymatic activity transferring the gamma-phosphate from GTP to GDP, presumably at G protein alpha-subunits, via a phosphohistidine intermediate.