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-32P]GTP in membranes of human leukemia (HL-60) cells. The phosphoprotein comigrated with beta-subunits of heterotrimeric GTP-binding proteins (G proteins) in different gel systems. Upon solubilization of the phosphorylated membranes, the phosphoprotein could be immunoprecipitated by a G protein beta-subunit-specific antiserum. The beta-subunit phosphorylation was transient and was found to be specific for GTP and its analog, guanosine 5'-O-(gamma-thio)triphosphate. When phosphorylated membranes were incubated with various nucleotides, the bound phosphate was specifically removed by GDP, suggesting that the phosphate can be retransferred onto GDP. Divalent cations, preferentially Mg2+ and Mn2+, were required for both phosphorylation and dephosphorylation. The phosphorylation was stable against treatment with NaOH but sensitive to treatment with heat, HCl, and hydroxylamine. Moreover, treatment of the membranes 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.
Wieland
T
T
Institut für Pharmakologie, Universität GH Essen, Germany.
Nürnberg
B
B
Ulibarri
I
I
Kaldenberg-Stasch
S
S
Schultz
G
G
Jakobs
K H
KH
D001692:Biological Transport; D002462:Cell Membrane; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006639:Histidine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D014407:Tumor Cells, Cultured
D006150:Guanine Nucleotides; D006898:Hydroxylamines; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D019811:Hydroxylamine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008345:Manganese; D006639:Histidine; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D008274:Magnesium; D004047:Diethyl Pyrocarbonate; C005436:phosphohistidine