Activation of Heterotrimeric G Proteins by a High Energy Phosphate Transfer via Nucleoside Diphosphate Kinase (NDPK) B and Gβ Subunits COMPLEX FORMATION OF NDPK B WITH Gβγ DIMERS AND PHOSPHORYLATION OF His-266 IN Gβ

Abstract G protein βγ dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue in the β subunit. The phosphate is high energetic and can be transferred onto GDP leading to formation of GTP. Purified Gβγ dimers do not display autophosphorylation, indicating the involvement of a separate protein kinase. We therefore enriched the Gβ-phosphorylating activity present in preparations of the retinal G protein transducin and in partially purified Gi/oproteins from bovine brain. Immunoblots, autophosphorylation, and enzymatic activity measurements demonstrated enriched nucleoside diphosphate kinase (NDPK) B in both preparations, together with residual Gβγ dimers. In the retinal NDPK B-enriched fractions, a Gβ-specific antiserum co-precipitated phosphorylated NDPK B, and an antiserum against the human NDPK co-precipitated phosphorylated Gβγ. In addition, the NDPK-containing fractions from bovine brain reconstituted the phosphorylation of purified Gβγ. For identification of the phosphorylated histidine residue, bovine brain Gβγ and Gtβγ were thiophosphorylated with guanosine 5′-O-(3-[35S]thio)triphosphate, followed by digestion with endoproteinase Glu-C and trypsin, separation of the resulting peptides by gel electrophoresis and high pressure liquid chromatography, respectively, and sequencing of the radioactive peptides. The sequence information produced by both methods identified specific labeled fragments of bovine Gβ1 that overlapped in the heptapeptide, Leu-Met-Thr-Tyr-Ser-His-Asp (amino acids 261–267). We conclude that NDPK B forms complexes with Gβγ dimers and contributes to G protein activation by increasing the high energetic phosphate transfer onto GDP via intermediately phosphorylated His-266 in Gβ1 subunits.