Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Complex formation of NDPK B with Gbeta gamma dimers and phosphorylation of His-266 IN Gbeta.
G protein betagamma dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue in the beta subunit. The phosphate is high energetic and can be transferred onto GDP leading to formation of GTP. Purified Gbetagamma dimers do not display autophosphorylation, indicating the involvement of a separate protein kinase. We therefore enriched the Gbeta-phosphorylating activity present in preparations of the retinal G protein transducin and in partially purified G(i/o) proteins from bovine brain. Immunoblots, autophosphorylation, and enzymatic activity measurements demonstrated enriched nucleoside diphosphate kinase (NDPK) B in both preparations, together with residual Gbetagamma dimers. In the retinal NDPK B-enriched fractions, a Gbeta-specific antiserum co-precipitated phosphorylated NDPK B, and an antiserum against the human NDPK co-precipitated phosphorylated Gbetagamma. In addition, the NDPK-containing fractions from bovine brain reconstituted the phosphorylation of purified Gbetagamma. For identification of the phosphorylated histidine residue, bovine brain Gbetagamma and G(t)betagamma were thiophosphorylated with guanosine 5'-O-(3-[(35)S]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 Gbeta(1) that overlapped in the heptapeptide, Leu-Met-Thr-Tyr-Ser-His-Asp (amino acids 261-267). We conclude that NDPK B forms complexes with Gbetagamma dimers and contributes to G protein activation by increasing the high energetic phosphate transfer onto GDP via intermediately phosphorylated His-266 in Gbeta(1) subunits.
Cuello
Friederike
F
Institut für Pharmakologie und Toxikologie, Fakultät für Klinische Medizin Mannheim, Universität Heidelberg, Maybachstrasse 14-16, D-68169 Mannheim, Germany.
Schulze
Rudiger A
RA
Heemeyer
Frank
F
Meyer
Helmut E
HE
Lutz
Susanne
S
Jakobs
Karl H
KH
Niroomand
Feraydoon
F
Wieland
Thomas
T
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D017886:Durapatite / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008958:Models, Molecular; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D010455:Peptides; D010710:Phosphates; D020033:Protein Isoforms; D011994:Recombinant Proteins; D017886:Durapatite; D009701:Nucleoside-Diphosphate Kinase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D019204:GTP-Binding Proteins; D004047:Diethyl Pyrocarbonate