Abstract
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.
Highlights
G protein ␥ dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue in the  subunit
G-Phosphorylating Activity in G protein transducin (Gt) and Complex Formation of Gt␥ with nucleoside diphosphate kinase (NDPK)—The  subunits of the retinal G protein Gt can be transientlyphosphorylated by GTP or GTP␥S and
To separate the two phosphoproteins from Gt␣, ϳ1 mg of protein from the second peak was applied onto a hydroxyapatite column, and proteins were eluted with increasing concentrations of potassium phosphate (0 – 400 mM)
Summary
G protein ␥ dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue in the  subunit. We conclude that NDPK B forms complexes with G␥ dimers and contributes to G protein activation by increasing the high energetic phosphate transfer onto GDP via intermediately phosphorylated His-266 in G1 subunits. There is amble evidence that nucleoside diphosphate kinase (NDPK) contributes to G protein activation by replenishment of GTP from ATP and GDP A phosphotransfer reaction that uses G subunits as phosphorylated intermediates has been observed in various tissues [12,13,14,15,16] In this reaction, the ␥-(thio)phosphate group of GTP or its analog GTP␥S is transferred onto a histidine residue of G. We report here that by the attempts to purify the co-factor from the retinal G protein transducin (Gt) or bovine brain membranes, we enriched the NDPK B isoform. We will further provide evidence for a complex formation of G␥ with NDPK B and for a specific phosphorylation of His-266 of G1 in this complex
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