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J. Biol. Chem., Vol. 278, Issue 9, 7220-7226, February 28, 2003

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

Friederike Cuello, Rüdiger A. Schulze^§, Frank Heemeyer^§, Helmut E. Meyer^¶, Susanne Lutz, Karl H. Jakobs^§, Feraydoon Niroomand, and Thomas Wieland^**

From the  Institut für Pharmakologie und Toxikologie, Fakultät für Klinische Medizin Mannheim, Universität Heidelberg, Maybachstrasse 14-16, D-68169 Mannheim, the ^§ Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, the ^¶ Institut für Physiologische Chemie, Ruhr-Universität Bochum, D-44780 Bochum, and the  Innere Medizin III-Kardiologie, Universität Heidelberg, D-69115 Heidelberg, Germany

G protein dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue in the  subunit. The phosphate is high energetic and can be transferred onto GDP leading to formation of GTP. Purified G dimers do not display autophosphorylation, indicating the involvement of a separate protein kinase. We therefore enriched the G-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 G dimers. In the retinal NDPK B-enriched fractions, a G-specific antiserum co-precipitated phosphorylated NDPK B, and an antiserum against the human NDPK co-precipitated phosphorylated G. In addition, the NDPK-containing fractions from bovine brain reconstituted the phosphorylation of purified G. For identification of the phosphorylated histidine residue, bovine brain G and G_t 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 G₁ that overlapped in the heptapeptide, Leu-Met-Thr-Tyr-Ser-His-Asp (amino acids 261-267). 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.

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