Cuello Friederike, Schulze Rudiger A, Heemeyer Frank, Meyer Helmut E, Lutz Susanne, Jakobs Karl H, Niroomand Feraydoon, Wieland Thomas
Institut für Pharmakologie und Toxikologie, Fakultät für Klinische Medizin Mannheim, Universität Heidelberg, Maybachstrasse 14-16, D-68169 Mannheim, Germany.
J Biol Chem. 2003 Feb 28;278(9):7220-6. doi: 10.1074/jbc.M210304200. Epub 2002 Dec 16.
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.
G蛋白βγ二聚体可在来自各种组织的膜中被GTP在β亚基的一个组氨酸残基处磷酸化。该磷酸基团具有高能，可转移到GDP上导致形成GTP。纯化的Gβγ二聚体不显示自磷酸化，表明有一个单独的蛋白激酶参与其中。因此，我们富集了视网膜G蛋白转导素制剂以及来自牛脑的部分纯化的G(i/o)蛋白中存在的Gβ磷酸化活性。免疫印迹、自磷酸化和酶活性测量表明，两种制剂中均富集了核苷二磷酸激酶（NDPK）B以及残留的Gβγ二聚体。在视网膜富含NDPK B的组分中，一种Gβ特异性抗血清共沉淀了磷酸化的NDPK B，而一种针对人NDPK的抗血清共沉淀了磷酸化的Gβγ。此外，来自牛脑的含NDPK的组分重建了纯化的Gβγ的磷酸化。为了鉴定磷酸化的组氨酸残基，将牛脑Gβγ和G(t)βγ用鸟苷5'-O-(3-[(35)S]硫代)三磷酸进行硫代磷酸化，然后分别用内肽酶Glu-C和胰蛋白酶消化，通过凝胶电泳和高压液相色谱分别分离得到的肽段，并对放射性肽段进行测序。两种方法产生的序列信息鉴定出牛Gβ(1)的特定标记片段，它们在七肽Leu-Met-Thr-Tyr-Ser-His-Asp（氨基酸261-267）中重叠。我们得出结论，NDPK B与Gβγ二聚体形成复合物，并通过增加经由Gβ(1)亚基中中间磷酸化的His-266将高能磷酸基团转移到GDP上，从而有助于G蛋白的激活。
