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
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
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DOI：
10.1074/jbc.m210304200
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发表时间：
2003-02-28
影响因子：
4.8
通讯作者：
Wieland, T
中科院分区：
文献类型：
--
作者：
Cuello, F;Schulze, RA;Wieland, T
G蛋白- γ二聚体可以在各种组织的膜上被GTP在β亚基上的组氨酸残基磷酸化。磷酸是高能量的，可以转移到GDP导致GTP的形成。纯化的伽马二聚体不显示自磷酸化，表明参与了单独的蛋白激酶。因此，我们在制备视网膜G蛋白转导蛋白和从牛脑中部分纯化的G（i/o）蛋白中富集了gβ -磷酸化活性。免疫印迹、自磷酸化和酶活性测量表明，两种制剂中都含有丰富的核苷二磷酸激酶（NDPK） B，以及残留的伽马二聚体。在视网膜NDPK B富集的部分中，一种gβ特异性抗血清共同沉淀磷酸化NDPK B，一种抗人NDPK共同沉淀磷酸化gbetagma的抗血清。此外，从牛脑中提取的含有ndpk的组分重建了纯化的gbetaggamma的磷酸化。为了鉴定磷酸化的组氨酸残基，用鸟苷5'- o -（3-[S-35]硫）三磷酸对牛脑Gbetagamma和G(t)betagamma进行硫代磷酸化，然后用内源性蛋白酶Glu-C和胰蛋白酶消化，分别用凝胶电泳和高压液相色谱分离所得肽，并对放射性肽进行测序。两种方法产生的序列信息鉴定了牛Gbeta(1)的特异性标记片段，这些片段重叠在七肽leu - met - thr - tyrr - ser - his - asp（氨基酸261-267）上。我们得出结论，NDPK B与伽马二聚体形成复合物，并通过Gbeta(1)亚基中磷酸化的His-266增加向GDP的高能磷酸盐转移，从而促进G蛋白的激活。
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-[S-35]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.