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The cloning and characterization of the 50 kDa protein which is associated with PDE3B and phosphorylated by insulin

与 PDE3B 相关并被胰岛素磷酸化的 50 kDa 蛋白的克隆和表征

基本信息

批准号：
17590939
负责人：
MAKINO Hideichi
金额：
$ 2.18万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590939/
关键词：
PDE3B adipocytes insulin phosphorylation insulin resistance interaction

项目摘要

We found the 50kDa protein (p50) which is associated with phosphodiesterase 3B(PDE3B) and phosphorylated by insulin in PI 3-kinase dependent manner on serine and threonine residues. To identify p50, we analyzed 80 clones which had been positive in the first screening of the yeast two-hybrid screening. The yeast two-hybrid screening had been performed to get PDE3B-associated protein, and 14-3-3 beta had been identified as PDE3B-associated protein by the screening.Of 80 clones, 11 clones were positive in the second screening of the yeast two-hybrid system. GST pull-down assay was performed to check the in vitro interaction between the positive clones and PDE3B using GST fusion protein of each positive clone as a bait and recombinant PDE3B synthesized by reticulocyte lysate system as a prey. The in vitro interaction between recombinant PBE3B and each of 11 clones was confirmed. GST fusion protein of 7 clones interacted with endogenous PDE3B of rat adipocytes. We made polyclonal antibodies to each 11 clones for the further analysis.On the other hand, we investigated the kinase of p50. The chromatographic analysis of cytosol fraction from insulin-stimulated rat adipocytes revealed that the same fraction phosphorylated both p50 and PDE3B and that the fraction included phosphorylated Akt.In this study, we got 7 clones as candidates of p50. Some kinds of proteins other than p50 and 14-3-3 beta are suggested to interact with PDE3B. The chromatographic analysis suggested that the kinase of p50 was Akt, which was identified as a PDE3B kinase..

我们发现与磷酸二酯酶3B（PDE3B）相关的50kDa蛋白（p50）被胰岛素磷酸化，以依赖于PI 3激酶的方式磷酸化丝氨酸和苏氨酸残基。为了鉴定p50，我们分析了80个在酵母双杂交筛选中首次筛选阳性的克隆。通过酵母双杂交筛选得到pde3b相关蛋白，筛选后14-3-3 β为pde3b相关蛋白。在80个无性系中，11个无性系在酵母双杂交系统的第二次筛选中呈阳性。采用GST下拉实验检测阳性克隆与PDE3B的体外相互作用，以每个阳性克隆的GST融合蛋白为诱饵，网状细胞裂解系统合成的重组PDE3B为猎物。证实了重组PBE3B与11个克隆的体外相互作用。7个克隆的GST融合蛋白与大鼠脂肪细胞内源性PDE3B相互作用。我们对11个克隆分别制备了多克隆抗体，用于进一步分析。另一方面，我们研究了p50的激酶。对胰岛素刺激大鼠脂肪细胞的胞浆组分进行色谱分析，发现同一组分同时磷酸化p50和PDE3B，且该组分中含有磷酸化的Akt。在本研究中，我们获得了7个p50候选克隆。除p50和14-3-3 β外，还有一些蛋白质可能与PDE3B相互作用。色谱分析表明p50的激酶为Akt，鉴定为PDE3B激酶。