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

我们发现50 kDa的蛋白质（p50）与磷酸二酯酶3B（PDE 3B）相关，并以PI 3-激酶依赖的方式被胰岛素磷酸化。为了鉴定p50，我们分析了在酵母双杂交筛选的第一次筛选中呈阳性的80个克隆。通过酵母双杂交系统筛选获得了PDE 3B相关蛋白，经鉴定14-3-3 β为PDE 3B相关蛋白，在80个克隆中，有11个克隆在酵母双杂交系统的第二次筛选中呈阳性。以各阳性克隆的GST融合蛋白为诱饵，以网织红细胞裂解物系统合成的重组PDE 3B为猎物，进行GST pull-down实验，检测阳性克隆与PDE 3B的体外相互作用。证实了重组PBE 3B与11个克隆中的每一个之间的体外相互作用。7个克隆的GST融合蛋白与大鼠脂肪细胞内源性PDE 3B相互作用。我们分别制备了11个克隆的多克隆抗体，并对p50激酶进行了研究。对胰岛素刺激的大鼠脂肪细胞的胞浆组分的色谱分析显示，同一组分磷酸化p50和PDE 3B，并且该组分包括磷酸化Akt。除p50和14-3-3 β外，还有一些蛋白质与PDE 3B相互作用。层析分析表明p50蛋白激酶为Akt，经鉴定为PDE 3B激酶。

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..