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

我们发现了与磷酸二酯酶3B(PDE3B)相关的50 kDa蛋白(P50)，它被胰岛素以依赖丝氨酸和苏氨酸残基的等电点3-激酶的方式磷酸化。为了鉴定p50，我们对酵母双杂交初筛阳性的80个克隆进行了分析。通过酵母双杂交筛选获得了PDE3B相关蛋白，经鉴定14-3-3β为PDE3B相关蛋白，在80个克隆中，11个克隆在酵母双杂交系统的二次筛选中呈阳性。以每一阳性克隆的GST融合蛋白为诱饵，以网织红细胞裂解产物系统合成的重组PDE3B为猎物，采用GST下拉实验检测阳性克隆与PDE3B的体外相互作用。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。我们对每11个克隆进行了多克隆抗体的进一步分析。另一方面，我们对p50的激酶进行了研究。对胰岛素刺激的大鼠脂肪细胞胞浆组分的层析分析表明，相同的组分对p50和PDE3B都有磷酸化作用，并且该组分含有磷酸化的Ak.在本研究中，我们获得了7个p50的候选克隆.建议除p50和14-3-3β以外的其他蛋白质与PDE3B相互作用。层析分析表明，p50的蛋白激酶为Akt，经鉴定为PDE3B。

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..