Biochemical characterization of small GTP binding protein in the brain of insect

Rab蛋白的小gtpase是膜运输的关键调节因子。据报道，家蚕的rabb 8 （BRab8）被蛋白激酶C （PKC）磷酸化，在哺乳动物细胞中表达并部分纯化（Uno等，2006）。为了确定BRab8的磷酸化氨基酸残基，7个丝氨酸残基和4个苏氨酸残基被显示为候选磷酸化氨基酸残基。因此，我们利用PCR技术构建了6个含有BRab8 cdna的缺失或突变片段。将获得的片段插入表达载体中。融合蛋白在大肠杆菌中表达，并用亲和层析法部分纯化。这些蛋白在体外被PKC和γ ^<32>P-ATP磷酸化，电泳并通过生物成像分析仪分析。结果，BRab8的一个丝氨酸-132被特异性磷酸化。进一步用rab8蛋白免疫分离得到多克隆抗体。通过免疫印迹和磷酸化蛋白染色，发现Rab8在体内被磷酸化。

Small GTPases of the Rab proteins are key regulators of membrane trafficking. It was reported that rab 8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C (PKC), which were expressed in mammalian cells and partially purified (Uno, et. al., 2006). To determine the phosphorylated amino acid residues of BRab8, seven serine and four threonin residues were shown as candidates to be phosphorylated. So, six deleted or mutated fragments containing cDNAs for BRab8 were constructed using PCR. The obtained fragments were inserted into expression vectors. Fusion-proteins were expressed in E.coli and partially purified using affinity chromatographies. These proteins were phosphorylated by PKC and gamma ^<32>P-ATP in vitro, electrophoresed and analyzed by a bioimaging analyzer. As a result, one serine-132 of BRab8 was specifically phosphorylated. Further, polyclonal antibody was isolated by immunizing with rab8 protein. From immunoblotting and staining phosphorylated proteins, Rab8 was found to be phosphorylated in vivo.