Regulation of cellular functions by the effector proteins of Ras

本研究试图阐明作用于Ras下游的大鼠信号通路的功能。(1)Ral下游分子的鉴定及其表征。我们确定POB 1作为一种新的RalBP 1结合蛋白。PCB 1与Grb 2结合，经酪氨酸磷酸化后与EGF受体形成复合物，其N端具有EH结构域。我们从牛脑中纯化了EH结构域结合蛋白Epsin和Eps 15。这两种蛋白质与AP-2和网格蛋白形成复合物，并且已知对于调节受体介导的内吞作用是重要的。(2)功能分析。Ral、RalBP 1和POB 1调节胰岛素和EGF受体的内吞作用，但不调节转铁蛋白受体的内吞作用，而Epsin参与这三种激动剂的受体介导的内吞作用。因此，Ral/RalBP 1/POB 1的信号传导复合物将信号从配体如胰岛素和EGF传递到Eps 15和Epsin，从而诱导配体依赖性内吞作用。(3)RalBP 1、POB 1、Epsin和Eps 15在有丝分裂期的磷酸化。检查了这些蛋白质的复合物的组装的调节。RalBP 1、POB 1、Epsin和Eps 15在CHO细胞中与AP-2的α-adaptin形成复合物，但在有丝分裂期形成减少。RalBP 1、POB 1、Epsin和Eps 15在有丝分裂期均被磷酸化。体外p34 cdc 2激酶可使POB 1和Epsin磷酸化。测定了它们的磷酸化位点（POB 1的411 S和Epsin的357 S）。磷酸化的Epsin和EpsinS 357 D与α-适配蛋白形成复合物的效率低于野生型Epsin。虽然POB 1的EH结构域直接结合Epsin，但Epsin的磷酸化抑制了结合。此外，EpsinS 357 D而不是EpsinS 357 A失去了Epsin对胰岛素依赖性内吞作用的影响。这些结果表明，有丝分裂期Epsin的磷酸化通过分解其与POB 1和α-adaptin的复合物来抑制受体介导的内吞作用。

In this study, we tried to clarify the functions of Rat signaling pathway which acts downstream of Ras.(1) Identification of downstream molecules of Ral and their characterization. We identified POB1 as a novel RalBP1-binding protein. PCB1 bound to Grb2, was tyrosine-phosphorylated, and formed a complex with EGF receptor in response to EGF.The N-terminal region of POB1 has an EH domain. We purified Epsin and Eps15 as EH domain binding proteins from bovine brain. Both proteins formed a complex with AP-2 and clathrin, and were known to be important for the regulation of receptor-mediated endocytosis.(2) Functional analyses. Ral, RalBP1, and POB1 regulated the endocytosis of the receptors for insulin and EGF but not for transferrin, while Epsin was involved in the receptor-mediated endocytosis of these three agonists. Therefore, the signaling complex of Ral/RalBP1/POB1 transmitted the signal from a ligand such as insulin and EGF to Eps 15 and Epsin, thereby induces the ligand-dependent endocytosis.(3) Phosphorylation of RalBP1, POB1, Epsin, and Eps15 in mitotic phase. The regulation of assembly of the complex of these proteins was examined. RalBP1, POB1, Epsin, and Eps15 formed a complex with a-adaptin of AP-2 in CHO cells, but the formation was reduced in mitotic phase. All of RalBP1, POB1, Epsin, and Eps15 were phosphorylated in mitotic phase. POB1 and Epsin were phosphorylated by p34cdc2 kinase in vitro. Their phosphorylation sites (411S of POB1 and 357S of Epsin) were determined. Phosphorylated Epsin and EpsinS357D formed a complex with a-adaptin less efficiently than wild-type Epsin. Although the EH domain of POB1 bound directly to Epsin, phosphorylation of Epsin inhibited the binding. Furthermore, EpsinS357D but not EpsinS357A lost the effect of Epsin on the insulin-dependent endocytosis. These results suggest that phosphorylation of Epsin in mitotic phase inhibits receptor-mediated endocytosis by disassembly of its complex with POB1 and α-adaptin.