Regulation of vecicler trans port and cell adhesion by small G protein Ral

在这项研究中，我们试图阐明POB 1和Epsin的新功能，它们调节Ral下游受体介导的内吞作用。(1)鉴定POB 1的结合蛋白。POB 1以前被鉴定为RalBP 1结合蛋白。POB 1和RalBP 1在小G蛋白Ral下游发挥作用，调节受体介导的内吞作用。为了寻找POB 1的其他功能，我们使用酵母双杂交方法筛选POB 1结合蛋白，发现POB 1与小鼠ASAP 1相互作用，ASAP 1是人类PAG 2同源物。PAG 2是具有ADP-核糖基化因子GTP酶激活蛋白活性的paxillin相关蛋白。在完整细胞中，POB 1与PAG 2形成复合物。含有POB 1的富含脯氨酸基序的羧基末端区域直接结合到包括PAG 2的SH 3结构域的羧基末端区域。用Ala（FOB 1（PA））取代Pro{super 423}和Pro{super 426}削弱了POB 1与PAG 2的结合。PAG 2的表达抑制了CHO-IR细胞的纤连蛋白依赖性迁移和桩蛋白募集到局灶性接触。与POB 1共表达而不与POB 1（PA）共表达抑制PAG 2对细胞迁移和桩蛋白定位的抑制作用。这些结果表明，POB 1通过其富含脯氨酸的基序与PAG 2相互作用，从而调节细胞迁移。(2)通过胰岛素刺激的Epsin的磷酸化。Epsin被鉴定为POB 1结合蛋白。当细胞用胰岛素处理时，CHO-IR细胞中的Epsin被磷酸化。TPA和钒酸盐处理也使Epsin磷酸化。胰岛素依赖的Epsin磷酸化被PI 3-K抑制剂 *ortmanin抑制，但TPA和钒酸盐依赖的磷酸化没有。TPA依赖的Epsin磷酸化被staurosporin抑制，但胰岛素依赖的磷酸化没有。这些结果表明Epsn是通过多种信号途径磷酸化的，至少是通过PI 3-K和PKC途径。

In this study, we tried to clarify the new functions of POB1 and Epsin, which regulate receptor-mediated endocytosis downstream of Ral.(1) Identification of a binding protein to POB1. POB1 was previously identified as a RalBP1-binding protein. POB1 and RalBP1 function downstream of small G protein Ral and regulate receptor-mediated endocytosis. To look for additional functions of POB1, we screened for POB1-binding proteins using a yeast two-hybrid method and found that POB1 interacts with mouse ASAP1, which is a human PAG2 homolog. PAG2 is a paxillin-associated protein with ADP-ribosylation factor GTPase-activating protein activity. POB1 formed a complex with PAG2 in intact cells. The carboxyl terminal region containing the proline-rich motifs of POB1 directly bound to the carboxyl terminal region including the SH3 domain of PAG2. Substitutions of Pro{super423} and Pro{super426} with Ala (FOB1(PA)) impaired the binding of POB1 to PAG2. Expression of PAG2 inhibited fibronectin-dependent migration and paxillin recruitment to focal contacts of CHO-IR cells. Co-expression with POB1 but not with POB1(PA) suppressed the inhibitory action of PAG2 on cell migration and paxillin localization. These results suggest that POB1 interacts with PAG2 through its proline-rich motif, thereby regulating cell migration.(2) Phosphorylation of Epsin by insulin stimulation. Epsin was identified as a POB1-binding protein. Epsin was phosphorylated in CHO-IR cells when the cells were treated with insulin. TPA and vanadate treatment also phosphorylated Epsin. Insulin-dependent phosphorylation of Epsin was inhibited by *ortmanin, a PI3-K inhibitor, but TPA- and vanadate-dependent phosphorylation were not. TPA-dependent phosphorylation of Epsin was suppressed by staurosporin, but insulin-dependent phospnorylation was not. These results suggest that Epsn is phosphoprylatea by several signaling patyhways, at least PI3-K and PKC pathways.