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

在本研究中，我们试图阐明POB1和Epsin调节Ral下游受体介导的内吞作用的新功能。(1)POB1结合蛋白的鉴定。POB1是一种RalBP1结合蛋白。POB1和RalBP1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POB1的额外功能，我们使用酵母双杂交方法筛选POB1结合蛋白，发现POB1与小鼠ASAP1相互作用，后者是人类PAG2的同源物。PAG2是一种具有ADP核糖化因子GTP酶激活活性的巴西林相关蛋白。在完整细胞中，POB1与PAG2形成复合体。含有POB1富含脯氨酸基序的羧基末端区域直接结合到包括PAG2的SH3结构域的羧基末端区域。用丙氨酸(FOB1(PA))取代Pro{Super423}和Pro{Super426}会削弱POB1与PAG2的结合。PAG2的表达抑制了纤维连接蛋白依赖的迁移和帕西林对CHO-IR细胞局部接触的募集。与POB1共表达但不与POB1(PA)共表达可抑制PAG2对细胞迁移和帕西林定位的抑制作用。这些结果表明，POB1通过其富含脯氨酸的基序与PAG2相互作用，从而调节细胞迁移。(2)胰岛素刺激使Epsin磷酸化。Epsin被鉴定为POB1结合蛋白。当CHO-IR细胞被胰岛素处理时，Epsin被磷酸化。TPA和钒酸盐处理也使Epsin磷酸化。PI3-K抑制剂*ortmanin可抑制胰岛素依赖的Epsin磷酸化，但不抑制TPA和钒依赖的磷酸化。星形孢菌素抑制依赖于TPA的Epsin的磷酸化，但不抑制依赖于胰岛素的磷酸化。这些结果表明，EPSN是通过多个信号通路，至少是PI3-K和PKC通路而形成的。

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.