Regulation of complex formation of POB1/epsin/adaptor protein complex 2 bymitotic phosphorylation

RalBP1 and POB1, the downstream molecules of small GTP-binding protein Pal, are involved in receptor-mediated endocytosis together with Epsin and Eps1 5. The regulation of assembly of the complex of these proteins wits examine d. RalBP1, POB1, Epsin, and Eps15 formed a complex with alpha-adaptin of AP -2 in Chinese hamster ovary cells, but the formation was reduced in mitotic phase. RalBP1, POB1, Epsin, and Eps15 were all phosphorylated in mitotic p hase. The phosphorylated forms of POB1 and Epsin were recognized by the ant ibody MPM2, which is known to detect mitotic phosphoproteins. POB1 and Epsi n were phosphorylated by p34(cdc2) kinase in vitro. Their phosphorylation s ites (Ser(411) of POB1 and Ser(357) Of Epsin) were determined. Phosphorylat ed Epsin and Epsin(S357D) formed a complex with alpha-adaptin less efficien tly than wild type Epsin. Although the EH domain of POB1 bound directly to Epsin, phosphorylation of Epsin inhibited the binding. Furthermore, Epsin(S 357D) but not Epsin(S357A) lost the effect of Epsin on the insulin-dependen t endocytosis, These results suggest that phosphorylation of Epsin in mitot ic phase inhibits receptor-mediated endocytosis by disassembly of its compl ex with POB1 and alpha-adaptin.