Regulation of complex formation of POB1/epsin/adaptor protein complex 2 by mitotic phosphorylation.
RalBP1 and POB1, the downstream molecules of small GTP-binding protein Ral, are involved in receptor-mediated endocytosis together with Epsin and Eps15. The regulation of assembly of the complex of these proteins was examined. 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 phase. The phosphorylated forms of POB1 and Epsin were recognized by the antibody MPM2, which is known to detect mitotic phosphoproteins. POB1 and Epsin were phosphorylated by p34(cdc2) kinase in vitro. Their phosphorylation sites (Ser(411) of POB1 and Ser(357) of Epsin) were determined. Phosphorylated Epsin and Epsin(S357D) formed a complex with alpha-adaptin less efficiently than wild type Epsin. Although the EH domain of POB1 bound directly to Epsin, phosphorylation of Epsin inhibited the binding. Furthermore, Epsin(S357D) but not Epsin(S357A) 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 alpha-adaptin.
Kariya
K
K
Department of Biochemistry, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Koyama
S
S
Nakashima
S
S
Oshiro
T
T
Morinaka
K
K
Kikuchi
A
A
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D016384:Consensus Sequence; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D007328:Insulin / Q000378:metabolism; D046911:Macromolecular Substances; D008938:Mitosis; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D033921:Vesicular Transport Proteins; D020725:ral GTP-Binding Proteins / Q000502:physiology
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D007328:Insulin; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D010750:Phosphoproteins; D033921:Vesicular Transport Proteins; C114154:epsin; D020725:ral GTP-Binding Proteins