Arrest of cell cycle by amida which is phosphorylated by Cdc2 kinase.
Amida was first isolated from a rat hippocampal cDNA library as an Arc-associated protein. Although previous studies have shown that Amida mRNA is predominantly expressed and developmentally regulated in rat testis and overexpression induces apoptosis, the function of Amida remains unclear. In this study, we found that overexpression of Amida inhibited cell growth. Flow cytometry analysis showed that Amida caused cell cycle inhibition in the S-phase and blocked cell cycle from entry into mitosis. Attempting to elucidate Amida effect on the cell cycle, we found that Amida was interacted with Cdc2 in mitosis and Amida's overexpression resulted in a decrease in Cdc2 kinase activity. In addition, Amida showed DNA-binding ability with DNA-affinity column chromatography. A region (aa, 76-189) between the two nuclear localization signals was found to be responsible for cell growth inhibition and DNA-binding activity, implying that DNA-binding activity may be necessary for Amida to repress cell cycle. Moreover, Amida was phosphorylated by Cdc2 kinase in vitro and Ser-180 of Amida was identified as the phosphorylation site. Furthermore, AmidaS180G (eliminate phosphorylation of Ser-180) showed stronger DNA-binding activity. Taken together, the data suggest that Amida may play an important role in cell cycle and may be partly regulated by Cdc2 kinase.
Gan
Yehua
Y
Department of Pharmacology, Osaka University Medical School, Yamada-oka, Suita, Osaka, Japan.
Taira
Eiichi
E
Irie
Yasuyuki
Y
Fujimoto
Takahiro
T
Miki
Naomasa
N
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D019556:COS Cells; D002453:Cell Cycle / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D008938:Mitosis; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D014162:Transfection
D018076:DNA, Complementary; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C404187:Tfpt protein, rat; D012694:Serine; D016203:CDC2 Protein Kinase