Annual Report

31. Phosphorylation Sites of NPAT by CyclinE/CDK2 Complex

Yasuharu Ninomiya, Akiyo Nisiyama, Masashi Sagara, Yoichi Taya* and Takashi Imai (* Natl. Cancer Center Research Institute)

Keywords: NPAT, ATM, cyclin/CDK, phosphorylation

The characteristic most widely studied in human genetic disorder ataxia telangiectasia (AT) is hypersensitivity to ionizing radiation. AT responsible gene, ATM could enhance survival of AT cells after irradiation exposure, decrease radiation-induced chromosome aberrations, reduce radioresistant DNA synthesis, and partially correct defective cell cycle checkpoints. We identified NPAT gene from 0.5 kb upstream of ATM. Because these two genes were transcribed in opposite direction and because our reporter assay revealed that the 0.5 kb nucleotide sequence flanked by the two genes had bi-directional promoter activity, we examined a possibility that the NPAT gene is also related to cellular responses to DNA damage or cell cycle control. And by retroviral study, the NPAT gene seems to have important role at cell cycle control. To investigate relationship between NPAT function and cell cycle control after irradiation, we first analyzed mRNA- and/or protein-expression pattern of the NPAT in each stage of the cell cycle in normal human fibroblast cells after irradiation. We found that both mRNA and protein of NPAT were not induced through cell cycle after 2 Gy irradiation.

Because NPAT was predicted to have multiple phosphorylation sites by protein kinases, which are function for cell cycle control, in vitro phosphorylation sites of NPAT by cyclin/CDKs using the GST fusion protein were examined. We identified the N-terminal 126 amino acid-region of NPAT was phospholylated by mainly both cyclin A/CDK2 and cyclin B/cdc2 while Thr1359 at C-terminal region of NPAT was phosphorylted by three types of cyclin/CDK complexes, cyclin A/CDK2, cyclin B/cdc2 and cyclin E/CDK2. Both Ser775 and Ser779 at the central region of NPAT were phosphorylated by mainly cyclin E/CDK2. In vitro phosphorylation by cyclin D1/CDK4 was not observed at any site of NPAT. These data suggest that the each phosphorylation site of NPAT was specifically recognized by cell cycle specific cyclin/CDKs and the phosphorylation of NPAT may be regulated through cell cycle specific manners.

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