세포 성장에 있어서 전사인자 NF-Y의 CDK2 의존성 인산화의 역할
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NF-Y, also known as CBF (CCAAT-bindingfactor), was first identified as a protein that binds to the Y box of MHC class II Ea promoter. Unique among transcription factors, NF-Y is comprised of three different subunits, YA, YB, and YC, that are collectively required to bind the CCAAT motif. With regard to the cell cycle, it has been shown that NF-Y binds to, and activates the promoters of cell cycle regulatory genes that are essential for DNA replication and entry into mitosis, such as cdk1, cdc25C, cyclin A and B, RNR R2, PCNA, E2F-1, and DNA pol a (19, 23). It was recently revealed that cdk2 phosphorylates two serine residues at 320 and 326 in YA subunit of NF-Y. This phosphorylation appeared to be critical for DNA binding of NF-Y. In this study, I constructed mutant forms of YA, in which two serine residues were replaced with glutamic acid or 0000, and then examined the functions of the YA mutants. The gel shift assay showed that both NF-Y with YA-DD and YA-EE have impaired DNA binding activity. Furthermore, expression of YA-DD or YA-EE resulted in growth inhibition. These results suggest that two serine residues, which is phosphorylated by cdk2, are critical for DNA binding of NF-Y complex and the cell cycle. The gel shift assay showed that DNA/NF-Y complex was significantly decreased in the cells expressing the YA mutant as compared with the cells expressing YA wild type. A dominant negative mutant of the NF-YA subunit, NF-YA 320326A/A, decreases the DNA binding activity at the cdc2 promoter. NF-YA 320326D/D also decrease the DNA binding activity. NF-YA D/D or NF-YA E/E mutant plasmid was transient transfection in HCT116 cells that had neomycin resistance marker. Resistant colonies were grown in the presence of neomycin(0.7mg/ml). NF-YA D/D or NF-YA E/E protein detected by anti-His antibody and NF-YA D/D or NF-YA E/E was not expressed in cells. Selected colonies number and size of the cell expressing YA mutant were same in compare with selected colonies of the cell expressing His-vecotor. Thus, YA phosphorylation mutant form were critical and toxic effected cell growth. This result implied that cdk2-dependent phosphorylation of the NF-Y transcription factor decreased DNA-NF-Y binding activity and were toxic effected cell growth.