Functional Interaction Between Mitosin And Activating Transcription Factor 4(ATF4) And Regulation On The CAMP-Responsive Elements Of Cellular And Viral Genes

Faithful segregation of chromosome to daughter cells during mitosis is critical for the stabilization of genetic materials and normal development of organism. Mitosin is a 35OkDa nuclearprotein that transiently localized to the outerplate of kinetochore and spindle pole in mitotic phase and its expression is cell-cycle dependent. Preliminary investigations have found that mitosin can regulate cell cycle and may acts as a mitotic checkpoint. In order to further investigate the function of mitosin, the yeast two-hybrid screening utilizing two kinetochore targeting domains of mitosin ATK(mitosin 2094?487+2792---2887) and TG(mitosin 2488? 113) as baits was performed against a placental cDNA library, two positive clones encoding the amino acid residue 56?5 I and 185?51 of ATF4/CREB2 was identified, then the full-length ATF4 was gained by PCR from the placental library. Deletion analysis indicated that the C-terminal domain of ATF4 containing basic and leucine zipper and the core region(2792?887) of mitosin was necessary for the interaction of the two proteins, further more, the cystein at 2864 was also indispensable for mitosin associated with ATF4, mutation of cystein to serine also abolished the interacton of the two proteins, suggesting heterodimer may happened between the Ieucine zipper. Full length and thc N-terminal truncated form of ATF4 can interact with mitosin 2488? 113 was also confirmed by GST-pull down assay, which suggested that same mechanism may underlied in the interaction between the two proteins and the interaction of mitosin with the kinetochore. Although the ATF/CREB protein family have similar bZip domain, but the homologous region of ATF proteins to the mitosin binding domain of ATF4 can not interact with mitosin in the yeast two-hybrid system which validated the specificity of the interaction between mitosin and ATF4. It was also shown that ATF4 expressed in E. coIl can specifically binds to the artificial probe containing consensus cAMP responsive element(CRE) TGACGTCA in electrophoretic mobility gel shift assay(EMSA), and mitosin 2488?644 can interfere with the interaction between ATF4 and CRE, while mitosin 2645-1 113 can form a ternary structure with 74 ATF4 and CRE, this indicate that mitosin may regulate ATF4 by a rather complicated machanism. By using a luciferase gene driven by the E-selectin promoter, ATF4 and mitosin can coordinately down-regulate the reporter gene, while mitosin can transactivate the luciferase gene driven by the HTLV- 1 LTR, this result presented a novel pathway from nuclear matrix protein to transcription factor, and indicate that mitosin may regulate cellular genes and may also play an important role in facilitating the changing of cell-cycle and the transformation of cells infected by virus containing CRE.