Study On The Interaction Between Nucleolar Protein Dnt1 And Tumor Suppressor Chfr's Homologue Dma1 In Fission Yeast

In the fission yeast Schizosaccharomyces pombe,the tumor suppressor Chfr's homologue Dma1 which is a spindle checkpoint protein and cytokinesis inhibitor, prevents cytokinesis by inhibiting the SIN.In the anaphase,Dma1-GFP signal was observed at both the division site and SPB.As a new inhibitor of SIN in early mitosis, nucleolar protein Dnt1 is localized in the nucleotus throughout the cell cycle,at anaphase it localizes to the SPB.Dnt1 shows sequence homology to Netl/Cfil in budding yeast.Based on the facts that both Dma1 and Dnt1 localize at SPB and involve in regulation of cytokinesis,we set out to examine the interaction between Dma1 and Dnt1 in fission yeast.We performed in vitro affinity assay using bacterially expressed recombinant protein MBP-Dma1 to incubate with cell extract from dnt1△cells carrying genomically integrated Dnt1-12myc.The result showed that Dnt1 can efficiently bind to Dma1,suggesting physical interaction between Dma1 and Dnt1 in vitro.We expressed the MBP-tagged Dma1 full-length and dma1^△FHA in E.coli and performed pull-down assay with Dnt1-13myc expressed in fission yeast.Western bolt suggested FHA domain was required for the interaction between Dma1 and Dnt1,And Dma1's FHA domain has been considered as a phosphopeptide binding domain.When protein was dephosphorylated with calf-intestinal alkaline phosphatase(CIAP) orλ-Protein phosphatase(λ-PPase),Our results strongly suggest that the phosphorylated form of Dnt1 binds Dma1.Because the phosphorylated form of Dnt1 binds Dma1 and the binding between these two proteins become the strongest at mitotic metaphase,we were suspicious that two major kinases,CDK1 and Plo1,might be responsible for phosphorylating Dnt1 when they have highest activity at metaphase.However,when we used the CDK1 sites mutants dnt1(7A) and dnt1(11A),metaphase-arrested plo1-24C and plo1-25 cells, and cut12.1 mutants which have compromised Plo1 activity respectively to test whether CDK1 and Plo1 have function in the interaction between Dma1 and Dnt1,we found that the interaction between these two proteins stayed intact.This suggests that there are must be other kinases which is involved in phosphorylating Dnt1 and enhance its binding capacity to Dma1.In this study,we have attacked the question that how Dma1 interacts with Dnt1 and how the interaction is regulated.We found that the phosphorylated form of Dnt1 binds strongly to Dma1 at metaphase through Dma1's FHA domain.Since Dma1 is the mammalian protein Chfr's homolog,our study on Dma1's regulation will certainly further our understanding of Chfr's function.