Analysis of protein kinase U (PKU) that may be recruited to the DNA double-strand breaks

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1)X-ray irradiation induced foci (IRIF) was observed in nuclei of HeLa or MCF7 cells when cells were immunostained by polyclonal anti-PKU-β (TLK1) antibodies. However, IRIF was not detected by monoclonal antibody. There is no significant difference in reacted proteins by two antibodies. Two of three proteins that recognized by antibodies in MCF7 cell lysate were identified as splicing variants.2)When exogenous HA-tagged PKU was introduced into cells, no IRIF was observed by anti-Ha antibody. However, we could not determine whether failure was due to overexpression.3)The siRNA significantly reduced the expression of PKU-beta at 3 days after transfedction. These cells, howver, still formed IRIF.4)MCF7 cells with low expression of PKU-beta mediated by siRNA was sensitive to either X-ray irradiation, UV-irradiation or genotoxic reagents such as mytomisin C.5)When analyzed by FACS, PKU-knockdown with MCF 7 cells lead to aneuploidy or polyploidy. The number of chromosome of MCF7 which usually contains 62 increased to 62-120 at three days after the siRNA transfection.6)RNAi-mediated disruption of PKU-beta in cells interferes with normal spindle assembly and positioning. SiRNA treatment blocked the separation and positioning of duplicated centrosomes, thereby preventing the migration of the microtubule asters to opposite sides of chromosomes. These results suggests that PKU-beta involves G2-M transition and plays an important role on chromosomal segregation.