Biochemical characterization of pKi67 with the identification of a mitotic-specific form associated with hyperphosphorylation and altered DNA binding.
Although widely used as an operational marker of proliferation, the cell cycle-regulated Ki67 protein is of unknown function. pKi67 is found predominantly in the nucleolus in cycling interphase cells and moves to become perichromosomal during mitosis. We have performed a detailed immunochemical analysis of pKi67 in HeLa cells and report the existence of a novel hyperphosphorylated form in mitosis. Two isoforms can be identified on immunoblots as a consequence of the previously described alternative splicing. In extracts from mitotic cells both these isoforms have considerably reduced mobility. Treatment with phosphatase converts the mitotic form to the interphase form. Immunoprecipitated pKi67 can be phosphorylated in vitro both by cdc2/cyclin B and by protein kinase C, and treatment by PKC leads to the full mobility shift. Treatment of nocodazole-arrested mitotic HeLa cells with staurosporine causes a dephosphorylation of pKi67 to the interphase state and a concomitant change in the localization of pKi67 with movement away from the perichromosomal layer to cytoplasmic dots that colocalize with nucleolin. These data indicate that pKi67 localization is regulated by the action of cell cycle-specific kinase(s) and phosphatase(s). The data presented here provide a starting point for the analysis of pKi67 function and regulation.
MacCallum
D E
DE
Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, United Kingdom.
Hall
P A
PA
D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle / Q000276:immunology / Q000502:physiology; D019926:Cyclin B / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019394:Ki-67 Antigen / Q000737:chemistry* / Q000378:metabolism*; D008938:Mitosis / Q000276:immunology / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism
D019926:Cyclin B; D019394:Ki-67 Antigen; D004247:DNA; D011493:Protein Kinase C; D016203:CDC2 Protein Kinase