title: The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B.
abstract: The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system. A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found. Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase.
authors.affiliation: Surgical Research Laboratory, Medical University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
authors.forename: M H H
authors.initials: MHH
authors.lastname: Schmidt
authors.forename: Rainer
authors.initials: R
authors.lastname: Broll
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Bruch
authors.forename: Oliver
authors.initials: O
authors.lastname: Bögler
authors.forename: Michael
authors.initials: M
authors.lastname: Duchrow
mesh_terms: D016203:CDC2 Protein Kinase / Q000032:analysis; D042846:CDC2-CDC28 Kinases; D002466:Cell Nucleolus / Q000235:genetics / Q000502:physiology*; D019925:Cyclin A / Q000032:analysis; D019926:Cyclin B / Q000032:analysis*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000032:analysis; D004265:DNA Helicases / Q000032:analysis; D005786:Gene Expression Regulation / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000032:analysis*; D019394:Ki-67 Antigen / Q000235:genetics / Q000502:physiology*; D008938:Mitosis / Q000235:genetics / Q000502:physiology*; D011233:Precipitin Tests / Q000379:methods; D017346:Protein Serine-Threonine Kinases / Q000032:analysis; D018160:Receptors, Cytoplasmic and Nuclear; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012269:Ribosomal Proteins / Q000032:analysis; D015398:Signal Transduction / Q000235:genetics; D014178:Translocation, Genetic / Q000235:genetics; D020798:Two-Hybrid System Techniques
chemical_list: D019925:Cyclin A; D019926:Cyclin B; C466996:IPO7 protein, human; D028884:Karyopherins; D019394:Ki-67 Antigen; D018160:Receptors, Cytoplasmic and Nuclear; D012269:Ribosomal Proteins; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D004265:DNA Helicases