Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase

In vertebrate cells, the nuclear entry of Cdc2–cyclin B1 (MPF) during prophase is thought to be essential for the induction and coordination of M-phase events. Phosphorylation of cyclin B1 is central to its nuclear translocation, but the kinases that are responsible remain unknown. Here we have purified a protein kinase from Xenopus M-phase extracts that phosphorylates a crucial serine residue (S147) in the middle of the nuclear export signal sequence of cyclin B1. We have identified this kinase as Plx1 (ref. 16), a Xenopus homologue of Polo-like kinase (Plk)-1 (refs 17, 18). During cell-cycle progression in HeLa cells, a change in the kinase activity of endogenous Plk1 toward S147 and/or S133 correlates with a kinase activity in the cell extracts. An anti-Plk1 antibody depletes the M-phase extracts of the kinase activity toward S147 and/or S133. An anti-phospho-S147 antibody reacts specifically with cyclin B1 only during G2/M phase. A mutant cyclin B1 in which S133 and S147 are replaced by alanines remains in the cytoplasm, whereas wild-type cyclin B1 accumulates in the nucleus during prophase. Co-expression of constitutively active Plk1 stimulates nuclear entry of cyclin B1. Our results indicate that Plk1 may be involved in targeting MPF to the nucleus during prophase.

[1]  A. N. Meyer,et al.  Nuclear localization of cyclin B 1 mediates its biological activity and is regulated by phosphorylation ( cytoplasmic retention signal y nuclear translocation ) , 1997 .

[2]  J. Pines,et al.  MPF localization is controlled by nuclear export , 1998, The EMBO journal.

[3]  S. Moncada,et al.  Nitric oxide: physiology, pathophysiology, and pharmacology. , 1991, Pharmacological reviews.

[4]  J. Pines,et al.  Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal , 1999, Current Biology.

[5]  A. Podtelejnikov,et al.  Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps. , 1996, Rapid communications in mass spectrometry : RCM.

[6]  L. Ignarro.  Nitric Oxide: A Unique Endogenous Signaling Molecule in Vascular Biology (Nobel Lecture) , 1999 .

[7]  F. McKeon,et al.  Human wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated cdc2 kinase , 1993, Cell.

[8]  R. Gerzer,et al.  Generation and characterization of a stable soluble guanylate cyclase-overexpressing CHO cell line. , 1999, Nitric oxide : biology and chemistry.

[9]  J. Brennan,et al.  Control of Cyclin B 1 localization through regulated binding of the nuclear export factor CRM 1 , 1998 .

[10]  J. Maller,et al.  Mitotic Effects of a Constitutively Active Mutant of the Xenopus Polo-Like Kinase Plx1 , 1999, Molecular and Cellular Biology.

[11]  T. Hunter,et al.  The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B. , 1994, The EMBO journal.

[12]  G. Schultz,et al.  Sensitizing soluble guanylyl cyclase to become a highly CO‐sensitive enzyme. , 1996, The EMBO journal.

[13]  U. Zabel,et al.  Human soluble guanylate cyclase: functional expression and revised isoenzyme family. , 1998, The Biochemical journal.

[14]  D. Longo,et al.  Polo-like Kinase Is a Cell Cycle-regulated Kinase Activated during Mitosis (*) , 1995, The Journal of Biological Chemistry.

[15]  Y. Zhao,et al.  Identification of histidine 105 in the beta1 subunit of soluble guanylate cyclase as the heme proximal ligand. , 1998, Biochemistry.

[16]  A. Kumagai,et al.  Purification and Molecular Cloning of Plx1, a Cdc25-Regulatory Kinase from Xenopus Egg Extracts , 1996, Science.

[17]  H. J. Ahr,et al.  Imaging Plate: Validation and First Experience with Quantitative Studies in Whole-Body Autoradiography during Drug Development. , 1993 .

[18]  David O. Morgan,et al.  Principles of CDK regulation , 1995, Nature.

[19]  J. Moore,et al.  Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1. , 1998, Genes & development.

[20]  T. Okano,et al.  Relocation and distinct subcellular localization of p34cdc2‐cyclin B complex at meiosis reinitiation in starfish oocytes. , 1992, The EMBO journal.

[21]  F. Murad.  Discovery of Some of the Biological Effects of Nitric Oxide and Its Role in Cell Signaling (Nobel Lecture) , 1999 .

[22]  E. Nigg.  Assembly-disassembly of the nuclear lamina , 1992, Current Biology.

[23]  G. Born,et al.  The aggregation of blood platelets , 1963, The Journal of physiology.

[24]  E. Nishida,et al.  Nuclear export of cyclin B1 and its possible role in the DNA damage‐induced G2 checkpoint , 1998, The EMBO journal.

[25]  D. Morgan,et al.  Nuclear Localization of Cyclin B1 Controls Mitotic Entry After DNA Damage , 1998, The Journal of cell biology.

[26]  S. Kornbluth,et al.  All aboard the cyclin train: subcellular trafficking of cyclins and their CDK partners. , 1999, Trends in cell biology.

[27]  A. Iwamatsu.  S‐Carboxymethylation of proteins transferred onto polyvinylidene difluoride membranes followed by in situ protease digestion and amino acid microsequencing , 1992, Electrophoresis.

[28]  Yunde Zhao,et al.  Localization of the heme binding region in soluble guanylate cyclase. , 1997, Biochemistry.

[29]  E. Nigg,et al.  Cell Cycle Regulation of the Activity and Subcellular Localization of Plk1, a Human Protein Kinase Implicated in Mitotic Spindle Function Finally, Genetic Studies Performed with Fungi and Flies Have Identified Multiple Protein Kinases , 1995 .

[30]  J. Stasch,et al.  Different effects of ANP and nitroprusside on cyclic GMP extrusion of isolated aorta. , 1989, European journal of pharmacology.

[31]  T. Hunter,et al.  Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport , 1991, The Journal of cell biology.

[32]  T. Hunt,et al.  Cyclins and their partners: from a simple idea to complicated reality. , 1991, Seminars in cell biology.

[33]  E. Nigg.  Polo-like kinases: positive regulators of cell division from start to finish. , 1998, Current opinion in cell biology.

[34]  J. Maller,et al.  Activated Polo-Like Kinase Plx1 Is Required at Multiple Points during Mitosis in Xenopus laevis , 1998, Molecular and Cellular Biology.

[35]  D. Glover,et al.  Polo-like kinases: a team that plays throughout mitosis. , 1998, Genes & development.

[36]  J. Maller,et al.  Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions , 1991, Molecular and cellular biology.

[37]  A. Hobbs.  Soluble guanylate cyclase: the forgotten sibling. , 1997, Trends in pharmacological sciences.

[38]  F. Murad,et al.  Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung. , 1990, The Journal of biological chemistry.

[39]  A. N. Meyer,et al.  Requirement for phosphorylation of cyclin B1 for Xenopus oocyte maturation. , 1995, Molecular biology of the cell.

[40]  Minoru Yoshida,et al.  CRM1 is responsible for intracellular transport mediated by the nuclear export signal , 1997, Nature.

[41]  U. K. Laemmli,et al.  Cleavage of structural proteins during , 1970 .

[42]  L. Meijer,et al.  Intra-M Phase-promoting Factor Phosphorylation of Cyclin B at the Prophase/Metaphase Transition* , 1999, The Journal of Biological Chemistry.

[43]  J. Pines,et al.  Four-dimensional control of the cell cycle , 1999, Nature Cell Biology.

[44]  A. Friebe,et al.  Mechanism of YC-1-induced activation of soluble guanylyl cyclase. , 1998, Molecular pharmacology.

[45]  B. Mayer,et al.  Novel guanylyl cyclase inhibitor potently inhibits cyclic GMP accumulation in endothelial cells and relaxation of bovine pulmonary artery. , 1996, The Journal of pharmacology and experimental therapeutics.

[46]  J. Pines,et al.  Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the Golgi apparatus. , 1995, The EMBO journal.

[47]  P. Nurse.  Universal control mechanism regulating onset of M-phase , 1990, Nature.