The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time

In Drosophila cells cyclin B is normally degraded in two phases: (a) destruction of the spindle-associated cyclin B initiates at centrosomes and spreads to the spindle equator; and (b) any remaining cytoplasmic cyclin B is degraded slightly later in mitosis. We show that the APC/C regulators Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1 bind to microtubules in vitro and associate with spindles in vivo. Fzy/Cdc20 is concentrated at kinetochores and centrosomes early in mitosis, whereas Fzr/Cdh1 is concentrated at centrosomes throughout the cell cycle. In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated cyclin B is degraded at the end of mitosis. A destruction box–mutated form of cyclin B (cyclin B triple-point mutant [CBTPM]–GFP) that cannot be targeted for destruction by Fzy/Cdc20, is no longer degraded on spindles in syncytial embryos. However, CBTPM–GFP can be targeted for destruction by Fzr/Cdh1. In cellularized embryos, which normally express Fzr/Cdh1, CBTPM–GFP is degraded throughout the cell but with slowed kinetics. These findings suggest that Fzy/Cdc20 is responsible for catalyzing the first phase of cyclin B destruction that occurs on the mitotic spindle, whereas Fzr/Cdh1 is responsible for catalyzing the second phase of cyclin B destruction that occurs throughout the cell. These observations have important implications for the mechanisms of the spindle checkpoint.

[1]  A. Murray,et al.  Cyclin is degraded by the ubiquitin pathway , 1991, Nature.

[2]  F. M. Yeong,et al.  Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20. , 2000, Molecular cell.

[3]  Daniel A Starr,et al.  Human Zw10 and ROD are mitotic checkpoint proteins that bind to kinetochores , 2000, Nature Cell Biology.

[4]  J. R. Daum,et al.  The spindle checkpoint of Saccharomyces cerevisiae responds to separable microtubule-dependent events , 2000, Current Biology.

[5]  J. R. Daum,et al.  Mammalian p55CDC Mediates Association of the Spindle Checkpoint Protein Mad2 with the Cyclosome/Anaphase-promoting Complex, and is Involved in Regulating Anaphase Onset and Late Mitotic Events , 1998, The Journal of cell biology.

[6]  R. Karess,et al.  Rough Deal and Zw10 are required for the metaphase checkpoint in Drosophila , 2000, Nature Cell Biology.

[7]  D. Glover,et al.  The A‐ and B‐type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2‐M transition. , 1990, The EMBO journal.

[8]  A. Hershko,et al.  Components of a system that ligates cyclin to ubiquitin and their regulation by the protein kinase cdc2. , 1994, The Journal of biological chemistry.

[9]  M. Kirschner,et al.  The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. , 2000, Genes & development.

[10]  S. Elledge,et al.  Cdk inhibitors in development and cancer. , 1996, Current opinion in genetics & development.

[11]  B. Alberts,et al.  Drosophila gamma-tubulin is part of a complex containing two previously identified centrosomal MAPs , 1993, The Journal of cell biology.

[12]  P. O’Farrell,et al.  Exit from mitosis in Drosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation. , 1998, Genes & development.

[13]  A Khodjakov,et al.  The centrosome in vertebrates: more than a microtubule-organizing center. , 2001, Trends in cell biology.

[14]  A. Brand,et al.  The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila , 1997, Current Biology.

[15]  S. Sazer,et al.  The Schizosaccharomyces pombe spindle checkpoint protein mad2p blocks anaphase and genetically interacts with the anaphase-promoting complex. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[16]  S. Artavanis-Tsakonas,et al.  The Drosophila cell cycle gene fizzy is required for normal degradation of cyclins A and B during mitosis and has homology to the CDC20 gene of Saccharomyces cerevisiae , 1995, The Journal of cell biology.

[17]  A. Spradling,et al.  The fusome organizes the microtubule network during oocyte differentiation in Drosophila. , 2000, Development.

[18]  M. Kirschner,et al.  The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. , 1998, Genes & development.

[19]  J. Grindlay,et al.  The Bub2p spindle checkpoint links nuclear migration with mitotic exit. , 2000, Molecular cell.

[20]  M. Yanagida,et al.  Control of metaphase-anaphase progression by proteolysis: cyclosome function regulated by the protein kinase A pathway, ubiquitination and localization. , 1999, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[21]  S. Prinz,et al.  CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. , 1997, Science.

[22]  C. Lehner,et al.  Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts , 1998, The EMBO journal.

[23]  W. Wells.  The spindle-assembly checkpoint: aiming for a perfect mitosis, every time. , 1996, Trends in cell biology.

[24]  J. Pines,et al.  Temporal and spatial control of cyclin B1 destruction in metaphase , 1999, Nature Cell Biology.

[25]  J. Pines,et al.  Cyclin a Is Destroyed in Prometaphase and Can Delay Chromosome Alignment and Anaphase , 2001, The Journal of cell biology.

[26]  P. Rørth.  Gal4 in the Drosophila female germline , 1998, Mechanisms of Development.

[27]  A. Murray,et al.  Budding yeast Cdc20: a target of the spindle checkpoint. , 1998, Science.

[28]  D. Burke,et al.  The spindle checkpoint: two transitions, two pathways. , 2000, Trends in cell biology.

[29]  A. Amon,et al.  A Mechanism for Coupling Exit from Mitosis to Partitioning of the Nucleus , 2000, Cell.

[30]  Tim Hunt,et al.  Anaphase-Promoting Complex/Cyclosome–Dependent Proteolysis of Human Cyclin a Starts at the Beginning of Mitosis and Is Not Subject to the Spindle Assembly Checkpoint , 2001, The Journal of cell biology.

[31]  J. Raff,et al.  The disappearance of cyclin B at the end of mitosis is regulated spatially in Drosophila cells , 1999, The EMBO journal.

[32]  E. Salmon,et al.  Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation , 2001, The Journal of cell biology.

[33]  K. Wood,et al.  CENP-E as an Essential Component of the Mitotic Checkpoint In Vitro , 2000, Cell.

[34]  J. Raff,et al.  The dynamic localisation of the Drosophila APC/C: evidence for the existence of multiple complexes that perform distinct functions and are differentially localised. , 2002, Journal of cell science.

[35]  C. Lehner,et al.  Drosophila fizzy-related Down-Regulates Mitotic Cyclins and Is Required for Cell Proliferation Arrest and Entry into Endocycles , 1997, Cell.

[36]  Jan-Michael Peters,et al.  Activation of the human anaphase-promoting complex by proteins of the CDC20/Fizzy family , 1998, Current Biology.

[37]  D. St Johnston,et al.  Miranda mediates asymmetric protein and RNA localization in the developing nervous system. , 1998, Genes & development.

[38]  Hongtao Yu,et al.  Mad2-Independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1. , 2001, Developmental cell.

[39]  P. Nurse,et al.  In vivo localisation of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis. , 2001, Journal of cell science.

[40]  Kim Nasmyth,et al.  Genes involved in sister chromatid separation are needed for b-type cyclin proteolysis in budding yeast , 1995, Cell.

[41]  V. Foe,et al.  Mitotic domains reveal early commitment of cells in Drosophila embryos. , 1989, Development.

[42]  M. Mann,et al.  Mitotic regulation of the APC activator proteins CDC20 and CDH1. , 2000, Molecular biology of the cell.

[43]  P. O’Farrell,et al.  Distinct molecular mechanism regulate cell cycle timing at successive stages of Drosophila embryogenesis. , 1994, Genes & development.

[44]  F. Gergely,et al.  D‐TACC: a novel centrosomal protein required for normal spindle function in the early Drosophila embryo , 2000, The EMBO journal.

[45]  A. Murray,et al.  A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts , 1994, Cell.

[46]  M. Kirschner,et al.  Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex. , 2001, Genes & development.

[47]  J. Raff,et al.  Centrosomes have a role in regulating the destruction of cyclin B in early Drosophila embryos , 2000, Current Biology.

[48]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[49]  G. Chan,et al.  Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2 , 2001, The Journal of cell biology.

[50]  J. Lis,et al.  Structure and expression of ubiquitin genes of Drosophila melanogaster , 1988, Molecular and cellular biology.

[51]  J. Labbé,et al.  Mps1 Is a Kinetochore-Associated Kinase Essential for the Vertebrate Mitotic Checkpoint , 2001, Cell.

[52]  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.

[53]  R. Karess,et al.  Kinetochore dynein: its dynamics and role in the transport of the Rough deal checkpoint protein , 2001, Nature Cell Biology.

[54]  F. Piano,et al.  The ZW10 and Rough Deal checkpoint proteins function together in a large, evolutionarily conserved complex targeted to the kinetochore. , 2001, Journal of cell science.

[55]  S. Kornbluth,et al.  Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control. , 1996, Current opinion in cell biology.

[56]  M. Bownes,et al.  Drosophila: A practical approach: edited by D. B. Roberts IRL Press, 1986. £26.00/$47.00 (xix + 295 pages) ISBN 0 94746 66 7 , 1987 .

[57]  M. Kirschner,et al.  A 20s complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B , 1995, Cell.

[58]  Stuart Tugendreich,et al.  CDC27Hs colocalizes with CDC16Hs to the centrosome and mitotic spindle and is essential for the metaphase to anaphase transition , 1995, Cell.

[59]  T. Hazelrigg,et al.  GFP expression in Drosophila tissues: time requirements for formation of a fluorescent product. , 1998, Developmental biology.

[60]  C. Rieder,et al.  Mitosis in vertebrate somatic cells with two spindles: implications for the metaphase/anaphase transition checkpoint and cleavage. , 1997, Proceedings of the National Academy of Sciences of the United States of America.