Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint

The spindle checkpoint delays anaphase until all chromosomes are properly attached to spindle microtubules. When the spindle checkpoint is activated at unattached kinetochores, the checkpoint proteins BubR1, Bub3 and Mad2 bind and inhibit Cdc20, an activator of the anaphase-promoting complex (APC). Here, we show that Xenopus laevis Cdc20 is phosphorylated at Ser 50, Thr 64, Thr 68 and Thr 79 during mitosis and that mitogen-activated protein kinase (MAPK) contributes to the phosphorylation at Thr 64 or Thr 68. Cdc20 mutants that are phosphorylation-deficient are able to activate the APC in X. laevis egg extracts. However, Cdc20 mutants in which any of the four phosphorylation sites were altered to Ala or Val failed to respond to the spindle checkpoint signal, owing to their reduced affinity for the spindle checkpoint proteins. This study demonstrates that the spindle checkpoint stops anaphase by inhibiting fully-phosphorylated Cdc20. Our results also have implications for the spindle checkpoint silencing mechanism.

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

[2]  Tomohiro Matsumoto,et al.  Fission yeast Slp1: an effector of the Mad2-dependent spindle checkpoint. , 1998, Science.

[3]  J. Ferrell,et al.  A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells , 1997, The Journal of cell biology.

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

[5]  A. Murray,et al.  Cell cycle extracts. , 1991, Methods in cell biology.

[6]  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.

[7]  A. Catling,et al.  Active MAP Kinase in Mitosis: Localization at Kinetochores and Association with the Motor Protein CENP-E , 1998, The Journal of cell biology.

[8]  J. Raff,et al.  The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time , 2002, The Journal of cell biology.

[9]  Andrew W. Murray,et al.  Spindle Checkpoint Protein Xmad1 Recruits Xmad2 to Unattached Kinetochores , 1998, The Journal of cell biology.

[10]  P. Hieter,et al.  The anaphase-promoting complex: new subunits and regulators. , 1999, Annual review of biochemistry.

[11]  T. Hunter,et al.  Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. , 1991, Methods in enzymology.

[12]  Emma Lees,et al.  Identification of an Overlapping Binding Domain on Cdc20 for Mad2 and Anaphase-Promoting Complex: Model for Spindle Checkpoint Regulation , 2001, Molecular and Cellular Biology.

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

[14]  Rey-Huei Chen,et al.  Spindle Checkpoint Protein Bub1 Is Required for Kinetochore Localization of Mad1, Mad2, Bub3, and Cenp-E, Independently of Its Kinase Activity , 2001, The Journal of cell biology.

[15]  Rey-Huei Chen,et al.  Spindle checkpoint requires Mad1-bound and Mad1-free Mad2. , 2002, Molecular biology of the cell.

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

[17]  G. V. Vande Woude,et al.  Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes * , 1998, Biology of the cell.

[18]  A. Musacchio,et al.  The spindle checkpoint: structural insights into dynamic signalling , 2002, Nature Reviews Molecular Cell Biology.

[19]  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.

[20]  Rey-Huei Chen,et al.  BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1 , 2002, The Journal of cell biology.

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

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

[23]  A. Hershko,et al.  Phosphorylation of Cdc20/fizzy negatively regulates the mammalian cyclosome/APC in the mitotic checkpoint. , 2000, Biochemical and biophysical research communications.

[24]  J. McIntosh,et al.  Activation of the MKK/ERK Pathway during Somatic Cell Mitosis: Direct Interactions of Active ERK with Kinetochores and Regulation of the Mitotic 3F3/2 Phosphoantigen , 1998, The Journal of cell biology.

[25]  E. Nishida,et al.  MAP Kinase Is Required for the Spindle Assembly Checkpoint but Is Dispensable for the Normal M Phase Entry and Exit in Xenopus Egg Cell Cycle Extracts , 1997, The Journal of cell biology.