Dual inhibition of sister chromatid separation at metaphase.
Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.
Stemmann
O
O
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Zou
H
H
Gerber
S A
SA
Gygi
S P
SP
Kirschner
M W
MW
D000705:Anaphase / Q000502:physiology; D000818:Animals; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002842:Chromatids / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism; D056744:Cyclin B1; D010450:Endopeptidases; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008677:Metaphase / Q000502:physiology*; D009687:Nuclear Proteins; D009865:Oocytes / Q000502:physiology; D010449:Peptide Mapping; D010750:Phosphoproteins; D010766:Phosphorylation; D029701:Saccharomyces cerevisiae Proteins; D064247:Separase; D014982:Xenopus laevis
C534699:CCNB1 protein, human; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D019926:Cyclin B; D056744:Cyclin B1; C490529:MCD1 protein, S cerevisiae; D009687:Nuclear Proteins; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; D016203:CDC2 Protein Kinase; D010450:Endopeptidases; C577986:ESP1 protein, S cerevisiae; C577987:ESPL1 protein, human; D064247:Separase