Abstract
Proper cell-cycle progression requires tight temporal control of the Anaphase Promoting Complex/Cyclosome (APC/C), a large ubiquitin ligase that is activated by one of two co-activators, Cdh1 or Cdc20. APC/C and Cdc20 are already present during interphase but APC/C–Cdc20 regulation during this window of the cell cycle, if any, is unknown. Here we show that cyclin A2–Cdk2 binds and phosphorylates Cdc20 in interphase and this inhibits APC/C–Cdc20 activity. Preventing Cdc20 phosphorylation results in pre-mature activation of the APC/C–Cdc20 and several substrates, including cyclin B1 and A2, are destabilized which lengthens G2 and slows mitotic entry. Expressing non-degradable cyclin A2 but not cyclin B1 restores mitotic entry in these cells. We have thus uncovered a novel positive feedback loop centred on cyclin A2–Cdk2 inhibition of interphase APC/C–Cdc20 to allow further cyclin A2 accumulation and mitotic entry.
Highlights
Proper cell-cycle progression requires tight temporal control of the Anaphase Promoting Complex/Cyclosome (APC/C), a large ubiquitin ligase that is activated by one of two co-activators, Cdh[1] or Cdc[20]
We first characterized the specificity of the antibodies by purifying yellow-fluorescent protein (YFP)-tagged Cdc[20] wild type (WT), Cdc[20] T59A and Cdc[20] T70A from mitotic cells using YFP affinity resin
We show for the first time that APC/C–Cdc[20] has activity in interphase and that phosphorylation of Cdc[20] by cyclin A2–cyclin-dependent kinase (Cdk)[2] is required to repress this activity to ensure efficient mitotic entry (Fig. 7)
Summary
Proper cell-cycle progression requires tight temporal control of the Anaphase Promoting Complex/Cyclosome (APC/C), a large ubiquitin ligase that is activated by one of two co-activators, Cdh[1] or Cdc[20]. Cdc[20] is inhibited by the spindle assembly checkpoint (SAC) during mitosis[20] and in addition studies have shown that phosphorylation of Cdc[20] by Cdk[21,22,23] on multiple sites can decrease APC/C–Cdc[20] activity by reducing APC/C–Cdc[20] interaction. These previous studies have focused on Cdc[20] phosphorylation in mitotic APC/C–Cdc[20] regulation but radioactive labelling of Cdc[20] suggested that it is phosphorylated already before mitosis[24]. We have uncovered a novel mechanism that inhibits Cdc[20] before the activation of the SAC in mitosis
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