Role of phosphorylation of Cdc20 in the regulation of the action of APC/C in mitosis.

Abstract

The ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) is essential for the control of mitosis, and its activity is subject to tight regulation. In early mitosis, APC/C is inhibited by the mitotic checkpoint system, but subsequently it regains activity and promotes metaphase-anaphase transition by targeting cyclin B and securin for degradation. The phosphorylation of APC/C by the mitotic protein kinase Cdk1-cyclin B facilitates its interaction with its coactivator Cdc20, while the phosphorylation of Cdc20 inhibits its binding to APC/C. This raises the question of how Cdc20 binds to APC/C under conditions of high Cdk1 activity. It seemed possible that the opposing action of protein phosphatases produces a fraction of unphosphorylated Cdc20 that binds to APC/C. We found, however, that while inhibitors of protein phosphatases PP2A and PP1 increased the overall phosphorylation of Cdc20 in anaphase extracts from <i>Xenopus</i> eggs, they did not decrease the levels of Cdc20 bound to APC/C. Searching for alternative mechanisms, we found that following the binding of Cdc20 to APC/C, it became significantly protected against phosphorylation by Cdk1. Protection was mainly at threonine sites at the N-terminal region of Cdc20, known to affect its interaction with APC/C. A model is proposed according to which a pool of unphosphorylated Cdc20, originating from initial stages of mitosis or from phosphatase action, combines with phosphorylated APC/C and thus becomes stabilized against further phosphorylation, possibly by steric hindrance of Cdk1 action. This pool of APC^Cdc20 appears to be required for the regulation of APC/C activity at different stages of mitosis.