Nek2A destruction marks APC/C activation at the prophase-to-prometaphase transition by spindle-checkpoint restricted Cdc20

Abstract

Nek2A is a presumed APC/CCdc20 substrate, which, like cyclin A, is degraded in mitosis while the spindle checkpoint is active. Cyclin A prevents spindle checkpoint proteins from binding to Cdc20 and is recruited to the APC/C in prometaphase. We found that Nek2A and cyclin A avoid stabilization by the spindle checkpoint in different ways. First, enhancing mitotic checkpoint complex (MCC) formation by nocodazole treatment inhibited the degradation of geminin and cyclin A while Nek2A disappeared at normal rate. Secondly, depleting Cdc20 effectively stabilized cyclin A but not Nek2A. Nevertheless, Nek2A destruction critically depended on Cdc20 binding to the APC/C. Thirdly, in contrast to cyclin A, Nek2A was recruited to the APC/C before the start of mitosis. Interestingly, the spindle checkpoint very effectively stabilized an APC/C-binding mutant of Nek2A, which required the Nek2A KEN box. Apparently, in cells, the spindle checkpoint primarily prevents Cdc20 from binding destruction motifs. Nek2A disappearance marks the prophase-to-prometaphase transition, when Cdc20, regardless of the spindle checkpoint, activates the APC/C. However, Mad2 depletion accelerated Nek2A destruction, showing that spindle checkpoint release further increases APC/CCdc20 catalytic activity.