Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C–Cdc20 complex

Abstract

The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.

The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing

“…Cells were either control treated or depleted of Cdc20 by RNAi and the expression of the different VenusCdc20 proteins induced by doxycycline. In vitro ubiquitination assays with in vitro translated Cyclin B1 1-86 was carried out as previously described 42 .…”

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

“…Although Nek2A is an APC/C substrate, conclusive evidence that its destruction in mammalian cells depends only on APC/C Cdc20 , or that a different proteasomal targeting pathway contributes to its degradation, too, is lacking. Furthermore, the role of Cdc20 in directing APC/C-mediated Nek2A degradation is under debate (Kimata et al, 2008;Sedgwick et al, 2013). In contrast to cyclin A, even at high levels Nek2A, has not been found to interfere with the ability of BubR1 to bind Cdc20 (Sedgwick et al, 2013), indicating that Nek2A and cyclin A might differ in the way their destruction escapes control by the spindle checkpoint.…”

Degradation of Ndd1 by APC/CCdh1 generates a feed forward loop that times mitotic protein accumulation

“…Recent studies have revealed intricate mechanisms that regulate the temporal pattern of degradation by the APC/C Cdc20 . Different substrates are degraded at different times, sometimes only minutes apart 3,9,47 . While the timing of degradation is obviously critical, especially for highly complex events during mitosis, we still know little about what happens at the other end-reaccumulation of these ARTICLE proteins during the S and G2 phases.…”