Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C

Abstract

The temporal control of mitotic protein degradation remains incompletely understood. In particular, it is unclear why the mitotic checkpoint prevents the anaphase-promoting complex/cyclosome (APC/C)-mediated degradation of cyclin B and securin in early mitosis, but not cyclin A. Here, we show that another APC/C substrate, NIMA-related kinase 2A (Nek2A), is also destroyed in pro-metaphase in a checkpoint-independent manner and that this depends on an exposed carboxy-terminal methionine-arginine (MR) dipeptide tail. Truncation of the Nek2A C terminus delays its degradation until late mitosis, whereas Nek2A C-terminal peptides interfere with APC/C activity in an MR-dependent manner. Most importantly, we show that Nek2A binds directly to the APC/C, also in an MR-dependent manner, even in the absence of the adaptor protein Cdc20. As similar C-terminal dipeptide tails promote direct association of Cdc20, Cdh1 and Apc10-Doc1 with core APC/C subunits, we propose that this sequence also allows a substrate, Nek2A, to directly bind the APC/C. Thus, although Cdc20 is required for the degradation of Nek2A, it is not required for its recruitment and this renders its degradation insensitive to the mitotic checkpoint.

Alternative Splicing Controls Nuclear Translocation of the Cell Cycle-regulated Nek2 Kinase

“…After mitotic entry, Nek2A is specifically targeted for proteasomal degradation as a result of APC/C-mediated ubiquitylation, whereas Nek2B remains stable as it lacks the degradation motifs present in the C terminus of Nek2A (24,25). These degradation motifs, a KEN-box and the C-terminal MR dipeptide, are present in Nek2C, and we predicted that this isoform would also be degraded in mitosis.…”

“…This is followed by a short motif (amino acids 333-370) shown to be required for centrosome targeting and microtubule binding (22). However, the position of splicing means that a binding site for protein phosphatase 1 (PP1 3 ; amino acids 383-386) and two APC/Cdependent degradation motifs, a KEN box (amino acids 391-399), and an MR-tail (amino acids 444 -445), are present in Nek2A but missing from Nek2B (23)(24)(25). This falls in line with observations that Nek2A is degraded upon mitotic entry, whereas Nek2B remains stable.…”

Structure and Regulation of the Human Nek2 Centrosomal Kinase

“…At the G 1 /S transition vertebrates express two major splice variants, Nek2A and Nek2B (19,20). These variants differ in their extreme C termini, which has important implications for their regulation, as the C terminus of Nek2A, but not Nek2B, contains both a binding site for protein phosphatase 1 and motifs that target the protein for ubiquitin-mediated degradation after mitotic entry (21)(22)(23).…”

APOLLON Protein Promotes Early Mitotic CYCLIN A Degradation Independent of the Spindle Assembly Checkpoint

“…SAC-independent Nek2A degradation requires the carboxyl-terminal dipeptide of Nek2A (Met-Arg), and the di-peptide mediates the recruitment of Nek2A to APC/C to activate ubiquitylation (58,59). Thus, Nek2A is degraded independently of the SAC.…”