title: Nek2A destruction marks APC/C activation at the prophase-to-prometaphase transition by spindle-checkpoint-restricted Cdc20.
abstract: Nek2 isoform A (Nek2A) is a presumed substrate of the anaphase-promoting complex/cyclosome containing Cdc20 (APC/C(Cdc20)). Nek2A, 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 being stabilized 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, whereas Nek2A disappeared at a normal rate. Second, depleting Cdc20 effectively stabilized cyclin A but not Nek2A. Nevertheless, Nek2A destruction crucially depended on Cdc20 binding to the APC/C. Third, 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/C(Cdc20) catalytic activity.
authors: Division of Cell Biology I (B5) and Division of Molecular Carcinogenesis (B7), The Netherlands Cancer Institute (NKI-AvL), 1066 CX Amsterdam, The Netherlands.
authors: Michiel
authors: M
authors: Boekhout
authors: Division of Cell Biology I (B5) and Division of Molecular Carcinogenesis (B7), The Netherlands Cancer Institute (NKI-AvL), 1066 CX Amsterdam, The Netherlands Department of Clinical Genetics (Division of Oncogenetics), VUmc and VUmc Cancer Center Amsterdam, CCA/V-ICI Research Program Oncogenesis, VUmc Medical Faculty, 1081 HV Amsterdam, The Netherlands r.wolthuis@vumc.nl.
authors: Rob
authors: R
authors: Wolthuis
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome / Q000378:metabolism; D064199:Cdc20 Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D019925:Cyclin A / Q000378:metabolism; D064248:Geminin / Q000378:metabolism*; D006801:Humans; D059566:M Phase Cell Cycle Checkpoints; D000072379:NIMA-Related Kinases; D015739:Nocodazole / Q000737:chemistry; D049468:Prometaphase; D011418:Prophase; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D064199:Cdc20 Proteins; D019925:Cyclin A; C577995:GMNN protein, human; D064248:Geminin; C577488:CDC20 protein, human; D064173:Anaphase-Promoting Complex-Cyclosome; C089441:NEK2 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases; D015739:Nocodazole
keywords: APC/C; Cdc20; Cyclin A; Nek2A; Spindle checkpoint