Abstract
Two mitotic cyclin types, cyclin A and B, exist in higher eukaryotes, but their specialised functions in mitosis are incompletely understood. Using degron tags for rapid inducible protein removal, we analyse how acute depletion of these proteins affects mitosis. Loss of cyclin A in G2‐phase prevents mitotic entry. Cells lacking cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown, which requires a decisive shift in the balance of cyclin‐dependent kinase Cdk1 and PP2A:B55 activity. Beyond this point, cyclin B/Cdk1 is essential for phosphorylation of a distinct subset of mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how cyclin A, cyclin B and Greatwall kinase coordinate mitotic progression by increasing levels of Cdk1‐dependent substrate phosphorylation.
Highlights
Cdk1 phosphorylates over 1,000 proteins (Daub et al, 2008; Dephoure et al, 2008) within the brief 20–30 min window of mitotic entry
Precise and uniform depletion is critical to investigate the functions of mitotic cyclins within a single cell cycle
We have recently reported that a combination of mAID- (Natsume et al, 2016) and SMASh- (Chung et al, 2015) tags results in highly efficient induced degradation (Lemmens et al, 2018), and applied this strategy to comprehensively analyse the functions of cyclin A2 and B1 in a non-transformed, h-TERT immortalised human epithelial cell line, RPE-1
Summary
Cdk phosphorylates over 1,000 proteins (Daub et al, 2008; Dephoure et al, 2008) within the brief 20–30 min window of mitotic entry. Rising levels of Cdk activity correlate with centrosome separation and chromosome condensation in prophase, followed by nuclear envelope breakdown (NEBD) and mitotic spindle formation in prometaphase, and the alignment of bi-oriented sister chromatids at the metaphase plate (Gavet & Pines, 2010). Binding of a cyclin partner is critical for allosteric activation of Cdks. Despite the central importance of these proteins for cell cycle control, the functional specialisation of mammalian A- and Btype cyclins remains unclear. Specific binding partners for cyclin A2 and B1 have been identified (Pagliuca et al, 2011), but the precise contributions of these two cyclins to mitotic entry remain unclear
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