Fig. 2.
An unidentified degron conserved among mammals is present in cyclin A2. a Mutation of all three known degrons (KEN box, D1 box and ABBA motif) in cyclin A2 did not impair cyclin A2 ubiquitination by APC/CCdc20 (compare lanes 1 and 4). However, the mutant cyclin A2ΔKD1A is subject to MCC-imposed inhibition (lanes 4–6), similar to Cdk2-cyclinB1-Cks2 (lanes 7–9). The remaining degrons in cyclin A2 after the mutation are labelled in dark red. b Replacing overlapping segments within residues 60–80 of cyclin A2 with GSA-linkers identified that residues 70–80 may contain an unidentified degron. Mutation of either residues 65–75 or 70–80 in addition to ΔKD1A severely impaired cyclin A2 ubiquitination by APC/CCdc20. c, d Degradation profiles of the eGFP-cyclin A2 mutants where either the KD1A degrons (leaving the D2 box present) or all four degrons were mutated in HEK cells under unperturbed mitosis (c) and a SAC arrest using STLC (d). Degradation pattern of cyclin B1 was recorded as a reference. The total eGFP fluorescence minus background fluorescence for each cell at each time point was normalized to NEBD, quantified and plotted over time. The timepoints of NEBD and reversine addition to release the SAC arrest are marked, respectively. Error bars indicate mean ± standard deviation of n = 62 wild-type cyclin A2, 24 ΔKD1A, 37 Δall and 45 wild-type cyclin B1 cells under unperturbed mitosis and 13 wild-type cyclin A2, 11 ΔKD1A and 19 Δall cells under a SAC arrest from three experiments. e Addition of a peptide modelled on residues 60–80 of cyclin A2 inhibited ubiquitination of the cyclin A2 ΔKD1A mutant, confirming that this region targets cyclin A2 for ubiquitination. f, g Sequence alignment of the N-terminal domain of cyclin A2 and phylogenic tree. Positions of the four degrons are indicated. All cyclin A2 sequences used in the sequence alignment contain the D1 box (except for mouse and rat), whereas the D2 box appeared at a later stage of evolution. Source data are provided as a Source Data file