Fig. 1.
Mutation of individual cyclin A2 degrons did not abolish its ubiquitination and degradation. a Domain organization of human cyclin A2 and cyclin B1, with their respective degrons indicated. DQEN and NAEN are the putative KEN boxes. b–g Ubiquitination assays of single degron mutations in cyclin A2 by APC/CCdh1 (b), APC/CCdc20 (d) and APC/CMCC (f). In vitro phosphorylated APC/C was used for complex formation with Cdc20 and the MCC, while unphosphorylated APC/C was used for Cdh1. The remaining degrons in cyclin A2 after the mutation are labelled in dark red. Ubiquitination reactions were analysed by Western blotting with an anti-His antibody to detect the ubiquitin-modified substrates. Control gels showing the unmodified substrate for representative reactions are shown in Supplementary Fig. 2. The western-blots were quantified, with error bars indicating standard deviation. The APC/C activity towards cyclin A2 mutants is normalized to ubiquitination of wild-type cyclin A2 and the significance is calculated using unpaired Student’s t-test (indicated with stars, n = 3 for c and 2 for e and g, Supplementary Table 2). h, i Degradation profiles of single degron mutation constructs of eGFP-cyclin A2 in HEK cells during unperturbed mitosis (h) and a SAC arrest using STLC (i). Under unperturbed mitosis, n = 62 wild-type, 49 ΔK, 47 ΔD1, 44 ΔD2 and 37 ΔA cells were analysed, whereas 13 wild-type, 14 ΔK, 15 ΔD1, 22 ΔD2 and 12 ΔA cells were analysed for conditions under a SAC arrest. The timepoints of NEBD and reversine addition to release the SAC arrest are marked, respectively. j Exemplary still images from time courses between NEBD and anaphase of eGFP-cyclin A2 destruction in HEK cells of wild-type cyclin A2 and the ΔD2 mutant. The chromosomes are coloured in magenta and cyclin A2 in green, with the outline of the cells marked with dashed yellow lines. Time is given as hh:mm. Scale bar 10 μm. Degradation of cyclin A2 is slightly delayed in the absence of the D2 box (compare the two cells at 15 and 21 min). The complete time courses are shown as Supplementary Movies 1 and 2. Source data are provided as a Source Data file