KEN and D box mutants stabilize Cdr2.
A. Cdr2 schematic depicting acidic, coiled coil (CC) and leucine zipper (LZ) motifs and KEN and D (D1, D2) boxes. Lower panels, alignment of consensus KEN and D box motifs with human (h), mouse (m) and rat (r) cdr2 and mutants. Amino acid residue numbers are from human Cdr2. B. Upper, HEK293s transfected with T7cdr2 or T7cdr2 KEN/D1D2, released from G2/M block and blotted with T7 or γ-tubulin antisera. Lower, quantitation of T7cdr2 and T7cdr2 KEN/D1D2 levels normalized to γ-tubulin. *p<0.01. C. Upper, autoradiographs of degradation assays of 35S-labeled cyclinB1, T7cdr2 and T7cdr2 KEN/D1D2 at indicated times. Lower, quantitation of raw cyclinB1, T7cdr2 and T7cdr2 KEN/D1D2 levels. *p<0.05. D. Left, anti-HA blot of T7 immunoprecipitates from HEK293s transfected with T7cdr2 or T7cdr2 KEN/D1D2 and HA-Ub (see Fig. S2); right, line graph of band intensity (x-axis) for adjacent western blot and molecular weight (y-axis); T7cdr2 (blue), KEN/D1D2 (red). E. Left, autoradiographs of 35S-T7cdr2 ubiquitination reaction with the APC/C, APC/C/HACdc20, APC/C/HACdh1, or control (Protein A beads) for indicated times. Right, quantitation of T7cdr2-Ub conjugates.
