Figure 5

Monomeric Fbw7 is active. A: Turnover of cyclin E by dimerization mutants of Fbw7. 293A cells were transfected either with active cyclin E (wild-type CDK2) or inactive cyclin E (dominant-negative CDK2) together with various versions of Fbw7 as indicated. Steady-state protein abundance of cyclin E was monitored by immunoblotting (upper panel). The same membrane was subsequently blotted for Fbw7 and CDK2 (middle panel). The asterisks mark cross-reactions from the 12CA-5 antibody used to detect HA-tagged CDK2. A shorter exposure of CDK2 was chosen and is shown in the lower panel. B: Cyclin E turnover at altered Fbw7 stoichiometry. 0.5 μg of cyclin E plasmid was co-expressed with titrated amounts of either wild-type or dimerization-deficient Fbw7α (3, 1, 0.3, and 0.1 μg, respectively) and blotted for steady-state protein abundance as indicated. C: Cyclin E turnover by Fbw7 at altered stoichiometry. 2 μg of Fbw7 plasmid or the non-dimerizing mutant were co-expressed with increasing amounts of cyclin E (0.5, 1, 2, and 4 μg) and protein levels analyzed as above. In both experiments 2 μg of CDK2 were co-expressed (not shown). D: Fbw7 dimerization is not required for c-Myc degradation. c-Myc and Fbw7 truncation mutants were co-transfected as indicated and their protein levels analyzed by western blotting. ΔN refers to N-terminal-less Fbw7 and corresponds to the common region of Fbw7. All truncation mutants start at the indicated methionines within the common region. The M158 mutant no longer contains the F-box and is therefore inactive.