FIGURE 3.

Dynamic association between Cdc34 and the SCF^βTrCP1-IκBα-Ub complex. A, kinetic parameters of polyubiquitination of IκBα-(1–54)-Ub by SCF^βTrCP2 and Cdc34. ³²P-Labeled IκBα-(1–54)-Ub (2pmol) was incubated with SCF^βTrCP2 (∼2 pmol) for 10 min. Separate mixtures of Cdc34∼Ub at differing concentrations (5.67, 1.87, 0.63, and 0.21 μm) of Cdc34 were incubated at 37 °C for 5 min. The two mixtures were combined, and reactions were stopped by the addition of SDS-loading buffer after 20, 40, 60, or 80 s. Substrate utilization of three independent data sets was calculated and shown graphically. K_m and K_cat were estimated by fitting to Michaelis-Menten kinetics and shown in the inset box. B, the competition scheme. The detailed procedure is described under “Experimental Procedures.” C, the inhibition of Cdc34-Ub^Cys on Cdc34-catalyzed elongation of ³²P-IκBα-Ub. The inhibitory effects were reversed by high concentrations of Cdc34. A short exposure of the input substrate is shown to better reveal its reduction with reaction time. D, the competition reaction. The effects of Cdc34-Ub^Cys, which was added after the reaction had been initiated, are shown. The substrate conversion was quantified and is shown graphically.