MID1 Catalyzes the Ubiquitination of Protein Phosphatase 2A and Mutations within Its Bbox1 Domain Disrupt Polyubiquitination of Alpha4 but Not of PP2Ac
Figure 5
XLOS-observed mutations within Bbox1 disrupted polyubiquitination of alpha4.
A. Ubiquitination assay of alpha4 was performed with the RB1B2 construct containing (i) C142S, (ii) C145T, and (iii) C195F. B. Ubiquitination of alpha4 by RB1B2 with the (i) A130T, (ii) A130V and (iii) A130S mutations within the Bbox1 domain. The amount of alpha4 used for the assay with the A130T RB1B2 mutant was higher than that used for the A130V mutant assay. Nonetheless, the overall results for the two RB1B2 mutants are very similar. For comparison, wild type RB1B2 was shown to catalyze polyubiquitination of alpha4, as indicated by the smear/ladder pattern designated with the labeled alpha4∼Ubn. Negative controls in which RB1B2 protein is omitted are shown for each reaction, as well. At the bottom of each figure, the ability of the mutant RB1B2 constructs to catalyze the ubiquitination of alpha4 is qualitatively noted. Antibody was specific for the C-terminal region of alpha4.