Pulling a Ligase out of a “HAT”: pCAF Mediates Ubiquitination of the Class II Transactivator

Figure 6

pCAF enhances ubiquitination of CIITA acetylation null mutants. (a) Acetylation null CIITA mutants have increased levels of degradative ubiquitination in the presence of pCAF. In vivo ubiquitination assay. COS cells were cotransfected with Flag-K141R, K144R, K141/144R CIITA, and Myc-pCAF. Eighteen hours following transfections, MG132 was added to indicated samples in order to inhibit the 26S proteasome. Cells were harvested, lysed, precleared, and immunoprecipitated with anti-Flag antibody (lane 2 negative control, immunoprecipitated with IgG). Western blots were performed and immunoprecipitated samples were immunoblotted using antiubiquitin antibodies. Lysate controls (bottom two panels) demonstrate expression of WT Flag CIITA, Flag-K141R, K144R, K141/144R, and Myc-pCAF. Data shown are cropped images from one immunoprecipitation gel and one lysate gel and are representative of three individual experiments. (b) CIITA acetylation null mutants have increased levels of K48 specific linkage ubiquitination in the presence of pCAF. 15 μL from the same sample was immunoprecipitated with anti-Flag antibody and immunoblotted for anti-K48 specific ubiquitin antibody. (c) Densitometry and quantification of data in (a). Densitometry was performed on three independent experiments, mean ± SD, . (d) Densitometry and quantification of data in (b). Densitometry was performed on three independent experiments, mean ± SD, . Area for densitometry analysis was selected from CIITA’s molecular weight of 150 KDa as labeled and above.

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