FIG 3.

HPV-31 E6 is differentially phosphorylated by PKA and AKT. (A) Purified GST fusion proteins of HPV-31 E6 wild-type and PBM mutants, delPDZ and T145AdelPDZ, either left untreated (−) or incubated with PKA (+) and ³²P-γATP as indicated. Proteins were analyzed by SDS-PAGE and autoradiography. The upper portion shows the autoradiogram, and the lower portion shows the Coomassie stain of the gel. (B) Experiment similar to that for panel A, but GST fusion proteins were either left untreated (−) or incubated with AKT (+). (C) HPV-18, -31, and -58 E6-GST fusion proteins, either left untreated (−) or incubated with PKA (+) in the presence of cold ATP. These were detected by Western blotting using anti-phospho-E6-specific antibody. The lower portion shows the Ponceau stain of the nitrocellulose membrane confirming equal levels of protein loading. (D) A similar analysis of GST fusion proteins of HPV-16 E6, HPV-31 E6, and the HPV-31 E6 mutants, delPDZ and T145AdelPDZ, either left untreated (−) or incubated with AKT (+) and detected by Western blotting using anti-phospho-E6-specific antibody. Arrows indicate the relevant GST proteins.