Mutant B56γ3 is unable to promote p53 Thr55 dephosphorylation. (A) Lysates of U2OS cells transfected with HA-tagged B56γ3, F395C, or Q392G/C398L (QC) mutant B56γ3, were either immunoprecipitated with anti-HA antibody, then analyzed by western blot against p53, PP2A A and C, HA, and vinculin (vinc) (left); or analyzed for p53 Thr55 phosphorylation, p21 protein levels, HA, p53, and vinculin (vinc) (right). Amino acid sequence of the p53-interaction domain of B56γ showing the cancer-derived F395C mutation is present on the top. (B) Lysates of U2OS cells transfected with empty vector control (EV), HA-tagged B56γ3, Q392G, C398L, Q392G/C398L (QC), or Q400S/Q401S (QQ) mutant B56γ3 were analyzed for p53 Thr55 phosphorylation, p21 protein levels, HA, p53, and vinculin (vinc). (C) Lysates of U2OS cells transfected with empty vector control (EV), HA-tagged B56γ3, or F395C mutant B56γ3 and either mock treated (M) or treated with ionizing radiation (IR) were analyzed for p53 Thr55 phosphorylation, HA, p53, and vinculin (vinc). (D) Lysates of U2OS cells transfected with varying amounts of HA-tagged F395C mutant B56γ3, were either mock (M) treated or treated with ionizing radiation (IR), then analyzed by western blot against endogenous (endo) and exogenous (exo) B56γ, as well as p53 and Thr55 phosphorylation. (E) Lysates of U2OS cells transfected with HA-tagged B56γ3 or F395C were immunoprecipitated with anti-HA antibody, then analyzed by western blot against p53, Cyclin G, ERK, Sgo, PP2A A, HA, and vinculin (vinc) antibodies.

Overexpression of cancer derived mutant B56γ3 is unable to block cell proliferation in a p53-dependent manner. Representatives of cell proliferation of the HCT116 human colon cancer cell lines (A) or the H1299 human lung cancer cell line (B) transfected with either wild type (WT), F395C, Q392G/C398L (QC), Q392G, C398L, Q400S/Q401S (QQ) mutant B56γ3, or a control CMV empty vector. Numbers of cells present at the 120 hour time point were normalized against the representative empty vector controls and plotted in a bar graph. Error bars show average +/− s.d. from triplicate plates in one representative experiment. Cell lysates were analyzed by immunoblotting of the transfected HA-B56γ3, endogenous B56γ3/B56γ2, p53 and p21 proteins. H1299 + WT: H1299 transfected with wild-type p53; H1299 + T55D: H1299 transfected with T55D. M: empty vector-transfected cell lysate at seeding.

Mapping of the B56γ domain required for interaction with p53. (A) Left: Diagram of amino acid sequences of B56α, B56γ and various B56γ deletion constructs aligned based on sequence homology. Shades of gray indicate increasing percentage of identical amino acids within that region. Right: Each of the B56γ constructs were expressed in bacteria as GST-fusion proteins, purified, and analyzed by SDS-PAGE (upper). U2OS cell lysates were incubated with the purified GST-fusion proteins indicated, then analyzed by western blot against p53, PP2A A and C, and vinculin (vinc) (lower). (B) U2OS cells were transfected with the HA-tagged B56γ constructs listed, lysed, and subjected to immunoprecipitation with anti-HA antibody. The precipitated proteins were analyzed by western blot against p53, PP2A A and C, HA, and vinculin (vinc). (C) ³⁵S-p53 was incubated with GST protein (GST), GST-B56γ3 protein, or GST-B56γ3 with peptides corresponding to amino acids 376 to 402 (B56γ), 376 to 388 (376), or 391 to 402 of B56γ3 (391), or amino acids 411 to 437 of B56α (B56α). Amino acid sequence of the p53-interaction domain of B56γ aligned with the homologous sequence of B56α is shown below. (D) Lysates of U2OS cells transfected with HA-tagged B56γ3, Q392G, C398L, Q392G/C398L (QC), Q400S/Q401S (QQ) mutant B56γ3, or B56α were either immunoprecipitated with anti-HA antibody, then analyzed by western blot against p53, PP2A A and C, HA, and vinculin (vinc) (left) or with anti-p53 antibody, then analyzed by western blot against HA, PP2A C, p53, and vinculin (vinc) (right).

Overexpression of cancer derived mutant B56γ3 is unable to block anchorage independent growth in a p53-dependent manner. (A) Anchorage-independent growth of HCT116 cells transfected with wild type (WT), F395C, Q392G/C398L (QC), Q392G, C398L, Q400S/Q401S (QQ), or an empty CMV vector control (EV). (B) Colony numbers. The values are the averages +/− standard deviations of the results of three representative experiments. (C) Immunoblots of the transfected HA-B56γ3 and endogenous B56γ3/B56γ2, p53, p21, and vinculin (vinc) proteins. -: HCT116 −/−; +: HCT116 +/+.