Characterization of the functional interaction between two tumor suppressors: p53 and B56Gamma-PP2A

The tumor suppressor protein p53 is a very important protein involved in the cellular response to DNA damage. During the course of its activation, p53 undergoes several post-translational modifications that promote its stabilization and activation. We previously demonstrated that p53 is kept at low levels inside the cell under growth conditions in part by the activity of TAF1, which phosphorylates p53 at Thr55. After DNA damage, p53 Thr55 phosphorylation decreases, leading to induction of p53 protein levels and promoting p53 tumor suppressive function.;In the present study, we identify PP2A as the phosphatase responsible for dephosphorylating p53 at Thr55 after DNA damage. PP2A is a heterotrimeric complex consisting of a catalytic (C), scaffolding (A), and regulatory (B) subunit. The B subunit is believed to regulate the substrate specificity and activity of the holoenzyme complex. We demonstrate that the B subunit, B56gamma is upregulated after DNA damage, leading to an overall increase in B567-PP2A complexes in the cell. We also demonstrate that B56gamma is responsible for directing PP2A activity to p53 and that this activity of PP2A is required for its p53-dependent tumor suppressive function.;During the course of the study, we observed both a p53-dependent and independent tumor suppressive function of B567-PP2A. In order to differentiate between the two, we isolated a small domain of B56gamma required for PP2A-p53 interaction. In addition, we characterized a previously identified cancer-derived mutation of B56gamma3, F395C, found on a residue within the p53 interaction domain. We found that this mutant protein, isolated from a human lung tumor, is unable to interact with p53 or promote PP2A p53-dependent tumor suppressive function, thereby strengthening the significance of B56gamma-PP2A regulation of p53 activity in tumorigenesis.;Finally, we uncovered Ser15 phosphorylation by ATM (Ataxia Telangiectasia Mutated) as a priming modification after DNA damage required to promote B56gamma-PP2A interaction with p53, subsequent dephosphorylation of p53 at Thr55, and p53-dependent tumor suppressive function. In an attempt to identify whether ATM is also able to regulate B56gamma directly, we demonstrate that ATM directly phosphorylates B56gamma at Ser510 leading to its upregulation after DNA damage. Taken together, our results provide evidence for a multi-step process by which ATM regulates B56gamma-PP2A activation, interaction with p53, and subsequent activation of p53 tumor suppressive functions after DNA damage.