ARTICLE ABSTRACTThe hetero-trimeric PP2A serine/threonine phosphatases containing the regulatory subunit B56, and in particular B56γ, can function as tumor suppressors. In response to DNA damage, the B56γ subunit complexes with the PP2A AC core (B56γ-PP2A) and binds p53. This event promotes PP2A-mediated dephosphorylation of p53 at Thr55, which induces expression of p21, and the subsequent inhibition of cell proliferation and transformation. In addition to dephosphorylation of p53, B56γ-PP2A also inhibits cell proliferation and transformation by a second, as yet unknown, p53-independent mechanism. Here, we interrogated a panel of B56γ mutations found in human cancer samples and cell lines and showed that these mutations lost B56γ tumor-suppressive activity by two distinct mechanisms: one is by disrupting interactions with the PP2A AC core and the other with B56γ-PP2A substrates (p53 and unknown proteins). For the first mechanism, due to the absence of the C catalytic subunit in the complex, the mutants are unable to mediate dephosphorylation of any substrate and thus failed to promote both the p53-dependent and -independent tumor-suppressive functions of B56γ-PP2A. For the second mechanism, the mutants lacked specific substrate interactions and thus partially lost tumor-suppressive function, i.e., either the p53-dependent or p53-independent contingent upon which substrate binding was affected. Overall, these data provide new insight into the mechanisms of tumor suppression by B56γ.Implications: This study further indicates the importance of B56γ-PP2A in tumorigenesis. Mol Cancer Res; 11(9); 995–1003. ©2013 AACR.