The B55α regulatory subunit targets PP2A holoenzyme to regulate Akt activity and cell growth Free

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Akt is involved in regulation of diverse cellular processes and plays a key role in cancer formation. Phosphorylation of Akt at regulatory residues Thr308 and Ser473 is catalyzed by PDK-1 and several candidate kinases, respectively, leading to its full activation. The protein serine/threonine phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity, and has been proposed to function as a tumor suppressor. The PP2A holoenzyme complex consists of a dimeric core enzyme, which includes the catalytic subunit (C) and the structural subunit (A), and a variable regulatory subunit (B). There are at least twelve B regulatory subunits in vertebrates, and they have been proposed to regulate substrate specificity and subcellular localization of the PP2A holoenzyme. At present, we report to identify the specific B regulatory subunit targeting PP2A holoenzyme to Akt, and to investigate the impact of this specific targeting on Akt activity. We identified endogenous association of PP2A AB55αC holoenzyme with Akt by anti-B55 co-immunoprecipitation analyses in pre-lymphoid FL5.12 cells. In addition, Akt was co-immunoprecipitated with exogenous HA-tagged B55α in NIH3T3 cells. The direct interaction between B55α and Akt1 was confirmed using in vitro pull-down analyses. Intriguingly, we found that overexpression of B55α subunit impaired phosphorylation at Thr308, and to a lesser extent at Ser473 of Akt in both FL5.12 cells and NIH3T3 cells. Silence of endogenous B55α expression in FL5.12 cells by shRNA-mediated RNA interference markedly increased phosphorylation at Thr308 but not at Ser473. Consistently, phosphorylation of several Akt substrates was significantly impaired by B55α overexpression in both FL5.12 and NIH3T3 cells. Furthermore, B55α overexpression retarded proliferation of NIH3T3 cells. Our data demonstrate that B55α subunit targets PP2A holoenzyme to regulate Akt activity and has an important role in cell growth.