Regulation of Phosphorylation of Thr-308 of Akt, Cell Proliferation, and Survival by the B55α Regulatory Subunit Targeting of the Protein Phosphatase 2A Holoenzyme to Akt

Abstract

Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit (A), catalytic subunit (C), and a variable regulatory subunit (B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55␣ subunit in NIH3T3 cells. The direct interaction between B55␣ subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55␣ subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55␣ overexpression in these cells. Silencing of B55␣ expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55␣C holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55␣ overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55␣ expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55␣-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival.

Turn motif phosphorylation negatively regulates activation loop phosphorylation in Akt

“…To determine which of these two phenomena is occurring, we tried to inhibit the A-loop phosphatase in vivo. We used okadaic acid (OA) to inhibit the A-loop phosphatase as protein phosphatase 2A has been reported to be an Aloop phosphatase in mammalian cells (Andjelkovicé t al., 1996;Sato et al, 2000;Borgatti et al, 2003;Ugi et al, 2004;Kuo et al, 2008). Microinjection of OA into unstimulated oocytes induced A-loop phosphorylation in the absence of 1-MeAde (Figure 5a), as expected from the constitutive activity of PDK1 (Alessi et al, 1997a, b;Hiraoka et al, 2004).…”

Protein phosphatase-1 regulates Akt1 signal transduction pathway to control gene expression, cell survival and differentiation

“…This is not surprising as previous studies have shown that PP-2A dephosphorylates Akt1 at Thr-308. 28,29 Immunocytochemistry analysis showed that both PP-1 and Akt1 displayed partial overlapping in both the cytoplasm and the nucleus within the HLECs (Figure 3c-f) and ARPE-19 cells (Figure 3g-j). In summary, our results showed that PP-1 can dephosphorylate Akt1 at Thr-450.…”

“…26,27 Dephosphorylation of Akt1 at Thr-308 is mediated by PP-2A. 28,29 On the other hand, dephosphorylation of Akt1 at Thr-450 remains unknown. In this study, we present both in vitro and in vivo evidence to show that PP-1 is a major phosphatase dephosphorylating Akt1 at Thr-450.…”

Protein Phosphatase 2A Isoforms Utilizing Aβ Scaffolds Regulate Differentiation through Control of Akt Protein

“…B subunits are thought to regulate PP2A function. Knockdown or mutation of particular B subunits leads to hyperactivation of specific substrates (7,8). In fact, there is evidence that B55␣ targets PP2A to dephosphorylate Akt at Thr-308 (7).…”

“…There are also two forms of the catalytic C subunit and four B subunit families, each with several members. B subunits target PP2A to particular locations or substrates (7,8). PP2A is being increasingly connected to cancer (9 -11).…”