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J. Biol. Chem., Vol. 283, Issue 4, 1882-1892, January 25, 2008

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Regulation of Phosphorylation of Thr-308 of Akt, Cell Proliferation, and Survival by the B55 Regulatory Subunit Targeting of the Protein Phosphatase 2A Holoenzyme to Akt^*

Yi-Chun Kuo, Kai-Yun Huang, Chung-Hsiang Yang, Yu-San Yang, Wen-Yu Lee, and Chi-Wu Chiang¹

From the Institute of Molecular Medicine, Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University Medical College, 701 Tainan, Taiwan

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 AB55C 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.

Received for publication, November 26, 2007

^* This work was supported by grants from the National Science Council 93-2320-B-006-086 and 95-2320-B-006-081. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Institute of Molecular Medicine, National Cheng Kung University Medical College, No.1 University Road, 701 Tainan, Taiwan. Tel.: 886-6-235-3535, ext. 3637; Fax: 886-6-209-5845; E-mail: chiangcw{at}mail.ncku.edu.tw.

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