Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55alpha regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt.
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 B55alpha subunit in NIH3T3 cells. The direct interaction between B55alpha subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55alpha 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 B55alpha overexpression in these cells. Silencing of B55alpha expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55alphaC holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55alpha overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55alpha expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55alpha-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival.
Institute of Molecular Medicine, Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University Medical College, 701 Tainan, Taiwan.
Yi-Chun
YC
Kuo
Kai-Yun
KY
Huang
Chung-Hsiang
CH
Yang
Yu-San
YS
Yang
Wen-Yu
WY
Lee
Chi-Wu
CW
Chiang
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D020868:Gene Silencing; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D041681:NIH 3T3 Cells; D010766:Phosphorylation / Q000187:drug effects; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*
D020035:Holoenzymes; C508596:IL3 protein, human; D007377:Interleukin-3; C512974:PPP2R2A protein, human; C494918:AKT1 protein, human; C514862:Akt1 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D054648:Protein Phosphatase 2