Identification and functional characterization of FoxO3A as a novel substrate of PP2A-B56γ3

摘要

Protein phosphatase 2A (PP2A) is a major eukaryotic serine/threonine phosphatase which consists of a scaffolding A subunit a catalytic C subunit and a variable regulatory B subunit The regulatory B subunits determine the substrate specificity and subcellular localization of PP2A We have studied the cellular roles of B56γ3 which belongs to the B’ (B56/PR61) family and has been shown to contribute to the major tumor suppressor activity of PP2A The forkhead box transcription factors of the O classification (FoxOs) in mammals belong to a family of transcription factors that bind conserved forkhead box DNA binding domain The mammalian FOXO subgroup consists of four members FoxO3A (FKHRL1) FoxO1 (FKHR) FoxO4 (AFX) and FOXO6 which play a role in regulating cellular proliferation/arrest metabolism cell survival/death and autophagy AKT catalyzes phosphorylation of FoxO3A at residue (S253 S315 Thr32) which promotes its cytoplasmic localization inactivation and degradation Phosphorylation of FoxO3A at S318 and S321 which is probably to enhance its rate of nuclear export was mediated by Casein kinase (CK) 1 On the other hand PP2A was also shown to regulate the subcellular localization and activity of FoxO3A; however the B subunit targeting PP2A to regulate FoxO3A remains unidentified In this report we investigated whether B56γ3 may target PP2A to regulate phosphorylation of FoxO3A We found that serum-stimulated phosphorylation of FoxO3A at Thr32 Ser253 and Ser318/321 were all decreased in NIH3T3 cells stably expressing B56γ3 as compared to that of NIH3T3 cells stably expressing vector only and also found that levels of phosphorylation of ectopically expressed FoxO3A at Thr32 Ser253 and Ser318/321 were all decreased by co-transfecting FoxO3A with 4HA-B56γ3 into NIH3T3 cells Besides we also found that NIH3T3 cells with stably expressing B56γ3 showed a significant decreased in levels of phospho-FoxO3A at Thr32 and to a lesser extent at Ser253 under both serum starvation and insulin stimulation as compared to cells carrying vector only In HeLa cells B56γ3 overexpression resulted in a significant reduction in the levels of serum-stimulated phosphorylation of FoxO3A at Thr32 and to a lesser extent at Ser253 and Ser318/321 as compared to cells carrying vector only Conversely HeLa cells with stable B56γ3 knockdown showed a significant increase in levels of phospho-FoxO3A at Thr32 and to a lesser extent at Ser253 and Ser318/321 under both serum starvation and serum stimulation as compared to cells carrying vector only Next we examined the role of B56γ3 in regulating FoxO3A localization and we found that cells with B56γ3 overexpression showed more FoxO3A accumulation in the nucleus upon insulin stimulation compared with cells overexpressing vector only suggesting that PP2A-B56γ3 counteracted insulin/PI3K/Akt signaling to regulate FoxO3A nuclear localization Furthermore we investigated the effect of B56γ3 overexpression on the localization of FoxO3A phosphorylation defective mutants (T32A S253A and S315A) We found that a significant increase in the distribution of FoxO3A in the nucleus when FoxO3A WT or S315A was applied and to a lesser extent when S253A or Thr32A was applied indicating that PP2A-B56γ3 may regulate nuclear localization by selectively dephosphorylating FoxO3A at Thr32 and S253 Together PP2A-B56γ3 may upregulate FoxO3A activity by increasing nuclear localization through dephosphorylating at Thr32 and S253 PP2A-B56γ3 may directly regulate FoxO3A phosphorylation without down-regulating AKT activity since the level of phospho-GSK-3?/β (Ser21/9) a downstream target of AKT was not downregulated by B56γ3 overexpression Furthermore we found that PP2A-B56γ3 can associate with FoxO3A by co-immunoprecipitation analysis Moreover FoxO3A transcriptional activity reporter assay in NIH3T3 cells with stably expressing B56γ3 and HeLa cells stably expressing shRNA for B56γ3 (shB56γ3) showed that PP2A-B56γ3 may upregulate FoxO3A activity as compared to cells carrying vector only In summary our results suggest that PP2A-B56γ3 counteracts insulin/PI3K/Akt signaling to regulate nuclear localization and expression of downstream target genes of FoxO3A by selectively dephosphorylating FoxO3A at Thr32

獎項日期	2016 2月 1
原文	English
監督員	Chi-Wu Chiang (Supervisor)