磷酸水解酶PP2A主要是真核細胞中的一種絲胺酸/蘇胺酸磷酸水解酶，其組成包含了構成結構骨架的A次單元，負責催化功能的C次單元，以及種類相當多樣的調節性B次單元。B次單元具有決定受質特異性和控制PP2A在細胞中所座落的位置。FoxOs在哺乳動物中是轉錄因子的其中一員且具有會與DNA結合的高度保留forkhead box區域，哺乳動物的FoxOs由四個成員組成，分別為FoxO3A (FKHRL1), FoxO1 (FKHR), FoxO4 (AFX) 以及FOXO6，扮演調節細胞增殖、細胞生長停滯、細胞代謝、細胞生存、細胞死亡以及細胞自噬的角色。FoxO3A功能主要受到磷酸化在絲胺酸253, 絲胺酸315以及蘇胺酸32的位點的調節磷酸化，會促使FoxO3A座落在細胞質、失去活性以及降解。此外，絲胺酸318/321的位點的調節磷酸化可能會藉由CK1增進出核的比率。另外一方面，PP2A已經被發現可以調節FoxO3A在細胞中所座落的位置和活性，然而哪一個PP2A的B次單元可以調節FoxO3A尚未清楚。在此研究中，我們探究是否PP2A的B次單元B56γ3可以調節FoxO3A的磷酸化。我們發現在NIH3T3過度表現B56γ3的細胞中相較於vector alone表現的細胞，在血清刺激下FoxO3A磷酸化在絲胺酸253，絲胺酸318/321以及蘇胺酸32位點的表現量呈現下降的情形，也發現當轉染FoxO3A和4HA-B56γ3到NIH3T3細胞時，FoxO3A磷酸化在絲胺酸253, 絲胺酸318/321以及蘇胺酸32位點表現量的程度皆全部下降，此外，我們也發現在NIH3T3過度表現B56γ3的細胞中相較於vector alone表現的細胞在給予胰島素刺激使FoxO3A磷酸化的程度在蘇胺酸32的位點顯著地下降最多，其次是FoxO3A磷酸化在絲胺酸253的位點。在HeLa過度表現B56γ3的細胞中相較於vector alone表現的細胞在血清刺激下FoxO3A磷酸化的程度在蘇胺酸32的位點顯著地下降最多，其次是FoxO3A磷酸化在絲胺酸253及絲胺酸318/321的位點。然而，當HeLa細胞以shRNA降低B56γ3表現，發現相較於vector alone表現的細胞在給予血清刺激下FoxO3A磷酸化的程度在蘇胺酸32的位點顯著地增加最多，其次是FoxO3A磷酸化在絲胺酸253的位點及絲胺酸318/321的位點。此外我們觀察到在HeLa 細胞在血清缺乏時B56γ3表現量增加，但是血清回補後卻減少。HeLa過度表現B56γ3的細胞在血清缺乏時FoxO3A表現量較vector alone表現的細胞高。接著我們探討B56γ3調節FoxO3A座落扮演的角色，我們發現細胞過度表現B56γ3中相較於vector alone表現的細胞在胰島素刺激下，FoxO3A聚集座落在細胞核的細胞明顯增加，顯示出PP2A的B次單元B56γ3可拮抗PI3K/Akt訊息傳遞來調節FoxO3A座落在細胞核。我們進一步地去探討在細胞過度表現B56γ3時，FoxO3A的突變型(蘇胺酸32位點突變成丙氨酸;T32A, 絲胺酸253位點突變成丙氨酸; S253A以及絲胺酸315位點突變成丙氨酸; S315A)的座落位置，我們發現細胞過度表現B56γ3時，FoxO3A WT以及FoxO3A S315A過度表現時相較於vector aloneFoxO3A會明顯地聚集座落在細胞核，其次是 FoxO3A S253A 或 Thr32A，表示PP2A-B56γ3 可能選擇性地去磷酸化FoxO3A在蘇胺酸32以及絲胺酸253的位點來調節FoxO3A座落在細胞核。因為Akt下游的受質GSK-3α/β (Ser21/9)磷酸化表現量並不因B56γ3表現量增加而下降，所以B56γ3有可能是直接調節FoxO3A磷酸化而不是透過負調節Akt所達成。此外，以免疫共沉澱方法分析後，發現PP2A的B次單元B56γ3可以與FoxO3A結合成複合體。此外，啟動子轉錄報導基因分析結果在NIH3T3細胞過度表現B56γ3以及HeLa細胞降低B56γ3表現時皆顯示PP2A-B56γ3可能可以增加FoxO3A的活性。總結來說，我們的結果發現了PP2A的B次單元B56γ3是調節FoxO3A座落在細胞中的位置，以及有選擇性地對FoxO3A磷酸化在蘇胺酸32的位點做去磷酸化。

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-3α/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.

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