磷酸水解酶PP2A是三個次單元所組成的複合體，包含結構蛋白A次單元，調節蛋白B次單元以及執行催化功能蛋白C次單元。其中B次單元決定受質特異性及引領PP2A完全酶到細胞內適當位置執行功能。最近的研究指出包含B56γ的PP2A完全酶有抑制腫瘤的角色。過去實驗室研究已經證明細胞在G1/S轉換期時，PP2A-B56γ3會集中分布在核，並調節G1/S的變換。PP2A-B56γ3透過調節p27KIP1表現量,在核內的分布及活性進而調控腫瘤細胞的增殖。而我們也發現線苷單磷酸活化蛋白激酶(AMPK)及酪蛋白激酶1-δ(CK1-δ)兩者皆會調控B56γ3在絲胺酸440位點的磷酸化，而這個位點對於B56γ3座落在核是重要的。為了探討絲胺酸440在調節B56γ3活性的角色，我們委託廠商製作針對磷酸化絲胺酸440的抗體來進行我們的研究。然而，我們實驗結果顯示磷酸化絲胺酸440的抗體在辨認絲胺酸(Ser)突變成磷酸化缺陷的丙胺酸(Ala)(S440A)的能力和辨識正常B56γ3 (wild-type)的能力相當。而意外的是，另一個磷酸化缺陷的突變，是在人類肺癌中發現的將絲胺酸(Ser)突變成異亮胺酸(Ile)(S440I)的突變，這個突變對於被磷酸化絲胺酸440的抗體的辨識能力較差。以免疫沉澱法分析的結果我們發現B56γ3-S440I與PP2A A次單元的結合大幅下降相較於和B56γ3-WT的結合，而B56γ3-S440A無明顯影響。我們透過雙分子螢光互補分析(BiFC)來探討絲胺酸440突變對於PP2A完全酶的影響，意外發現雙分子螢光互補分析訊號僅S440A與PP2A A次單元結合能力減弱，相較於B56γ3-WT，而B56γ3-S440I無明顯影響。另外我們透過生長曲線的實驗得知S440I及S440A的突變會削弱B56γ3對於細胞增殖的抑制效果。進一步我們在免疫共沉澱法(co-IP)的實驗發現B56γ3-S440I和B56γ3-S440A兩者突變皆影響對於p27的結合。此外，我們發現B56γ3-S440I和B56γ3-S440A皆削弱B56γ3對於p27在蘇胺酸157位點的磷酸化的調節效果。總結而言，我們證明絲胺酸440的角色在對於調控PP2A AC核心酶的結合及p27的調控。絲胺酸的突變可能造成PP2A- B56γ3對於腫瘤的抑制功能。

Protein phosphatase 2A (PP2A) is a trimeric complex that consists of a scaffolding A subunit, a catalytic C subunit, and a variable regulatory B subunit. The B subunits determine the substrate specificity and subcellular localization of the PP2A holoenzyme. Accumulated evidence indicates that the B56γ-containing PP2A holoenzyme (PP2A-B56γ) plays a tumor suppressor role. PP2A-B56γ3 reduces proliferation of an array of cancer cell lines through regulating levels, nuclear localization and activity of p27KIP1. Our lab previously found that both AMP-activated protein kinase (AMPK) and casein kinase I (CKI) regulate phosphorylation of B56γ3 at Ser440, which is critically involved in regulating the nuclear localization of B56γ3. To investigate the role of Ser440 in regulating function of B56γ3, we have obtained anti-phospho-Ser440 (p-S440) custom-made antibody (Ab) to pursue our study. However, our data showed that the anti-pS440 Ab equally recognized both wild-type B56γ3(B56γ3WT) and the phosphorylation defective mutant B56γ3 S440A. Interestingly, another phosphorylation defective mutant B56γ3 S440I, which was found in human lung carcinoma, was poorly recognized by the antibody. We found that B56γ3 S440I, but not B56γ3 S440A, showed significantly reduced association with the A subunit, suggesting a defect in recruitment into the PP2A holoenzyme. We performed bimolecular fluorescence complementation analysis (BiFC) to investigate the effect of Ser440 mutations on PP2A holoenzyme assembly and showed that BiFC signals were reduced when B56γ3S440A, but not S440I, bound with PP2A A subunit compared with B56γ3WT. We found that both S440I and S440A mutants showed impaired inhibitory function on cell proliferation. Besides, results of co-immunoprecipitation (co-IP) showed that both B56γ3 S440I and B56γ3 S440A had reduced interaction with p27 KIP1. In consistent with the co-IP data, we found that both B56γ3 S440I and B56γ3 S440A showed impaired activity in regulating levels of phosphorylation at Thr157 of p27KIP1 compared with B56γ3WT. In summary, we demonstrate that Ser440 plays a role in regulating assembly of B56γ3 with the AC core enzyme and in regulating interaction with p27KIP1. Therefore, it is possible that mutation at Ser440 may result in loss of tumor suppression activity of PP2A-B56γ3.

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