PP2A磷酸酶由三個次單元所構成，包含結構性次單元A，催化性次單元C，以及多樣的調節性次單元B。B次單元又可以分為四個家族包括B、B'、B'、以及B'。目前的研究認為多樣的B次單元可以影響PP2A磷酸酶對受質的專一性以及細胞中的分布表現。
我們首先透過螢光顯微鏡以及雙分子螢光互補作用(BiFC, Bimolecular fluorescence complementation)的方法來探討PP2A磷酸酶兩個次單元間的結合以及分布表現，透過將螢光蛋白半部接上次單元A，另一個螢光蛋白半部接上不同的次單元B，當兩個次單元結合時，兩個螢光半部也因互補作用而結合產生螢光。實驗結果顯示A會因結合不同的B而改變A/B聚合體的表現分布，這樣的結果也與透過免疫螢光染色法觀察不同B次單元在細胞中的分布表現相符。此外我們也透過雙分子螢光互補作用來探討其他與PP2A磷酸酶有互動的蛋白。如α4，又稱為IGBP1，目前已知與次單元C會結合一起，且在壓力刺激下可以促進次單元C的穩定度。BiFC結果顯示α4/Cα聚合物呈現全細胞分布表現情形，且α4增強了Aα/Cα聚合物的訊號。
接著，我們將雙分子螢光互補作用(BiFC)結合螢光共振能量轉移(FRET, Fluorescence resonance energy transfer)來探討PP2A三分子聚合物的結合情形。為了完整建立以BiFC合併FRET的分析方法，除了原先已經建構的部分A、B、C次單元的BIFC表現載體，再建構以N端或C端融合CFP (Cyan fluorescence protein)螢光蛋白到各個次單元的表現質體，但我們發現只有Cα-CFP和B55β2-CFP呈現出如預期的螢光與蛋白表現。透過BiFC和FRET的結合，我們成功看到Cα-CFP與Aα-B56γ3-YFP (BiFC)，以及B55β2-CFP與Aα-Cα-YFP (BiFC)之間產生共振能量轉移。也透過加入SV40 Small T antigen (ST)及其失去與次單元A鍵結的突變型來證明BiFC-FRET訊號的特異性。此外我們也看到失去與次單元A鍵結的B55β2-CFP 突變型無法與Aα-Cα-YFP (BiFC)產生FRET訊號。再次證明我們觀測到的FRET訊號的特異性。
總結來說，我們建立雙分子螢光互補作用(BiFC)與螢光共振能量轉移(FRET)結合的方式來探討PP2A磷酸酶次單元分子間的結合，也提供了一個可以探討活細胞中PP2A磷酸酶次單元分子動態結合與分布的新穎方法。

Protein phosphatase 2A (PP2A) is a heterotrimeric complex, including a scaffold subunit (PP2A/A), a catalytic subunit (PP2A/C), and a variable regulatory subunit (PP2A/B). There are four families of the B subunit including B, B’, B’’, and B’’’. These diverse B subunits are believed to determine the substrate specificity and subcellular localization of PP2A. However, there is no direct evidence of localization of PP2A controlled by the B subunits, and absence of tools for observing the trimeric PP2A holoenzyme complex in living cells has hampered the understanding on how the PP2A trimeric complex reacts to upstream stimuli in a spatial and temporal manner.
We have employed both immunofluorescence microscopy and bimolecular fluorescence complementation (BiFC) analysis to investigate the subcellular localization and association of the PP2A subunits. To investigate the role of the B subunits in determining the localization of the PP2A holoenzymes, we applied BiFC to analyze fluorescent signals generated by association of the fluorescence protein fragment (FPF)-fused A subunit with various FPF-fused B subunits, and results showed distinct subcellular distribution of different A/B complexes, similar to that detected by indirect immunofluorescence analysis of individual B subunits. We also investigated other complexes formed by PP2A and known PP2A interacting proteins in cells using BiFC analysis. Alpha 4, also named IGBP1 (immunoglobulin binding protein 1), has been shown to interact with the C subunit and stabilize the C subunit during stress stimulations. Results of BiFC analysis indicated that the α4/Cα complex is located to various subcellular locations, and that BiFC signals of Aα/Cα complexes were markedly enhanced by α4.
Next, we combined BiFC with fluorescence resonance energy transfer (FRET) to investigate the association of the PP2A trimeric complex. Firstly, we established expression constructs of different PP2A subunits fused to cyan fluorescence protein (CFP) for BiFC-FRET analysis, and found that, of all constructs made, only Cα-CFP and B55β2-CFP were reasonably expressed in transient transfection experiments. Further, we demonstrated that FRET signals were successfully generated between Cα-CFP and Aα-B56γ3-YFP (BiFC), and between B55β2-CFP and Aα-Cα-YFP (BiFC). The FRET signals were specific as shown by abolishment of FRET signals in the presence of wild-type small t antigen (ST) of simian virus 40 (SV40), but not by mutant ST which is truncated at the A subunit-binding domain. In addition, no FRET signals were observed between Aα-Cα-YFP (BiFC) and a mutant B55β2-CFP which is defective in binding to the A subunit.
In summary, we established a BiFC-based FRET to visualize the association of A, B, and C subunits in cells. And the findings provide a novel approach for investigating dynamics of the trimeric PP2A in living cells.

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