蛋白質磷酸水解酶2A型（以下簡稱PP2A）屬於絲氨酸/蘇氨酸蛋白磷酸水解酶家族，目前已知其參與調節許多細胞活性表現，例如細胞訊號傳遞、細胞生長、細胞週期、細胞自噬、細胞凋亡、轉錄和轉譯等等現象。結構部分，PP2A的完全酶是由催化次單元（C）、結構次單元（A）和具有高度多樣性的調節次單元（B）所組成的。其中，是由調節次單元（B）主導PP2A的受質特異性和其在細胞中座落位置。調節次單元（B）目前已知有四種亞型，包括B（B55 / PR55），B`（B56 / PR61），B'（PR72 / PR130）和B'（striatin / SG2NA）。根據已知文獻，PP2A可通過調節70 kDa核醣體蛋白S6激酶（p70S6K）之分子活性來調節生物體內的代謝和體內恆定機制，p70S6K是一種可接收營養和生長因子相關訊號傳遞路徑的蛋白激酶，並可把來自上游的mTOR分子訊號繼續往下傳遞。依據我們之前的研究成果，發現B56的其中一分型-B56γ3，參與了p70S6K的去磷酸化調控。然而，這兩分子間是透過什麼樣的互動機制來完成這樣的調控目前則尚不清楚。因此在本研究中，我們最主要的目標在於研究B56γ3與p70S6K之間交互作用的分子機制。首先，利用生物資訊學的分析，文獻中已有報告在PP2A-B56家族的幾種目前已知的受質上，皆發現一種高度保留的短線性的motif （簡稱為SLiM），利用此發現，我們預測了p70S6K上有兩個可能的B56γ3結合位點，分別位於E173和E424位置上。我們透過定點突變的實驗方法建構了p70S6K E424A和E173A兩種突變型表達載體，並用於探究E173A或E424A兩個突變位點對於p70S6K與B56γ3之間交互作用的影響。首先，我們利用HEK293T細胞株，將p70S6K WT，p70S6K E424A或p70S6K E173A表達載體與空載體或4HA-B56γ3表達載體共轉染至HEK293T細胞中，並進行了共免疫沉澱法的分析，透過這樣的實驗，發現p70S6K E424A和p70S6K E173A兩種突變型，相較於p70S6K WT型，均會顯著的降低p70S6K與PP2A-B56γ3之間相互作用。同樣地，我們接下來在NIH3T3，HeLa和HEK293T等細胞株中也進行了共轉染實驗，發現p70S6K的E424A和E173A的突變型均減弱了受到B56γ3所主導的，對於p70S6K的去磷酸化作用。另一方面，我們透過研究一系列B56γ3序列區段剔除突變型來比較對於p70S6K去磷酸化的效果，並試著找出在B56γ3分子上負責與p70S6K結合的相關位點或區域。利用相似的實驗方法，將p70S6K WT表達載體與空載體或4HA-B56γ3 WT（aa 1-514）、4HA-B56γ3 no NLS（aa 1-aa 405），4HA-B56γ3 no NLSΔ100 （aa 1-aa 305）、4HA-B56γ3 no NLSΔ200（aa 1-aa 205）和4HA- B56γ3 no NLSΔ300（aa 1-aa105）共轉染至細胞中，實驗結果顯示，在B56γ3上的NLS結構域（aa 406 – aa 514）的剔除後，減弱了B56γ3所主導的p70S6K去磷酸化作用，而再接續剔除B56γ3後面區段(分別剔除aa 106 至aa 404的區段)則發現p70S6K去磷酸化現象沒有進一步增加的趨勢，這樣的實驗數據指出，PP2A-B56γ3上位於aa 406 – aa 514區段的NLS結構域可能主導了B56γ3與p70S6K間的相關互動機制。總和來說，我們的實驗數據顯示，p70S6K的E424周圍和/或E173周圍的motif可以作為p70S6K上與B56γ3進行交互作用的區域。而在B56γ3分子結構中，與p70S6K交互作用位點則可能坐落於aa 406至aa 514的區域內。

Protein phosphatase 2A (PP2A) belongs to the serine/threonine protein phosphatase family, and is involved in regulating many cellular activities, such as cell signaling, cell growth, cell cycle, autophagy, apoptosis, transcription and translation. A PP2A holoenzyme is composed of a catalytic subunit (C), a structural subunit (A), and a highly variable regulatory subunit (B). The B subunit determines the substrate specificity and subcellular localization of PP2A. There are four subtypes of B subunits, including B(B55/PR55), B`(B56/PR61), B``(PR72/PR130), and B```(striatin/SG2NA). PP2A has been shown to regulate organismal metabolism and homeostasis by regulating the 70 kDa ribosomal protein S6 kinase (p70S6K), which functions as an integrator of nutrient and growth factor signals downstream of the mammalian target of rapamycin (mTOR) signaling. We and others have found that B56γ3, one of the B56 isoforms, is involved in PP2A- catalyzed dephosphorylation of the 70 kDa ribosomal S6 kinase (p70S6K). However, how B56γ3 interacts with p70S6K remains unclear. In this study, we aimed to investigate the molecular mechanism underlying the interaction between B56γ3 and p70S6K. In previous reports, bioinformatics analysis based on a conserved short linear motif (SLiM) found on several known substrates of PP2A-B56 family predicted two potential B56 binding sites, E424 and E173, of p70S6K. We created expression constructs of p70S6K E424A and p70S6K E173A mutant by site-directed mutagenesis and investigated the impact of mutation of E173A or E424A on p70S6K interaction with B56γ3. We performed co-IP analysis on lysates of HEK293T with co-transfection of expression vector of p70S6K WT, p70S6K E424A, or p70S6K E173A with empty vector or expression vector of 4HA-B56γ3 and found that both p70S6K E424A and p70S6K E173A showed reduced interaction with 4HA-B56γ3 as compared to p70S6K WT. Consistently, we performed transient co-transfection experiments and found that both mutation of E424A and E173A attenuated dephosphorylation of p70S6K by PP2A-B56γ3 in NIH3T3, HeLa, and HEK293T cells. On the other hand, we investigated the p70S6K binding domain of B56γ3 by investigating the effect of a series of deletion mutants of B56γ3 on mediating dephosphorylation of p70S6K. Results of co-transfection of expression vector of p70S6K WT with empty vector or expression vector of 4HA-B56γ3 WT(aa 1-aa 514), 4HA-B56γ3 no NLS (aa 1-aa 405), 4HA-B56γ3 no NLS Δ100 (aa 1-aa 305), 4HA-B56γ3 no NLS Δ 200 (aa 1-aa 205) and 4HA-B56γ3 no NLS Δ 300 (aa 1-aa 105) showed that loss of the NLS domain (aa 406-aa 514) attenuated the B56γ3-mediated p70S6K dephosphorylation and no further increases in p70S6K dephosphorylation with loss of the other domains (aa106-aa404) of B56γ3, suggesting that the NLS domain encompassing aa406-aa514 of B56γ3 may be involved in mediating p70S6K binding. In summary, our data showed that the motif surrounding E424 and/or the motif surrounding E173 of p70S6K may serve as a B56γ3 binding motif of p70S6K. In addition, the p70S6K interaction sites of B56γ3 may reside within the domain encompassing aa 406 to aa 514.

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