蛋白磷酸水解酶2A型 (簡稱PP2A) 是主要的絲氨酸/蘇氨酸磷酸酶之一，且在哺乳動物細胞中廣泛表達。PP2A完全酶由三個次單元所組成，包括構成結構骨架的A次單元、負責催化功能的C次單元以及種類多變並負責受質選擇的B次單元。根據先前研究，B56γ3調節次單元在PP2A的腫瘤抑制功能中扮演著重要角色。另一方面，由AKT、mTOR和p70S6K組成的訊息軸線是負責整合營養物質和生長因子對細胞的刺激反應的訊息傳遞，以驅動細胞生長，但經常在癌症中經常失去控制。我們的實驗室先前的研究發現在多種細胞中，在恆定狀況下過度表現B56γ3會提高AKT磷酸化程度。相反的，p70S6K的活化反而被B56γ3下調。在此研究中，在生長因子的刺激下，我們發現 B56γ3過度表現增強了AKT在Thr308與Ser473位點磷酸化的，這兩個磷酸化位點是AKT完全活化的標誌，相反的，將B56γ3穩定調低表現時，則降低了AKT在Thr308與Ser473位點磷酸化的程度。我們發現在胰島素刺激的NIH 3T3細胞中的AKT 磷酸化程度，顯著的受B56γ3過度表現而提高，但在表皮生長因子刺激的HCT116細胞中的結果並不明顯。相反的，相較於對照組細胞，在穩定降低表達B56γ3的HCT116細胞，以表皮生長因子刺激AKT磷酸化的程度則顯著的降低。同樣的，相較於對照組細胞，在穩定降低表達B56γ3的HeLa細胞及SW480細胞，以胰島素、表皮生長因子或胰島素類生長因子-1刺激AKT磷酸化的程度有顯著的降低。另一方面，相較於對照組細胞，B56γ3過度表達下的HCT116細胞，僅稍微降低表皮生長因子刺激的p70S6K Thr389磷酸化的程度。而在穩定調低表達的B56γ3的HeLa細胞中，在胰島素或表皮生長因子刺激下，但有顯示出一種上調了p70S6K Thr389位點的磷酸化的趨勢。另外，NIH3T3細胞中，B56γ3過度表達，在胰島素刺激或是胺基酸補充時，都顯著降低了磷酸化p70S6K的程度，而在SW480細胞中穩定降低表達B56γ3時，顯著增加了磷酸化 p70S6K的程度。此外，我們探討了 p70S6K所介導的生長因子訊息傳導負回饋調節在B56γ3上調 AKT 磷酸化中的角色，而我們的數據顯示，B56γ3過度表達導致的AKT 磷酸化程度增加，會因持續活化態的 p70S6K-E389D3E突變型的共表達而降低，但不活化態的p70S6K-K100R突變型則沒造成調降影響。另一方面，我們的數據顯示，透過使用p70S6K抑製劑 LY-2584702處理細胞，可以讓因穩定調低表現B56γ3導致的AKT磷酸化降低的程度回升。這些結果顯示B56γ3上調 AKT 磷酸化的現象，是可能藉由調控p70S6K所主導對生長因子訊息傳導的負回饋調節機制而造成。接著我們研究B56γ3促成的AKT激活是否可以促進化療藥處理後的癌細胞的存活，我們使用5-氟尿嘧啶 (5-FU) 處理大腸直腸癌細胞來探討B56γ3的影響。我們發現B56γ3過度表達促進了大腸直腸癌細胞的存活並提高了這樣的細胞對5-FU反應的半抑制濃度(IC50)，而這樣的存活提高現象在AKT 抑製劑MK-2206共同處理細胞時被抵消。如預期的，MK-2206處理減弱B56γ3過度表達所引起的AKT活化。相反的，B56γ3穩定調低表現讓大腸直腸癌細胞在5-氟尿嘧啶(5-FU)處理下，表現出增加5-氟尿嘧啶(5-FU)的藥物敏感性及降低對5-FU反應半抑制濃度(IC50)。綜合所得到的結果，我們認為具有B56γ3調節次單元的PP2A可將生長因子刺激而活化的p70S6K的活性調降，隨後藉減弱 p70S6K所主導對生長因子訊息傳導的負回饋調節機制而導致AKT 活化，從而促進接受化療藥物處理的大腸直腸癌細胞的存活。

Protein phosphatase 2A (PP2A) is one of the major serine/threonine phosphatases and widely expressed in mammalian cells. A PP2A holoenzyme consists of three subunits including a scaffolding A subunit, a catalytic C subunit, and a variable regulatory B subunit. The B56γ3 regulatory subunit of PP2A has been reported to play an important role in the tumor suppressor function of PP2A. On the other hand, the signaling axis composed of AKT, mammalian target of rapamycin (mTOR), and 70 kDa ribosomal S6 kinase (p70S6K) integrates stimuli of nutrient and growth factors to drive cell and organismal growth and is often deregulated in cancer. Our lab previously showed that B56γ3 plays a positive role in regulating AKT phosphorylation in several cell lines at steady state. In contrast with AKT, p70S6K activation was showed to be down-regulated by B56γ3. Here, we found that B56γ3 overexpression enhanced levels of phospho-AKT at both Thr308 and Ser473, hallmarks of a full AKT activation, whereas B56γ3 overexpression increased levels of phospho-AKT in insulin-stimulated NIH3T3 cells, but modestly in EGF-stimulated HCT116 B56γ3 overexpression cells. Knockdown of B56γ3 expression significantly decreased levels of phospho-AKT in EGF-stimulated HCT116 cells, insulin or EGF-stimulated HeLa cells, and IGF-1-stimulated SW480 cells. B56γ3 overexpression only modestly reduced the level of phospho-p70S6K at Thr389 in EGF-stimulated HCT116 cells, whereas knockdown of B56γ3 showed a trend, in up-regulating the level of phospho-p70S6K at Thr389 in insulin or EGF-stimulated HeLa cells. However, B56γ3 overexpression significantly reduced levels of phospho-p70S6K in insulin or amino acid-stimulated NIH3T3 cells, and knockdown of B56γ3 significantly increased levels of phospho-p70S6K in IGF-stimulated SW480 cells. Furthermore, we addressed the role of p70S6K-mediated negative feedback regulation in B56γ3 up-regulating AKT phosphorylation, and our data showed that increased levels of phospho-AKT by B56γ3 overexpression were decreased by co-expression of constitutively activated p70S6K-E389D3E mutant, but not by kinase-dead p70S6K-K100R mutant. On the other hand, decreases in levels of phospho-AKT in cells with B56γ3 knockdown were restored by treating cells with p70S6K inhibitor LY-2584702. To investigate whether B56γ3-mediated AKT activation can promote cancer cell survival upon chemotherapy, we treated colorectal cancer (CRC) cell lines with or without B56γ3 overexpression with 5-Fluorouracil (5-FU). We found that B56γ3 overexpression promoted survival of CRC cells and increased IC50 of the 5-FU treatment, which was abolished when cells were co-treated with AKT inhibitor MK-2206. As expected, MK-2206 treatment attenuated increases in levels of AKT phosphorylation by B56γ3 overexpression. On the other hand, CRC cells with B56γ3 knockdown showed increased sensitivity and decreased IC50 under 5-FU treatment. In sum, the B56γ3 regulatory subunit of PP2A down-regulates p70S6K activation in response to growth factor stimulation and subsequently causes up-regulation of AKT activation through attenuating p70S6K-mediated negative feedback regulation on growth factor-stimulated signaling, regulating in increased survival of CRC cells treated by chemotherapeutic drugs.

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