蛋白質磷酸酶2A型 (PP2A) 是真核細胞中一種主要的絲氨酸/蘇氨酸磷酸水解酶，在許多細胞功能調控過程中扮演必要的角色。磷酸酪胺酸磷酸酶活化分子 (PTPA)，又稱為PP2A磷酸水解酶活化分子，氨基酸序列分析結果顯示此分子從酵母菌到人類在演化中被高度保留，對活化態PP2A完全酶的產生是很重要的調控者，但是目前對於PTPA調控PP2A的活化確切機制以及PTPA在哺乳類動物細胞中所扮演的生理角色仍然是大部分未知的。在此，我們探討PTPA對調節細胞生長、運動的角色及其可能參與的調控路徑。首先我們運用雙分子螢光互補法 (BiFC) 的方法確認PP2Ac與PTPA所形成的複合體是以細胞核質均勻分佈為主。接著，我們也運用了免疫沉澱法 (co-IP) 驗證了內生性的PP2A/A能夠與構成BiFC的PP2Ac和PTPA形成複合體，推測內生性的PP2A/A、PP2Ac及PTPA可能會以三聚體的型式存在於細胞當中。雖然我們發現在NIH3T3、HeLa及Hep3B細胞中過度表現PTPA對於細胞增殖沒有顯著的變化，相反地，當以核醣核酸干擾法將PTPA表現減低時，會顯著減少細胞增殖速度。同時也發現在NIH3T3及HeLa細胞中將PTPA表現減低時，細胞呈現紡錘狀的數目增多，也伴隨細胞型態狹長的改變，並顯著減少actin stress fiber的形成，及大幅降低細胞的傷痕癒合運動能力。另一方面，當PTPA表現減低時，雖然Hep3B細胞actin stress fiber的形成改變是不明顯的，但對於細胞的傷痕癒合運動能力也是大幅降低的。在HeLa細胞中，我們也發現將PTPA表現減低時，能提高RhoAGTPase的活性，卻降低Rac1GTPase的活性。接著，我們探討PTPA可能是經由哪些相關的訊息傳遞路徑來造成這些細胞現象，在NIH3T3細胞中，過度表現與PP2Ac結合有缺陷的PTPA突變型 (N277K) 而非野生型的PTPA，會減少PP2Ac單元的表現量；減低PTPA表現會減少AKT的絲氨酸473與蘇氨酸308位點的磷酸化程度。此外，我們也探討了在NIH3T3細胞中PTPA對於mTOR下游p70S6K的影響，並發現減低PTPA表現會使p70S6K的蘇氨酸389位點的磷酸化降低。在HeLa細胞中，減低PTPA表現會顯著減少B56調節次單元家族的表現量。在Hep3B細胞中，PTPA過度或是減低表現僅會些微影響PP2A A、B和C次單元的表現量。在HeLa與Hep3B中PTPA表現量降低會提高AKT絲氨酸473與蘇氨酸308兩位點的磷酸化程度，在HeLa細胞中PTPA表現減低會使p70S6K的表現量降低。在Hep3B細胞中PTPA表現量降低會提高mTOR下游受質p70S6K與S6的磷酸化表現，同時也會提高AKT下游的受質GSK3的磷酸化表現量。過度表現野生型的PTPA而非與PP2Ac結合有缺陷的PTPA突變型 (N277K)，會提高E-cadherin的表現量，相反地，減低PTPA表現會降低E-cadherin的表現量。總結以上結果，我們發現PTPA對於PP2A/A、PP2Ac、AKT、AKT絲氨酸473與蘇氨酸308位點的磷酸化、p70S6K蘇氨酸389位點的磷酸化有不同的調控角色。在NIH3T3與HeLa細胞中，PTPA表現降低造成細胞生長、stress fiber生成與細胞運動的減少。除此，降低PTPA表現量會提高細胞型態的轉變，增加紡錘狀的細胞型態，降低E-cadherin的表現量程度，可能參與上皮細胞間質轉化 (EMT) 的過程，因此，PTPA在細胞中所扮演的角色可能是具有多效性功能的。

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase that plays an essential role in many cellular processes in eukaryotic cells. Phosphotyrosyl phosphatase activator (PTPA), also known as PP2A phosphatase activator is highly conserved from yeast to human and is critical for forming active PP2A holoenzymes, but the exact mechanism underlying PTPA-mediated activation of PP2A and whether PTPA plays other roles in cells are largely unknown. Here, we report on the investigation of the role of PTPA in cell proliferation and motility, and the role in regulating the signaling pathways involved in controlling cell growth, proliferation, and motility. Bimolecular fluorescence complementation (BiFC) analysis was applied to investigate the formation and subcellular localization of the complex formed between PP2Ac and PTPA. The results of BiFC analysis showed that the complex of PP2Ac and PTPA displayed a ubiquitous pattern throughout the entire cell. Results of co-immunoprecipitation (co-IP) showed that the endogenous PP2A/A was also found to associate with the BiFC complex of PP2Ac and PTPA, suggesting that PP2A/A, PP2Ac and PTPA may form heterotrimeric complexes in cells. Furthermore, we found that PTPA overexpression showed modest effect on cell proliferation, but PTPA knockdown notably reduced cell proliferation of NIH3T3, HeLa and Hep3B cells. NIH3T3 and HeLa cells with stable PTPA knockdown showed increased numbers of cells with an elongated spindle-shaped morphology and showed markedly reduced amounts of stress fibers. In agreement with the reduced amounts of stress fibers, PTPA knockdown significantly reduced cell wound-healing migration of both NIH3T3 and HeLa cells. On the other hand, Hep3B cells displayed very few stress fibers and had no obvious changes of stress fiber formation by PTPA knockdown, but significantly reduced cell wound-healing migration. In HeLa cell, PTPA knockdown further increased the level of active RhoA, but decreased the level of active Rac1. Next, we investigated the signaling molecules regulated by PTPA. In NIH3T3 cells, overexpression of the PP2Ac-binding defective mutant form of PTPA (N277K), but not the wild-type PTPA, significantly reduced the levels of C subunits. PTPA knockdown decreased phosphorylation levels of AKT at both T308 and S473. In addition, we investigated the effect of PTPA on mTOR downstream target p70S6K, and found that PTPA knockdown reduced the phosphorylation of p70S6K at T389. In HeLa cells, PTPA knockdown significantly decreased levels of B56 family subunits. In Hep3B cells, PTPA overexpression and knockdown slightly modulated levels of PP2A A, B, and C subunits. In addition, in HeLa and Hep3B cells, PTPA knockdown increased phosphorylation levels of AKT at both S473 and T308. In HeLa cells, PTPA knockdown only reduced the p70S6K protein level. In Hep3B cells, PTPA knockdown increased phosphorylation levels of mTOR downstream target p70S6K and S6, and the AKT downstream target GSK3-β. Overexpression of wild-type PTPA, but not the PP2Ac-binding defective mutant PTPA (N277K), increased the level of E-cadherin, whereas PTPA knockdown reduced the level of E-cadherin. Our data demonstrate that PTPA differentially regulates levels of PP2A/A, PP2Ac, AKT, p70S6K, phospho-AKT Thr308, phospho-AKT Ser473, phospho-p70S6K Thr389 in a cell context-dependent manner. PTPA knockdown results in reduced cell proliferation, reduced amounts of stress fiber, and reduced cell motility in both NIH3T3 and HeLa cells. In addition, PTPA knockdown increased cells switching to spindle-shape morphology and reduced E-cadherin levels, suggesting a role in epithelial to mesenchymal transition (EMT). Together, PTPA has pleiotropic functions in various aspects of cell processes, ranging from cell proliferation to EMT.

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