Investigate the cellular functions of PP2A phosphatase activator (PTPA)

  • 楊 宛璇

Student thesis: Master's Thesis


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
Date of Award2016 Feb 1
Original languageEnglish
SupervisorChi-Wu Chiang (Supervisor)

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