Study the tumor suppression mechanism of the B56γ3 regulatory subunit of PP2A

  • 賴 泰佑

Student thesis: Doctoral Thesis


PP2A is a heterotrimer protein complex composed of a structural A subunit a catalytic C subunit and one variable regulatory B subunits and accounts for most serine/threonine phosphatase activity in mammalian cells Previous studies have shown that A and B56 subunits play important roles in tumor suppression Here we investigated how B56γ3 delays the G1/S transition and S phase progression During the time course when NIH3T3 cells were stimulated to re-enter cell cycle cells with B56γ3 overexpression showed higher protein levels and reduced phosphorylation of p27KIP1 at Thr187 (phospho-Thr187) whose phosphorylation promotes degradation of p27KIP1 compared with cells expressing vector control Similarly the levels of p27KIP1 were increased by B56γ3 overexpression and decreased by B56γ3 knockdown The co-immunoprecipitation and in vitro pull down assay showed that B56γ3 directly interacts with p27KIP1 In vitro PP2A-B56γ3 catalyzed dephosphorylation of phospho-Thr187 in a dose-dependent manner Together we demonstrate that PP2A-B56γ3 enhances the p27 level to delay the G1/S transition and S phase progression In addition to p27KIP1 stability the subcellular localization of p27KIP1 also determines its role in cancer progression The cytoplasmic mislocalization of p27KIP1 cooperates with Ras to promote tumor progression B56γ3 overexpression enhanced nuclear localization of p27KIP1 whereas knockdown of B56γ3 decreased p27KIP1 nuclear localization B56γ3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157) whose phosphorylation promotes cytoplasmic localization of p27KIP1 whereas B56γ3 knockdown significantly increased the level of phospho-Thr157 In vitro PP2A-B56γ3 catalyzed dephosphorylation of phospho-Thr157 in a dose-dependent and okadaic acid-sensitive manner B56γ3 did not increase p27KIP1 nuclear localization by down-regulating the upstream kinase Akt activity and outcompeted a myristoylated constitutively active Akt (Aktca) in regulating Thr157 phosphorylation and subcellular localization of p27KIP1 In addition results of interaction domain mapping revealed that both the N-terminal and C-terminal domains of p27 and a domain at the C-terminus of B56γ3 are required for interaction between p27 and B56γ3 Furthermore we demonstrated that p27KIP1 levels are positively correlated with B56γ levels in both non-tumor and tumor parts of a set of human colon tissue specimens However positive correlation between nuclear p27KIP1 levels and B56γ levels was found only in the non-tumor parts but not in tumor parts of these tissues implicating a dysregulation in PP2A-B56γ3-regulated p27KIP1 nuclear localization in these tumor tissues Together we demonstrate that PP2A-B56γ3 suppresses tumor progression through enhancing p27KIP1 stability and nuclear localization of p27KIP1by catalyzing the dephosphorylation of p27KIP1 at Thr187 and Thr157 respectively Previous reports have implicated that PP2A-B56γ holoenzymes participate in regulating cellular metabolism through down-regulating p70S6K1 activity Thus we investigated whether PP2A-B56γ3 can dephosphorylate p70S6K1 We found that overexpression of B56γ3 reduced p70S6K phosphorylation at Thr389 whereas knockdown of B56γ3 resulted in increased p70S6K phosphorylation at Thr389 in steady state EGF or LY294002 treatment As expected the phosphorylation of p70S6K1 downstream substrate S6 was decreased by B56γ3 overexpression but increased by B56γ3 knockdown The cell size of cells with B56γ3 overexpression was reduced compared to that in cells with vector control whereas the cell size of cells expressing shB56γ3 was increased compared to that in cells expressing shLuc Results of co-immunoprecipitation in vitro pull down assay and in vitro dephosphorylation assay demonstrated that PP2A-B56γ3 directly catalyzed p70S6K1 dephosphorylation in a dose-dependent and okadaic acid-sensitive manner In contrast the phospho-Ser2448 of mTOR and phospho-S65 of 4EBP1 were not reduced by PP2A-B56γ3 Altogether our study provides novel mechanisms by which the PP2A-B56γ3 holoenzyme plays its tumor suppressor role
Date of Award2016 Mar 21
Original languageEnglish
SupervisorChi-Wu Chiang (Supervisor)

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