帶有B56γ3調節次單元的PP2A磷酸水解酶(PP2A-B56γ3)已知可以扮演一個抑癌的角色。我們發現了PP2A-B56γ3會透過以下的機制來抑制癌症:減緩細胞生長、延遲細胞週期進行、增加p27KIP1的穩定表現及使其入核來降低CDK2的活性、及降低p70S6K1的磷酸化及活性來抑制mTOR/p70S6K1 signaling pathway。在NIH3T3細胞重新進入細胞週期時，當有B56γ3大量表現，會有延遲G1/S transition和S phase progression的現象。同時，在serum starvation後加回serum的實驗中，我們發現隨著時間進程，有B56γ3大量表現的細胞，p27KIP1表現量會增加，且p27KIP1在蘇氨酸187位點的磷酸化會有減少的現象。蘇氨酸187位點的磷酸化會導致p27KIP1的穩定性下降，進而減少表現量。同樣的，在HeLa細胞，當B56γ3減少表現的時候，p27KIP1的表現量也會下降。進一步的，我們透過Co-immunoprecipitation、in vitro pull down analysis和in vitro dephosphorylation等實驗方法，發現了B56γ3確實會直接與p27KIP1有交互作用，且p27KIP1確實是PP2A-B56γ3的直接受質。
PP2A-B56γ3除了會增加p27KIP1的穩定性之外，我們也發現當B56γ3大量表現的時候會增加在細胞核中的p27KIP1；反之，當B56γ3減少表現的時候，就會減少細胞核中的p27KIP1。大量表現的B56γ3會減少p27KIP1在蘇氨酸157的磷酸化；反之，減少表現的B56γ3則會增加p27KIP1在蘇氨酸157的磷酸化。已知蘇氨酸157位點的磷酸化會促進p27KIP1坐落在細胞質中，相反的，蘇氨酸157的去磷酸化會促進p27KIP入核，我們以in vitro dephosphorylation的實驗，發現PP2A-B56γ3對於p27KIP在蘇氨酸157的去磷酸化的催化作用是dose-dependent及okadaic acid-sensitive manner。進一步的，我們發現B56γ3並不是透過降低Akt的活性來減少p27KIP1在絲氨酸157的磷酸化及使得p27KIP1進入細胞核中。而在Domain mapping的實驗中，我們發現p27KIP1的N-端及C-端，以及B56γ3的C-端對於兩者之間的交互作用非常重要。而在臨床檢體中，透過Immunohistochemistry的方法，我們發現p27KIP1的表現量和B56γ的表現量無論是在非腫瘤或腫瘤組織中皆呈現正相關。然而，細胞核中p27KIP1的表現量僅在非腫瘤的部分會和B56γ呈現正相關；在腫瘤組織的部分則沒有關係。這樣的結果暗示了在這些tumor的組織中，PP2A- B56γ3對於p27KIP1坐落在細胞核中的調控失衡了。
接著我們證明了PP2A-B56γ3會去磷酸化p70S6K1的蘇氨酸389這個位點，並且同樣不是透過降低Akt的方式來降低p70S6K1的磷酸化。我們發現當大量表現B56γ3確實可以降低p70S6K1在蘇氨酸389的磷酸化；而降低細胞內的B56γ3表現量則可以提升p70S6K1在蘇氨酸389的磷酸化，且這樣的調控能力，不僅是在Steady state下，在處理EGF或LY294002的情況下也一樣。但是，PP2A-B56γ3並不降低mTOR和4EBP1的磷酸化。同樣的，S6是p70S6K1的直接下游受質，其磷酸化也會在大量表現B56γ3時下降，降低表現B56γ3時上升。而且co-immunoprecipitation、in vitro pull down analysis和in vitro dephosphorylation assay等分析結果證明了PP2A-B56γ3磷酸水解酶確實是直接將p70S6K1去磷酸化，且這樣的作用是does-dependent manner及okadaic acid-sensitive manner。總之，我們的研究結果對於PP2A-B56γ3 holoenzyme如何扮演一個抑癌分子的角色提供了新的機制與發現。

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.

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