蛋白質磷酸酶 2A 型 (PP2A) 屬於一種三元複合體的酵素，由三種次單元所構成，依據次單元功能分別為定義為結構的 A 次單元，代表催化的 C 次單元以及具有調節能力的 B 次單元。磷酸酶 PP2A 目前被認為為在癌症中扮演了重要的抑癌角色。在許多關於調節性次單元 B 的研究當中，B56γ 次單元在過去被認為在 PP2A 調節癌化過程中扮演了一個很重要的角色，其可以透過調控細胞的生長以及運動來達到抑癌的作用。儘管如此，截至目前為止 B56γ 如何調控細胞運動的機制仍然不是很清楚，而且個別的 B56γ 次單元異構體是否扮演不同的角色也不是很確定。因此，我們想要探究帶有 B56γ3 調節次單元的磷酸酶 PP2A 對於細胞的運動機制的影響。首先，我們用免疫螢光分析法觀察；發現調節次單元 B56γ3 會與一個參與在形成細胞焦點黏著斑 (focal adhesion) 上的蛋白分子樁蛋白 (paxillin) 共同坐落在細胞焦點黏著斑上，接著利用共同免疫沉澱法我們也證明帶有 B56γ3 調節次單元的磷酸酶 PP2A 與樁蛋白有共同存在於一個複合體。之後，我們利用共同免疫沉澱法及試管中的蛋白質交互作用沉降方法證明 B56γ3 調節次單元可以透過樁蛋白之蛋白分子上的 LD 區段與其直接的結合。當進一步探討 PP2A 調節次單元 B56γ3 對樁蛋白的兩個分別調節細胞的型態以及爬行的重要的磷酸化位點，包括絲胺酸 83 以及絲胺酸 178，我們發現樁蛋白上的絲胺酸 178 之磷酸化可以被調節次單元 B56γ3 選擇性地調控，並且這樣的調控不是透過調控樁蛋白上游的 JNK 激酶的訊息傳遞路徑而達到。此外我們也發現樁蛋白的絲胺酸 83 的磷酸化會被 B56γ1 調節次單元所調控。相反的是，絲胺酸 83 以及絲胺酸 178 的磷酸化都不會被 B56α 所調控。更進一步，我們利用試管中去磷酸化反應實驗證明樁蛋白上磷酸化的絲胺酸 178 可以被帶有 B56γ3 調節次單元的 PP2A 去磷酸。因為樁蛋白上的磷酸化位點絲胺酸 178 對於細胞骨架蛋白分子的重組或細胞焦點黏著斑上的形成扮演了很重要的角色，所以，我們發現當 B56γ3 於細胞中過度表現的時候，細胞的一般運動以及在體外傷痕癒合分析下的運動能力會下降。相反地，當以核醣核酸干擾法將 B56γ3 表現減低的時候，細胞的運動以及在傷痕癒合的運動速度會上升。這些現象我們在不同類型的細胞株，包括 NIH3T3，HeLa 以及食道癌細胞 TE7 以及 BIC 皆得到相似的結果。此外我們也發現當 B56γ3 過度表現的時候，在以 transwell 侵襲移行運動分析法分析 NIH3T3 以及 HeLa 細胞穿透模板的侵襲會受到抑制，相反地，當 B56γ3 表現下降的時候，細胞的侵襲移行運動能力會上升。總而言之，我們的結果證明調節次單元 B56γ3 可以選擇性地調控樁蛋白絲胺酸 178 的磷酸化表現，並且進一步的調控細胞運動以及移行的能力。

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme composed of a structural A subunit, a catalytic C subunit, and a variable regulatory B subunit, and is thought to play a tumour suppressor role. Among various B subunits studied, the B56γ subunits of PP2A play an important role in tumour suppressor function of PP2A by regulating cell proliferation and motility. Previous studies have shown that mouse B56γ subunits modulate phosphorylation of paxillin and regulate cell motility. However, the role of B56γ in regulating motility and migration of human cancer cells and the molecular mechanism by which B56γ regulated paxillin phosphorylation has not yet been identified. Herein, we elucidate the mechanism for the regulation of cell motility by B56γ3-containing PP2A. We showed co-localization of B56γ3 and paxillin, a key scaffold protein of focal adhesion, and demonstrated association of paxillin and B56γ3-containing PP2A by co-immunoprecipitation (Co-IP). By Co-IP and in vitro pull-down analysis, we found that paxillin directly interacted with B56γ3 through the LD domain. Next we investigated the role of B56γ3 in regulation of two key residues of paxillin, Ser83 and Ser178, were previously shown to be involved in regulating cell morphology and migration, respectively, and found that B56γ3 selectively regulated phosphorylation of Ser178, but not Ser83, without affecting the activity of upstream kinase JNK. In comparison to B56γ3, paxillin phosphorylation of Ser83 was preferentially regulated by B56γ1, and neither phosphorylation at Ser83 or Ser178 was affected by B56α. Furthermore, we demonstrated B56γ3-containing PP2A directly catalyzed dephosphorylation of paxillin Ser178 by in vitro dephosphorylation assay. Consistent with the role of paxillin phosphorylation at Ser178 in regulating focal adhesion and cytoskeleton rearrangement, cells overexpressing B56γ3 displayed reduced stress fibers. Furthermore, B56γ3 overexpression reduced cell motility and wound-healing migration, whereas silencing B56γ3 expression markedly enhanced motility and migration in NIH3T3, HeLa, and human esophageal cancer cells. We also demonstrated that the ability of cell invasion was reduced when cells were overexpressed with B56γ3, whereas silencing B56γ3 expression markedly enhanced cell invasion in NIH3T3 and HeLa cells. In conclusion, our data demonstrate that the B56γ3 subunit selectively directs PP2A to regulate phosphorylation of paxillin at Ser178 and regulate cell motility and migration.

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