磷酸酶PP2A是一種絲胺酸/蘇胺酸磷酸酶，組成PP2A的三個次單元分別是構成結構鷹架的A次單元，負責催化功能的C次單元，以及種類相當多樣的調節性B次單元。這些種類繁多的B次單元可以決定PP2A全酶的活性、受質特異性，以及其在細胞中的位置。我們的研究主要聚焦在探討具有腫瘤抑制功能的B56γ3調節次單元，座落於細胞核的分子機制。我們實驗室之前已經發現，在NIH3T3細胞中，當細胞處在細胞間期時，B56γ3主要是核質均勻的分布在整個細胞中。而當細胞被同步化在細胞週期的S時期時，B56γ3則會集中在核。根據序列分析，我們預測了至少四個核定位信號(nuclear localization signals, 以下簡稱NLSs), 分別是NLS1, 2, 3, 與4。這四段NLS彼此相當靠近且座落於B56γ3的C端。我們的結果顯示，刪除這四段可能的NLS都不影響 B56γ3於核的座落。而更進一步刪除預測之NLS1上游的100個胺基酸，則會將B56γ3侷限在細胞質，與此結果一致的是，包含這100個胺基酸的蛋白序列足以驅使EGFP-PK進入細胞核內。另一方面，依序截斷這些可能的NLS及其周邊的序列後發現，刪除預測的NLS2會促進B56γ3在細胞間期時，集中在核的比例，進一步刪除位於NLS1與NLS2中的連接序列，則會阻止在細胞間期以及細胞週期S時期，B56γ3集中在核的比例。此外，探討調節B56γ3於細胞週期S時期，集中在核的訊息傳遞路徑，我們發現，抑制AMPK的活性會降低 B56γ3不論是在細胞間期或細胞週期S期，集中在核的比例，而這樣核集中比例下降的情形，則與AMPK抑制劑的濃度呈現正相關。與此一致地，突變位於NLS1與NLS2連接序列上，可能的AMPK磷酸化位點—絲胺酸440，使其無法再被磷酸化，則會減少B56γ3於細胞週期S時期，集中在核的情形。入核轉運受體(以下稱importin)之一的importin-α是一種轉接蛋白(adaptor protein)，透過importin-α結合帶有NLS的貨物蛋白與importin-，使得帶有NLS的貨物能被運輸至細胞核內。如同預期，胞外pull down分析結果顯示，B56γ3會與importin-α及importin-β有交互作用。綜合實驗結果，首先，我們發現，B56γ3有一段區域對於其細胞週期S時期的進核極為關鍵，同時，我們的結果也說明了B56γ3可能藉由importin-α仰賴抑或是importin-α不相關的方式進核。

Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase which comprises three subunits: the scaffolding A subunit, the catalytic C subunit, and the variable regulatory B subunit. The B subunits are involved in determining the activity, substrate specificity, and subcellular localization of PP2A. Our study mainly focused on characterization of molecular mechanisms underlying nuclear localization of B56γ3, which has been reported to function as a tumor suppressor regulatory subunit of PP2A. We have found that in NIH3T3 cells, B56γ3 is distributed homogenously throughout the cell in steady state, and becomes enriched in the nucleus during S phase in cell cycle-synchronized cells. We have predicted at least four nuclear localization signals (NLSs), NLS1, 2, 3, 4, located closely to each other at the C terminus within B56γ3 by sequence analyses, but found that deletion of these putative NLSs did not impair B56γ3 nuclear localization. Further truncation of 100 amino acids upstream of the putative NLS1 confined the B56γ3 in cytosol, and the domain encompassing the 100-amino acid drove fused EGFP-PK into the nucleus. Serial truncation analyses of the putative NLSs and their flanking sequences showed that loss of the putative NLS2 after deletion of putative NLS3 and NLS4 resulted in enhanced levels of nuclear localized B56γ3 in steady-state cells. Subsequent deletion of the linker between the putative NLS1 and NLS2 abolished the B56γ3 nuclear enrichment phenotype in both asynchronous and S-phase synchronized cells. To identify the signaling pathway regulating the S-phase nuclear enrichment of B56γ3, we found that inhibition of AMPK reduced nuclear localization of B56γ3 in both steady state and S phase in a dose-dependent manner, and phosphorylation defective mutation at Ser440, the potential AMPK targeting site within the linker region, impaired the S-phase nuclear enrichment of B56γ3. Further, in vitro pull down analyses showed that B56γ3 interacts with both importin-α and importin-β. In addition, co-immunoprecipitation analyses revealed that B56γ3 was associated with importin-β in cells. In summary, we found domains crucial for steady state and S-phase nuclear enrichment of B56γ3, and our data also suggest that B56γ3 may enter the nucleus through an importin-α-dependent and/or -independent mechanism.

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