磷酸酶PP2A是一種絲胺酸/蘇胺酸磷酸酶，並且調節許多細胞的功能，組成PP2A的三個次單元分別是構成結構的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。雖然B次單元被認為可以控制PP2A在細胞中座落的位置，但其本身在細胞內座落位置如何調節的分子機制還是未知的。這裡我們報告有關利用缺失突變與雙分子螢光互補作用分析出調節B56γ3調節次單元座落在細胞核的功能位與訊息傳遞路徑。我們發現至少有兩個區域影響了B56γ3座落在核的分佈，包括預測的NLS1和NLS2之間的連接序列(aa 413 to aa461) 與 預測的NLS1上游的100個胺基酸(aa306 to aa405) ，因為去掉這兩個區域之一都會增加座落以細胞質為主的B56γ3。
入核轉運受體importin-α是一種轉接蛋白(adaptor protein)，透過importin-α結合帶有NLS的分子與importin-β結合，使得帶有NLS的分子能被運輸至細胞核內。在in vitro pull down的實驗中顯示全長的B56γ3會分別與importin -α and –β有交互作用，而當失去aa306-aa405這100個胺基酸則會降低B56γ3與importin -α and –β的交互作用但失去在aa41到461的區域則不會影響importin -α及importin –β的的結合。為了瞭解調節B56γ3的進核的分子機制，進一步的利用處理訊息傳遞激脢抑制劑篩選出可能參與在調節B56γ3座落在核的訊息傳遞路徑，我們發現了CK1 的抑制劑顯著的減少B56γ3在核的分佈，接著我們利用shRNA降低CK1δ表現，發現其的確降低了B56γ3在核的分佈。免疫沉澱實驗證明了B56γ3與CK1δ有交互作用，並且在in vitro CK1δ可以磷酸化B56γ3在Ser440，S440是一個被序列分析預測可能的CK1催化的磷酸位點，且座落在我們發現可調節B56γ3進核的NLS1和NLS2之間連接序列區域中。於是我們研究B56γ3的磷酸化缺陷突變(S440A) 的影響，發現其增加了B56γ3在細胞質的分佈，而模仿磷酸化的突變(S440D)則大量增加在細胞核的分佈。此外，我們也發現抑制CK1時，會降低了B56γ3的蛋白質量，而處理了蛋白水解脢抑制劑MG132則恢復了受CK1抑制而降低的B56γ3蛋白的量。此外，磷酸化的突變S440A及S440D則對由抑制CK1導致的降解產生抗性。總結 ， 我們的結果發現了CK1δ透過磷酸化調節PP2A的B56γ3調節次單元的進核並且控制其蛋白質穩定度。

Protein phosphatase 2A (PP2A) is a major cellular serine/threonine phosphatase and regulates many cellular processes. The PP2A holoenzyme consists of a scaffolding A subunit, a catalytic C subunit, and a variable regulatory B subunit. Although the regulatory B subunit has been proposed to control subcellular localization of PP2A, the mechanism underlying subcellular localization of the regulatory B subunit remains largely unknown. Here, we report the investigation on the functional motifs and signaling pathways regulating nuclear localization of the B56γ3 regulating subunit using deletion mutation and bimolecular fluorescence complementation (BiFC) analysis. We found at least two domains involved in nuclear localization of the B56γ3, including the linker region (aa 413 to aa 461) between the putative NLS1 and NLS2 and the region encompassing aa 306 to aa 405. Loss of either of the two domains impaired B56γ3 nuclear localization. More, B56γ3 directly interacts with both importin -α and –β in vitro, and loss of the region encompassing aa 306 to aa 405, but not the linker region, markedly abolished the interaction. Further, we screened a panel of kinase inhibitors for signaling pathways which may be involved in nuclear localization of B56γ3 and found that inhibition of casein kinase I (CK1), but not CDK2, MEK2, and PI3K/AKT, significantly reduced nuclear localization of B56γ3. Consistently, knockdown of CK1δ with short hairpin RNA(shRNA) reduced B56γ3 nuclear localization. Reciprocal co-immunoprecipitation analysis demonstrated that B56γ3 was associated with CK1δ, and CK1δ catalyzed phosphorylation of B56γ3 at Ser440 in vitro. Overexpression of CK1δ increased levels of phosph-S440 of B56γ3, whereas knockdown of CK1δ reduced levels of phospho-S440 of B56γ3. Since Ser440 is located within the linker domains found to be involved in regulating B56γ3 nuclear localization, we investigated the phosphorylation defective mutant (S440A) of B56γ3 and found markedly increased cytoplasmic localization of B56γ3 by this mutation. In contrast, a phosphorylation mimetic mutant (S440D) of B56γ3 showed substantially increased nuclear localization as compared to wildtype B56γ3. Inhibition of CK1 reduced the phosphorylation level of phospho-Ser440, and unexpectedly, also reduced the protein level of B56γ3. Treatment of cells with protease inhibitor MG132 restored the B56γ3 protein levels, whereas both B56γ3S440A and B56γ3S440D were resistant to CK1 inhibition-mediated degradation and were not affected by MG132 treatment. In summary, our data demonstrate that CK1δ phosphorylates B56γ3 at Ser440 to regulate the nuclear localization of B56γ3 and may control B56γ3 protein stability.

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