探討STK40如何調控YAP的活性與其在細胞核質的移動

Abstract

細胞內的運作機制非常複雜，通常需要經過不同pathway的crosstalk分工才能完成。包含常見的cell migration.我們實驗室在2019年透過two-hit screen的方式找到一個組合，可以更好的干擾細胞遷移，並命名為合成阻動(synthetic dysmobility)。同時使用 MEK 抑制劑 Trametinib 抑制 MAPK 信號通路，並敲低絲胺酸/蘇胺酸激酶 40（STK40）。結果顯示，雙重處理後的細胞遷移能力顯著降低。細胞遷移受 Yes相關蛋白 (YAP)控制，我們也發現在敲低STK40後，YAP會從細胞核移位到細胞質。由於目前關於STK40的研究很少，且STK40如何調控 YAP 的機制尚不清楚。基於此，我們對STK40對YAP的調控方式初步研究。在先前的研究數據顯示，和一般隨著細胞密度增加，而YAP會隨之從細胞核移位至細胞質不同。敲低 STK40後的細胞，即使增加細胞密度，細胞核內 YAP 水平無顯著降低。而當過表達 STK40 時，YAP 似乎與 STK40 共定位，這意味著 STK40 可能影響 YAP 的分布。此外，高細胞密度會將 STK40 和 YAP 轉運到細胞質中。我們進一步假設了幾種 STK40 調控 YAP 的機制。第一個機制涉及 Hippo 通路， Hippo 通路的核心成員 MST1/2 和 LATS1/2 可促進YAP磷酸化，使其轉移到細胞質。我們使用小分子抑制劑抑制它們的活性，並同時使用 shRNA 慢病毒敲低 STK40，結果顯示部分 YAP 仍然轉移到細胞質，這表明可能有其他因子可以影響STK40 調控 YAP 的移位。第二個假設是 STK40 通過核運輸蛋白影響 YAP 分布。蛋白質需要核運輸蛋白來進出細胞核，內輸蛋白和外輸蛋白負責這些任務。內輸蛋白將蛋白質運輸進細胞核，而 外輸蛋白則執行相反的功能。我們使用小分子抑制劑 Leptomycin B（針對外輸蛋白）和 Ivermectin（針對內輸蛋白），並搭配 shRNA 慢病毒同時敲低 STK40，觀察到 Leptomycin B 處理組的結果與 LATS 抑制劑處理組類似，而同時敲低STK40並加 Ivermectin 的組別與單純敲低STK40的組別則沒有在核質分布上有顯著改變。這強烈表明 STK40 可能通過影響內輸蛋白來影響 YAP 的分布。最後，在以添加小分子抑制劑Y27632（ROCK 抑制劑）模擬細胞密度上升時，細胞骨架張力減弱對 STK40 的影響時，結果顯示 YAP 轉移到細胞質。有趣的是，我們發現 ROCK 似乎是調控 YAP 核質轉運的最下游且最關鍵的因子。此外，ROCK 也不受STK40所調控。我們的發現表明了一條新的 STK40 介導的 YAP 調控途徑。目前，我們正在識別 STK40 的主要靶標，希望揭示細胞密度如何控制 YAP 活動以實現接觸抑制的機制力。

The cellular mechanisms are highly complex, often requiring crosstalk among different pathways to accomplish tasks such as cell migration. In 2019, our laboratory identified a combination that could more effectively interfere with cell migration through a two-hit screen approach, which we named synthetic dysmobility. This involved using the MEK inhibitor Trametinib to inhibit the MAPK signaling pathway and knocking down Serine/Threonine Kinase 40 (STK40). The results showed that cell migration significantly decreased after the dual treatment. Since cell migration is regulated by Yes- associated protein (YAP), we also found that knocking down STK40 caused YAP to translocate from the nucleus to the cytoplasm. Given the limited research on STK40 and the unclear mechanism of how STK40 regulates YAP, we conducted preliminary studies to explore this regulation. In our preliminary data, we observed that, unlike typical cells where YAP translocate from the nucleus to the cytoplasm with increasing cell density, STK40-knockdown cells did not show a significant decrease in nuclear YAP levels even with increased cell density. Additionally, when STK40 was overexpressed, YAP appeared to colocalize with STK40, suggesting that STK40 might influence YAP distribution. Furthermore, high cell density facilitated the translocation of both STK40 and YAP to the cytoplasm. We hypothesized several mechanisms through which STK40 might regulate YAP. The first mechanism involves the Hippo pathway, where core members MST1/2 and LATS1/2 promote YAP phosphorylation, leading to its translocation to the cytoplasm. Using small molecule inhibitors to inhibit their activity and simultaneously knocking down STK40 with shRNA lentivirus, we observed that some YAP still translocated to the cytoplasm, indicating that other factors might influence STK40’s regulation of YAP translocation. The second hypothesis is that STK40 affects YAP distribution through karyopherins. Proteins require karyopherin to shuttle in and out of the nucleus, with importin and exportin performing these tasks. Importin transports proteins into the nucleus, while exportin handles the reverse function. We used small molecule inhibitors Leptomycin B (targeting exportin) and Ivermectin (targeting importin) in combination with shRNA lentivirus to knock down STK40. The results showed that the Leptomycin B-treated group’s outcome was similar to the LATS inhibitor-treated group, whereas the group with both STK40 knockdown and Ivermectin treatment did not show significant changes in nuclear-cytoplasmic distribution compared to the STK40 knockdown alone group. This strongly suggests that STK40 might influence YAP distribution by affecting importin. Finally, simulating increased cell density with the addition of the small molecule inhibitor Y27632 (a ROCK inhibitor), we observed weakened cytoskeletal tension's effect on STK40, resulting in YAP translocation to the cytoplasm. Interestingly, we found that ROCK seems to be the most downstream and crucial factor regulating YAP nuclear-cytoplasmic transport, and it is not regulated by STK40. Our findings indicate a novel STK40-mediated pathway regulating YAP. Currently, we are identifying the primary targets of STK40, aiming to reveal how cell density controls YAP activity to achieve contact inhibition.