探索SLK和ROCK在細胞遷移的拮抗作用

Investigation of SLK/ROCK antagonism in cell migration

周彥萱(Yen-Hsuan Chou)

指導教授 ： 蔡丰喬

國立臺灣大學/醫學院/藥理學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202103249

摘要

細胞遷移在許多生理程序中都扮演重要角色，因此研究細胞如何遷移成為一項重要的議題。我們實驗室先前利用「two-hit shRNA screening assay」，將119個與細胞遷移相關的基因個別敲減(knockdown)，並分別搭配給予三種訊息鏈的抑制劑。結果顯示ROCK抑制劑Y27632搭配敲減SLK基因，會大幅增加人類臍靜脈內皮細胞(HUVEC)的爬行速度。SLK(Ste20-like kinase)是一種絲氨酸/蘇氨酸蛋白激酶，目前已知會影響細胞週期、附著力、細胞凋亡與細胞骨架。然而其中詳細作用機轉還未被研究清楚。
我們實驗室先前利用人類口腔上皮癌細胞SAS研究，發現knockdown SLK會使細胞爬行速度及細胞爬行的方向性都降低；另外，有文獻指出SLK在細胞爬行的前緣會產生聚集的現象。因此我們提出假說：SLK聚集在細胞前緣的現象可能會調控爬行方向性。以免疫螢光染色、活細胞影像及膜蛋白分離技術，我們證實過度表現的SLK的確會聚集在遷移細胞的前緣。未來計畫利用CRIPSR knockin綠螢光蛋白，以觀察內生性SLK的分布位置。
另一方面我們也致力於釐清SLK與ROCK之間的交互作用，目前我們認為SLK與ROCK並無直接的調控關係。近來數篇報導提出。近年來數篇報導提出SLK與ROCK同為RhoA下游的effector蛋白，意味著兩者可能存在間接的競爭關係。而其中一篇報導更使我們注意到lymphocyte-oriented kinase(LOK)蛋白與SLK在演化上與功能上的高度相似性。目前我們以西方墨點法(western blot)與免疫螢光染色觀察過度表現或knockdown SLK時，搭配其他調控RhoA/ROCK訊息鏈的藥物，觀察應力纖維(stress fiber)及黏著斑(focal adhesion)訊號隨之產生的變化。

關鍵字

SLK ； 細胞遷移 ； 細胞前緣 ； RhoA ； ROCK

並列摘要

Cell migration is required in many biological processes. Therefore, figuring out how cell migration is regulated becomes an important project. Our lab used a “two-hit shRNA screening assay” to find candidates among 119 genes involved in cell migration. As a result, SLK knockdown combined with ROCK inhibitor (Y27632) treatment would dramatically raise the migration speed of HUVEC (Human umbilical vein endothelial cells). SLK (Ste20-like kinase) is a serine/threonine kinase, which has roles in cell cycle, adhesion, apoptosis, and cytoskeleton. However, the mechanisms in these processes have not been fully understood by now.
Our lab members had found that SLK knockdown reduced migration speed and polarity in SAS. Furthermore, a previous study had shown the recruitment of SLK to the leading edge of migrating cells. Hence, we hypothesize: SLK at leading edge is essential for cell migration polarity. In this study, immunofluorescence, live cell images and membrane protein extraction were used to demonstrate that overexpressed SLK accumulate at leading edge in SAS and HUVEC. We are looking forward to exploring endogenous SLK by CRISPR knockin GFP.
We also have been working on the interaction between SLK and ROCK signaling. So far, SLK had been verified that it does not modulate ROCK directly. Recent papers described both SLK and ROCK as effectors of RhoA, and thus there might be competition of binding with active RhoA between SLK and ROCK. Moreover, LOK((lymphocyte-oriented kinase), a homolog of SLK, was underlined in one of the recent studies. We are trying to clarify the effect of SLK overexpression/knockdown, which would also be combined with drug treatment, on stress fiber/focal adhesion by using immunofluorescence and western blot.

並列關鍵字

SLK ； cell migration ； leading edge ； RhoA ； ROCK

參考文獻

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4. Yamada, E., et al., Molecular cloning and characterization of a novel human STE20-like kinase, hSLK. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2000. 1495(3): p. 250-262.

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