探討絲氨酸／蘇氨酸蛋白激酶40與絲裂原活化蛋白激酶的交互作用對細胞遷移之影響

Abstract

細胞遷移對於胚胎發育、血管新生和癌細胞轉移都佔有一席之地，近期研究找出一些和細胞遷移相關的基因，不過並不清楚這些基因之間是如何交互作用的，為了了解基因交互作用對細胞遷移之影響，我們使用了短髮夾RNA和小分子抑制劑的方式來建立雙擊篩選平台，釐清分子之間交互作用，在這個篩選平台中，令我們感到有趣的組合是STK40和MAPK的交互作用，先前研究便有指出敲弱STK40會改變細胞遷移，而在我們的結果中，STK40和MAPK雙重抑制時會顯著性降低細胞遷移，因此我們認為細胞遷移中STK40和MAPK之間是有交互作用的存在。 首先我們使用單一細胞遷移追蹤平台，進而得知STK40敲弱會使細胞速度和協調性減低的趨勢，在雙重抑制實驗中，發現到STK40會改變細胞骨架-黏著斑，於是我們便進一步探討，發現STK40會增加黏著斑的訊號，為了更了解這之間的機制，我們會有兩大方向: (1)了解STK40對於黏著斑影響 (2)還有研究STK40的功能，上述這些方向能讓我們知道STK40如何影響細胞遷移和MPAK之間的交互作用。 在這篇論文中我們證明了，(1)STK40會透過牽引力-黏著斑這條路徑調控細胞遷移 (2)STK40會和MAPK交互作用而影響黏著斑以及細胞遷移，目前我們發現到STK40可能是個接頭蛋白，改變p-ERK的位置，進而影響黏著斑以及細胞遷移，於是我們致力於尋找更多證據來闡述STK40是否為接頭蛋白。

Cell migration is important for embryonic development, angiogenesis and cancer metastasis. Recent study identified migration-related gene, but how they interact with each other remains elusive. To find out their interaction, we using “two-hit” screen to identify interaction by short hairpin RNA (shRNA) and small molecule inhibitor. The screen generated several candidate shRNA-inhibitor pairs, among which Serine-Threonine Kinase 40 (STK40)-MAPK specifically caught our attention. STK40 is a putative serine/threonine kinase. Previous study showed that STK40 knockdown (shSTK40) increased cell migration. However, our two-hit screen shSTK40 plus MAPK inhibitor (MAPKi) dramatically reduced cell migration, indicating that co-inhiition of STK40 and MAPK disrupted cell migration in a synergistic manner. Therefore, we hypothesize that STK40 interacts with MAPK pathway to regulate cell migration. First, we conducted single cell migration assays and find out shSTK40 decreased cell motility and coordination. In double knockdown experiments, we discovered that STK40 control cell migration via cell-matrix adhesion (Paxillin). To find out its mechanism, we (1) examined how STK40 affect focal adhesion (FA) and (2) studied STK40 function. In this master thesis, we discovered that (1)STK40 regulate cell migration via traction force-focal adhesion pathway and (2)STK40-MAPK interaction affect focal adhesion and cell migration. In our recent data showed that STK40 may be a adaptor protein. STK40 alter p-ERK localization to regulate focal adhesion and cell migration. Thus, we need more evidence to find out STK40 function.