人類腺嘌呤核苷二磷酸核醣化相似因子四之二聚化與轉譯後修飾特性探討

Abstract

腺嘌呤核苷二磷酸核醣化相似因子(Arf-like, Arl) 是隸屬於大鼠肉瘤 (Ras) 超家族中二磷酸腺苷核糖基化因子 (Arfs) 家族的小分子鳥嘌呤核苷酸結合蛋白 (Small GTP binding protein)。其中，Arl4次家族的三個成員，Arl4A，Arl4C及Arl4D，都能透過與特定蛋白質結合以促進細胞遷移。近年來已有許多文獻指出轉譯後修飾參與調控小分子鳥嘌呤核苷酸結合蛋白的表型及其下游訊息傳遞路徑的活性。其中，轉譯後修飾也已被證實對於小分子鳥嘌呤核苷酸結合蛋白的二聚化至關重要。為了進一步釐清Arl4的調控機制，我們首先探討轉譯後修飾對於Arl4功能的影響，並接著研究Arl4是否具有寡聚能力。在第一部份，我們主要觀察已知的Arl4轉譯後修飾位點在進行模擬和缺陷型突變後會對Arl4表現型造成何種影響。另一方面，我們也著重於尋找何種生理環境下可能會改變Arl4的轉譯後修飾情形。首先，我們藉由穩定同位素標記氨基酸細胞培養技術結合質譜儀 (SILAC-MS) 分析發現在纖連蛋白促進Arl4A的細胞膜定位時，Arl4A絲氨酸143的磷酸化似乎會有所增加，但經由免疫螢光染色法發現S143D磷酸化模擬突變體並不會影響Arl4A的細胞膜定位。接著，在Arl4C部分，我們發現兩個轉譯後修飾突變體，K181R泛素化缺陷型和S185D磷酸化模擬型，無法誘導細胞產生適當長度的絲狀偽足，並導致較低的細胞遷移能力。最後，在Arl4D中，我們通過免疫螢光染色法及放線菌酮追蹤檢測 (cycloheximide chase assay) 發現Arl4D的細胞核定位會加速其降解，因此我們想進一步分析Arl4D易位入細胞核的過程是否受到轉譯後修飾的調控。然而，沒有已鑑定的泛素化缺陷位點會影響Arl4D的核定位，且到目前為止我們所測試的生理條件中，僅發現Arl4D在氯化亞鈷誘導的缺氧環境下會異位至細胞核周圍，但不會進入核內。在第二部分中，我們首先運用酵母菌雙雜交篩選法 (yeast two hybrid)、試管結合實驗 (in vitro binding assay) 及免疫共沉降實驗 (Co-IP) 驗證Arl4成員之間存在同聚及異聚作用。同時，我們也發現Arl4D之間的寡聚作用與其鳥嘌呤核苷酸結合狀態沒有直接關聯性。我們接著透過試管結合實驗發現Arl4D的尾端對於其同聚作用來說是重要的。另一方面，透過免疫螢光染色法我們觀察到在細胞中Arl4能夠彼此募集並共同坐落於細胞膜上。功能探討部分，我們經由細胞爬行能力實驗發現Arl4A介導的細胞遷移需要Arl4D的參與。以上結果顯示轉譯後修飾與Arl4的亞細胞定位及功能密切相關，並揭示了Arl4寡聚對其功能的潛在影響，詳細的調控機制值得進一步探討。

Arf-like (Arl) proteins are small guanine-nucleotide binding (G) proteins, which belong to the Sar1/Arf family of Ras superfamily. Arl4s can be classified into three isoforms, Arl4A, Arl4C, and Arl4D. Each of them has been reported to promote cell migration via cooperating with specific interacting partners. Recently, literatures have indicated that post-translational modification (PTM) can modulate the phenotype of small GTPases and the activity of their downstream signaling pathway. Moreover, PTM has been reported to be essential for GTPase dimerization. To further characterize the regulatory mechanisms of Arl4s, we firstly study the functional effect of PTM on Arl4s and secondly clarify whether Arl4s can be dimerized. In the first part, we examine the phenotype of Arl4s by using Arl4s ubiquitin or ubiquitin-like defective, and phospho-mimetic or defective mutants. We also verify possible physiology condition that could affect the PTM profile of Arl4s. Since fibronectin treatment enhance the plasma membrane localization of Arl4A, we analyze the change of Arl4A PTM profile by SILAC, and shows that phosphorylation of Arl4A Ser143 seems to increase upon fibronectin treatment. However, our immunofluorescence data showed that S143D phospho-mimetic mutant is inefficient to increase the plasma membrane localization of Arl4A. In Arl4C, We found that two PTM mutants, ubiquitin-defective K181R and phospho-mimetic S185D, fail to generate filopodia with proper length, and exhibit lower cell migration ability. For Arl4D, we could not identified ubiquitin-defective sites to be critical for its nuclear localization. We could not observe nucleus-localized Arl4D among any physiology conditions we screened either. In the second part, we clarify the homo- and hetero-oligomerization of Arl4s by yeast two hybrid system, in vitro binding assay, and co-immunoprecipitation. We found that the interaction between Arl4D is independent of its nucleotide-binding state and seems to depend on its C-terminal but not N-terminal tail. Similarly, our immunofluorescence data shows that Arl4s are able to be recruited by each other to the plasma membrane. Furthermore, Arl4D depletion compromises Arl4A mediated cell migration, supporting that Arl4s might hetero-cooperate to induce downstream functions. In conclusion, our observations indicated that PTM plays pivotal roles in regulating the subcellular localization and function of Arl4s, and also reveals potential effect of Arl4s oligomerization on its function.