搜索和描述Mst3的關聯性蛋白質

Abstract

Mammalian Sterile 20-like kinase 3(Mst3) 屬於germinal center kinase(GCK) Ⅲ家族的成員，是一個分子量52kDa 左右的絲氨酸/酥氨酸的蛋白質激脢。在初步的研究顯示，Mst3會和Apoptosis-activating factor以及 Endonuclease G產生作用，而且可能在扮演調控細胞凋亡的角色。所以我們觀察到Mst3會和細胞內的蛋白質結合形成複合體，那對於Mst3有重要的功能。所以我們利用蛋白質體學的技術來找出在細胞內可能會和Mst3產生關聯性的蛋白質。蛋白質體學的技術提供深入的觀察蛋白質與蛋白質之間的作用，以及細胞中各種的修飾作用。從全細胞萃取物以及細胞質和粒線體的碎片中取得的蛋白質先和Mst3的抗體進行免疫沉澱的反應。將免疫沉澱的分離物利用二維電泳來分離，接著用質譜儀鑑定蛋白質的身分。有17個蛋白質在質譜儀的分析中被鑑定其身分並且可能會和Mst3產生作用。在這些蛋白質中，β-TrcP 被發現可以和Mst3免疫沉澱。因此β-TrcP 可能參與調控Mst3 的訊息傳遞路徑。

Mammalian Sterile 20-like protein kinase 3 (Mst3), a member of the germinal center kinase (GCK) Ⅲ family, is a Ser/ Thr protein kinase with a molecular mass of 52 kDa. In a preliminary study Mst3 was demonstrated to interact with apoptosis –activating factor and endonuclease G and might play a role in mediating apoptosis. This result suggests that Mst3 may form a complex with intracellular proteins that may be important for the function of Mst3. Proteomics is then employed to find out the possible intracellular proteins that may associated with Mst3. Proteomic technique provides insight into protein-protein interaction and various modifications within cells. Proteins from whole cell extract were first immunoprecipitated with Mst3-specific antibody. The isolated immunocomplex was then separated sequentially on isoelectric focusing (first dimension) and SDS-PAGE (second dimension), followed by analyzing with mass spectrometry. Seventeen proteins were identified in the MS analysis, which exhibit potential to interact with Mst3. Among these proteins β-TrcP was found to be immunoprecipitated with Mst3 by both antibodies specific to Mst3 and β-TrcP. This result suggests that β-TrcP may participate in the Mst3-mediated signaling pathway.