辨識Mst3 domain在細胞內分布區域之研究

Abstract

Mst3 (Mammalian Ste20-like protein kinase3)是隸屬人類類Ste20蛋白激酶家族的成員之一，但其生理上的功能仍尚待釐清。近年來，Mst3被認為在調控細胞凋亡的過程中扮演一個重要的角色。在細胞實驗中，大量表現Mst3將會導致染色質濃縮、DNA片段化以及出現明顯細胞凋亡的特徵。過去的研究也已證實Mst3是一個新興存在於粒線體內的絲氨酸/蘇氨酸蛋白激酶，而且同時在粒線體與細胞質中皆可發現Mst3。有趣的是，Mst3分布在粒線體內外膜間的腔室裡並且它與啟動細胞凋亡的蛋白分子， 例如AIF(apoptotic-inducing factor)和EndoG (endonuclease G)，會形成pro-apoptotic 複合體。在細胞凋亡的過程中，這些pro-apoptotic蛋白分子會從粒線體被釋出到細胞質以及細胞核引發核內染色體濃縮聚集和DNA的分裂。 本研究主要是尋找Mst3的粒線體target sequence。Mst3KR以及Mst3KRΔ314被用來作為研究Mst3在細胞內分佈的指標。為了要執行這些實驗，許多不同長度的eGFP-HA-Mst3質體已被建構。這些實驗結果將可以讓我們了解到更多關於Mst3本身結構與功能上的相關性以及Mst3如何調控粒線體功能的分子機制。

Mst3 (mammalian Ste20-like protein kinase 3) is a member of human Ste20-like protein kinase family with unknown physiological function. It has been postulated recently to play a role in mediating programmed cell death (or apoptosis) in response to environmental cues. Overexpression of Mst3 could induce chromosome condensation and DNA fragmentation, characteristics of apoptosis. Preliminary studies also show that Mst3 is a novel serine/threonine protein kinase that resides in both mitochondria and cytoplasm. Interestingly, in mitochondria, Mst3 is present in the intermembrane space and associates with pro-apoptotic proteins, including apoptosis-inducing factor (AIF) and Endonuclease G (EndoG), forming a pro-apoptotic complex. During apoptosis, mitochondrial pro-apoptotic proteins translocate from mitochondria to cytosol and nucleus and trigger the chromosome condensation and DNA cleavage. This work is to identify the mitochondrial-targeting sequence in Mst3. Mst3KR, a kinase-dead form, and Mst3KRΔ314, a truncated form of Mst3KR, were used as models to study their subcellular distribution in HeLa cells. To carry out this experiment, various C-terminal deleted mutants of EGFP-HA-Mst3 were constructed. The distribution of these EGFP/Mst3 mutants may allow us to understand more about the structure-function relationship of Mst3 and the molecular mechanism of Mst3 in regulating mitochondrial functions.