TRB2交互作用蛋白之功能研究

Abstract

Tribbles－絲胺酸/蘇胺酸蛋白質磷酸酶家族成員－已經被證明在果蠅胚胎發育過程中協調細胞增生、遷移以及形態發生。雖然在演化上Tribbles是被高度保留的，但是哺乳類同源基因的生理功能大多還是未知的。最近我們實驗室已經證明人類Tribbles同源基因2型(TRB2) 參與人類紅白血病TF-1細胞在去除顆粒細胞－巨噬細胞聚落刺激因子(GM-CSF)所引起的細胞凋亡之中。隨著去除GM-CSF，TRB2的訊息核醣核酸會被高度誘導表現，而且過量表現TRB2會促使許多造血系統來源的細胞株進行細胞凋亡。然而過量表達TRB2所引起細胞凋亡中的機制則需要更進一步的研究。為了達到這個目的，我們首先在這裡證實TRB2促進細胞凋亡的功能並不需要其推測的磷酸酶活性，而且其蛋白質的表達量是受到蛋白酶體－依賴性途徑所調控。其次，先前我們利用酵母菌雙雜交篩選實驗找到了數個與TRB2的交互作用蛋白。在這篇論文中，我們進一步在試管內及哺乳類細胞內去驗證其中三個蛋白－MNK2b、γ-Parvin及UBC9－與TRB2之間的交互作用。TRB2與MNK2b或與γ-Parvin之間的交互作用可以在GST pull-down實驗中被證明。利用免疫共同沉澱法和哺乳類雙雜交實驗更加確定了TRB2可以跟γ-Parvin在細胞內結合。相對地，TRB2與UBC9的交互作用則沒有在試管內及細胞內被證實。最後我們探討這些蛋白在細胞凋亡中可能扮演的生理功能，並且發現MNK2b與γ-Parvin會部分地干擾TRB2所促進的細胞凋亡功能。總結來說，我們的研究指出γ-Parvin可能是TRB2生理的結合蛋白，而且可能在TRB2引起的細胞凋亡中扮演角色。

Tribbles, a serine/threoine protein kinase family member, has been shown to coordinate cell proliferation, migration and morphogenesis during the development of Drosophila embryo. Although Tribbles are highly conserved throughout the evolution, the physiological function of mammalian orthologs is largely unknown. Recently, our laboratory has demonstrated that human Tribbles ortholog 2 (TRB2) is involved in GM-CSF withdrawal-induced apoptosis of TF-1 cells, a human erytholeukemia cell line. TRB2 mRNA is highly induced upon removal of GM-CSF, and ectopic expression of TRB2 promotes apoptosis of cells mainly of the hematopoietic origin. However, the underlying mechanism of TRB2-induced apoptosis needs to be further elucidated. For this purpose, we firstly report here that the putative kinase activity is not required for the pro-apoptotic function of TRB2, and its protein level is regulated through the proteasome- dependent pathway. Second, a yeast two-hybrid screen was previously applied and several candidate genes were identified. In this dissertation, we further characterized the interactions of three of these proteins, MNK2b, γ-Parvin and UBC9, with TRB2 in vitro and in mammalian cells. The interaction of TRB2 with MNK2b or γ-Parvin could be demonstrated in GST pull-down experiments. The co-immunoprecipiation and the mammalian two-hybrid assay were further used to confirm TRB2 associates with γ-Parvin in cells. In contrast, the interaction between TRB2 and UBC9 was not detected both in vitro and in vivo. Last, we explored the biological effect of these interactions on apoptosis and found that MNK2b and γ-Parvin partially interfere with the pro-apoptotic function of TRB2. Taken together, our data suggest that γ-Parvin is likely a physiological binding protein of TRB2 and may play a role in TRB2-induced apoptosis.