臺灣博碩士論文加值系統

腺苷酸2A亞型受體(A2A adenosine receptor; 簡稱為A2A-R)在腦中很多區域皆有分佈，並且能調控神經細胞的生理功能。我們之前的研究發現一個會與A2A-R C端交互作用的蛋白-Translin X (TRAX)。TRAX最初是以會與Translin相結合的面貌被發現，並被認為可能參與DNA的修復與重組機制。我們更進一步證明，活化A2A-R後可透過其與TRAX的相互作用來恢復因p53被抑制而停止的神經突觸生長現象。在目前的研究結果中，兩個會與TRAX交互作用的蛋白質-C1D和KIF2A被發現在這個恢復作用中扮演關鍵的角色。我們對KIF2A特別感興趣，因為KIF2A是參與微管蛋白去聚合化的一種驅動蛋白質，而且已發現和神經生長因子所誘導的神經突觸生長有關。實驗結果亦顯示若大量表現KIF2A，就如同TRAX一樣，可以恢復因p53被抑制而停止的神經突觸生長現象。但若調降KIF2A的表現量，則會抑制TRAX本身的恢復作用，因此TRAX有可能經由和KIF2A的相互作用來達成其功能。為了證實這個假設，共同免疫沉澱實驗顯示，抑制p53的活性很顯著地降低了TRAX和KIF2A之間的結合，由此可推論TRAX和KIF2A之間的交互作用對TRAX的恢復作用是很重要的。除此之外，研究顯示KIF2A位在細胞質時才能調控神經突觸的發展。而當p53被抑制時，KIF2A被留在細胞核中因而無法發揮作用。有趣的是，活化A2A-R會讓KIF2A從細胞核中轉移到細胞質發揮作用，可能因此恢復了因破壞p53而受損的神經突觸生長現象。總結以上實驗結果,我們推論A2A-R可能是經由TRAX和KIF2A之間的直接交互作用，來復原由於抑制p53而受傷的神經突觸生長。

The A2A adenosine receptor (A2A-R) is located in many different areas of the brain and has been implicated in a wide variety of neuronal functions. We previously reported that the C terminus of A2A-R binds Translin associated protein X (TRAX). TRAX was first identified as an associating protein of translin, which plays an important role in DNA repair and recombination. We further demonstrated that, via interacting with TRAX, activation of the A2A-R rescued the impairment of nerve growth factor (NGF)-induced neurite outgrowth caused by p53 inhibition in PC12 cells. In the present study, we found that two TRAX-interacting proteins (C1D and KIF2A) play a critical role in the rescuing action of TRAX. In particular, KIF2A is of great interest because it is a member of the microtubule-depolymerizing kinesins family and has been implicated in the NGF-evoked neurite outgrowth. Overexpression of KIF2A rescued the NGF-neurite outgrowth upon p53 blockage as did TRAX, while down-regulation of KIF2A suppressed the TRAX-mediated rescue effect. TRAX therefore might mediate its action through interacting with KIF2A. In line with this hypothesis, results of co-immunoprecipitation revealed that suppression of p53 markedly reduced the binding of TRAX to KIF2A, suggesting that a functional interaction between TRAX and KIF2A is required for the TRAX-rescue effect. In addition, KIF2A might exert its function in the cytosol since blocking p53, which suppressed the NGF-induced neurite outgrowth, retained KIF2A in the nucleus. In contrast, stimulation of the A2A-R, which rescues the NGF-induced neurite outgrowth upon p53 blockage, translocates KIF2A from the nucleus to the cytosol. Collectively, our findings suggest that activation of the A2A-R rescued the p53-impaired neurite outgrowth in PC12 cells through a functional interaction between TRAX and KIF2A.

Table of Contents……………………………Ⅰ
List of Table…………………………………Ⅱ
List of Figures………………………………Ⅲ
Abbreviations…………………………………Ⅳ
Chinese abstract……………………………Ⅴ
English abstract……………………………VI


Introduction…………………………………………………1
1.A2A adenosine receptor
2.TRAX
3.C1D
4.KIF2A
5.Purpose

Materials and Methods……………………8
1.Reagents
2.Cell Culture
3.Transfection, and neuronal differentiation
4.SDS-Polyacrylamide Gel Electrophoresis and Western Blotting
5.Flow Cytometry and Cell Sorting
6.Coimmuoprecipitation
7.Immunocytochemistry

Results…………………………………………13
1.Modulation of the A2A-rescue effect during p53 blockage via TRAX and its two interacting proteins, KIF2A and C1D
2.Characterization of the molecular mechanism underlying the action of the TRAX signalosome in mediating the A2A rescue effect


Discussion………………………………………22
1.Two TRAX-interacting proteins contribute to the A2A-R/TRAX rescue effect
2.Cytosolic distribution of KIF2A and TRAX is critical for neurite outgrowth
3.The functional interaction between TRAX and KIF2A is required for rescue of neurite outgrowth
4.The potential molecular mechanism involved in action of KIF2A and C1D underling the A2A-R/TRAX pathway
5.Conclusion and perspectives

References…………………………………………29

Table and Figures…………………………………36

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