研究在內吞作用當中Nak和AP2的關連性

Association of Nak with AP2 in Endocytosis

賴紫庭(Tzu-Ting Lai)

指導教授 ： 邱繼輝

共同指導教授 ： 簡正鼎

國立臺灣大學/生命科學院/生化科學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.00096

摘要

內吞作用在生物體內是一種非常重要的機制,其中包括了調節在細胞膜上的受體活性的調控以及因應細胞外訊息而調節特殊物質的吸收.內吞作用主要可以分成三個步驟:1.囊胞形成,2.囊胞內陷及從細胞膜上分離,3.相關作用蛋白的剝落.在第一個步驟囊胞形成時需要兩個重要的蛋白質,分別是clathrin以及clathrin輔助蛋白AP2(clathrin adaptor protein 2),clathrin輔助蛋白含有四個不同的組成,分別是alpha蛋白,beta蛋白,AP50蛋白,以及sigma蛋白,在之前的研究指出AAK1(adaptor protein kinase 1)在進行囊泡形成時會磷酸化AP50,幫助細胞膜上受體的回收,在果蠅中AAK1的相似蛋白稱為Nak(Numb-associated protein), Nak在1998年經由酵母菌雙雜合系統(yeast two hybrid)以numb為餌調到的作用蛋白,在實驗室之前的研究發現在Nak核酸干擾果蠅中發現對於神經樹突發育有影響,在此篇論文便是探討在果蠅中Nak是否扮演跟在哺乳動物中AAK1在內吞作用當中扮演一樣的角色或是亦在其他步驟仍有功能.
本論文搭配質譜技術的分析以及免疫沈澱技術法的配合,研究Nak在內吞作用中扮演的角色,實驗結果發現此蛋白在質譜分析之下會和clathrin輔助蛋白作用,但其中和AP50蛋白的作用力最弱,因此我們推測Nak蛋白可能會影響clathrin輔助蛋白的組成,此外利用在哺乳系統中dynamin的相似蛋白shibire之溫度調控突變株發現,Nak的確不是作用在囊泡內陷這個步驟,有可能參與在內吞作用當中較後面的步驟,配合本論文對於Nak和clathrin輔助蛋白的研究,可以更進一步瞭解Nak在內吞作用當中對於果蠅樹突發育的影響.

關鍵字

內吞作用,Nak,AP2

並列摘要

Endocytosis is a fundamental step to regulate a diverse array of cellular activities. In response to signals, the process of endocytosis can mediate membrane receptor recycling, and cargos delivery to intracellular organelles. The phosphorylation state of endocytic components, such as the adaptor protein complex 2 (AP2) and the engulfed membrane, can modulate clathrin-coated pit formation and cargo selection. The ARK family of serine/threonine kinases is shown to regulate endocytosis through the phosphorylation of various endocytic components. Numb associated kinase (Nak), the only ARK member in Drosophila, bears a serine/threonine kinase domain at the amino-terminus and various endocytic motifs, including DLL for interacting with clathrin and DPF for α-adaptin. To investigate possible signaling pathways and cellular mechanisms regulated by Nak, I have performed immunoprecipitation for Nak and identified proteins associated with Nak by Mass-Spectrometry (MS). Through the MS analysis, I have identified α-adaptin, β-adaptin and the u2 subunit AP50 of the AP2 complex. With the complex association in the dynamin mutant, shibire, I suggest that Nak may take part in late steps of endocytosis. I also performed a male recombination experiment to screen for nak mutants for further functional analysis. Several hypomorphic alleles obtained in this screening show reductions in mRNA and protein levels.

並列關鍵字

endocytosis,Nak,AP2

參考文獻

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