邱繼輝臺灣大學：生化科學研究所賴紫庭Lai, Tzu-TingTzu-TingLai2007-11-262018-07-062007-11-262018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/52748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.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.Table of contents Table of contents……………………………………………………………………..I Abbreviation…………………………………………………………………………1 Abstract………………………………………………………………………………2 Chapter 1. Introduction 1. Clathrin-mediated endocytosis…………………………………………………….3 2. Molecular architecture of adaptor proteins…………………………………….......4 3. Adaptor protein complexes in the Drosophila melanogaster………………………5 4.Shibire temperature sensitive mutants specifically block the clathrin-coated vesicles.………………………………………………………………………………5 5. Regulation of clathrin-mediated endocytosis by phosphorylation………………...6 6. Nak, a member of the Ark family……………………………………………….....6 7. Mass spectrometry…………………………………………………………………7 8. Specific aims………………………………………………………………………10 Chapter 2. Materials and Methods Materials and methods 1. Constructs…………………………………………………………………………11 2. Antibodies…………………………………………………………………………11 3. Site-directed mutagenesis and molecular cloning………………………………...12 4. RT-PCR……………………………………………………………………………13 5. Cell Culture……………………………………………………………………......13 6. S2 cell transient transfection………………………………………………………13 7. SDS-PAGE and immunobloting…………………………………………………..14 8. Immunoprecipitation………………………………………………………………14 9. Silver staining and Sypro-Ruby staining………………………………………..15 10. Sample preparation for Mass spectrometry……………………………………..16 11. Fly strains and male recombination mutagenesis……………………………….16 Chapter 3: Results 1. Two subunits of adaptor proteins were specifically immunoprecipitated by myc-Nak in S2 cells………………………………………………………………….18 2. Association of AP50 to Nak………………………………………………………19 3. AP50 was immunoprecipitated by Flag-Nak in the Drosophila embyo…………..20 4. α-adaptin binding motifs in Nak is important for the interaction with AP2………21 5. Nak may participate in late step of the endocytosis…………………..…………..22 6. Analysis of the Nak recombination hypomorph alleles…………………………..23 Chapter 4. Discussion 1. Why only AP2 complexes are identified in mass spectrometry analysis?...............25 2. The modification of AP50 in the endocytosis……………………………………..26 3. Whether Nak affects the subunit stochiometry of the AP2 complex ?…………………………………………………………………………….27 4. The effect of NakmDPF mutant…………………………………………………..28 5. The P-element line GE10537 is a hypomorphic allele of nak…………………….29 6. Possible functions of Nak in the endocytosis……………………………………..29 Reference……………………………………………………………………………31 Appendix Table Table1. Mass spectrometrically identified Drosophila Nak interacting proteins ( I ) Table2. Mass spectrometrically identified Drosophila Nak interacting proteins ( II ) Table3. Mass spectrometrically identified Drosophila Nak interacting proteins ( III ) Table4. Hypomorphs generated in the one round of male recombination experiment Figure Fig.1 Fly Nak associates with potentially AP2 complex…………………………….35 Fig.2 Fly Nak associates with potentially AP2 complex in vivo…………………….36 Fig.3 Nak can interact with α-adaptin………………..…………………………….37 Fig.4 The different components of AP2 complex binds with Nak…………………..38 Fig.5Double-confirm the association between Nak and AP2 complex in fly embryo……………………………………………………………………………….39 Fig.6 Schematic representation of flag-Nak, flag-NakmDPF1/2 constructs used…...40 Fig.7 The association between AP50 and Nak is dependent on α-adaptin………...41 Fig.8 The procedure of the male recombination experiment………………………..43 Fig.9 The male recombination lines, MR2~MR8, was a hypomorphic allele of Nak…………………………………………………………………………………..44 Fig.10 Nak might take part in the late steps of the endocytosis……………………..46en-US內吞作用,Nak,AP2endocytosis,Nak,AP2other