探討腺嘌呤核苷二磷酸核糖化因子相似蛋白四(Arl4s)之功能

Functional Characterization of ADP-Ribosylation Factor-Like Protein 4s (Arl4s)

林新晉(Shin-Jin Lin)

指導教授 ： 李芳仁

國立臺灣大學/醫學院/分子醫學研究所/博士(2021年)

https://doi.org/10.6342/NTU202104031

摘要

章節一
胞內體上的Arl4A透過與VPS36的結合進而抑制EGFR的降解
Arl4Ａ (ADP-ribosylation factor-like 4A; Arl4Ａ)是屬於腺嘌呤核苷二磷酸核醣化因子相似蛋白 (Arf-like) 之一。這個蛋白質家族廣為人知的功能主要參與在囊泡傳遞運輸、細胞骨架重組、和胞器的形成等等。其中Arl4A又特別被報導了參與在細胞膜皺褶的形成、細胞骨架的調控、高基氏體的形成等等功能之中；然而Arl4A在囊泡傳遞運輸中扮演何種角色，目前還不明白。配體 (EGF) 誘導的EGFR(受器)內吞作用，並且驅動下游訊息路徑的傳遞，這一連串的過程控制了細胞許多功能。我們發現Arl4A可以透過與VPS36結合，降低了EGFR受器的降解。我們也觀察到Arl4A的敲除，會加速EGFR快速的運輸至溶酶體內進行降解。我們更進一步分析，Arl4A與VPS36蛋白質上氨基酸序列351-356進行結合。我們將此序列突變後導致Arl4A無法與VPS36結合，透過這個突變的VPS36，我們發現EGFR的降解速度會比起野生型來得快。我們的結果顯示著，Arl4A會負調控VPS36的功能，並且減慢EGFR去泛素化的速度，進而減緩EGFR降解的速度。
章節二
Arl4D與EB1的結合有助於EB1招引至中心粒以及幫助微管生成
Arl4D (ADP-ribosylation factor-like 4D; Arl4D)是屬於腺嘌呤核苷二磷酸核醣化因子相似蛋白 (Arf-like) 之一。目前所知，Arl4D可以調控細胞型態、細胞遷移與細胞骨架的重組。EB1是一種位於微管（microtubule）正端（plus end tip）以及中心粒(centrosome)上的蛋白，EB1在中心粒上的成核作用（microtubule nucleation）扮演很重要的角色。在本研究中，我們發現活化態的Arl4D（GTP bound form）會藉由SxLP motif與EB1直接的結合。我們也發現Arl4D會與中心粒有共同定位外，當我們調降（knockdown）Arl4D表達後，微管自中心粒的成核作用會受到抑制，也會影響EB1定位到中心粒的能力。進一步，降低Arl4D與EB1的結能力，也會降低EB1定位於中心粒上與抑制中心粒上的微管成核作用。此外，我們也發現了Arl4D會促進EB1與p150之間的交互作用，此交互作用對於微管的成核與穩定有很大的幫助。總而言之‚我們的結果顯示Arl4D影響EB1與p150在中心粒上的結合，進而調控中心粒上的微管成核作用的新機制。

關鍵字

腺嘌呤核苷二磷酸核醣化相似因子4 ； 表皮生長因子受體 ； 微管 ； 微管尖端蛋白

並列摘要

Chapter-I
Endosomal Arl4A attenuates EGFR degradation via interacting with ESCRT-II component VPS36
ADP-ribosylation factor (Arf)-like protein 4A (Arl4A), an Arf small GTPase, functions in cytoskeleton remodeling and cell migration. Arl4A localizes at the plasma membrane, trans-Golgi network and endosomal compartments, however, the role of Arl4A at the endosomes remains unclear. Ligand-induced epidermal growth factor receptor (EGFR) endocytosis following by endosome-located active EGFR signaling and lysosomal degradation play important roles in controlling multiple biological processions. Here, we report that Arl4A attenuates EGFR degradation via interacting with endosomal sorting complex for transport (ESCRT)-II component Vps36. We first observed that Arl4A-depletion enhance the transport of endocytosed EGFR from endosomes to lysosomes. Arl4A-depletion accelerates the EGF-stimulated EGFR degradation. Expression of Arl4A delays the EGFR de-ubiquitination at the late endosomes and its transport to the lysosomes. We show that Arl4A directly interacts with VPS36 and amino acid residues 351-356 of it are necessary for this interaction. Similar to that in Arl4-depleted cells, expression of Arl4A-binding-defective VPS36 accelerates EGFR degradation in VPS36-depleted cells. Thus, we propose that Arl4A binding to VPS36 negatively regulates EGFR degradation through modulation of de-ubiquitination of EGFR at the late endosome/MVB.
Chapter-II
Arl4D recruits EB1 to centrosome and contributes to EB1 and p150 assembly
ADP-ribosylation factor (Arf)-like 4D (Arl4D), one of the Arf-like small GTPases, functions in the regulation of cell morphology, cell migration, and actin cytoskeleton remodeling. End-binding 1 (EB1) is a microtubule (MT) plus-end tracking protein that preferentially localizes at the tips of the plus ends of growing MTs and at the centrosome. EB1 depletion results in many centrosome-related defects. Here, we report that Arl4D promotes the recruitment of EB1 to the centrosome and regulates MT nucleation. We first showed that Arl4D interacts with EB1 in a GTP-dependent manner. This interaction is dependent on the C-terminal EB homology region of EB1 and partially dependent on an SxLP motif of Arl4D. We found that Arl4D colocalized with γ-tubulin in centrosomes and the depletion of Arl4D resulted in a centrosomal MT nucleation defect. We further demonstrated that abolishing Arl4D-EB1 interaction decreased MT nucleation rate and diminished the centrosomal recruitment of EB1 without affecting MT growth rate. In addition, Arl4D binding to EB1 increased the association between the p150 subunit of dynactin and the EB1, which is important for MT stabilization. Together, our results indicate that Arl4D modulates MT nucleation through regulation of the EB1–p150 association at the centrosome.

並列關鍵字

Arl4 ； Epidermal growth factor receptor ； Microtubule ； End-binding protein 1

參考文獻

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