Arl4 介導之訊號傳遞功能性特徵分析與膜動態調控機制

Abstract

Arl4 小型 GTP 酶（腺嘌呤核苷二磷酸核醣化相似因子四, Arl4）在調控膜相關訊號傳遞與細胞骨架動態中扮演關鍵角色，對於細胞遷移至關重要。然而，調控 Arl4 活化、訊號傳遞以及效應分子調控的分子機制仍未被充分釐清。本研究探討 Arl4 蛋白如何透過自我交互作用、支架蛋白功能以及上游活化機制來協調膜上的訊號傳遞。我們發現，纖連蛋白（fibronectin）刺激可促進質膜上磷脂醯肌醇 4,5-雙磷酸（phosphatidylinositol 4,5-bisphosphate, PI(4,5)P₂）的累積，進而增強 Arl4D 的自我交互作用。此自我交互作用促進下游效應蛋白 p21 活化激酶 1（p21-activated kinase 1, Pak1）的募集，並建立一個正回饋迴路，其中 Pak1 所介導的磷酸化進一步穩定 Arl4D 在質膜上的定位，從而促進細胞遷移。此外，我們證實 Arl4D 作為一個支架蛋白，連結細胞外訊號調節激酶 1/2（extracellular signal-regulated kinase 1/2, Erk1/2）與 Pak1。在血小板衍生生長因子（platelet-derived growth factor, PDGF）刺激下，Arl4D 將 Erk1/2 招募至細胞膜，使 Erk1/2 能夠磷酸化 Pak1 的第 212 位蘇胺酸（Thr212），進而增強細胞遷移相關的訊號。 此外，我們鑑定出含 FYVE、RhoGEF 與 PH 結構域蛋白 1（FYVE, RhoGEF and PH domain–containing protein 1, FGD1）為 Arl4A/D 先前未被辨識的鳥嘌呤核苷酸交換因子（guanine nucleotide exchange factor, GEF）。FGD1 在 PDGF 訊號下調控 Arl4A/D 的活化，建立了生長因子訊號與 Arl4 活化之間的分子連結。總結而言，本研究揭示了一個由膜脂質、激酶訊號以及上游 GEF 活性所組成的整合性調控網絡，用以控制 Arl4 依賴的訊號傳遞與細胞遷移，並提供小型 GTP 酶如何達成空間性組織與訊號特異性的嶄新見解。

Arl4 small GTPases regulate membrane-associated signaling and cytoskeletal dynamics that are critical for cell migration. However, the molecular mechanisms regulating Arl4 activation, signaling transduction, and effector regulation remain poorly understood. In this study, we investigated how Arl4 proteins coordinate membrane signaling through self-interaction, scaffolding functions, and upstream activation mechanisms. We found that fibronectin stimulation promotes the accumulation of PI(4,5)P₂ at the plasma membrane, which enhances Arl4D self-interaction. This self-association facilitates the recruitment of the downstream effector Pak1 and establishes a positive feedback loop in which Pak1-mediated phosphorylation further stabilizes Arl4D on the plasma membrane, thereby promoting cell migration. In addition, we demonstrate that Arl4D functions as a scaffolding protein that bridges Erk1/2 and Pak1. Under PDGF stimulation, Arl4D recruits Erk1/2 to the plasma membrane, enabling Erk1/2-mediated phosphorylation of Pak1 at Thr212, which enhances migratory signaling. Furthermore, we identify FGD1 as a previously unrecognized guanine nucleotide exchange factor (GEF) for Arl4A/D. FGD1 regulates Arl4A/D activation under PDGF signaling, providing a mechanistic link between growth factor signaling and Arl4 activation. Together, these findings reveal a coordinated regulatory network in which membrane lipids, kinase signaling, and upstream GEF activity integrate to control Arl4-dependent signaling and cell migration, and provide new insight into how small GTPases achieve spatially organized and signal-specific functions.