探討腺嘌呤核苷二磷酸核醣化因子相似蛋白四 A之功能

Abstract

ARL4 (ARF-Like 4) small G protein subfamily belongs to ADP-Ribosylation Factor (Komander et al.) of Ras small GTPase superfamily. ARL4 subfamily consists of ARL4A, ARL4C and ARL4D. Transient expression patterns of ARL4s have been reported responded to physiological conditions, such as embryonic development, tissue morphogenesis and stress. Recent studies show that ARL4s are regulators of cellular cytoskeleton and upstream of ARF6 signaling pathways. Cell migration participates in development of functional organ in embryonic development and systematic balance when adult. Coordination of multiple signaling pathways involving in cytoskeleton and membrane dynamics, cell adhesion and cell polarity are required for promoting cell migration. In this study, we explore signaling pathways of ARL4A in membrane protrusion, cell adhesion and cell migration. We found ARL4A was mainly localized at membrane through both targeting signals of N-terminal myristoylation and C-terminal polybasic motif. ARL4A promoted membrane protrusion on fibronectin and increased cell spreading in nucleotide binding dependent manner. ARL4A also induced focal adhesion dynamics. Depletion of ARL4A affected focal adhesion morphology. It implies that ARL4A plays a role in cell migration. Our previous study shows that ELMO is ARL4A interacting protein. Interaction of ELMO with ARL4A through N-terminal Ras-binding domain (RBD) affects ARL4A-induced cell spreading and actin remodeling. Here, we identify p21-activating kinase (PAK) as novel interacting protein of ARL4A. Similar to ELMO-RBD interaction, ARL4A interacted with PAK through p21-binding domain (PBD), which is required for active Cdc42 and Rac1 binding. Expression of ARL4A promoted PAK/β-PIX /GIT1 complexes to membrane protrusion. Disruption β-PIX interaction and depletion PAK1 diminished ARL4A-induced membrane protrusion. Moreover, we found that residues R105 and K114 in the C-terminal PBD domain were required for ARL4A and PAK interaction. Replacement of R105 and K114 to alanine abolished ARL4A interaction and cell migration. Taken together, in this study, we reveal ARL4A function in membrane protrusion, cell adhesion and cell migration through interaction with PAK in integrin-mediated signaling pathway.