ARRDC5 cooperates with ACAP4 to regulate ARF6 activity at the cell membrane (964.1)

The ARF6 GTPase is essential for regulating multiple cellular processes, such as, secretion, endocytosis, membrane trafficking, cell migration. ACAP4 is an ARF6 GTPase‐activating protein is critical EGF‐elicited cell migration (Mol. Cell Proteomics. 5, 1437‐49). However, how ACAP4 regulates proper levers of active ARF6 is not well understood. Here we show that, arrestin domain‐containing 5 (ARRDC5), a novel protein of α‐ arrestin family, competes with ARF6 for binding ACAP4. ARRDC5 can inhibit ACAP4 mediated inactivation of ARF6 in a dose‐dependent manner. Knock‐down of ARRDC5 decreases cellular ARF6‐GTP, whereas ARRDC5 overexpression increases cellular ARF6‐GTP. Surprisingly, EGF stimulation of cells causes phosphorylation of ARRDC5 at Ser247 by AKT1, which is essential for cell motility. Significantly, our biochemical characterization demonstrated that the phosphorylation of Ser247 strengthens ACAP4‐ARRDC5 association. The co‐localization of ACAP4 with ARRDC5 occurred in ruffling membranes formed upon EGF stimulation. In addition, expression of the phospho‐mimicking mutant of ARRDC5 promotes ARF6 activation and cell migration, knock‐down of AKT1 inhibits ARF6 activation and cell migration. Our date thus uncover ARRDC5 as a modifier of cellular ARF6‐GTP through regulation of ACAP4. AKT1‐mediated ARRDC5 phosphorylation facilitates cell migration by sequestering ACAP4 at the cell membrane to stabilize active ARF6 in response to EGF stimulation. Grant Funding Source : DK56292