Identification And Functional Characterization Of A Novel Arf6-specific GTPase Activating Protein-ACAP4

The ADP-ribosylation factors (Arfs) belong to the Ras superfamily members and are a family of ubiquitous and highly conserved GTP-binding proteins in eukaryotes. Based on biochemical activities and sequence similarity, proteins within the Arf family can be divided into Arf and Arf-like proteins. The Arf proteins have been further subdivided into class I (Arf1, Arf2, and Arf3), class II (Arf4 and Arf5), and class III (Arf6) Arfs. Similar to other GTP binding proteins, Arfs function as molecular switches, cycling between GTP-bound state and GDP-bound state, with multiple targets affecting a variety of cellular events. Arf6 is the sole Arf protein function on plasma membrane, the GTP-binding protein Arf6 regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. A key determinant of Arf6 function is the lifetime of the GTP-bound active state, which is orchestrated by GTP-GDP exchanging factors and GTPase-activating proteins. GTPase-activating proteins(GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. It has reported that Arf6 is essential to regulate breast cancer cell invasion dependent on Arf6 GTPase cycle. However, very little is known about the molecular mechanisms underlying Arf6-mediated cell migration.To systematically analyze proteins that regulate Arf6 activity during cell migration, we perform a proteomic analysis of proteins selectively interacted with active Arf6 using mass Spectrometry. Here, we report the identification and characterization of a novel Arf GAPs. We named it as ACAP4 according to its conservedly structual characterization. Together with previously described Arf GAPs, ASAP1, PAP/PAG3 and ACAP1/2, ACAP4 can be grouped into AZAP protein family defined by several common structural motifs including two coiled coils, one PH domain, one GAP motif, and two ankyrin repeats.Our biochemical characterization demonstrates that ACAP4 has a phosphoinositide dependent GAP activity, ACAP4 functioned as Arf6 GAPs. In vitro, ACAP4 preferred Arf6 as a substrate, rather than Arf1 and Arf5. In HeLa cells, overexpression of ACAP4...