The PH Domain of ASAP1 Binds N terminus of Arf1 in Presence of PIP2 for Efficient GTPase‐activating Protein Activity

Abstract

ASAP1 is a multi‐domain GTPase‐activating protein (GAP) for ADP‐ribosylation factor (Arf) guanine nucleotide binding proteins. ASAP1 has been found to affect cell behaviors dependent on adhesions and actin, including the proliferation, invasion and metastasis of cancer cells. The cellular function of ASAP1 depends on its highly regulated and robust Arf GAP activity, which refers to the enzymatic activity of converting Arf•GTP to Arf•GDP. Arf GAP activity requires the PH domain together with the catalytic Arf GAP domain and is modulated 4 orders of magnitude by the membrane phospholipid phosphatidylinositol 4,5‐ bisphosphate (PIP2) binding to the PH domain. However, the mechanistic basis of PIP2‐stimulated GAP activity is not completely understood. Here we found that the N terminal extension of the substrate Arf1 is necessary for the PIP2‐stimulated activity of ASAP1 in GTP hydrolysis assays. A peptide 2–17 from N terminal Arf1 inhibited GAP activity with full length Arf1 as substrate. Using fluorescence anisotropy and Forster resonance energy transfer (FRET), we found binding between the N‐terminal 2–17 amino acids of Arf1 and ASAP1. Binding was confined to the ASAP1 PH domain and depended on presence of PIP2. Limited proteolysis and crosslinking experiments indicate conformational changes in ASAP1 PH domain on binding PIP2. Mutational studies involving deletion of the putative interacting residues on the PH domain results in loss of binding to N terminal Arf1 as well as ASAP1 GAP activity. We propose that PIP2 binding to PH domain brings conformational changes that facilitate binding to N terminal Arf1 and then the later interaction favorably orients Arf GAP domain with the substrate Arf1 enhancing GAP activity of ASAP1.Support or Funding InformationThis work is supported by NIH Intramural Program (Project BC 007365).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.