Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4,5-bisphosphate [Signal Transduction]

Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Δ17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1's activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP2), stimulated GAP activity on an N terminus–containing variant, [L8K]ARF1, but only marginally affected activity on [Δ17]ARF1. A peptide comprising residues 2–17 of ARF1 ([2–17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first β-strand of the PH domain. Point mutations in either the linker or the C-terminal α-helix of the PH domain decreased [2–17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 ...
Source: Journal of Biological Chemistry - Category: Chemistry Authors: Tags: Enzymology Source Type: research