Abstract
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 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.
Highlights
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
To explore the role of the N terminus of Arf1 for PIP2-stimulated GAP activity of ASAP1, PZA was titrated into reaction mixtures containing myrArf11⁄7GTP, nonmyrArf1, or [⌬17]Arf11⁄7GTP and large unilamellar vesicles (LUVs) with or without incorporated phosphatidylserine (PS), which reduces the concentration of PIP2 needed for maximum activation (14)
GAP activity is reported as the C50, which is the concentration of PZA needed to induce 50% of the GTP bound to Arf1 to be hydrolyzed in 3 min and is inversely related to enzymatic power (Fig. 1B and Table 1) (47, 48)
Summary
We found that PIP2-stimulated activity of a recombinant protein composed of the PH, Arf GAP, and ankyrin repeat domains of ASAP1, [325–724]ASAP1, referred to as PZA (for PH, Zinc binding, which comprises the Arf GAP catalytic domain, and Ankyrin repeat domains, see Fig. 1A) by more than 10,000-fold (14). We found the FRET signal was saturable with a half-maximum effect at 2.3 Ϯ 0.14 M [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]Arf1-EDANS (Fig. 4D), similar to the concentration dependence observed for inhibition of GAP activity. There was a 100-fold difference in activity between the proteins, measured with a GAP assay based on the difference in tryptophan fluorescence between Arf11⁄7GTP and Arf11⁄7GDP (Fig. 7F) (61) Based on these results, the linker might contribute to binding, but it is not the major determinant.
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