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Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4,5-bisphosphate.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2019 Nov 15; Vol. 294 (46), pp. 17354-17370. Date of Electronic Publication: 2019 Oct 06. - Publication Year :
- 2019
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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 (PIP <subscript>2</subscript> ). The mechanistic basis of PIP <subscript>2</subscript> -stimulated GAP activity is incompletely understood. Here, we investigated whether PIP <subscript>2</subscript> 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 PIP <subscript>2</subscript> has little effect on ASAP1's activity. A soluble PIP <subscript>2</subscript> analog, dioctanoyl-PIP <subscript>2</subscript> (diC8PIP <subscript>2</subscript> ), 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 PIP <subscript>2</subscript> -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 PIP <subscript>2</subscript> regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.
- Subjects :
- ADP-Ribosylation Factor 1 chemistry
ADP-Ribosylation Factors chemistry
Actins chemistry
Actins genetics
Adaptor Proteins, Signal Transducing chemistry
GTPase-Activating Proteins chemistry
GTPase-Activating Proteins genetics
Humans
Neoplasms genetics
Phosphatidylinositol 4,5-Diphosphate chemistry
Pleckstrin Homology Domains genetics
Point Mutation genetics
Protein Binding genetics
ADP-Ribosylation Factor 1 genetics
ADP-Ribosylation Factors genetics
Adaptor Proteins, Signal Transducing genetics
Phosphatidylinositol 4,5-Diphosphate genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 294
- Issue :
- 46
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 31591270
- Full Text :
- https://doi.org/10.1074/jbc.RA119.009269