Journal of Biological Chemistry
Volume 294, Issue 46, 15 November 2019, Pages 17354-17370
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Enzymology
Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4,5-bisphosphatePIP2 regulates Arf1 binding to the ASAP1 PH domain

https://doi.org/10.1074/jbc.RA119.009269Get rights and content
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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.

ADP ribosylation factor (ARF)
allosteric regulation
GTPase-activating protein (GAP)
cell signaling
protein–protein interaction
actin remodeling
ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1)
integrin adhesion
phosphatidylinositol 4,5-bisphosphate (PIP2)
pleckstrin homology domain

Cited by (0)

This work was supported by NCI Intramural Program Project BC-007365 from the National Institutes of Health (to R. A. B., M. D. H., M. E. Y., and P. A. R.), an Alex's Lemonade Stand Foundation Young Investigators Award, and by the NCI Rasopathies Initiative from the National Institutes of Health (to M. E. Y.). The authors declare that they have no conflicts of interest with the contents of this article.

This article contains Figs. S1–S3.

7

We determined the critical micelle concentration for diC8PIP2 to be >1 mm, using the method described in Ref. 57.

8

We also observed binding of diC8PIP2 to [2–17]Arf1 by circular dichroism spectrometry (not shown).

9

The differences between GAP activity in LUVs and with PIP2 analogs are described in more detail in a manuscript currently in preparation (N. S. Roy, X. Jian, R. Luo, P. A. Randazzo, and M. E. Yohe).

10

High throughput screens for inhibitors using this assay are ongoing.

11

PH, PTB, EVH1, and RanBD have a common fold, referred to as the PH domain superfold (63).

1

Present address: Dept. of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298.

2

Present address: Dept. of Biology, Wake Forest University, Winston-Salem, NC 27109.

3

Present address: Novo Nordisk, Beijing 100020, China.

6

The abbreviations used are:

    GAP

    GTPase-activating protein

    Arf

    ADP-ribosylation factor

    Arl

    Arf-like GTPase

    BAR

    Bin/amphiphysin/RVS

    CDR

    circular dorsal ruffles

    DH

    Dbl homology

    diC8PIP2

    dioctanoyl PIP2

    EDANS

    5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid

    EDC

    1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

    HVR

    hypervariable region

    HMQC

    heteronuclear multiple quantum correlation

    LUV

    large unilamellar vesicle

    MSP

    membrane scaffolding protein

    PA

    phosphatidic acid

    PDB

    Protein Data Bank

    PDGF

    platelet-derived growth factor

    PH

    pleckstrin homology

    PIP2

    phosphatidylinositol 4,5-bisphosphate

    PS

    phosphatidylserine

    PTB

    phosphotyrosine binding

    TAMRA

    carboxylic acid of tetramethylrhodamine

    ND

    nanodisc.