Journal of Biological Chemistry
EnzymologyInteraction 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
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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.
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We determined the critical micelle concentration for diC8PIP2 to be >1 mm, using the method described in Ref. 57.
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We also observed binding of diC8PIP2 to [2–17]Arf1 by circular dichroism spectrometry (not shown).
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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).
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High throughput screens for inhibitors using this assay are ongoing.
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Present address: Dept. of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298.
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Present address: Dept. of Biology, Wake Forest University, Winston-Salem, NC 27109.
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Present address: Novo Nordisk, Beijing 100020, China.
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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.