Phosphoinositide-dependent Activation of the ADP-ribosylation Factor GTPase-activating Protein ASAP1

doi:10.1074/jbc.275.13.9653

Phosphoinositide-dependent Activation of the ADP-ribosylation Factor GTPase-activating Protein ASAP1

EVIDENCE FOR THE PLECKSTRIN HOMOLOGY DOMAIN FUNCTIONING AS AN ALLOSTERIC SITE*

From the Division of Basic Sciences, NCI and ^¶NHLBI, National Institutes of Health, Bethesda, Maryland 20892, ^§Royal Free and University College Medical School, Royal Free Campus, London NW32PF, United Kingdom, and ^‖Nutrimed Biotech, Langmuir Laboratory, Ithaca, New York 14850

Abstract

The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin β family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin β4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P₂) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P₂binding to the PH domain; however, in contrast to the Arf exchange factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF-induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P₂ binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf.

Footnotes

↵* This work was supported by Public Health Service/National Institutes of Health (NIH) Grants GM 49594 and GM 51138 (to R. A. for the synthesis of phosphoinositides) and the Division of Basic Sciences, NCI, NIH.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ These two authors contributed equally to this work.
↵** To whom correspondence should be addressed: Laboratory of Cellular Oncology, Division of Basic Sciences, National Cancer Institute, 37 Convent Dr. MSC 4255, Bethesda, MD 20892. Tel.: 301-496-3788; Fax: 301-496-5839; E-mail: randazzo@helix.nih.gov.
↵2 T. R. Jackson, L. Foroni, K. Miura, and P. A. Randazzo, manuscript in preparation.
↵3 R. Aneja, S. G. Aneja, A. P. Parra, and P. T. Ivanova, manuscript in preparation.
↵4 The phosphoinositide dependence of the generation of the 8-kDa product could result from a phosphoinositide-protein interaction dependent on SDS and occurring in the SDS-containing sample buffer used for electrophoresis.
↵5 J. Gruschus and J. Ferretti, unpublished observations.
Abbreviations:

Arf

ADP-ribosylation factor

ASAP1

Arf GAP containing Src homology 3, ANK repeat, and PH domains

GAP

GTPase-activating protein

GEF

guanine nucleotide exchange factor

PA

phosphatidic acid

PC

phosphatidylcholine

PtdIns

phosphatidylinositol

PtdIns-4P

phosphatidylinositol 4-phosphate

PtdIns-4

5-P₂ or PtdIns-P₂, phosphatidylinositol 4,5-bisphosphate

PtdIns-3

4-P₂, phosphatidylinositol 3,4-bisphosphate

PtdIns-3

4,5-P₃ or PtdIns-P₃, phosphatidylinositol 3,4,5-trisphosphate

bbPtdIns-4

5-P₂, phosphatidylinositol 4,5-bisphosphate purified from bovine brain

diC16PtdIns-4

5-P₂ or diC16PtdIns-P₂, dipalmitoyl phosphatidylinositol 4,5-bisphosphate

PH

pleckstrin homology

PDGF

platelet-derived growth factor
- Received November 3, 1999.
- Revision received January 11, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.