title: Phosphoinositide-dependent activation of the ADP-ribosylation factor GTPase-activating protein ASAP1. Evidence for the pleckstrin homology domain functioning as an allosteric site.
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 beta 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 beta4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P(2) binding 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-P(2) 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.
authors.affiliation: Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Kam
authors.forename: K
authors.initials: K
authors.lastname: Miura
authors.forename: T R
authors.initials: TR
authors.lastname: Jackson
authors.forename: J
authors.initials: J
authors.lastname: Gruschus
authors.forename: P
authors.initials: P
authors.lastname: Roller
authors.forename: S
authors.initials: S
authors.lastname: Stauffer
authors.forename: J
authors.initials: J
authors.lastname: Clark
authors.forename: R
authors.initials: R
authors.lastname: Aneja
authors.forename: P A
authors.initials: PA
authors.lastname: Randazzo
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D001798:Blood Proteins; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D010716:Phosphatidylinositols; D010750:Phosphoproteins; C039077:platelet protein P47; D020727:ADP-Ribosylation Factors