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.
Highlights
The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton
Four members of this family have been identified2 [13, 14], and one member of the family, ASAP1, was purified as an Arf GTPase-activating proteins (GAPs) that is coordinately activated by phosphatidic acid (PA) and phosphoinositides [13]
Activation of ASAP1 by Phospholipids Is Specific for PtdIns4,5-P2— ASAP1 and Arf exchange factors are both regulated by phosphoinositides, differences in the particular phosphoinositide could provide independent regulation
Summary
Protein Expression in and Purification from Bacteria—To express the PH domain, ZA and PZA, reading frames for proteins consisting of residues 338 – 431 (PH domain), 452–724 (referred to here as ZA), and 325–724 (PZA) of ASAP1 were ligated into the NdeI/XhoI sites of pet19c (Novagen) to express histidine-tagged proteins and pet (Novagen) to express proteins without additional residues. GAP activity of PZA (circles and inverted triangles) and ZA (triangles) at the indicated concentrations was determined in the presence of 1.4 Triton X-100 (open symbols) or Triton X-100 containing 360 M PA and 90 M bovine brain PtdIns-P2 (filled symbols) using either wild type. The PtdIns-P2 dependence of GAP activity of 0.5 nM PZA using either wild type Arf or the indicated Arf mutants in the presence of Triton X-100 micelles containing 360 M PA and the indicated concentration of PtdIns-P2 was determined. Lipid binding to Arf [12] and PZA [32] was determined as described in the same buffer used to measure GAP activity. The Arf GAP activity for 0.5 nM of the indicated proteins was determined using mixed micelles of Triton X-100, 360 M PA, and the indicated concentrations of bbPtdIns-P2.
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