Phosphoinositide-dependent activation of the ADP-ribosylation factor GTPase-activating protein ASAP1 - Evidence for the pleckstrin homology domain functioning as an allosteric site

The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regula tors of membrane traffic and the actin cytoskeleton. Both negative and posi tive regulators of Arf, the centaurin beta family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain plecks trin homology (PH) domains and are activated by phosphoinositides, To under stand how the activities are coordinated, we have examined the role of phos phoinositide binding for Arf GAP function using ASAP1/centaurin beta 4 as a model, In contrast to Arf exchange factors, phosphatidylinositol 4,5-bisph osphate (PtdIns-4,5-P-2) specifically activated Arf GAP, D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P-2 b inding to the PH domain; however, in contrast to the Arf exchange factors a nd 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) a ctivation and recruitment could be uncoupled; (iii) the PH domain was neces sary for activity even in the absence of phospholipids; and (iv) the Arf GA P domain influenced localization and lipid binding of the PH domain. Furthe rmore, PtdIns-4,5-P-2 binding to the PH domain caused a conformational chan ge 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 sugge st a model in which feedforward and feedback loops involving lipid metaboli tes coordinate GTP binding and hydrolysis by Arf.