ASAP1, A PHOSPHOLIPID-DEPENDENT ARF GTPASE-ACTIVATING PROTEIN THAT ASSOCIATES WITH AND IS PHOSPHORYLATED BY SRC

Membrane trafficking is regulated in part by small GTP-binding protein s of the ABP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and c loned two variants of a 130-kDa phosphatidylinositol 4,5-biphosphate ( PIP2)-dependent Arf1 GTPase-activating protein (GAP), which we call AS AP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zin c finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 bind ing motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP2-de pendent GAP activity on Arf1 and Arf5, less activity on Arf6, and no d etectable activity on Ar12 in vitro. The cDNA for ASAP1 was independen tly identified in a screen for proteins that interact with the SH3 dom ain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on ty rosine residues in cells expressing activated Src. Both coimmunoprecip itation and tyrosine phosphorylation depend on the same proline-rich c lass II Src SH3 binding site required for in vitro association. By dir ectly interacting with both Arfs and tyrosine kinases involved in regu lating cell growth and cytoskeletal organization, ASAP1 could coordina te membrane remodeling events with these processes.