title: ASAP1, a phospholipid-dependent arf GTPase-activating protein that associates with and is phosphorylated by Src.
abstract: Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4, 5-biphosphate (PIP2)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP2-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain 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 tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
authors.forename: M T
authors.initials: MT
authors.lastname: Brown
authors.forename: J
authors.initials: J
authors.lastname: Andrade
authors.forename: H
authors.initials: H
authors.lastname: Radhakrishna
authors.forename: J G
authors.initials: JG
authors.lastname: Donaldson
authors.forename: J A
authors.initials: JA
authors.lastname: Cooper
authors.forename: P A
authors.initials: PA
authors.lastname: Randazzo
mesh_terms: D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D018909:src Homology Domains / Q000502:physiology; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D019061:src-Family Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors