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.

ACKNOWLEDGMENTS

We thank Douglas Lowy, Dan Cassel, James Battey, and Patrick Donohue for discussions and advice; W. S. Lane, R. Robinson, J. Neveu, and D. Arnelle of the Harvard Microchemistry Facility for expertise in HPLC, mass spectrometry, and peptide sequencing; Jenny Clark for expertise in nucleotide sequencing; S. Stauffer and J. Kam for technical assistance; S. Hollenberg, B. Mayer, F. Gertler, B. Howell, C. Sachsenmaier, S. M. Thomas, P. Cicchetti, E. M. Eddy, R. Braun, J. Wang, S. Feller, G. Myles, and R. Bourette for libraries, strains, and plasmids; Douglas Lowy for expertise in etymology; and Dan Cassel for initiating contact between our groups.

This work was supported by grant CA41072 from the U.S. Public Health Service (J.A.C.) and the Division of Basic Sciences, National Cancer Institute (P.A.R.). M.T.B. was supported by a postdoctoral fellowship from the National Institutes of Health (CA62598).