DEF-1/ASAP1 is a GAP for ARF1 that Enhances Cell Motility Through a GAP-Dependent Mechanism Craig Furman § , Sarah M. Short ! , Romesh R. Subramanian § , Bruce R. Zetter ! , and Thomas M. Roberts § ; From the § Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School and ! Department of Surgical Research, Childrens Hospital, Harvard Medical School, Boston, Massachusetts, 02115 ; To whom correspondence should be addressed. Tel.: 617-632-3049; Fax: 617-632-4770; E-mail: thomas_roberts@dfci.harvard.edu. DEF-1/ASAP1 is an ARF GAP 1 that localizes to focal adhesions and is involved in cytoskeletal regulation. In this paper, we use a cell-based ARF GAP assay to demonstrate that DEF-1 functions as a GAP for ARF1 and not ARF6 in vivo. This degree of substrate preference was unique to DEF-1, as other ARF GAP proteins, ACAP1, ACAP2, and ARFGAP1 were able to function on both ARF1 and ARF6. Since transient overexpression of DEF-1 has been shown to interfere with focal adhesion formation and PDGF-induced membrane ruffling, we investigated whether NIH 3T3 cells stably expressing DEF-1 have altered cell motility. Here we report that ectopic DEF-1 enhances cell migration toward PDGF as well as IGF-1. This chemotactic effect appears to result from a general increase in cell motility, as DEF-1 expressing cells also exhibit enhanced levels of basal and chemokinetic motility. The increase in cell motility is dependent on DEF-1 GAP activity since a DEF-1 mutant lacking the GAP domain failed to stimulate motility. This suggests that DEF-1 alters cell motility through the deactivation of ARF1. In contrast, the inhibition of cell spreading by DEF-1 was not dependent on GAP activity, indicating that spreading and motility are altered by DEF-1 through different pathways. ADP-ribosylation factors (ARFs) represent a family of small G proteins that are involved in the regulation of membrane trafficking and the actin cytoskeleton (for reviews see (1, 2)). There are six different mammalian isoforms of ARF which have been divided into three classes based on sequence similarities. Class 1 consists of ARF1, ________________________ 1 Abbreviations used are: ARF GAP, ADP-ribosylation factor GTPase activating protein; PDGF, platelet- derived growth factor; ER, endoplasmic reticulum; FAs, focal adhesions; PH, pleckstrin homology; ANK, ankyrin; SH3, src homology 3; FAK; focal adhesion kinase. - 1 - 1 Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc. JBC Papers in Press. Published on December 31, 2001 as Manuscript M109149200 by guest on May 10, 2017 http://www.jbc.org/ Downloaded from