title: DEF-1/ASAP1 is a GTPase-activating protein (GAP) for ARF1 that enhances cell motility through a GAP-dependent mechanism.
abstract: DEF-1/ASAP1 is an ADP-ribosylation factor GTPase-activating protein (ARF GAP) 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 platelet-derived growth factor-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.
authors.affiliation: Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Craig
authors.initials: C
authors.lastname: Furman
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Short
authors.forename: Romesh R
authors.initials: RR
authors.lastname: Subramanian
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Zetter
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Roberts
mesh_terms: D016475:3T3 Cells; D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology*; D002460:Cell Line; D002465:Cell Movement; D002469:Cell Separation; D003598:Cytoskeletal Proteins / Q000096:biosynthesis; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D051419:Paxillin; D010750:Phosphoproteins / Q000096:biosynthesis; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012190:Retroviridae / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D016335:Zinc Fingers; D018909:src Homology Domains
chemical_list: D000089982:ADP-Ribosylation Factor 6; C117891:ASAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; C485546:Asap1 protein, mouse; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D020690:GTPase-Activating Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; C496062:Pxn protein, mouse; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715727:Arf6 protein, mouse