Mislocalization or reduced expression of Arf GTPase-activating protein ASAP1 inhibits cell spreading and migration by influencing Arf1 GTPase cycling.
ADP-ribosylation factor (Arf) family of small GTP-binding proteins plays a central role in membrane trafficking and cytoskeletal remodeling. ASAP1 (Arf-GAP containing SH3, ankyrin repeats, and PH domain) is a phospholipid-dependent Arf GTPase-activating protein (Arf-GAP) that binds to protein-tyrosine kinases Src and focal adhesion kinase. Using affinity chromatography and mass spectrometry (MS), we identified the adaptor protein CD2-associated protein (CD2AP) as a candidate binding partner of ASAP1. Both co-immunoprecipitation and GST pull-down experiments confirmed that CD2AP stably interacts with ASAP1 through its N-terminal SH3 domains. Using a mislocalization strategy, we show that sequestration of endogenous ASAP1 to mitochondria with a CD2AP SH3-mito fusion protein (the three N-terminal SH3 domains of CD2AP fused to Listeria monocytogenes ActA mitochondria-targeting sequence) inhibited REF52 cell spreading and migration in response to fibronectin stimulation. Using an alternative strategy we show that suppressing ASAP1 expression with small interfering RNA duplexes also significantly retarded cell spreading and inhibited cell migration. Furthermore, abrogation of ASAP1 function using either small interfering RNAs or mislocalization approaches caused an increase of GTP loading on Arf1 and loss of paxillin from adhesions. These results taken together with our previous observations that overexpression of ASAP1 inhibits cell spreading and alters paxillin localization to adhesions (Liu, Y., Loijens, J. C., Martin, K. H., Karginov, A. V., and Parsons, J. T. (2002) Mol. Biol. Cell. 13, 2147-2156) suggest that the recruitment of certain adhesion components such as paxillin requires dynamic GTP/GDP turnover of Arf1 GTPase.
Liu
Yunhao
Y
Department of Microbiology and Cancer Center, University of Virginia Health System, Charlottesville, Virginia 22908, USA.
Yerushalmi
Gil M
GM
Grigera
Pablo R
PR
Parsons
J Thomas
JT
D020823:ADP-Ribosylation Factor 1 / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000502:physiology; D006801:Humans; D051379:Mice; D051419:Paxillin; D010750:Phosphoproteins / Q000502:physiology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C485546:Asap1 protein, mouse; D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D020823:ADP-Ribosylation Factor 1