Abstract
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.
Highlights
Cell migration is a cyclic process comprising of four basic steps: adhesion, attachment, translocation, and retraction of the rear [1, 2]
We show that sequestration of endogenous ASAP1 to mitochondria with a CD2-associated protein (CD2AP) SH3-mito fusion protein inhibited REF52 cell spreading and migration in response to fibronectin stimulation
We show that CD2AP, a ubiquitously expressed adaptor protein, stably associates with the ADP-ribosylation factors (Arfs) GTPase-activating protein ASAP1 through its N-terminal SH3 domains
Summary
ADP-ribosylation factor; ASAP1, Arf-GAP containing SH3, ankyrin repeats and pleckstrin homology domain; CD2AP, CD2-associated protein; FAK, focal adhesion kinase; GAP, GTPase-activating protein; GST, glutathione S-transferase; mAb, monoclonal antibody; PH, pleckstrin homology; REF, rat embryo fibroblasts; SH3, Src homology 3; siRNA, small interfering RNA; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; GFP, green fluorescent protein; EGF, epidermal growth factor. These seemingly contradictory observations raise the possibility that the regulation of Arf GTPase by ASAP1 plays an important role in the stabilization of paxillin in newly formed adhesions
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.