Jing-Ming Dong1, Felicia Pei-Ling Tay1 Hannah Lee-Foon Swa2, Jayantha Gunaratne2,5, Thomas Leung1, Brian Burke3 and Ed Manser1,3,4*
1 sGSK Group, Institute of Molecular & Cell Biology, Proteos Building, 61 Biopolis Drive 138673, Singapore.
2 Quantitative Proteomics Group. Institute of Molecular & Cell Biology
3 Institute of Medical Biology (IMB), 8A Biomedical Grove, #06-06 Immunos Building 138648, Singapore.
4 Department of Pharmacology, National University of Singapore, 117597.
5 Yong Loo Lin School of Medicine, National University of Singapore, 117597.
* Corresponding author.
Published in Science Signaling on 14 June 2016.
Focal adhesions are protein complexes that link metazoan cells to the extracellular matrix through the integrin family of transmembrane proteins. Integrins recruit many proteins to these complexes, referred to as the “adhesome.” We used proximity-dependent biotinylation (BioID) in U2OS osteosarcoma cells to label proteins within 15 to 25 nm of paxillin, a cytoplasmic focal adhesion protein, and kindlin-2, which directly binds beta-integrins. Using mass spectrometry analysis of the biotinylated proteins, we identified 27 known adhesome proteins and 8 previously unknown components close to paxillin. However, only seven of these proteins interacted directly with paxillin, one of which was the adaptor protein Kank2. The proteins in proximity to beta-integrin included 15 of the adhesion proteins identified in the paxillin BioID data set. BioID also correctly established kindlin-2 as a cell-cell junction protein. By focusing on this smaller data set, new partners for kindlin-2 were found, namely, the endocytosis-promoting proteins liprin b1 and EFR3A, but, contrary to previous reports, not the filamin-binding protein migfilin. A model adhesome based on both data sets suggests that focal adhesions contain fewer components than previously suspected and that paxillin lies away from the plasma membrane. These data not only illustrate the power of using BioID and stable isotope–labeled mass spectrometry to define macromolecular complexes but also enable the correct identification of therapeutic targets within the adhesome.
Figure Legend : Suggested localization of FA proteins in FAs in U2OS cells based on BioID data. The schematic shows the protein-protein interactions we detected between paxillin or kindlin-2 and other FA proteins and that have been selected published literature. To simplify the drawing, we have added multiple integrin dimers. Kindlin-2 can interact with the integrin in the presence of talin (32) and likely with Cd98h. Proteins thought to interact directly with integrin are in pink. Transmembrane or peripheral membrane proteins are in blue.
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