Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale.
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 β 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 β 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 β1 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.
sGSK Group, Institute of Molecular and Cell Biology, Agency for Science Technology and Research, Proteos Building, 61 Biopolis Drive, Singapore 138673, Singapore.
Jing-Ming
JM
Dong
sGSK Group, Institute of Molecular and Cell Biology, Agency for Science Technology and Research, Proteos Building, 61 Biopolis Drive, Singapore 138673, Singapore.
Felicia Pei-Ling
FP
Tay
Quantitative Proteomics Group, Institute of Molecular and Cell Biology, Agency for Science Technology and Research, Singapore 138673, Singapore.
Hannah Lee-Foon
HL
Swa
Quantitative Proteomics Group, Institute of Molecular and Cell Biology, Agency for Science Technology and Research, Singapore 138673, Singapore. Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore.
Jayantha
J
Gunaratne
sGSK Group, Institute of Molecular and Cell Biology, Agency for Science Technology and Research, Proteos Building, 61 Biopolis Drive, Singapore 138673, Singapore.
Thomas
T
Leung
Institute of Medical Biology, 8A Biomedical Grove, #06-06 Immunos Building, Singapore 138648, Singapore.
Brian
B
Burke
sGSK Group, Institute of Molecular and Cell Biology, Agency for Science Technology and Research, Proteos Building, 61 Biopolis Drive, Singapore 138673, Singapore. Institute of Medical Biology, 8A Biomedical Grove, #06-06 Immunos Building, Singapore 138648, Singapore. Department of Pharmacology, National University of Singapore, Singapore 117597, Singapore. ed.manser@imcb.a-star.edu.sg.
Ed
E
Manser
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D019921:Biotinylation; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism; D022001:Focal Adhesions / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; C473794:FBLIM1 protein, human; C477322:FERMT3 protein, human; C000613889:Kank2 protein, human; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C449803:PPFIBP1 protein, human; D025521:Tumor Suppressor Proteins