Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer.
Microscopy-based fluorescence resonance energy transfer (FRET) provides an opportunity to monitor molecular processes in the natural environment in live cells. Here we studied molecular interactions and tyrosine phosphorylation of paxillin, Crk-associated substrate (CAS), and focal adhesion kinase (FAK) in focal adhesions. For that purpose, these focal adhesion phosphoproteins, fused to cyan or yellow fluorescent proteins (CFP or YFP) were expressed in cultured fibroblasts. To assess the dynamics of tyrosine phosphorylation we used YFP- or CFP-tagged SH2 domain of pp60(src) (dSH2), which specifically binds to phosphotyrosine residues. FRET measurements, combined with immunolabeling with phosphospecific antibodies revealed that FAK, CAS and paxillin are tyrosine phosphorylated in early matrix adhesions and that FAK is in FRET proximity to CAS and paxillin in focal complexes and focal adhesions. Data suggest that paxillin incorporation into nascent focal complexes precedes its tyrosine phosphorylation, which then gradually increases. In cells treated with Rho-kinase inhibitors or expressing constitutively active Rac, focal complexes showed similar levels of paxillin tyrosine phosphorylation as seen in mature focal adhesions. Dynamic FRET-based examination indicated that paxillin phosphorylation occurs in specific areas (hotspots) within focal adhesions, whereas FAK phosphorylation is broadly distributed.
Ballestrem
Christoph
C
Department of Molecular Cell Biology, The Weizmann Institute of Science, 76100 Rehovot, Israel.
Erez
Noam
N
Kirchner
Joachim
J
Kam
Zvi
Z
Bershadsky
Alexander
A
Geiger
Benjamin
B
D000818:Animals; D050719:Crk-Associated Substrate Protein / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000737:chemistry / Q000502:physiology*; D022001:Focal Adhesions / Q000502:physiology*; D051379:Mice; D041681:NIH 3T3 Cells; D051419:Paxillin / Q000502:physiology; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D014443:Tyrosine / Q000737:chemistry / Q000502:physiology*; D018909:src Homology Domains
D050719:Crk-Associated Substrate Protein; D051419:Paxillin; D014443:Tyrosine; D051416:Focal Adhesion Protein-Tyrosine Kinases