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. 2015 Feb 18;290(13):8360–8372. doi: 10.1074/jbc.M115.638874

FIGURE 7.

FIGURE 7.

Arg interacts directly with integrin β1 in cells. A, diagram of expression constructs used for FRET-FLIM assays and summary of the interaction results. B, Arg interacts directly with integrin β1 in cells. Integrin β1−/− mouse embryonic fibroblasts were transiently transfected with WT integrin β1-GFP alone or with Arg-mCherry or cortactin-mCherry and allowed to adhere and spread on fibronectin-coated coverslips for 45 min. FRET-FLIM analysis was performed, and fluorescence lifetimes are shown in the heat map corresponding to the values in the tau color scale legend. C, average FRET efficiencies for the direct Arg-β1 interaction. Data from three independent experiments, 8 cells/experiment/condition. Error bars represent S.E. from all 24 cells. *, p < 0.05. D, FRET was performed on integrin β1−/− mouse embryonic fibroblasts transiently co-transfected with WT β1-GFP with either Arg-RFP, N-term Arg-RFP, or C-term Arg-RFP and plated on fibronectin. Fluorescence lifetimes are shown in the heat map corresponding to the values in the tau color scale legend in B. Fluorescence lifetimes were calculated from two independent experiments of 10 cells/experiment/condition, and FRET efficiencies were quantified in E where error bars represent S.E. from all 20 cells. *, p < 0.05; **, p < 0.01. These experiments show that the Arg N-terminal half is necessary and sufficient to mediate interactions with integrin β1 in cells. F, Arg kinase- and SH2 domain-mediated interactions are both important mediators for the β1-Arg interaction in cells. Integrin β1−/− mouse embryonic fibroblasts were transiently co-transfected with WT integrin β1-GFP or 4KA β1-GFP, and Arg-mCherry or Arg R198K-mCherry and plated on fibronectin for 45 min, and FRET efficiencies were calculated from two independent experiments of nine cells/experiment/condition and reported in G where error bars represent S.E. from all 18 cells. **, p < 0.01; ***, p < 0.001. Fluorescence lifetimes are shown in the heat map corresponding to the values in the tau color scale legend in B. H, a model for Arg kinase activation by integrin β1. Upon integrin activation, Arg binds a lysine-rich region in the β1 tail through its kinase domain, and this partially activates Arg kinase activity. Arg can then phosphorylate the membrane-proximal Tyr-783 site on the integrin β1 tail, possibly in cis or trans, creating a second interface for the Arg SH2 domain. Engagement of these interaction interfaces enhances Arg kinase activation. N-term, N-terminal; C-term, C-terminal.