Fig. 2: Src is a regulatory kinase for the ITGB1 NPxY motifs.
From: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells
a, A schematic representation of the possible conformations for the ITGB1 intramolecular FRET biosensor (Illusia), where the mTurquoise2–YPet FRET pair is separated by the PTB from Dok1, a linker and the cytoplasmic domain (aa772–798) from ITGB1 (including the two NPxY motifs). Illusia is recruited to the membrane through an acylation substrate sequence derived from Lyn kinase. Ex, excitation; Em, emission. b, Representative FLIM images (left) and quantification of apparent FRET efficiency (right) of MM231 cells stably expressing Illusia after Dox-inducible overexpression of either Src(WT), kinase-dead Src(K295R) or constitutively active Src(Y527F)/Src(E378G) (n = 60 cells in all conditions with the exception of 70 for Src(Y527F) (−)Dox and 65 for Src(Y527F) (+)Dox pooled from three biological replicates; one-way analysis of variance (ANOVA) with a Šidák correction for multiple comparisons). Scale bars, 10 μm. c, Representative western blot of MM231 cells after Dox-inducible Src(WT), Src(K295R), Src(Y527F) or Src(E378G) overexpression. d, Densitometry analysis of western blots from c (n = 4 biological replicates; one-sample two-tailed t-test against the normalized control values for each cell line without Dox). The data are mean ± s.e.m. e, A scheme of an ELISA for pY. f, An ELISA for changes in ITGB1 phosphorylation using recombinant ITGB1 peptide and Src kinase in the absence or presence of ATP and the Src inhibitor Sara (1 μM; n = 3 biological replicates; triplicate wells/replicates; one-way ANOVA with a Šidák correction for multiple comparisons). Unphosphorylated ITGB1 and phosphorylated ITGB1 p(Y783) peptides were included as negative and positive controls, respectively. The data are the mean ± s.e.m. g, Representative FLIM–FRET images (left) and quantification (right) of MM231 and TIF cells with stable expression of Illusia and Sara treatment (1 μM) for 24 h (MM231s, n = 129 (DMSO) and 125 (Sara) cells pooled from five biological replicates; TIFs, n = 74 (DMSO) and 75 (Sara) cells pooled from three biological replicates; unpaired two-tailed Student’s t-test with a Welch’s correction). Scale bars, 10 μm. The boxplots represent the median and IQR. The whiskers extend to the minimum and maximum values. The grey areas on the boxplots highlight the IQR of the control conditions. NS, not significant. *P < 0.05, ***P < 0.001.
