Fig. 3
From: Sharpin suppresses β1-integrin activation by complexing with the β1 tail and kindlin-1
Kindlin-1 directly interacts with sharpin (via its F0 subdomain) and recruits sharpin to inhibit β1-integrin activation. a The kindlin family members (K1, K2 and K3) were fused with the DNA-binding domain of Gal4 in pGBKT7 vector and sharpin (SH) was fused with the transcriptional activation domain of Gal4 in pGADT7 vector. The interaction between kindlin and sharpin was evaluated using the Matchmaker™ Gold yeast two-hybrid system by a serial dilution method on selection media. Two molecules known for interacting with each other (Bop1/Bop2) were used as a positive control and the empty vectors were used as a negative control. Growth of yeast cells on SD2 selection media indicates successful transformation; growth of yeast cells on SD4 selection media indicates a positive protein-protein interaction. b Glutathione Sepharose beads were loaded with GST and GST-fused sharpin (GST-SH) proteins and used to incubate with his-tagged kindlins (His-K1, His-K2 and His-K3). The loading of GST and GST-SH on the beads was measured by Coomassie blue (C. blue) staining. Binding of His-kindlins to GST proteins was analyzed by immunoblotting (IB). c Purified GST and GST-β1CT proteins were coupled to Glutathione Sepharose and used to incubate with flag-tagged talin head (Flag-TH) in the presence or absence of his-tagged sharpin (His-SH) and/or kindlin-1 (K1). After incubation, beads were extensively washed. The loading of GST proteins was evaluated by Coomassie blue (C. blue) staining. Precipitated protein samples on the beads, including Flag-TH, His-SH and K1, were evaluated by SDS-PAGE followed by immunoblotting (IB). d Glutathione Sepharose beads were loaded with GST and GST-fused kindlin-1 (GST-K1) proteins and used to incubate with his-tagged N-terminus (His-SH-N) or C-terminus (His-SH-C) of sharpin. The loading of GST and GST-K1 on the beads was measured by Coomassie blue (C. blue) staining. Binding of His-SH-N or His-SH-C to GST proteins was analyzed by immunoblotting (IB). e Kindlin-1 (K1) and its mutants, including K1ΔF0, K1ΔF1, the N-terminal fragment (F0 + F1) of kindlin-1 (K1N), and the kindlin-1 QW/AA mutant (K1AA), were expressed and purified with a his tag. Interaction of GST or GST-fused sharpin (GST-SH) with these kindlin-1 proteins were evaluated by pull-down assays followed by immunoblotting (IB). f Interaction of sharpin with kindlin-1 and its mutants were also evaluated using the Matchmaker™ Gold yeast two-hybrid system, same as described in (a). g The effects of kindlin-1 mutants, including K1AA and K1ΔF0, on integrin α5β1 activation in CHO cells were evaluated by co-transfection and the GST-Fn-III binding assay. The results represent the mean ± SD of at least 3 experiments. (MFI: median of fluorescence intensity; ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001)