Fig. 5: Kindlin-2 interacts with both LATS1 and YAP and impedes the interaction between LATS1 and YAP.

A Total lysates were extracted from TM4 cell lines (without treatments) for co-immunoprecipitation (Co-IP) experiments. Equal amount of proteins was immunoprecipitated separately with IgG or anti-Kindlin-2 antibody. Western blot displayed the expression of YAP, LATS1 and Kindlin-2 with specific antibodies. B FLAG and FLAG-Kindlin-2 expression vector were transfected into TM4 cell lines separately. 48 h after transfection, total cell lysates were immunoprecipitated with anti-FLAG antibody and protein G-Sepharose. The expression of YAP, LATS1, and FLAG were detected with specific antibodies by Western blot analysis. C Anti-YAP antibody was used for Co-IP experiment and the expression of Kindlin-2 was detected by specific antibodies. D Anti-LATS1 antibody was used for Co-IP experiment and the expression of Kindlin-2 was detected by specific antibody. E GFP-Kindlin-2 and FLAG-YAP expression vector were transfected into HEK293T cell lines separately. 48 h after transfection, total cell lysates were immunoprecipitated with anti-FLAG antibody and protein G-Sepharose. The expression of YAP and FLAG were detected with specific antibodies by Western blot analysis. F 15P-1 cells were stained with an anti-YAP mouse (red) and an anti-Kindlin-2 rabbit (green). Nuclei were stained with DAPI (blue), followed by visualization with confocal microscopy. Scale bars, 10μm. Magnification were shown. G 15P-1 cells were stained with an anti-LATS1 mouse (red) and an anti-Kindlin-2 rabbit (green). Nuclei were stained with DAPI (blue), followed by visualization with confocal microscopy. Scale bars, 10 μm. Magnification were shown. H–J Indicated truncates of Kindlin-2, YAP and LATS1 were constructed according to the functional domains respectively. YAP contains two WW domains in the middle and a transactivation domain at the C terminus. LATS1 contains a kinase region in the middle part. K HEK293 cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were incubated with GST or GST-YAP in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. L Purified His-Kindlin-2 protein was incubated with purified GST or GST-YAP fragments at 4 °C overnight. Beads were washed and the remaining proteins were resolved by SDS-PAGE and further analyzed by Western blot analysis using anti-Kindlin-2 antibody (upper panel). The GST and GST-YAP were stained by Coomassie blue (lower panel). M HEK293 cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were incubated with GST or GST-LATS1-Kinase region in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. N HEK293 cells were co-transfected with GFP-Kindin-2 and the indicated truncates of FLAG-LATS1. Cell lysates were incubated with anti-FLAG antibody and protein G-Sepharose. The expression of GFP and FLAG were detected with specific antibodies by Western blot analysis. O, P Kindlin-2 isolates the interaction between LATS1 and YAP. O GFP or GFP-Kindlin-2 were co-transfected with FLAG-LATS1 into HEK293T cell lines. P Control siRNA or Kindlin-2 siRNA were co-transfected with FLAG-LATS1 into HEK293T cell lines. Co-immunoprecipitation was performed after 48 h transfection with FLAG-M2 beads. Western blot showed the expression of immunoprecipitation lysis YAP and FLAG and unprocessed lysis YAP, FLAG and Kindlin-2. Q Within the red rectangle is the brief working model of Kindlin-2 inactivating Hippo signaling pathway by interacting with LATS1 and YAP to inhibit the phosphorylation of YAP and to promote YAP nuclear localization. Outside the red rectangle is widely known Hippo signaling activation process.