Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation

Smurf1 mediates Kindlin-2 proteasomal degradation. (A) GFP-Kindlin-2 plasmid (2 µg) was transfected into HEK293T cells together with increasing amounts of Smurf1 WT plasmid. Kindlin-2 expression was determined by immunoblotting with an anti-GFP antibody 24 h after transfection. (B) HEK293T cells were transfected with increasing amounts of Smurf1 expression plasmid, and endogenous protein levels of Kindlin-2 were determined. (C) GFP-Kindlin-2 was transfected into HEK293T cells together with Smurf1 WT or C699A plasmid, and Kindlin-2 expression was examined. (D) HEK293T cells with transfection of GFP-Kindlin-2 and Flag-Smurf1 plasmids were treated with a proteasome inhibitor MG132 (20 µM) or DMSO for 6 h. Kindlin-2 expression was measured. (E) HEK293T cells were transfected with control siRNA or Smurf1 siRNA, and the expression of Kindlin-2 was detected. (F) Flag-Smurf1 was transfected into HeLa cells together with GFP-Kindlin-2, and cells were treated with CHX at 100 µg/ml for the indicated times. The half-life of GFP-Kindlin-2 was measured by Western blot. Quantification of the Kindlin-2 half-life was performed, and each point is represented as the mean ± SD of triplicate experiments. (G) HeLa cells were transfected with Flag-Smurf1, and cells were treated with CHX at 100 µg/ml for the indicated times. The half-life of endogenous Kindlin-2 protein was measured by Western blot and analyzed. Each point represents the mean ± SD of triplicate experiments. (H) CHX-chase experiments of Kindlin-2 in MDA-MB-231 cells transfected with control siRNA or Smurf1 siRNA are shown. Quantification of Kindlin-2 half-life was performed, and each point is represented as the mean ± SD (n = 3).

Smurf1 induces polyubiquitination of Kindlin-2. (A) Flag-Kindin-2 and HA-Ub were cotransfected into HEK293T cells together with control vector, Myc-Smurf1 (WT), or Myc-Smurf1 (C699A) expression plasmid. Kindlin-2 ubiquitination was detected by immunoprecipitation with anti-Flag M2 beads and immunoblotting with an anti-HA antibody. (B) E1, UbcH5c (E2), HA-Ub, GST-Smurf1 (expressed and purified from bacteria), and His-Kindlin-2 (expressed and purified from bacteria) were incubated at 30°C for 2 h in ubiquitination reaction buffer. Ubiquitinated Kindlin-2 was visualized by immunoblotting with an anti-HA antibody. (C) MDA-MB-231 cells were transfected with indicated siRNAs for 48 h and pretreated with proteasome inhibitor MG132 (10 µM) for 12 h. Polyubiquitination of endogenous Kindlin-2 was detected by anti-ubiquitin antibody. (D) HEK293T cells were transfected with Flag-Kindlin-2, Myc-Smurf1, and various Ub mutant plasmids. 24 h after transfection, an in vivo ubiquitination assay was performed. (E) HA-Ub WT or mutants of K27 and K33 were transfected into HEK293 cells together with Flag-Kindlin-2 and Myc-Smurf1 or control vector. Kindlin-2 ubiquitination was detected. (F) HEK293T cells were transfected with HA-Ub, Myc-Smurf1, and various Kindlin-2 mutant plasmids, and an in vivo ubiquitination assay was performed. (G) HEK293T cells were transfected with Myc-Smurf1 and Kindlin-2 mutant plasmid, and Kindlin-2 levels were assessed. (H) Myc-Smurf1 was transfected into HeLa cells together with Flag-Kindlin-2 WT or Flag-Kindlin-2 K153/154+187R mutant, and cells were treated with 100 µg/ml CHX for the indicated times. The half-life of Flag-Kindlin-2 was measured by Western blot. Quantification of Kindlin-2 half-life was performed, and each point is represented as mean ± SD of triplicate experiments.

Smurf1 has no effect on Talin-H degradation. (A) CFP-Talin-H plasmid (2 µg) was transfected into HEK293T cells together with increasing amounts of Smurf1 expression vector. Talin-H expression was determined by immunoblotting with an anti-GFP antibody 24 h after transfection. (B) Flag-Smurf1 was transfected into HEK293T cells together with Talin-H or mutants of Talin-H, and Talin-H expression was examined. (C) HEK293T cells transfected with Flag-Talin-H were treated with proteasome inhibitor MG132 (20 µM) or DMSO for 6 h, and Smurf1 and Flag-Talin-H protein expression were detected. (D) Flag-Talin-H and HA-Ub plasmids were cotransfected into HEK293T cells together with control vector, Myc-Smurf1 (WT), or Myc-Smurf1 (C699A) expression plasmid. Talin-H ubiquitination was detected by immunoprecipitation with anti-Flag M2 beads and immunoblotting with anti-HA antibody. (E) HEK293T cells were transfected with Flag-Talin-H, Myc-Smurf1, and K48R or K63R Ub mutant plasmids, and after 24 h, an in vivo ubiquitination assay was performed. (F) HEK293T cells were transfected with Flag-Talin-H, Myc-Smurf1, and different linkage Ub plasmids, and after 24 h, an in vivo ubiquitination assay was performed. (G) HEK293T cells were transfected with HA-Ub, Myc-Smurf1, and Flag-Talin-H K83+357 mutant plasmid, and then an in vivo ubiquitination assay was performed.

Smurf1 interacts with Kindlin-2 in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody or normal IgG followed by immunoblotting using Smurf1 antibody. (B) The endogenous interaction between Kindlin-2 and Smurf1 was analyzed by coIP. (C) Fusion protein His-MBP-Kindlin-2 was incubated with GST or GST-Smurf1 in vitro for MBP pull-down assays. Affinity matrices for MBP were used. (D) HEK293T cells were cotransfected with Flag-Smurf2 and GFP-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using GFP antibody. (E) Colocalization of endogenous Smurf1 and Kindlin-2 was analyzed by immunofluorescence staining. The image was merged. Bars, 10 µm. (F) Indicated truncates of Smurf1 and Kindlin-2 were constructed according to their functional domains. (G and H) HEK293T cells were transfected with the indicated truncates of Smurf1. Cell lysates were immunoprecipitated with anti-Flag antibody (G) or Kindlin-2 antibody (H) followed by immunoblotting using an anti–Kindlin-2 (G) or Myc (H) antibody. (I) HEK293T cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were then incubated with GST or GST-Smurf1 in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. (J) HEK293T cells were transfected with the indicated truncates of Flag-Kindlin-2, and cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Myc antibody. (K) The PY motif mutant of Kindlin-2 or Kindlin-2 WT was cotransfected with Smurf1 into HEK293T cells. CoIP was performed with an anti-Flag antibody followed by immunoblotting using an anti-Myc antibody.

Smurf1 suppresses cell spreading and adhesion. (A–D) For dynamic spreading assays, indicated siRNAs or plasmids were transfected into HeLa cells, and the cells were seeded onto 5 µg/ml FN–coated nontreated six-well plates and maintained at 37°C and 5% CO₂ on the microscope stage. Images were collected by a confocal microscopy. Bars, 50 µm. Expression levels of Smurf1 and Kindlin-2 protein were measured by Western blot (B). Flattened and well-spread cells at indicated times were counted. Values are mean ± SD of three independent experiments; *, P < 0.05 vs. Flag group (C and D). (E–G) WT, Smurf1^−/−, or Smurf1 rescue MEFs were plated on FN-coated coverslips for 30 min. Spreading phenotypes of cells are shown. Bar, 50 µm. Expression levels of Smurf1 and Kindlin-2 protein were measured by Western blot (F). Graphic presentation of the spreading cells is shown; values are mean ± SD of three independent experiments; *, P < 0.05 vs. WT group; #, P < 0.05 vs. Smurf1^−/− group (G). (H) Indicated plasmids were transfected into HeLa cells, the cells were plated on FN-coated coverslips, and attachment of the cells on FN was analyzed at 30 or 60 min from three independent experiments. Values are mean ± SD of three independent experiments; *, P < 0.05 vs. GFP group; #, P < 0.05 vs. GFP + Flag-Kindlin-2 group. (I) Attachment of HeLa cells transfected with control siRNA, Kindlin-2 siRNA, or Smurf1 siRNA on FN was analyzed. Values are mean ± SD of three independent experiments; *, P < 0.05 vs. control group. (J) WT, Smurf1^−/−, or KO rescue MEFs were plated on FN-coated coverslips, and attachment of the cells on FN was analyzed at 30 min. Values are mean ± SD of three independent experiments; *, P < 0.05 vs. WT group; #, P < 0.05 vs. Smurf1^−/− group.

Smurf1 inhibits focal adhesion formation. (A) Indicated plasmids were transfected into HeLa cells, then the cells were plated on FN-coated coverslips for 30 or 60 min and immunoreacted with anti-Paxillin and anti–Kindlin-2 or Flag antibodies. Expression and localization of GFP-Smurf1, Kindlin-2, and paxillin were observed under a confocal microscope with 63× objective. Bars, 5 µm. (B and C) Numbers of paxillin-staining focal adhesions per cell (B) and unit area (C) were quantified. Values are mean ± SD of three independent experiments. **, P < 0.01 vs. GFP + Flag group; ##, P < 0.01 vs. GFP + Flag-Kindlin-2 group. *, P < 0.05 vs. GFP + Flag group; #, P < 0.05 vs. GFP + Flag-Kindlin-2 group. Smurf1CA, Smurf1 C699A. (D–F) WT, Smurf1^−/−, or Smurf1 rescue MEFs were plated on FN-coated coverslips for 30 min and immunoreacted with antibody to Kindlin-2 and Paxillin. Expression of Kindlin-2 and Paxillin was determined by confocal microscopy under 63× objective. Bars, 5 µm. Numbers of paxillin-staining focal adhesions per cell and unit area were quantified (E and F). Values are mean ± SD of three independent experiments. *, P < 0.05 vs. WT group; #, P < 0.05 vs. Smurf1 KO group. (G–J) HeLa cells expressing EGFP-Zyxin were transfected with control siRNA or Smurf1 siRNA, plated on FN until fully spread (5 µg/ml), and then analyzed using time-lapse confocal microscopy with 100× objective. Bars, 10 µm. (G) Rate (per minute) of assembly (H), disassembly (I), and lifespan (J) of FAs as measured by change in GFP fluorescence over time for control siRNA and Smurf1 siRNA cells. Values are mean ± SD of three independent experiments; *, P < 0.05 vs. control group.