Fig 2: SMURF1 bound MMP9 to promote its degradation, thereby inhibiting BC migration and invasion. (A) Mass spectrometric analysis was performed to identify MMP9-associated proteins. (B and C) Expression of HSPA8, CBL, SMURF1, and MMP9 in the indicated cell extracts, as determined by western blotting. (D and E) Immunoprecipitation was performed on 293T cell lysates with control IgG (D), anti-Myc, or (E) anti-FLAG antibody. The immunoprecipitate was assayed for MMP9 and SMURF1 levels. (F) T24T (anti-miR-516a/shPHLPP2 #2/vector) cells and T24T (anti-miR-516a/shPHLPP2 #2/Flag-SMURF1) cells were lysed, and overexpression efficiency of SMURF1 was evaluated by western blotting. (G and H) Invasion and migration of T24T (anti-miR-516a/shPHLPP2 #2/vector) and T24T (anti-miR-516a/shPHLPP2 #2/Flag-SMURF1) cells were evaluated by transwell assay. Data were expressed as mean ± SD, and the asterisk (*) indicates a significant difference. (I) The degradation rate of MMP9 was monitored in T24T (anti-miR-516a/shPHLPP2 #2/vector) and T24T (anti-miR-516a/shPHLPP2 #2/Flag-SMURF1) cells by western blotting. ImageJ was used to quantify MMP9 expression relative to ACTB

Fig 3: NADK inhibits the ubiquitination and degradation of BMPR1A by interacting with Smurf1. Figure 4A-B. Co-IP was performed to detect the interaction between the exogenously expressed Flag-NADK and HA-Smurf1. 4C. The interaction between endogenously expressed NADK and Smurf1 was detected by coimmunoprecipitation in A549 cells. 4D. The interaction between NADK and Smurf1 was induced by BMP2. Cells treated with BMP-2 recombined protein were harvested at different time points for Co-IP. 4E. Co-IP was used to determine the binding domain of NADK for the interaction with Smurf1. The full length NADK and truncation domain were shown. Smurf1 and NADK or its truncation mutants were co-transfected into HEK293T cells. After 36 hours, Co-IP was performed. 4F. Ubiquitin assay were performed to detect the effect of NADK on the ubiquitination of BMPR1A by Smurf1. 4G. The effect of overexpression of NADK in A549 cells on the half-life of BMPR1A protein level was detected. Cells were treated with CHX (80µg/ml) for different periods of time, and the BMPR1A protein was measured by western blot. 4H. Graphic summary. N-terminal of NADK interacted with Smurf1, and impaired the degradation of BMPR1A.

Fig 4: Graphic abstract. NADK activates BMPs/ID1 signalling by binding Smurf1 and inhibiting the degradation of BMPR1A, thus promoting the growth and LN metastasis of NSCLC cells.