Figure 5. BRCC3-ALK2 axis regulates BMP signaling by BRCC3 deubiquitination of ALK2 Lys-472 and Lys-475.

PASMCs transfected with or without Ad-BRCC3 were cultured under normoxia or hypoxia for 48 hours. Cell extracts were immunoprecipitated (IP) with anti-ALK2 and immunoblotted (IB) with anti-Ub K63 in A, anti-BRCC3 in E, and anti-SMURF1 in F. The input controls were immunoblotted with various antibodies as indicated. Cell lysates from cultured PASMCs were immunoprecipitated with anti-BRCC3 in C or anti-ALK2 in D and immunoblotted with anti-ALK2, anti-BRCC3, or anti-SMURF1. The input controls were lysates immunoblotted with anti-BRCC3, anti-ALK2, anti-SMURF1, or anti–β-actin. Lung tissues from WT/Nor, WT/SuHx, and BRCC3-Tg/SuHx groups of mice were immunoprecipitated with anti-ALK2 and immunoblotted with anti-Ub K63 in B and anti-SMURF1 in G. H, Schematic diagram of the competitive binding of BRCC3 and SMURF1 with ALK2. I, Schematic diagram of BRCC3 interaction with ALK2. ALK2 is depicted in blue and BRCC3 in green. The moiety highlighted in red in ALK2 are experimentally verified ubiquitination sites.⁴² ALK2 K472 and K475 were predicted to be in the interaction interface, which includes all residue pairs within 5.0 Å between the interacting molecules. J through L, HEK293 cells were transfected with expression plasmid encoding ALK2-WT, ALK2-K472R, ALK2-K475R, or ALK2-K472R/K475R (KR) together with Ub plasmids. Cell extracts were immunoprecipitated with anti-ALK2 and immunoblotted with anti-Ub K63 in J. The input protein was immunoblotted with various as indicated in K. L, PASMCs were transfected with ALK2-WT or ALK2-K472R/K475R plasmids. Nuclear translocation was quantified by immune fluorescence staining with pSmad1/5 (red), and nuclei were counterstained with DAPI (blue). Scale bar=50 μm. Data in A through L are mean±SEM from 3 to 7 independent experiments. Normally distributed data were analyzed by 1-way ANOVA in K and 2-way ANOVA in L between multiple groups. Data in K and L, *P<0.05 vs ALK2-WT or ALK2-WT in normoxia group. #P<0.05 vs ALK2-WT in hypoxia group. ALK2 indicates activin receptor-like kinase-2; BMP, bone morphogenetic protein; BRCC3, BRCA1/BRCA2-containing complex subunit 3; PASMC, pulmonary arterial smooth muscle cell; SMURF1, Smad ubiquitination regulatory factor 1; SuHx, Sugen5416/hypoxia; and WT, wild-type.