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. Author manuscript; available in PMC: 2021 Jul 8.
Published in final edited form as: Nat Cell Biol. 2012 Jun 24;14(7):727–737. doi: 10.1038/ncb2528

Figure 5.

Figure 5

c-Abl phosphorylates BMPRIA, which plays a role in osteoblast expansion. (a) Co-expression with c-Abl led to BMPRIA tyrosine phosphorylation. BMPRIA (HA tagged) was expressed alone or co-expressed with c-Abl in Cos7 cells. BMPRIA was immunoprecipitated (IP) using anti-HA antibodies and its tyrosine phosphorylation was determined by immunoblotting (IB). KD, kinase dead. (b) Reduced tyrosine phosphorylation of BMPRIA in c-Abl−/− osteoblasts. Endogenous BMPRIA was immunoprecipitated from the cell lysate of the mutant and control cells, and its tyrosine phosphorylation was determined by immunoblotting. Right panel: quantification. (c) The in vitro kinase assay shows that immunoprecipitated c-Abl, but not kinase-dead c-Abl, was able to phosphorylate purified BMPRIA on tyrosine residues. Right panel: quantification. (d) Interaction between endogenous c-Abl and BMPRIA in osteoblasts. Osteoblasts were treated with 100 ng ml−1 BMP2 for 1 h or untreated; cell lysates were divided into three parts for analysis of BMPRIA and c-Abl expression by western blot (WB; left panel), immunoprecipitation of BMPRIA (upper right) or immunoprecipitation of c-Abl (lower right), with IgG as control antibodies. (e) A diagram showing the alignment of the C-terminal regions of BMPRIA of different species. (f) Mutagenesis revealed that all four tyrosine residues could be phosphorylated with Tyr 453/467 being more preferably phosphorylated by c-Abl. BMPRIA with different combinations of tyrosine to phenylalanine mutations was co-expressed with c-Abl and their phosphorylation status was determined by western blot analysis. m1, Y453/457/458F; m2, Y457/458/467F; m3, Y453/467F; m4, 453/457/458/467F (see Supplementary Fig. S5e lower panel for positioning of these mutations). (g) Bmpr1a−/− osteoblasts expressing YF BMPRIA showed a decrease in lifespan when compared with cells expressing WT BMPRIA. For the levels of WT and mutant BMPRIA, see Supplementary Fig. S6a. Data are means ± s.e.m. (n = 3). *P < 0.05, compared with WT BMPRIA. Uncropped images of blots are shown in Supplementary Fig. S8.