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Fig. 3. The BMP type I receptor is the substrate of PP1. (A) C2C12 cells transfected with different combinations of genes were labeled with [32P]orthophosphate in the presence or absence of BMP2 as indicated. ALK3-HA was immunoprecipitated from lysates of treated cells and separated by 8.5% SDS-PAGE. Gels were dried and exposed to Biomax Mr film (Eastman Kodak). PBD (F872A) is a mutant with the disrupted PP1c-binding domain. Phosphatase inhibitor-1 is an inhibitor for PP1. (B) BMP type I receptors interact with PP1 regulatory subunit GADD34. COS1 cells transfected with HA-tagged type I receptors (ALK3 and ALK6) and Flag-tagged GADD34. Lysates were subjected to co-immunoprecipitation with anti-Flag and the presence of ALK3 and ALK6 were probed with antibody against HA. (C) Unlike in the TGF
signaling pathway, GADD34 interacts with the BMP type I receptor without the bridging of inhibitory Smads. The GADD34-ALK3/6 complex was further confirmed by a sequential immunoprecipitation. COS1 cells were first co-transfected with ALK3/6-HA with or without Flag-Smad6/7 and GADD34. The lysates were subjected to immunoprecipitation with HA antibody, and the resultant precipitates were eluted from protein-G-Sepharose beads by HA peptide competition and then subjected to a second immunoprecipitation with anti-Flag antibody. The final precipitates were immunoblotted with antibodies against all these components.
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