Figure 4.

Dephosphorylation of TβRI by Smad7-recruited PP1 complex. (a and b) In vitro dephosphorylation assay. (a) GST–TβRI was phosphorylated by an in vitro phosphorylation reaction. GST–TβRI–³²P was incubated with different immunoprecipitates (anti-HA) from lysates of COS1 cells transfected with different combinations of genes, in the absence or presence of phosphatase inhibitor (I-1) or recombinant rabbit PP1c (rR-PP1c) as indicated. (b) The relative ³²P phosphorylation level of the type I receptor in a, normalized to input of GST–TβRI–³²P, is plotted as the mean ± SD from three experiments. (c and d) In vivo dephosphorylation assay. (c) Mv1Lu cells transfected with different combination of genes were labeled with [³²P]orthophosphate in the presence or absence of TGFβ-1 as indicated. TβRI–HA was immunoprecipitated from lysates of treated cells and separated by 8.5% SDS-PAGE. Gels were dried and exposed to Biomax M _r film (Eastman Kodak). ΔGADD34 is a mutant without the PP1c binding domain and ΔSmad7 is absent in its TβRI binding site. OA is an inhibitor for both PP1 and PP2. (d) The relative ³²P phosphorylation level of TβRI in c was plotted as the mean ± SD from three experiments.