Figure 3.
TGF-β inhibits p70s6k and antagonizes EGFR signaling pathways at the level of p70s6k. (A) p70s6k kinase activity in EpH4 cells arrested in G1 and exposed to TGF-β for various times. Sixty-five percent inhibition of p70s6k activity by TGF-β was observed after 5 min, persisting for more than 1 h. (B) p70s6k kinase activity was determined in epithelial cells, arrested in G1 by contact inhibition, released into cell cycle, and treated with TGF-β or left untreated. TGF-β inhibits the increase of p70s6k activity in early G1. (♦) Control; (□) plus 5ng/mL TGF-β. (C) p70s6k activity in response to TGF-β and an EGFR kinase inhibitor in epithelial EpH4 cells after 20 min. The inhibitory effects of a combination of TGF-β and PD 153.035 are synergistic. (D) p70s6k activity in response to TGF-β and TGF-α in unsynchronized cycling cells. TGF-β inhibits the p70s6k; TGF-α activates p70s6k and partially neutralizes the inhibitory action of TGF-β on p70s6k activity. (Open bars) 5 ng/mL of TGF-α; (gray bars) TGF-α + TGF-β; (black bars) 5ng/mL of TGF-β. (E,F) Immunoprecipitation and Western blot of p70s6k transiently cotransfected in 293 cells with wild-type TβRI and II or inactive mutants (TβRI +II dn). (E) TGF-β inhibits the hyper-phosphorylation of p70s6k in dependence on an activatable TGF-β–TβR complex. Serum-induced hyperphosphorylated forms of p70s6k (p70s6k pp) disappear only after TGF-β stimulation of the coexpressed wild-type TβRI and TβRII (lanes 6,7), but not by stimulation of kinase inactivated TβRI/II (lane 2,3). (F) Corresponding p70s6k kinase activities determined in immunoprecipitates shown in E. The decrease of p70s6k pp (Fig. 4a, lane 7) is accompanied by a reduction of p70s6k kinase activity by more than 60%.