Rapamycin-insensitive regulation of 4E-BP1 in regenerating rat liver

In cultured cells, growth factor-induced phosphorylation of two translation modulators, p70 S6 kinase and eukaryotic initiation factor 4E-binding prot ein 1 (4E-BP1), is blocked by nanomolar concentrations of the immunosuppres sant rapamycin. Rapamycin also attenuates liver regeneration after partial hepatectomy, but it is not known if this growth-suppressive effect is due t o dephosphorylation of p70 S6 kinase and/or 4E-BP1. We found that partial h epatectomy induced a transient increase in liver p70 S6 kinase activity and 4E-8P1 phosphorylation as compared with sham-operated rats. The amount of p70 S6 kinase protein in regenerating liver did not increase, but active ki nase from partially hepatectomized animals was highly phosphorylated. Phosp horylated 4E-BP1 from regenerating liver was unable to form an inhibitory c omplex with initiation factor 4E. Rapamycin blocked the activation of p70 S 6 kinase in response to partial hepatectomy in a dose-dependent manner, but 4E-BP1 phosphorylation was not inhibited. By contrast, functional phosphor ylation of 4E-BP1 induced by injection of cycloheximide or growth factors w as partially reversed by the drug. The mammalian target of rapamycin (mTOR) has been proposed to directly phosphorylate 4E-BP1. Western blot analysis using phospho-specific antibodies showed that phosphorylation of Thr-36/45 and Ser-64 increased in response to partial hepatectomy in a rapamycin-resi stant manner. Thus, rapamycin inhibits p70 S6 kinase activation and liver r egeneration, but not functional phosphorylation of 4E-BP1, in response to p artial hepatectomy. These results indicate that the effect of rapamycin on 4E-BP1 function in vivo can be significantly different from its effect in c ultured cells.