The immunosuppressant rapamycin induces inactivation of p70(s6k) through dephosphorylation of a novel set of sites

The immunosuppressant rapamycin selectively abolishes phosphorylation and activation of p70(s6k)/p85(s6k) at concentrations that either block or suppress cell growth. The four sites of phosphorylation associated with p70(s6k)/p85(s6k) activation all display Ser/Thr-Pro motifs and are closely clustered within a putative autoinhibitory domain of the enzyme. To produce a constitutively active, rapamycin-resistant form of the kinase, these four sites were converted to either Asp or Glu. When overexpressed in human 293 cells, the activity of the mutant is similar to that of the parent enzyme, under conditions where the parent is phosphorylated and active. Unexpectedly, however, the mutant remains sensitive to rapamycin and is inactivated in vitro by protein phosphatase 2A. Peptide maps reveal that rapamycin abolishes the activity of the overexpressed p70(s6k) through the dephosphorylation of a novel set of sites distinct from those associated with mitogenic activation.