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 p70s6k/p85s6k at concentrations that either block or suppress cell growth. The four sites of phosphorylation associated wi th p70s6k/p85s6k activation all display Ser/Thr-Pro motifs and are clo sely clustered within a putative autoinhibitory domain of the enzyme. To produce a constitutively active, rapamycin-resistant form of the ki nase, these four sites were converted to either Asp or Glu. When overe xpressed in human 293 cells, the activity of the mutant is similar to that of the parent enzyme, under conditions where the parent is phosph orylated and active. Unexpectedly, however, the mutant remains sensiti ve to rapamycin and is inactivated in vitro by protein phosphatase 2A. Peptide maps reveal that rapamycin abolishes the activity of the over expressed p70s6k through the dephosphorylation of a novel set of sites distinct from those associated with mitogenic activation.