THE PRINCIPAL TARGET OF RAPAMYCIN-INDUCED P70(S6K) INACTIVATION IS A NOVEL PHOSPHORYLATION SITE WITHIN A CONSERVED HYDROPHOBIC DOMAIN

The immunosuppressive agent rapamycin induces inactivation of p70(s6k) with no effect on other mitogen-activated kinases, Here we have emplo yed a combination of techniques, including mass spectrometry, to demon strate that this effect is associated with selective dephosphorylation of three previously unidentified p70(s6k) phosphorylation sites: T229 , T389 and S404, T229 resides at a conserved position in the catalytic domain, whose phosphorylation is essential for the activation of othe r mitogen-induced kinases. However, the principal target of rapamycin- induced p70(s6k) inactivation is T389, which is located in an unusual hydrophobic sequence outside the catalytic domain, Mutation of T389 to alanine ablates kinase activity, whereas mutation to glutamic acid co nfers constitutive kinase activity and rapamycin resistance. The impor tance of this site and its surrounding motif to kinase function is emp hasized by its presence in a large number of protein kinases of the se cond messenger family and its conservation in putative p70(s6k) homolo gues from as distantly related organisms as yeast and plants.