Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site

A critical step in S6 kinase 1 (S6K1) activation is Thr²²⁹ phosphorylation in the activation loop by the phosphoinositide-dependent protein kinase (PDK1). Thr²²⁹ phosphorylation requires prior phosphorylation of the Ser/Thr-Pro sites in the autoinhibitory domain and Thr³⁸⁹ in the linker domain, consistent with PDK1 more effectively catalyzing Thr²²⁹ phosphorylation in a variant harboring acidic residues in these positions (S6K1-E389D₃E). S6K1-E389D₃E has high basal activity and exhibits partial resistance to rapamycin and wortmannin, and its activity can be further augmented by mitogens, effects presumably mediated by Thr²²⁹ phosphorylation. However, PDK1-induced Thr²²⁹ phosphorylation is reported to be constitutive rather than phosphatidylinositide 3,4,5-trisphosphate-dependent, suggesting that S6K1-E389D₃E activity is mediated through a distinct site. Here we use phosphospecific antibodies to show that Thr²²⁹ is fully phosphorylated in S6K1-E389D₃E in the absence of mitogens and that regulation of S6K1-E389D₃E activity by mitogens, rapamycin, or wortmannin parallels Ser³⁷¹ phosphorylation. Consistent with this observation, a dominant interfering allele of the mammalian target of rapamycin, mTOR, inhibits mitogen-induced Ser³⁷¹ phosphorylation and activation of S6K1-E389D₃E, whereas wild type mTOR stimulates both responses. Moreover, in vitro mTOR directly phosphorylates Ser³⁷¹, and this event modulates Thr³⁸⁹ phosphorylation by mTOR, compatible with earlier in vivo findings.