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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 Thr(229) phosphorylation in the activation loop by the phosphoinositide-dependent protein kinase (PDK1). Thr(229) phosphorylation requires prior phosphorylation of the Ser/Thr-Pro sites in the autoinhibitory domain and Thr(389) in the linker domain, consistent with PDK1 more effectively catalyzing Thr(229) phosphorylation in a variant harboring acidic residues in these positions (S6K1-E389D(3)E). S6K1-E389D(3)E 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 Thr(229) phosphorylation. However, PDK1-induced Thr(229) phosphorylation is reported to be constitutive rather than phosphatidylinositide 3,4,5-trisphosphate-dependent, suggesting that S6K1-E389D(3)E activity is mediated through a distinct site. Here we use phosphospecific antibodies to show that Thr(229) is fully phosphorylated in S6K1-E389D(3)E in the absence of mitogens and that regulation of S6K1-E389D(3)E activity by mitogens, rapamycin, or wortmannin parallels Ser(371) phosphorylation. Consistent with this observation, a dominant interfering allele of the mammalian target of rapamycin, mTOR, inhibits mitogen-induced Ser(371) phosphorylation and activation of S6K1-E389D(3)E, whereas wild type mTOR stimulates both responses. Moreover, in vitro mTOR directly phosphorylates Ser(371), and this event modulates Thr(389) phosphorylation by mTOR, compatible with earlier in vivo findings.

Pubmed ID: 11914378


  • Saitoh M
  • Pullen N
  • Brennan P
  • Cantrell D
  • Dennis PB
  • Thomas G


The Journal of biological chemistry

Publication Data

May 31, 2002

Associated Grants


Mesh Terms

  • Androstadienes
  • Animals
  • Antibiotics, Antineoplastic
  • Binding Sites
  • Cell Line
  • Enzyme Inhibitors
  • Genes, Dominant
  • Glutathione Transferase
  • Humans
  • Immunoblotting
  • Insulin
  • Phosphorylation
  • Plasmids
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins
  • Ribosomal Protein S6 Kinases
  • Serine
  • Sirolimus
  • Threonine
  • Transfection