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Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro.

p70 S6 kinase alpha (p70alpha) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70alpha activation in a manner that can be overcome by coexpression of p70alpha with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70alpha phosphorylation in vitro is accompanied by a substantial restoration in p70alpha kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70alpha by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70alpha activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined.

Pubmed ID: 10567431


  • Isotani S
  • Hara K
  • Tokunaga C
  • Inoue H
  • Avruch J
  • Yonezawa K


The Journal of biological chemistry

Publication Data

November 26, 1999

Associated Grants

  • Agency: NCI NIH HHS, Id: CA73818
  • Agency: NIDDK NIH HHS, Id: DK17776

Mesh Terms

  • 3-Phosphoinositide-Dependent Protein Kinases
  • Cell Line
  • Culture Media, Serum-Free
  • Enzyme Activation
  • Humans
  • Immunoblotting
  • Mitogens
  • Mutation
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor)
  • Precipitin Tests
  • Protein Kinases
  • Protein-Serine-Threonine Kinases
  • Recombinant Fusion Proteins
  • Ribosomal Protein S6 Kinases
  • TOR Serine-Threonine Kinases