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.
Isotani
S
S
Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
Hara
K
K
Tokunaga
C
C
Inoue
H
H
Avruch
J
J
Yonezawa
K
K
D064413:3-Phosphoinositide-Dependent Protein Kinases; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D008934:Mitogens / Q000494:pharmacology; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000235:genetics / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases
D016895:Culture Media, Serum-Free; D008934:Mitogens; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C546842:MTOR protein, human; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases