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.
Saitoh
Masao
M
Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.
Pullen
Nicholas
N
Brennan
Paul
P
Cantrell
Doreen
D
Dennis
Patrick B
PB
Thomas
George
G
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005799:Genes, Dominant; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology*; D013912:Threonine / Q000737:chemistry; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D000903:Antibiotics, Antineoplastic; D004791:Enzyme Inhibitors; D007328:Insulin; D011993:Recombinant Fusion Proteins; D013912:Threonine; D012694:Serine; D005982:Glutathione Transferase; D019893:Ribosomal Protein S6 Kinases; D020123:Sirolimus; D000077191:Wortmannin