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

Masao Saitoh, Nicholas Pullen, Paul Brennan, Doreen Cantrell, Patrick B. Dennis, George Thomas (Lead / Corresponding author)

    Research output: Contribution to journalArticle

    133 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)20104-20112
    Number of pages9
    JournalJournal of Biological Chemistry
    Volume277
    Issue number22
    DOIs
    Publication statusPublished - 31 May 2002

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