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

Saitoh, Masao, Pullen, Nicholas, Brennan, Paul ORCID: https://orcid.org/0000-0001-8792-0499, Cantrell, Doreen, Dennis, Patrick B and Thomas, George 2002. Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site. Journal of Biological Chemistry 277 (22) , 20104-20112.. 10.1074/jbc.M201745200

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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 Thr229is fully phosphorylated in S6K1-E389D3E in the absence of mitogens and that regulation of S6K1-E389D3E activity by mitogens, rapamycin, or wortmannin parallels Ser371phosphorylation. 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 Thr389phosphorylation by mTOR, compatible with earlier in vivofindings. •Abbreviations: S6K1 p70 S6 kinase 1 PDK1 phosphoinositide-dependent kinase 1 mTOR mammalian target of rapamycin GST glutathione S-transferase FKBP12 FK506-binding protein 12 PI3K phosphatidylinositol 3-kinase HAhemagglutinin MOPS 4-morpholinepropanesulfonic acid PKB protein kinase B

Item Type:	Article
Status:	Published
Schools:	Medicine
Subjects:	R Medicine > R Medicine (General)
ISSN:	00219258
Last Modified:	25 Oct 2022 09:05
URI:	https://orca.cardiff.ac.uk/id/eprint/57197

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