Immunopurified Mammalian Target of Rapamycin Phosphorylates and Activates p70 S6 Kinase α in Vitro

Shuji Isotani,Kenta Hara,Chiharu Tokunaga,Hitomi Inoue,Joseph Avruch,Kazuyoshi Yonezawa

doi:10.1074/jbc.274.48.34493

Shuji Isotani, Kenta Hara + Show 4 more

Open Access

https://doi.org/10.1074/jbc.274.48.34493

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Abstract

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.

Highlights

P70 S6 kinase ␣ (p70␣),1 whose major substrate is the 40 S ribosomal protein S6, plays a critical role in the translation of a subclass of mRNAs that contain a short oligopyrimidine sequence immediately following the transcriptional start site [1]. p70␣ is activated in response to insulin/mitogens in vivo through a multisite phosphorylation of serine and threonine residues [2]
To obtain a precisely folded full-length p70␣ polypeptide that was expressed in mammalian cells and dephosphorylated and inactivated in vivo, HEK293 cells were transiently transfected with a full-length HA-tagged p70␣, and cells were deprived of serum and treated with rapamycin (0.2 ␮M) for 30 min prior to harvest
MTORcatalyzed p70␣ phosphorylation was detectable with all washing conditions, the mammalian target of rapamycin (mTOR) autophosphorylation and kinase activity toward p70␣ is substantially enhanced when the immunoprecipitate is washed with the high salt wash buffer containing 1% Nonidet P-40 or the RIPA buffer, compared with that prepared after washing with the high salt wash buffer without detergent

Summary

Introduction

P70 S6 kinase ␣ (p70␣),1 whose major substrate is the 40 S ribosomal protein S6, plays a critical role in the translation of a subclass of mRNAs that contain a short oligopyrimidine sequence immediately following the transcriptional start site [1]. p70␣ is activated in response to insulin/mitogens in vivo through a multisite phosphorylation of serine and threonine residues [2]. Activation of p70␣ by mTOR in Vitro catalyzed phosphorylation on the eukaryotic initiation factor-4E binding protein 1 (eIF-4E BP1) reside in Ser/Thr-Pro motifs [18, 19].

Results

Conclusion