Steady-state kinetic and inhibition studies of the mammalian target of rapamycin (mTOR) kinase domain and mTOR complexes

Biochemistry. 2010 Oct 5;49(39):8488-98. doi: 10.1021/bi100673c. Epub 2010 Sep 8.

Abstract

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase and a major controller of cell growth. In cells, mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2. The mTORC1 complex can phosphorylate 4EBP1 and S6K1, two key regulators of translation initiation, whereas mTORC2 phosphorylates AKT1, an event required for AKT1 activation. Here, we expressed and purified human mTORC1 and mTORC2 from HEK-293 cells using FLAG-M2 affinity chromatography. Western blotting analysis using phospho-specific antibodies indicated that recombinant mTORC1 and mTORC2 exhibit distinct substrate preferences in vitro, consistent with their roles in cells. To improve our understanding of the enzymatic properties of mTOR alone and mTOR in its complex form, steady-state kinetic profiles of truncated mTOR containing the kinase domain (residues 1360-2549) and mTORC1 were determined. The results revealed that mTORC1 is catalytically less active than truncated mTOR, as evidenced by 4.7- and 3.1-fold decreases in catalytic efficiency, k(cat)/K(m), for ATP and 4EBP1, respectively. We also found that truncated mTOR undergoes autophosphorylation through an intramolecular mechanism. Mass spectrometric analysis identified two novel mTOR autophosphorylation sites, Ser2454 and either Thr2473 or Thr2474, in addition to the previously reported Ser2481 site. Truncated mTOR and mTORC1 were completely inhibited by ATP competitive inhibitors PI103 and BEZ235 and partially inhibited by rapamycin/FKBP12 in a noncompetitive fashion toward ATP. All inhibitors tested exhibited similar inhibitory potencies between mTORC1 and truncated mTOR containing the kinase domain. Our studies presented here provide the first detailed kinetic studies of a recombinant mTOR complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / antagonists & inhibitors
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / isolation & purification
  • Adaptor Proteins, Signal Transducing / metabolism
  • Amino Acid Sequence
  • Carrier Proteins / antagonists & inhibitors
  • Carrier Proteins / genetics
  • Carrier Proteins / isolation & purification
  • Carrier Proteins / metabolism
  • Cell Line
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Kinetics
  • Mechanistic Target of Rapamycin Complex 1
  • Metals / metabolism
  • Molecular Sequence Data
  • Multiprotein Complexes
  • Phosphorylation
  • Proteins / antagonists & inhibitors
  • Proteins / genetics
  • Proteins / isolation & purification
  • Proteins / metabolism
  • Rapamycin-Insensitive Companion of mTOR Protein
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Regulatory-Associated Protein of mTOR
  • Sirolimus / pharmacology
  • Substrate Specificity
  • TOR Serine-Threonine Kinases / antagonists & inhibitors*
  • TOR Serine-Threonine Kinases / genetics
  • TOR Serine-Threonine Kinases / isolation & purification
  • TOR Serine-Threonine Kinases / metabolism*
  • Up-Regulation
  • mTOR Associated Protein, LST8 Homolog

Substances

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • Enzyme Inhibitors
  • Intracellular Signaling Peptides and Proteins
  • MAPKAP1 protein, human
  • MLST8 protein, human
  • Metals
  • Multiprotein Complexes
  • PRR5 protein, human
  • Proteins
  • RICTOR protein, human
  • RPTOR protein, human
  • Rapamycin-Insensitive Companion of mTOR Protein
  • Recombinant Proteins
  • Regulatory-Associated Protein of mTOR
  • mTOR Associated Protein, LST8 Homolog
  • MTOR protein, human
  • Mechanistic Target of Rapamycin Complex 1
  • TOR Serine-Threonine Kinases
  • Sirolimus