title: Expression, enzyme activity, and subcellular localization of mammalian target of rapamycin in insulin-responsive cells.
abstract: The role of the mammalian target of rapamycin (mTOR) was investigated in insulin responsive cell lines. mTOR was expressed at high levels in insulin responsive cell types and in 3T3-L1 cells mTOR expression levels increased dramatically as cells differentiated from fibroblasts into insulin responsive adipocytes. mTOR localized to membrane fractions in all cells tested and in 3T3-L1 adipocytes mTOR was specifically localized to microsomal membranes. Protein kinase activity directed towards mTOR was tightly associated with mTOR immunoprecipitates and this kinase activity was inhibited by FKBP12-rapamycin indicating it was due to an autokinase activity present in mTOR. The mTOR autokinase and the protein kinase activity of the p110 alpha isoform of PI 3-kinase shared several notable similarities; (a) both were maximally active in the presence of Mn2+ but also showed significant activity in the presence of Mg2+ (b) neither were inhibited by the presence of non-ionic detergent and (c) both were inhibited by wortmannin and LY294002, known inhibitors of the PI 3-kinase lipid kinase activity. These data taken together indicate the autokinase activity lay in the PI 3-kinase homology domain. In summary mTOR is a membrane anchored protein kinase that is active in conditions encountered in vivo and the fact it is highly expressed in insulin responsive cell types is consistent with a role in insulin signalling.
authors.affiliation: Department of Biochemistry and Molecular Biology, University College London, United Kingdom.
authors.forename: D J
authors.initials: DJ
authors.lastname: Withers
authors.forename: D M
authors.initials: DM
authors.lastname: Ouwens
authors.forename: B T
authors.initials: BT
authors.lastname: Nave
authors.forename: G C
authors.initials: GC
authors.lastname: van der Zon
authors.forename: C M
authors.initials: CM
authors.lastname: Alarcon
authors.forename: M E
authors.initials: ME
authors.lastname: Cardenas
authors.forename: J
authors.initials: J
authors.lastname: Heitman
authors.forename: J A
authors.initials: JA
authors.lastname: Maassen
authors.forename: P R
authors.initials: PR
authors.lastname: Shepherd
mesh_terms: D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007328:Insulin / Q000502:physiology*; D051379:Mice; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000378:metabolism*; D011090:Polyenes / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus; D013347:Subcellular Fractions / Q000201:enzymology; D058570:TOR Serine-Threonine Kinases
chemical_list: D007328:Insulin; D011090:Polyenes; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C546842:MTOR protein, human; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus