EXPRESSION, ENZYME-ACTIVITY, AND SUBCELLULAR-LOCALIZATION OF MAMMALIAN TARGET OF RAPAMYCIN IN INSULIN-RESPONSIVE CELLS

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 lev els increased dramatically as cells differentiated from fibroblasts in to insulin responsive adipocytes. mTOR localized to membrane fractions in all cells tested and in 3T3-L1 adipocytes mTOR was specifically lo calized to microsomal membranes. Protein kinase activity directed towa rds mTOR was tightly associated with mTOR immunoprecipitates and this kinase activity was inhibited by FKBP12-rapamycin indicating it was du e to an autokinase activity present in mTOR. The mTOR autokinase and t he protein kinase activity of the p110 alpha isoform of PI 3-kinase sh ared several notable similarities; (a) both were maximally active in t he presence of Mn2+ but also showed significant activity in the presen ce of Mg2+ (b) neither were inhibited by the presence of non-ionic det ergent and (c) both were inhibited by wortmannin and LY294002, known i nhibitors of the PI 3-kinase lipid kinase activity. These data taken t ogether indicate the autokinase activity lay in the PI 3-kinase homolo gy 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 i n insulin signalling. (C) 1997 Academic Press.