CHARACTERIZATION OF THE PHOSPHOPROTEINS AND PROTEIN-KINASE ACTIVITY IN MTOR IMMUNOPRECIPITATES

The mammalian target of rapamycin, mTOR, has been shown to be an upstr eam regulator of translational effecters. In the present study, in ord er to detect potential molecules involved in the mTOR signaling, an in vitro phosphorylation assay using mTOR immunoprecipitates from HEK293 cells was carried out. In addition to the autophosphorylation of mTOR , P-32 incorporation into 80-kDa (pp80) and 175-kDa (pp175) bands was observed in mTOR immunoprecipitates. The protein kinase activity towar d the recombinant eIF-4E binding protein 1 (4E-BP1) was also detected as previously described. When mTOR immunoprecipitates from HEK293 cell s were prepared in the presence of a detergent, Nonidet P-40, the 4E-B P1 kinase activity and P-32 incorporation into pp175 dramatically dimi nished, while the phosphorylation of mTOR and P-32 incorporation into pp80 did not change. These results raised a possibility that mTOR may associate with protein cofactors, some of which may be involved in the regulation of kinase activities associated with mTOR. (C) 1998 Academ ic Press.