IndraLab

"To test whether mTOR would also phosphorylate Ser371 in vitro, Myc-S6K-WT derived from 293 cells pretreated with rapamycin was used as a direct substrate for HA-tagged mTOR immunopurified from 293 cells. The results show that Myc-mTOR-WT induces increased Ser371 phosphorylation in vitro as assessed by Western blot analysis with the Ser371 anti-phosphospecific antibody following electrophoresis on low acrylamide gels (Fig. 5A), consistent with in vivo findings (Fig. 2A). In contrast, HA-mTOR-KI had no effect. More importantly, in the presence of rapamycin and FKBP12, but not in the presence of either component alone, phosphorylation of Ser371 by mTOR is abolished (Fig. 5A), consistent with the in vivo finding (Fig. 4). In parallel, incubation of S6K1-E389D3E with HA-mTOR-WT also led to increased S6K1-E389D3E activation and Ser371 phosphorylation, in an FKBP-12/rapamycin-sensitive manner (Fig. 5B and data not shown). Furthermore, the extent of both responses was similar to those observed in vivo (compare Figs. 4B and 5B). Therefore, Ser371 phosphorylation appears to be directly regulated by mTOR in vitro and in vivo. (From full text) Incubation of either S6 kinase variant with wild type, but not kinase-inactive, mTOR led to increased Thr389 phosphorylation, with the extent of Thr389 phosphorylation much higher in S6K1-S371A than in wild type S6K1 (Fig. 6B). However, to achieve the same level of activity as S6K1-WT, S6K1-S371A apparently requires much higher levels of Thr389 phosphorylation (Fig. 6B), consistent with detailed titration studies (data not shown). Although unexpected, these findings are compatible with Ser371 phosphorylation regulating Thr389 phosphorylation and with its ability to directly affect S6K1 activity. (From full text)"

"Effect of Ser371 on S6K1 Activation-- As shown previously (13), substitution of an alanine or an aspartate for Ser371 blocks serum- or insulin-induced Thr389 phosphorylation and S6K1 activation (Fig. 6A). However, substitution of an acidic residue at Thr389 in the S6K1-E389D3E background fails to rescue kinase activity (13), suggesting that Ser371 phosphorylation contributes directly to S6K1 activation independent of its role in regulating Thr389 phosphorylation. To test this possibility in vitro, either S6K1 or S6K1-S371A, from 293 cells pretreated with rapamycin, were incubated with either HA-mTOR-WT or HA-mTOR-KI. Both S6K1 variants displayed basal levels of phosphorylated Thr229, which were not altered by incubation with either mTOR variant (Fig. 6B). However, incubation of either S6 kinase variant with wild type, but not kinase-inactive, mTOR led to increased Thr389 phosphorylation, with the extent of Thr389 phosphorylation much higher in S6K1-S371A than in wild type S6K1 (Fig. 6B). However, to achieve the same level of activity as S6K1-WT, S6K1-S371A apparently requires much higher levels of Thr389 phosphorylation (Fig. 6B), consistent with detailed titration studies (data not shown). Although unexpected, these findings are compatible with Ser371 phosphorylation regulating Thr389 phosphorylation and with its ability to directly affect S6K1 activity. (From full text)"