IndraLab

"the treatment of SV40-immortalized human corneal epithelial cells (HCE-T cells) with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), widely used as an AMPK activator, inhibits p70 S6 kinase a (p70a) activities. AICAR treatment also inhibits phosphorylation of Thr-412 in p70a, which is indispensable for its activity."

"the treatment of SV40-immortalized human corneal epithelial cells (HCE-T cells) with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), widely used as an AMPK activator, inhibits p70 S6 kinase a (p70a) activities. AICAR treatment also inhibits phosphorylation of Thr-412 in p70a, which is indispensable for its activity."

"conditional inactivation of Cdc2 reduces phosphorylation of S6K1 at S/TP sites while simultaneously increasing phosphorylation of Thr(389) and of the S6K1 substrate, RPS6."

"Although the p70s6kE389 construct has high basal activity, its overexpression in the absence of mitogen stimulation does not induce the translational up-regulation of eEF-1a transcripts, indicating that p70s6k activation is not sufficent to induce this response."

"Although the p70s6kE389 construct has high basal activity, its overexpression in the absence of mitogen stimulation does not induce the translational up-regulation of eEF-1a transcripts, indicating that p70s6k activation is not sufficent to induce this response."

"We report here that a mammalian recombinant p70alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. "

"##from text## Phosphorylation of p70 S6 kinase on Thr389 is essential for the activation of the kinase [22]. In the presence of insulin, the intensity of the Thr389 p70 S6 kinase bands was significantly increased after 15 min exposure and reached 2.02 ± 0.31 after 15 min and 1.36 ± 0.17 after a 16 hour incubation period when compared to respective controls which were made equal to one (mean ± SE, t-test, p < 0.05, n = 4)."

"We report here that a mammalian recombinant p70alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412."

"altered glucose availability, which regulates AMPK activity, also modulates the activity of p70 S6k. AICAR treatment also inhibits phosphorylation of Thr-412 in the p70 S6 kinase (p70 S6k), which is indispensable for the activity. In addition to the mTOR signal acting as a priming switch that modulates p70 S6k activation, AMPK appears to provide an overriding switch linking p70 S6k regulation to cellular energy metabolism."

"Furthermore, overexpression of either hVps34 or the associated hVps15 protein kinase in the insulin-responsive Chinese hamster ovary line GRC+LR-73 (42) lead to an increase in phosphorylation of S6K1 at Thr389 (Fig. 1B), which correlates with S6K1 activation (45)."

"from full text - Collectively, these data suggest that p70S6K phosphorylated at T389 is among the most hyperphosphorylated isoforms and is a reliable marker of full p70S6K activation as suggested previously (22, 23)."

"altered glucose availability, which regulates AMPK activity, also modulates the activity of p70 S6k. AICAR treatment also inhibits phosphorylation of Thr-412 in the p70 S6 kinase (p70 S6k), which is indispensable for the activity. In addition to the mTOR signal acting as a priming switch that modulates p70 S6k activation, AMPK appears to provide an overriding switch linking p70 S6k regulation to cellular energy metabolism."

"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)"