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J Biol Chem. 1998 Jun 26;273(26):16621-9.

Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies.

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Diabetes Unit and Medical Services and the Department of Molecular Biology, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114, USA.


The p70 S6 kinase is activated by diverse stimuli through a multisite phosphorylation directed at three separate domains as follows: a cluster of (Ser/Thr) Pro sites in an autoinhibitory segment in the noncatalytic carboxyl-terminal tail; Thr-252 in the activation loop of the catalytic domain; and Ser-394 and Thr-412 in a segment immediately carboxyl-terminal to the catalytic domain. Phosphorylation of Thr-252 in vitro by the enzyme phosphatidylinositol 3-phosphate-dependent kinase-1 or mutation of Thr-412 --> Glu has each been shown previously to engender some activation of the p70 S6 kinase, whereas both modifications together produce 20-30-fold more activity than either alone. We employed phospho-specific anti-peptide antibodies to examine the relative phosphorylation at several of these sites in wild type and various p70 mutants, in serum-deprived cells, and in response to activators and inhibitors of p70 S6 kinase activity. Substantial phosphorylation of p70 Thr-252 and Ser-434 was present in serum-deprived cells, whereas Thr-412 and Thr-444/Ser-447 were essentially devoid of phospho-specific immunoreactivity. Activation of p70 by insulin was accompanied by a coordinate increase in phosphorylation at all sites examined, together with a slowing in mobility on SDS-PAGE of a portion of p70 polypeptides. Upon addition of rapamycin or wortmannin to insulin-treated cells, the decrease in activity of p70 was closely correlated with the disappearance of anti-Thr-412(P) immunoreactivity and the most slowly migrating p70 polypeptides, whereas considerable phosphorylation at Ser-434 and Thr-252 persisted after the disappearance of 40 S kinase activity. The central role of Thr-412 phosphorylation in the regulation of kinase activity was further demonstrated by the close correlation of the effects of various deletions and point mutations on p70 activity and Thr-412 phosphorylation. In conclusion, although p70 activity depends on a disinhibition from the carboxyl-terminal tail and the simultaneous phosphorylation at both Thr-252 and Thr-412, p70 activity in vivo is most closely related to the state of phosphorylation at Thr-412.

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