title: Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies.
abstract: 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.
authors.affiliation: 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.
authors.forename: Q P
authors.initials: QP
authors.lastname: Weng
authors.forename: M
authors.initials: M
authors.lastname: Kozlowski
authors.forename: C
authors.initials: C
authors.lastname: Belham
authors.forename: A
authors.initials: A
authors.lastname: Zhang
authors.forename: M J
authors.initials: MJ
authors.lastname: Comb
authors.forename: J
authors.initials: J
authors.lastname: Avruch
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001665:Binding Sites / Q000276:immunology; D007328:Insulin / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010748:Phosphopeptides / Q000276:immunology*; D010766:Phosphorylation; D017354:Point Mutation; D011090:Polyenes / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000235:genetics / Q000378:metabolism*; D020123:Sirolimus; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D000906:Antibodies; D007328:Insulin; D010748:Phosphopeptides; D011090:Polyenes; D011994:Recombinant Proteins; D019869:Phosphatidylinositol 3-Kinases; D019893:Ribosomal Protein S6 Kinases; D020123:Sirolimus; D000077191:Wortmannin