3 Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro

Background: The p70 S6 kinase, an enzyme critical for cell-cycle progression through the G1 phase, is activated in vivo by insulin and mitogens through coordinate phosphorylation at multiple sites, regulated by signaling pathways, some of which depend on and some of which are independent of phosphoinositide 3-kinase (Pi 3-kinase). It is not known which protein kinases phosphorylate and activate p70. Results: Go-expression of p70 with 3-phosphoinositide-dependent protein kinase 1 (PDK1), a protein kinase that has previously been shown to phosphorylate and activate protein kinase B (PKB, also known as c-Akt), resulted in strong activation of the S6 kinase in vivo. In vitro, PDK1 directly phosphorylated Thr252 in the activation loop of the p70 catalytic domain, the phosphorylation of which is stimulated by PI 3-kinase in vivo and is indispensable for p70 activity. Whereas PDK1-catalyzed phosphorylation and activation of PKB in vitro was highly dependent on the presence of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3), PDK1 catalyzed rapid phosphorylation and activation of p70 in vitro, independent of the presence of PtdIns(3,4,5)P-3. The ability of PDK1 to phosphorylate p70 Thr252 was strongly dependent on the phosphorylation of the p70 noncatalytic carboxy-terminal tail (amino acids 422-525) and of amino acid Thr412. Moreover, once Thr252 was phosphorylated, its ability to cause activation of the p70 S6 kinase was also controlled by the p70 carboxy-terminal tail and by phosphorylation of p70 Ser394, and most importantly, Thr412. The overriding determinant of the absolute p70 activity was the strong positive cooperativity between Thr252 and Thr412 phosphorylation; both sites must be phosphorylated to achieve substantial p70 activation. Conclusions: PDK1 is one of the components of the signaling pathway recruited by Pi a-kinase for the activation of p70 S6 kinase as well as of PKB, and serves as a multifunctional effector downstream of the Pi 3-kinase.