Abstract
Protein kinase B and p70 S6 kinase are members of the cyclic AMP-dependent/cyclic GMP-dependent/protein kinase C subfamily of protein kinases and are activated by a phosphatidylinositol 3-kinase-dependent pathway when cells are stimulated with insulin or growth factors. Both of these kinases are activated in cells by phosphorylation of a conserved residue in the kinase domain (Thr-308 of protein kinase B (PKB) and Thr-252 of p70 S6 kinase) and another conserved residue located C-terminal to the kinase domain (Ser-473 of PKB and Thr-412 of p70 S6 kinase). Thr-308 of PKBalpha and Thr-252 of p70 S6 kinase are phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in vitro. Recent work has shown that PDK1 interacts with a region of protein kinase C-related kinase-2, termed the PDK1 interacting fragment (PIF). Interaction with PIF converts PDK1 from a form that phosphorylates PKB at Thr-308 alone to a species capable of phosphorylating Ser-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. Here we demonstrate that PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. We study the effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase. Surprisingly, we find that PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412.
Highlights
P70 S6 kinase (p70 S6K)1 is activated by insulin and growth p70 S6 kinase (p70 S6K) is activated by insulin and growth factors, through a phosphoinositide 3-kinase-dependent pathway and becomes phosphorylated on at least 7 Ser/Thr residues in response to these agonists
Phosphorylation of p70 S6K by PDK1 Is Inhibited by PDK1 interacting fragment (PIF)— PDK1 binds with submicromolar affinity to a region of protein kinase C-related kinase-2 (PRK2) termed PIF [22]
PIF is situated C-terminal to the kinase domain of PRK2, and the binding of this region of PRK2 to PDK1 is mediated by a consensus motif similar to that encompassing Thr-412 of p70 S6K, except that the residue at this position is Asp (Asp-978), rather than Thr or Ser
Summary
P70 S6 kinase (p70 S6K)1 is activated by insulin and growth p70 S6K is activated by insulin and growth factors, through a phosphoinositide 3-kinase-dependent pathway and becomes phosphorylated on at least 7 Ser/Thr residues in response to these agonists. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412.
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