Abstract
3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells.
Highlights
It remains to be determined whether and how Phosphoinositide-dependent kinase-1 (PDK1) activity is regulated in cells
While phosphorylation in the activation loop has been shown to be essential for PDK1 activity, it remains to be determined whether phosphorylation at this site is regulated in cells
Insulin Stimulates Autophosphorylation of PDK1 at Ser244— We previously found that insulin had no significant effect on PDK1 phosphorylation at Ser244 when the kinase is overexpressed in cells [10]
Summary
It remains to be determined whether and how PDK1 activity is regulated in cells. Unlike its substrates such as Akt and p70 S6 kinase, the in vitro kinase activity of PDK1 immunoprecipitated from cells is not significantly affected by prior treatment of the cells with either growth factors or pharmacological inhibitors. PDK1 activity has been found to be stimulated by insulin [5, 6] or sphingosine [7], suggesting that the activity of the kinase may be regulated in certain cell types. In addition to Ser244, PDK1 undergoes growth factor-stimulated phosphorylation at additional site(s) [10], suggesting a potential mechanism for regulating PDK1 activity.
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