Mechanism of Phosphorylation of Protein Kinase B/Akt by a Constitutively Active 3-Phosphoinositide-dependent Protein Kinase-1

Ramon A. Riojas,Michael J. Wick,Fresnida J. Ramos,Lily Q. Dong,Feng Liu

doi:10.1074/jbc.m003937200

Abstract

Phosphorylation of Thr(308) in the activation loop and Ser(473) at the carboxyl terminus is essential for protein kinase B (PKB/Akt) activation. However, the biochemical mechanism of the phosphorylation remains to be characterized. Here we show that expression of a constitutively active mutant of mouse 3-phosphoinositide-dependent protein kinase-1 (PDK1(A280V)) in Chinese hamster ovary cells overexpressing the insulin receptor was sufficient to induce PKB phosphorylation at Thr(308) to approximately the same extent as insulin stimulation. Phosphorylation of PKB by PDK1(A280V) was not affected by treatment of cells with inhibitors of phosphatidylinositol 3-kinase or by deletion of the pleckstrin homology (PH) domain of PKB. C(2)-ceramide, a cell-permeable, indirect inhibitor of PKB phosphorylation, did not inhibit PDK1(A280V)-catalyzed PKB phosphorylation in cells and had no effect on PDK1 activity in vitro. On the other hand, co-expression of full-length protein kinase C-related kinase-1 (PRK1/PKN) or 2 (PRK2) inhibited PDK1(A280V)-mediated PKB phosphorylation. Replacing alanine at position 280 with valine or deletion of the PH domain enhanced PDK1 autophosphorylation in vitro. However, deletion of the PH domain of PDK1(A280V) significantly reduced PDK1(A280V)-mediated phosphorylation of PKB in cells. In resting cells, PDK1(A280V) localized in the cytosol and at the plasma membrane. However, PDK1(A280V) lacking the PH domain localized predominantly in the cytosol. Taken together, our findings suggest that the wild-type PDK1 may not be constitutively active in cells. In addition, activation of PDK1 is sufficient to phosphorylate PKB at Thr(308) in the cytosol. Furthermore, the PH domain of PDK1 may play both positive and negative roles in regulating the in vivo function of the enzyme. Finally, unlike the carboxyl-terminal fragment of PRK2, which has been shown to bind PDK1 and allow the enzyme to phosphorylate PKB at both Thr(308) and Ser(473), full-length PRK2 and its related kinase PRK1/PKN may both play negative roles in PKB-mediated downstream biological events.

Highlights

Phosphorylation of Thr308 in the activation loop and Ser473 at the carboxyl terminus is essential for PKB activation [2, 3]
Co-expression of PDK1A280V with PKB resulted in a marked increase in PKB phosphorylation at Thr308 to a level approximately equal to that attained by insulin treatment (Fig. 1A, top panel, lane 3 versus lane 5)
Phosphorylation of PKB at Thr308 in the activation loop by PDK1 is considered essential for PKB activation

Summary

Introduction

Phosphorylation of Thr308 in the activation loop and Ser473 at the carboxyl terminus is essential for PKB activation [2, 3]. We show that expression of a constitutively active mutant of mouse 3-phosphoinositidedependent protein kinase-1 (PDK1A280V) in Chinese hamster ovary cells overexpressing the insulin receptor was sufficient to induce PKB phosphorylation at Thr308 to approximately the same extent as insulin stimulation. Western blot analysis using phospho-specific antibodies directed against either Thr308 or Ser473 of PKB showed little PKB phosphorylation under basal conditions or in cells coexpressing wild-type PDK1 or the kinase-defective mutant PDK1K114G (Fig. 1A, top and second panels, lanes 1, 2, and 4).

Results

Conclusion