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J. Biol. Chem., Vol. 275, Issue 51, 40400-40406, December 22, 2000

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

Michael J. Wick, Lily Q. Dong, Ramon A. Riojas^§, Fresnida J. Ramos, and Feng Liu^§¶

From the Departments of  Pharmacology and ^§ Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229

Phosphorylation of Thr³⁰⁸ in the activation loop and Ser⁴⁷³ 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 Thr³⁰⁸ 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. C₂-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 Thr³⁰⁸ 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 Thr³⁰⁸ and Ser⁴⁷³, full-length PRK2 and its related kinase PRK1/PKN may both play negative roles in PKB-mediated downstream biological events.

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