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, Feng Liu

Research output: Contribution to journalArticlepeer-review

118 Scopus citations


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 (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. Phosphorylation of PKB by PDK1A280V 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 PDK1A280V-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 PDK1A280V-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 PDK1A280V significantly reduced PDK1A280V-mediated phosphorylation of PKB in cells. In resting cells, PDK1A280V localized in the cytosol and at the plasma membrane. However, PDK1A280V 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 carboxylterminal 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.

Original languageEnglish (US)
Pages (from-to)40400-40406
Number of pages7
JournalJournal of Biological Chemistry
Issue number51
StatePublished - Dec 22 2000

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Cell Biology


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