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Molecular and Cellular Biology, August 2000, p. 5712-5721, Vol. 20, No. 15
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Effect of Phosphoinositide-Dependent Kinase 1 on Protein Kinase B Translocation and Its Subsequent Activation

Nathalie Filippa,1 Carol L. Sable,1 Brian A. Hemmings,2 and Emmanuel Van Obberghen1,*

INSERM U145, IFR 50, Faculté de Médecine, 06107 Nice Cedex 2, France,1 and Friedrich Miescher Institute, CH 4002 Basel, Switzerland2

Received 2 August 1999/Returned for modification 23 September 1999/Accepted 17 April 2000

In this report we investigated the function of phosphoinositide-dependent protein kinase 1 (PDK1) in protein kinase B (PKB) activation and translocation to the cell surface. Wild-type and PDK1 mutants were transfected into HeLa cells, and their subcellular localization was analyzed. PDK1 was found to translocate to the plasma membrane in response to insulin, and this process did not require a functional catalytic activity, since a catalytically inactive kinase mutant (Kd) of PDK1 was capable of translocating. The PDK1 presence at the cell surface was shown to be linked to phospholipids and therefore to serum-dependent phosphatidylinositol 3-kinase activity. Using confocal microscopy in HeLa cells we found that PDK1 colocalizes with PKB at the plasma membrane. Further, after cotransfection of PKB and a PDK1 mutant (Mut) unable to translocate to the plasma membrane, PKB was prevented from moving to the cell periphery after insulin stimulation. In response to insulin, a PKB mutant with its PH domain deleted (Delta PH-PKB) retained the ability to translocate to the plasma membrane when coexpressed with PDK1. Finally, we found that Delta PH-PKB was highly active independent of insulin stimulation when cotransfected with PDK1 mutants defective in their PH domain. These findings suggest that PDK1 brings PKB to the plasma membrane upon exposure of cells to insulin and that the PH domain of PDK1 acts as a negative regulator of its enzyme activity.

* Corresponding author. Mailing address: INSERM U 145, IFR 50, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 2, France. Phone: 33-4-93-81-54-47. Fax: 33-4-93-81-54-32. E-mail: vanobbeg{at}unice.fr.

Molecular and Cellular Biology, August 2000, p. 5712-5721, Vol. 20, No. 15
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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