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Molecular and Cellular Biology, September 2002, p. 6247-6260, Vol. 22, No. 17
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.17.6247-6260.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Multiple Phosphoinositide 3-Kinase-Dependent Steps in Activation of Protein Kinase B

Michael P. Scheid,1 Paola A. Marignani,1 and James R. Woodgett1,2*

Department of Experimental Therapeutics, University Health Network,1 Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada2

Received 5 February 2002/ Returned for modification 26 March 2002/ Accepted 4 June 2002

The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist-induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, we employed two distinct models of posttranslational inducible activation and membrane recruitment. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3' phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation.

* Corresponding author. Mailing address: Department of Medical Biophysics, University of Toronto, 610 University Ave., Toronto, Ontario M5G 2M9, Canada. Phone: (416) 946-2962. Fax: (416) 946-2984. E-mail: jwoodget{at}uhnres.utoronto.ca.

Molecular and Cellular Biology, September 2002, p. 6247-6260, Vol. 22, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.17.6247-6260.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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