Akt/Protein Kinase B Is Regulated by Autophosphorylation at the Hypothetical PDK-2 Site

Alex Toker,Alexandra C Newton

doi:10.1074/jbc.275.12.8271

Alex Toker, Alexandra C Newton

Open Access

https://doi.org/10.1074/jbc.275.12.8271

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Abstract

The function of Akt (protein kinase B) is regulated by phosphorylation on two sites conserved within the AGC kinase family: the activation loop (Thr-308) in the kinase core and a hydrophobic phosphorylation site on the carboxyl terminus (Ser-473). Thr-308 is phosphorylated by the phosphoinositide-dependent kinase-1, (PDK-1), whereas the mechanism of phosphorylation of the hydrophobic site, tentatively referred to as the PDK-2 site, is unknown. Here we report that phosphorylation of the hydrophobic motif requires catalytically competent Akt. First we show that a kinase-inactive construct of Akt fails to incorporate phosphate at Ser-473 following IGF-1 stimulation in vivo but does incorporate phosphate at Thr-308 and a second carboxyl-terminal site, Thr-450; this ligand triggers the phosphorylation of both sites in wild-type enzyme. Neither does a catalytically inactive construct in which phosphorylation at the activation loop is blocked, T308A, become phosphorylated on the hydrophobic site in response to stimulation. Second, we show that Akt autophosphorylates on the hydrophobic site in vitro: phosphorylation of the activation loop by PDK-1 triggers the phosphorylation of the hydrophobic site in kinase-active, but not thermally inactivated, Akt alpha. Thus, Akt is regulated by autophosphorylation at the Ser-473 hydrophobic site.

Highlights

From the ¶Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0640 and the ‡Signal Transduction Group, Boston Biomedical Research Institute, Boston, Massachusetts 02114
The hydrophobic site has generally been accepted to be regulated by a heterologous kinase, referred to as PDK-2 [14] or Ser-473 kinase [9]
A yeast twohybrid screen led to the identification of the carboxyl terminus of the protein kinase C-related kinase, PRK2, as a module interacting with PDK-1 [28]

Summary

Introduction

From the ¶Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0640 and the ‡Signal Transduction Group, Boston Biomedical Research Institute, Boston, Massachusetts 02114. The function of Akt (protein kinase B) is regulated by phosphorylation on two sites conserved within the AGC kinase family: the activation loop (Thr-308) in the kinase core and a hydrophobic phosphorylation site on the carboxyl terminus (Ser-473). The mitogen sensitivity of this site, like that of the activation loop, has led to the proposal that it, too, is phosphorylated by an agonistsensitive kinase, tentatively named PDK-2 [14] Such a kinase has remained refractory to molecular or biochemical identification [17]. In addition to the mitogen-sensitive sites, 32P labeling studies have revealed that Akt is constitutively phosphorylated at Thr-450 in vivo [18] This position corresponds to the turn motif in the protein kinase Cs as it has the consensus motif T/SPXD [21]

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