title: Activation of a GST-tagged AKT2/PKBbeta.
abstract: The protein kinase AKT is a key regulator for cell growth, cell survival and metabolic insulin action. However, the mechanism of activation of AKT in vivo, which presumably involves membrane recruitment of the kinase, oligomerization, and multiple phosphorylation events, is not fully understood. In the present study, we have expressed and purified dimeric GST-fusion proteins of human protein kinase AKT2 (DeltaPH-AKT2) in milligram quantities via the baculovirus expression system. Treatment of virus-infected insect cells with the phosphatase inhibitor okadaic acid (OA) led to phosphorylation of the two regulatory phosphorylation sites, Thr309 and Ser474, and to activation of the kinase. Likewise, phosphorylation of Thr309 in vitro by recombinant PDK1 or mutation of Thr309 and Ser474 to acidic residues rendered the kinase constitutively active. However, even though the specific activity of our AKT2 was increased 15-fold compared to previous reports, GST-mediated dimerization alone did not lead to an activation of the kinase. Whereas both mutagenesis and phosphorylation led to an increase in the turnover number of the enzyme, only the latter resulted in a marked reduction (20-fold) of the apparent Km value for the exogenous substrate Crosstide, indicating that this widely used mutagenesis only partially mimics phosphorylation. Kinetic analysis of GST-AKT2 demonstrates that phosphorylation of Thr309 in the activation loop of the kinase is largely responsible for the observed reduction in Km and for a subsequent 150-fold increase in the catalytic efficiency (k(cat)/Km) of the enzyme. Highly active AKT2 constructs were used in autophosphorylation reactions in vitro, where inactive AKT2 kinases served as substrates. As a matter of fact, we found evidence for a minor autophosphorylation activity of AKT2 but no significant autophosphorylation of any of the two regulatory sites, Thr309 or Ser474.
authors.affiliation: Institute of Biochemistry, University of Cologne, Cologne, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Baer
authors.forename: Ivonne
authors.initials: I
authors.lastname: Lisinski
authors.forename: Monika
authors.initials: M
authors.lastname: Gompert
authors.forename: Dominik
authors.initials: D
authors.lastname: Stuhlmann
authors.forename: Katja
authors.initials: K
authors.lastname: Schmolz
authors.forename: Helmut W
authors.initials: HW
authors.lastname: Klein
authors.forename: Hadi
authors.initials: H
authors.lastname: Al-Hasani
mesh_terms: D016367:Baculoviridae / Q000235:genetics; D003001:Cloning, Molecular; D019281:Dimerization; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D019319:Okadaic Acid / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: C505590:DeltaPH-AKT2 protein; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C505591:crosstide peptide; D019319:Okadaic Acid; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases