Crystal structure of an activated Akt/Protein Kinase B ternary complex with GSK3-peptide and AMP-PNP

Abstract

The protein kinase Akt/PKB is stimulated by the phosphorylation of two regulatory residues, Thr 309 of the activation segment and Ser 474 of the hydrophobic motif (HM), that are structurally and functionally conserved within the AGC kinase family. To understand the mechanism of PKB regulation, we determined the crystal structures of activated kinase domains of PKB in complex with a GSK3beta-peptide substrate and an ATP analog. The activated state of the kinase was generated by phosphorylating Thr 309 using PDK1 and mimicking Ser 474 phosphorylation either with the S474D substitution or by replacing the HM of PKB with that of PIFtide, a potent mimic of a phosphorylated HM. Comparison with the inactive PKB structure indicates that the role of Ser 474 phosphorylation is to promote the engagement of the HM with the N-lobe of the kinase domain, promoting a disorder-to-order transition of the alphaC helix. The alphaC helix, by interacting with pThr 309, restructures and orders the activation segment, generating an active kinase conformation. Analysis of the interactions between PKB and the GSK3beta-peptide explains how PKB selects for protein substrates distinct from those of PKA.

Drug discovery approaches targeting the PI3K/Akt pathway in cancer

“…The elucidation of the three-dimensional cocrystal structures of PKA, Akt and PDK1 bound to ATP, ATP analogs or kinase inhibitors (Biondi et al, 2000;Yang et al, 2002a;Komander et al, 2004) may provide initial insights into how PDK1 kinase selectivity might be achieved with ATP-competitive inhibitors. We can expect that these structure-based design efforts combined with new screening methods (for example, inverse in silico screening; Zahler et al, 2007) may provide the basis for the identification and development of a new generation of PDK1 modulators with the desirable activity/selectivity profile and pharmacological properties.…”

Structure and function of Polo-like kinases

“…The model of the core kinase domain is based primarily on the structures of Aurora-A and -B (Bayliss et al, 2003;Cheetham et al, 2002;Nowakowski et al, 2002). The activation loop was modelled in an active conformation using the structure of the Akt/PKB : AMP-PNP : GSK3-peptide ternary complex (Yang et al, 2002). ATP and a substrate peptide (Gly-Ala-Ala-Ala-Glu-Ala-Ser-Phe-Ala-Ala) are shown in stick representation and were also modelled based on the Akt/PKB : AMP-PNP : GSK3-peptide ternary complex structure (Yang et al, 2002).…”

“…The kinase domain of human Plk1 was therefore modelled using the SWISS-MODEL server (Guex and Peitsch, 1997) based on the crystal structures of Aurora-A (Bayliss et al, 2003) (PDB codes 1Ol5 and 1Ol7), (Nowakowski et al, 2002) (PDB code 1MQ4), Aurora-B (Cheetham et al, 2002) (PDB code 1MUO), and Akt/ PKB (Yang et al, 2002) (PDB code 1O6K) ( Figure 5a, b). This Plk1 model was subsequently used as a template to model the Plk2, Plk3, and Plk4 kinase domains (Figure 5c-e).…”

Molecular alterations in apoptotic pathways after PKB/Akt-mediated chemoresistance in NCI H460 cells

“…Phosphorylation of Akt results in its activation and dissociation from the membrane. The resolution of the crystal structures of activated Akt and inactive Akt2 has provided further clues about their regulation and activation (Yang et al, 2002;Huang et al, 2003). Substrates of Akt are phosphorylated within the consensus sequence RXRXXS/T (Obata et al, 2000).…”