Abstract
Protein kinase B (PKB/Akt) belongs to the AGC superfamily of related serine/threonine protein kinases. It is a key regulator downstream of various growth factors and hormones and is involved in malignant transformation and chemo-resistance. Full-length PKB protein has not been crystallised, thus studying the molecular mechanisms that are involved in its regulation in relation to its structure have not been simple. Recently, the dynamics between the inactive and active conformer at the molecular level have been described. The maintenance of PKB's inactive state via the interaction of the PH and kinase domains prevents its activation loop to be phosphorylated by its upstream activator, phosphoinositide-dependent protein kinase-1 (PDK1). By using a multidisciplinary approach including molecular modelling, classical biochemical assays, and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM), a detailed model depicting the interaction between the different domains of PKB in its inactive conformation was demonstrated. These findings in turn clarified the molecular mechanism of PKB inhibition by AKT inhibitor VIII (a specific allosteric inhibitor) and illustrated at the molecular level its selectivity towards different PKB isoforms. Furthermore, these findings allude to the possible function of the C-terminus in sustaining the inactive conformer of PKB. This study presents essential insights into the quaternary structure of PKB in its inactive conformation. An understanding of PKB structure in relation to its function is critical for elucidating its mode of activation and discovering how to modulate its activity. The molecular mechanism of inhibition of PKB activation by the specific drug AKT inhibitor VIII has critical implications for determining the mechanism of inhibition of other allosteric inhibitors and for opening up opportunities for the design of new generations of modulator drugs.
Highlights
Protein kinase B (PKB/Akt) is a key regulator downstream of various growth factors and hormonal signals
The initial docking model of the protein kinase B (PKB) pleckstrin homology (PH) and kinase domains interaction prior to performing molecular dynamics revealed that the residue Trp 80, critically positioned at the tip of the VL3 loop of the PH domain, was buried deep inside the PKB kinase domain cleft in the PH-in conformer
The refined dynamic model showed that through this PH-induced cavity, the Trp 80 residue, located at its centre, was accessible from outside, whereas PKB remained in its inactive conformer (Figure 1A-b)
Summary
Protein kinase B (PKB/Akt) is a key regulator downstream of various growth factors and hormonal signals. The phosphorylation of Thr 308 in the kinase domain of PKBa/Akt 1 by phosphoinositidedependent protein kinase-1 (PDK1) [6] and Ser 473 in the hydrophobic motif by mTORC2 complex [7] and/or DNAPK [8], is central for PKB activation [9]. These phosphorylations were shown to be dependent on the colocalisation of PKB and PDK1 at the plasma membrane through the interaction of their PH domains with PtdIns (3,4,5) P3 and PtdIns (3,4) P2 for the former [10,11,12] and PtdIns (3,4,5) P3 for the latter [13]. The dephosphorylation of these residues by okadaic acidsensitive and -insensitive phosphatases was shown to deactivate PKB [14,15]
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