High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site

Abstract

3-phosphoinositide dependent protein kinase-1 (PDK1) plays a key role in regulating signalling pathways by activating AGC kinases such as PKB/Akt and S6K. Here we describe the 2.0 A crystal structure of the PDK1 kinase domain in complex with ATP. The structure defines the hydrophobic pocket termed the "PIF-pocket", which plays a key role in mediating the interaction and phosphorylation of certain substrates such as S6K1. Phosphorylation of S6K1 at its C-terminal PIF-pocket-interacting motif promotes the binding of S6K1 with PDK1. In the PDK1 structure, this pocket is occupied by a crystallographic contact with another molecule of PDK1. Interestingly, close to the PIF-pocket in PDK1, there is an ordered sulfate ion, interacting tightly with four surrounding side chains. The roles of these residues were investigated through a combination of site-directed mutagenesis and kinetic studies, the results of which confirm that this region of PDK1 represents a phosphate-dependent docking site. We discuss the possibility that an analogous phosphate-binding regulatory motif may participate in the activation of other AGC kinases. Furthermore, the structure of PDK1 provides a scaffold for the design of specific PDK1 inhibitors.

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit,

“…The B helix structure is conserved among the PKA, PDK1, and PKB structures (39,40), all members of the AGC family; therefore, the B helix is likely Proximal to the conserved Gly-rich loop (residues 50-55, red), the B helix (residues 76-80, red) and the C-terminal tail (residues 330-334, red) are variable between the two protein kinases and may account for the difference in inhibition potency of the analogues. In PKA, a stretch of Glu residues in the C-terminal tail is not present in PKC.…”

The survival pathways phosphatidylinositol-3 kinase (PI3-K)/phosphoinositide-dependent protein kinase 1 (PDK1)/Akt modulate liver regeneration through hepatocyte size rather than proliferation

“…To confirm the role of PDK1-Akt/mTOR pathway in liver regeneration, we employed the "pif-pocket" mutant of PDK1, which allows PDK1 to signal exclusively to Akt but not to p70 S6K or others. 30,[34][35][36] Adenovirus-mediated introduction of the pif-pocket mutant (L155E) did re-phosphorylate Akt (Thr308), but not p70 S6K (Thr389), 4 hours after PH in L-Pdk1KO mice (Fig. 5A).…”

Novel Small Molecule Inhibitors of 3-Phosphoinositide-dependent Kinase-1

“…To investigate further how the inhibitors bind to PDK1, we prepared crystals of the PDK1 kinase domain (residues 51-359), as described by Biondi et al (30). We then introduced compounds into PDK1 crystals and determined the structures of enzymeinhibitor complexes by x-ray diffraction methods.…”