Ricardo M. Biondi

The PIF‐binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB

PKB/Akt, S6K1 and SGK are related protein kinases activated in a PI 3‐kinase‐dependent manner in response to insulin/growth factors signalling. Activ ation entails phosphorylation of these kinases at two residues, the T‐loop and the hydrophobic motif. PDK1 activates S6K, SGK and PKB isoforms by phosphorylating these kinases at their T‐loop. We demonstrate that a pocket in the kinase domain of PDK1, termed the ‘PIF‐binding pocket’, plays a key role in mediating the interaction and phosphorylation of S6K1 and SGK1 at their T‐loop motif by PDK1. Our data indicate that prior phosphorylation of S6K1 and SGK1 at their hydrophobic motif promotes their interaction with the PIF‐binding pocket of PDK1 and their T‐loop phosphorylation. Thus, the hydrophobic motif phosphorylation of S6K and SGK converts them into substrates that can be activated by PDK1. In contrast, the PIF‐binding pocket of PDK1 is not required for the phosphorylation of PKBα by PDK1. The PIF‐binding pocket represents a substrate recognition site on a protein kinase that is only required for the phosphorylation of a subset of its physiological substrates.

Signalling specificity of Ser/Thr protein kinases through docking-site-mediated interactions

Signal transduction pathways use protein kinases for the modification of protein function by phosphorylation. A major question in the field is how protein kinases achieve the specificity required to regulate multiple cellular functions. Here we review recent studies that illuminate the mechanisms used by three families of Ser/Thr protein kinases to achieve substrate specificity. These kinases rely on direct docking interactions with substrates, using sites distinct from the phospho-acceptor sequences. Docking interactions also contribute to the specificity and regulation of protein kinase activities. Mitogen-activated protein kinase (MAPK) family members can associate with and phosphorylate specific substrates by virtue of minor variations in their docking sequences. Interestingly, the same MAPK docking pocket that binds substrates also binds docking sequences of positive and negative MAPK regulators. In the case of glycogen synthase kinase 3 (GSK3), the presence of a phosphate-binding site allows docking of previously phosphorylated (primed) substrates; this docking site is also required for the mechanism of GSK3 inhibition by phosphorylation. In contrast, non-primed substrates interact with a different region of GSK3. Phosphoinositide-dependent protein kinase-1 (PDK1) contains a hydrophobic pocket that interacts with a hydrophobic motif present in all known substrates, enabling their efficient phosphorylation. Binding of the substrate hydrophobic motifs to the pocket in the kinase domain activates PDK1 and other members of the AGC family of protein kinases. Finally, the analysis of protein kinase structures indicates that the sites used for docking substrates can also bind N- and C-terminal extensions to the kinase catalytic core and participate in the regulation of its activity.

Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C‐terminal residues of PKA

The 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1) phosphorylates and activates a number of protein kinases of the AGC subfamily. The kinase domain of PDK1 interacts with a region of protein kinase C‐related kinase‐2 (PRK2), termed the PDK1‐interacting fragment (PIF), through a hydrophobic motif. Here we identify a hydrophobic pocket in the small lobe of the PDK1 kinase domain, separate from the ATP‐ and substrate‐binding sites, that interacts with PIF. Mutation of residues predicted to form part of this hydrophobic pocket either abolished or significantly diminished the affinity of PDK1 for PIF. PIF increased the rate at which PDK1 phosphorylated a synthetic dodecapeptide (T308tide), corresponding to the sequences surrounding the PDK1 phosphorylation site of PKB. This peptide is a poor substrate for PDK1, but a peptide comprising T308tide fused to the PDK1‐binding motif of PIF was a vastly superior substrate for PDK1. Our results suggest that the PIF‐binding pocket on the kinase domain of PDK1 acts as a ‘docking site’, enabling it to interact with and enhance the phosphorylation of its substrates.

A phosphoserine/threonine‐binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation

The growth factor‐activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C‐terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBα, MSK1 and SGK1 contain a similar phosphate‐binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism in which the phosphorylated hydrophobic motif and activation loop act on the αC‐helix of the kinase structure to induce synergistic stimulation of catalytic activity. Sequence conservation suggests that this mechanism is a key feature in activation of >40 human AGC kinases.

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

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 Å 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.

A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta ) and PKC-related kinase 2 by PDK1.

Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKCζ, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKCζ and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKCζ and PKCι, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKCζ, full-length PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in vivo. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKCζ. These findings indicate that the hydrophobic motif of PRK2 and PKCζ acts as a “docking site” enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.

Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1.

Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation-dependent activation of AGC kinases. The AGC kinase PDK1 is activated by the docking of a phosphorylated motif from substrates. Here we present the crystallography of PDK1 bound to a rationally developed low-molecular-weight activator and describe the conformational changes induced by small compounds in the crystal and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present molecular details of the allosteric changes induced by small compounds that trigger the activation of PDK1 through mimicry of phosphorylation-dependent conformational changes.

Allosteric activation of the protein kinase PDK1 with low molecular weight compounds

Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF‐pocket and have the ability to allosterically activate phosphoinositide‐dependent protein kinase 1 (PDK1) by modulating the phosphorylation‐dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogs, interaction‐displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode of action and may inspire future rational development of compounds with the ability to modulate phosphorylation‐dependent conformational transitions in other proteins.

Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation

The growth factor/insulin‐stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C‐terminal tail region are well characterized. We investigated the role of the third, so‐called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr‐binding site above the glycine‐rich loop within the kinase domain, promoting zipper‐like association of the tail with the kinase domain, serving to stabilize the HM in its kinase‐activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM‐mediated stabilization of the αC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.

Differential Stability of Cell-Free Circulating microRNAs: Implications for Their Utilization as Biomarkers

Background MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study. Methods The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied. Results The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles. Conclusions Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.