REGULATION OF PDK1 KINASE BY SMALL MOLECULES AND METABOLITES: BIDIRECTIONAL ALLOSTERY AND BEYOND

SACERDOTI M; HICKS KG; LEROUX AE; GHODE A; ANAND GS; GROSS LZF; RUTTER J; BIONDI RM

Conferencia; Translational Chemical Biology Nature Conference; 2020

Nature Conferences

Phosphoinositide-dependent protein kinase 1 (PDK1) is a master kinase of the PI3-kinase signalling pathway that phosphorylates at least 23 other evolutionary related AGC kinases. Phosphorylation by PDK1 is required for the activity of all substrates: they are phosphorylated either constitutively or with different timing upon PI3-kinase activation. The mechanism of activation of PDK1, mediated by the PIF-pocket regulatory site, is conserved within the large group of AGC kinases, including the isoforms of PKC, Akt, SGK, S6K, RSK, MSK, etc. Over the years, our laboratory has used a chemical and structural biology approach to study and characterize the bidirectional allosteric regulation between the PIF-pocket and the ATP-Binding site on the catalytic domain of PDK1. Compounds that we have developed to bind at the PIF-pocket activate or inhibit AGC kinases by allosterically affecting the ATP-Binding site. Furthermore, small compounds, i.e. PS653, and drugs developed by the pharmaceutical industry to bind at the ATP-Binding site can allosterically enhance or inhibit interactions at the PIF-pocket. Indeed, the same bidirectional allosteric mechanism of regulation that drives conformational changes from a regulatory site to the ATP-Binding site can be exploited by drugs targeting the latter to disrupt protein kinase interactions. We next employed biochemical and structural techniques, in silico molecular dynamics and hydrogen/deuterium exchange (HDX) approaches to investigate whether metabolites could also bind at the ATP-Binding site and allosterically modulate protein kinase interactions. We found that Adenosine, which binds at the ATP-Binding site, allosterically enhances interactions at the PIF-pocket. We demonstrate that Adenosine and PIFtide (a peptide derived from the hydrophobic motif of a PDK1 substrate that binds to the PIF-pocket) synergistically enhance the interaction of one another. Finally, we investigate the effect of distinct metabolites, which bind at the ATP-binding site, on properties of full length PDK1. Our in vitro studies suggest that the allosteric mechanism of regulation could possibly sense the cellular metabolic state and alter the dynamic formation of protein kinase complexes, physiologically modulating cellular signaling by a novel mechanism.