Study of PDK1 regulation by metabolites and interaction with kinase substrates (Póster)

LISSY GROSS; MARIANA SACERDOTI; KEVIN G. HICKS; JARED RUTTER; ALEJANDRO E. LEROUX; SEBASTIÁN KLINKE; RICARDO M. BIONDI

Mendoza

Congreso; LVIII Reunión Anual de la SAIB; 2022

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

Protein kinases are very important ON-OFF switches in cells. Dysregulation of their activities often lead to diseases, such as cancer or diabetes. Therefore, protein kinases are tightly and selectively regulated. Phosphoinositide-dependent protein kinase 1 (PDK1) is a master AGC kinase of the PI3K signalling pathway that phosphorylates at least other 23 AGC kinases in mammals, including isoforms of Akt/PKBs, S6Ks, SGKs, PKCs, PRKs, among others. However, PDK1 is present not only in mammals but throughout the eukaryotic evolution with orthologs in plants, yeasts and protozoa. Over the years, our laboratory used a chemical and structural biology approach to study and characterize in detail the regulation of PDK1. We demonstrated an allosteric regulation within the protein kinase domain, from a regulatory site termed PIF-pocket to the active site, as well as the existence of the reverse process. This bidirectional allosteric mechanism of regulation between both pockets can be modulated by small molecules that bind to the active orthosteric site and either enhance or inhibit the interaction at the PIF-pocket allosteric regulatory site. As a probe directed to the PIF-pocket, we employed the polypeptide PIFtide, which corresponds to the last 23 residues of the protein kinase PRK2. We showed that the metabolite adenosine can enhance the interaction of PDK1 with PIFtide and therefore asked ourselves if other metabolites could bind at the active site of protein kinases and physiologically regulate the formation of protein kinase complexes. In this work we present the effect of diverse metabolites and compounds on the allosteric interaction of PDK1 with the polypeptide probe PIFtide and the interaction between PDK1 and PDK1 substrates. We also validate and analyze the binding mode of such metabolites by obtaining the crystal structures of the catalytic domain of PDK1 in complex with them. Finally, we will discuss the potential impact of the studies on new mechanisms by which metabolites binding at the active site could allosterically modulate protein kinase conformations, formation of protein kinase complexes and thereby, be modulators of cellular signalling.