The PDK1 conformational landscape (Póster)

MARIANA SACERDOTI; LISSY GROSS; KARIN FROESE; ABHIJEET GHODE; ANDREW M. RILEY; SEBASTIÁN KLINKE; GANESH S. ANAND; KRISTINA PARIS; EVELYN SUESS; MARÍA V. CAPELLARI; PEDRO F. ARAMENDÍA; ALEJANDRO E. LEROUX; BARRY V. POTTER; CARLOS J. CAMACHO; RICARDO M. BIONDI

Evento online debido a la pandemia de coronavirus

Congreso; European Chemical Biology Symposium ECBS 2021 Meeting; 2021

European Chemical Society, EU-OPENSCREEN, and Società Chimica Italiana

Phosphoinositide-dependent protein kinase 1 (PDK1) is a master kinase of the PI3-kinase signaling pathway that phosphorylates at least 23 other evolutionary related AGC kinases. It has an N-terminal kinase domain (KD), a linker region and a C-terminal PH domain. Most work on PDK1 regulation has focused on the KD, but recent work suggests the existence of PDK1 dimers. Over the years, our laboratory has used a chemical and structural biology approach to characterize the bidirectional allosteric regulation between the PIF-pocket, a regulatory site located on the small lobe of the KD, and the ATP-Binding site of PDK1. Phosphorylation by PDK1 is required for the activity of all substrates and most, like S6K, SGK, PKC, rely on a docking interaction where a C-terminal hydrophobic motif interacts with the PIF-pocket of PDK1. Interestingly, this interaction is not a requirement for the phosphorylation of PKB/Akt after PI3-kinase activation, but both proteins have a PH domain that binds PIP3 at the cell membrane and colocalize there. Recent discoveries show that PKB/Akt can also be phosphorylated by PDK1 in a PIP3 independent context, suggesting the existence of alternative mechanisms of interaction.Inositol polyphosphorylated molecules derived from InsP5 inhibit PDK1 activity in vitro. However, we found that 2-O-Bz-InsP5 (HYG8) does not inhibit the phosphorylation of a peptide substrate termed T308tide. Moreover, we show that HYG8 can selectively inhibit the phosphorylation and activation of PKB/Akt by PDK1 in vitro, but does not inhibit the phosphorylation and activation of SGK. HYG8 disrupts PDK1 dimers, measured by AlphaScreen and corroborated by single molecule fluorescence microscopy. H/D exchange experiments indicate that HYG8 stabilizes a monomeric conformation of PDK1, where the linker-PH domain region protects the "back" of the KD (monomeric conformation I).  We performed a small scale screening to find additional small molecules that regulate the formation of PDK1 dimers. Results suggest the existence of a second monomer (monomeric conformation II).  We conclude that PDK1 could exist as a dynamic equilibrium of conformations, which appear physiologically relevant because of the impact on the selective interactions and substrate phosphorylation. Such regulatory mechanisms could lead to novel approaches for the design of protein kinase inhibitors, targeting protein interactions to regulate the phosphorylation of a subset of substrates.