In vitro study of the interaction between the protein kinase PDK1 and two kinase substrates

GROSS, LZF; GALCERAN, F; SACERDOTI, M; RINALDI, J; KLINKE, S; OTERO, L; LEROUX, AE; BIONDI, RM

Rosario

Congreso; LIX Reunión Científica anual; 2023

Sociedad Argentina de Investigacion Bioquimica y Biologia Molecular (SAIB)

Protein kinases serve as crucial cellular switches, governing essential ON-OFF functions within cells. The improper regulation of these kinases frequently plays a role in the development of diseases like cancer and diabetes. Consequently, stringent and precise control mechanisms are in place to regulate their activities.Phosphoinositide-dependent protein kinase 1 (PDK1) plays a pivotal role as a master AGC kinase in the PI3K signaling pathway. It phosphorylates the activation loop of over 23 other AGC kinases, including Akt/PKB, S6K, SGK, PKC, PRK, and more. This phosphorylation is important because it is required for the activity of all these protein kinases. 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 in the past that the metabolite adenosine can enhance the interaction of PDK1 with PIFtide, while the ortosteric inhibitor GSK2334470 displaces it. However, detailed information regarding the regulation and structure of a complex between PDK1 and its substrate kinases is still missing. We will present the initial studies on the in vitro interaction of PDK1 with two protein kinase substrates, S6K and PRK2 using AlphaScreen technology. PDK1 interacted with high affinity with both kinases and these interactions were inhibited by PIFtide. Using this assay, we demonstrate how small compounds and metabolites can modulate these protein-protein interactions, exerting orthosteric as well as allosteric effects on the protein complex. It was curious that the already phosphorylated PRK2 interacted with high affinity with PDK1, because our standard model suggested that the active form would not have the PIF-sequence exposed. Interestingly, the interaction of PRK2 with PDK1 mediated by the C-terminal PIF-sequence was compatible with PRK2 activity. Finally, we will provide preliminary information on the PDK1-substrate complex using transmission electron microscopy (negative‐stain TEM). Our study increases our understanding of the PDK1 regulation and specificity, based on the regulated docking interaction with substrates.