Molecular insights into the regulatory landscape of PKC-related kinase-2 (PRK2/PKN2) using targeted small compounds

• Published in: The Journal of biological chemistry 2024-08, Vol.300 (8), p.107550, Article 107550
• Main Authors: Gross, Lissy Z.F., Winkel, Angelika F., Galceran, Facundo, Schulze, Jörg O., Fröhner, Wolfgang, Cämmerer, Simon, Zeuzem, Stefan, Engel, Matthias, Leroux, Alejandro E., Biondi, Ricardo M.
• Format: Article
• Language: English
• Subjects: AGC kinases; allosteric regulation; chemical biology; PDK1; PKN; PRK; protein conformation; protein kinase; small molecule; substrate specificity
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1016/j.jbc.2024.107550

The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.