Protein kinases play major roles in all eukaryotic cells under normal cellular conditions and during the development of human diseases due to mutations, deletions, or gene fusions. Thus, the protein kinase family has become a primary target in the drug discovery industry. One of the main challenges in studying how protein kinases are activated and regulated, which is of paramount importance for the development of potent and selective therapeutic measures, is their complex architecture and domain organization. Protein kinases exist as large macromolecular complexes composed of a catalytic subunit and regulatory domains that control kinase activity allosterically. In the described work, single molecule optical tweezers will be employed in combination with mutagenesis, biochemical activity assays, isothermal titration calorimetry, analytical ultracentrifugation and steered molecular dynamic simulations to dissect the allosteric activation mechanisms in the cAMP-dependent protein kinase A (PKA). The PKA regulatory subunit harbors cAMP binding domains that experience conformational plasticity depending on whether it is interacting with the catalytic subunit or with cAMP. How does the CNB domain fold adapt to bind either cAMP or the kinase catalytic subunit? Which parameters that control the protein fold are perturbed by mutations found in disease states, and how is it related to deregulation of kinase activity? To answer these questions, we proposed two specific aims: (1) to define the mechanisms of domain communication between CNB domains in the PKA regulatory subunit; (2) to identify the allosteric communication networks between the regulatory and catalytic subunits of PKA. Completion of this project will shed light onto the molecular mechanisms by which the ubiquitous and structurally conserved CNB domain is able adapt its fold and underlying energy landscape to drive the PKA holoenzyme assembly process or the cAMP-dependent activation.
Mutations or gene fusions in Protein Kinase A (PKA) are associated to numerous human diseases, including Carney Complex disease, acrodysostosis-1, and fibrolamellar hepatocellular carcinoma. At the core of PKA activation and regulation is the cyclic nucleotide binding (CNB) domain, a regulatory domain used ubiquitously in all forms of life, from bacteria to humans. Therefore, understanding how the CNB domains regulate PKA under normal condition (wild type) or disease states (mutants) is of biomedical and basic scientific importance. !
