NMR Structural/Functional Characterization of an Oncogenic Mutant of cAMP-Dependent Protein Kinase A: PRKACA-DNAJB1

Cyclic AMP (cAMP)-dependent Protein kinase A (cAMP-PKA) is involved in regulating a multitude of biological processes including cell growth and division, cell differentiation, as well as metabolism and immune responsiveness, as such misregulation of PKA has been implicated in tumorigenesis. Recent genomic studies have identified that the single driver of the progression of fibrolamellar hepatocellular carcinoma (FL-HCC) is a mutant of the catalytic subunit of cAMP-dependant Protein Kinase A (PKA) fused with the DNAJB1 chaperons on the N-terminus (PKA-DNAJB1). This result in upregulation of kinase activity in vivo; however, the underlying molecular mechanism for the progression of the FL-HCC cancer by PKA-DNAJB1 is unknown. Our activity assays demonstrate that there is no significant difference in activity between the wild type enzyme and PKA-DNAJB1, however there is a substantial difference in affinity by the heat stable protein kinase A inhibitor (PKI). To investigate this difference we study the structure and dynamics of the wild type and PKA-DNAJB1 using NMR spectroscopy. We find that the kinase core is largely intact, but there are allosteric changes propagated to the DNAJB1 fusion upon ligand binding. Nuclear spin relaxation measurements show that for PKA-DNAJB1 the two constructs move independently in the ps-ns timescale. Our future work will focus on understanding how differential conformational dynamics in the μs-ms timescale leads to differential specificity of binding of PKI, providing potential explanation for the constitutive activity of PKA in FL-HCC.