Pathogenic Mutations in Catalytic Subunit Isoforms of the cAMP-Dependent Protein Kinase

Protein kinase A (PKA) is an integral part in a myriad of cellular pathways and a main effector of the second messenger cAMP. Since its discovery in the late 1960s, PKA has become one of the best-studied eukaryotic protein kinases in terms of enzymatic and structural features. Pathological implications of PKA have just started to emerge. There are three main isoforms (α, β, γ) of the catalytic subunit of PKA (PKA-C) which constitute a holoenzyme (R2C2) by association with a regulatory subunit (PKA-R) dimer. While most of the research in recent decades has been focused on PKA-Cα, little research has been done on PKA-Cβ. Lately, disease-associated mutations highlight the non-redundant features of the PKA-Cα/Cβ isoforms, in particular the exceptional role of PKA-Cβ. This dissertation focussed on the detailed biochemical and in cell characterization of pathological mutations of PKA-Cα/Cβ to understand the molecular mechanisms of these mutations, and to illustrate the non-redundancy of PKA-Cα/Cβ. With a direct link to Hedgehog (Hh) signalling, the mutations in PKA-Cα (G136R) and in PKA-Cβ1 (H87N, H87R, G234R) are related to cardioacrofacial dysplasia 1 and 2, respectively. PKA-Cα G136R and PKA-Cβ1 G234R contribute to inhibitor and substrate binding, while PKA-Cβ1 H87N and H87R alter an important interaction with the phosphorylation site pT197 in the activation loop. The PKA-Cβ1 mutations displayed decreased catalytic activity, reduced PKIα inhibitor affinity and altered enzymatic parameters of (co-)substrates (ATP, Kemptide). Biochemical and biophysical data point to H87R being the most severe mutation followed by H87N and G234R. In contrast, PKA-Cα G136R displayed mainly an increased (!) affinity towards the inhibitor PKIα. PKA-Cβ1 G234R is depending on the presence of the PDK1 in E. coli expression for proper autophosphorylation of the two residues T197 and S338. Real-time BRET assays were employed to study PKA holoenzyme dynamics and regulation in HEK293 cells. All mutations except for PKA-Cα G136R showed a drastic increase in cAMP-dependent holoenzyme dissociation of PKA-RI and PKA-RII isoforms. When using the physiological β-adrenergic agonist isoproterenol, the hypothesis of labile PKA-C mutant holoenzymes was reassured. The mutations had similar effects in the splice variant PKA-Cβ2, which shows unique tissue expression. So far unpublished PKA-Cα disease-associated mutants (A20P, ΔN289-I291) also showed more labile PKA holoenzymes. In this dissertation, intramolecular effects of the PKA-C mutations on regulation and enzymatic parameters were characterized. These disease-associated mutations are located at key positions, thereby altering kinase function and regulation. It can be shown that aberrant Hh signalling is mainly due to increased cAMP-sensitivity of the resulting PKA-C mutant holoenzymes.