Benjamin-Zukerman, T.; Pane, V.; Safadi-Safa, R.; Solomon, M.; Lev-Ram, V.; Aboraya, M.; Dakwar, A.; Bertinetti, D.; Hoy, A.; Mol, M.; van Swieten, J.; Herberg, F. W.; Maillard, R.; Ilouz, R.

Protein kinase A (PKA) is a crucial signaling enzyme in neurons, with its dysregulation being implicated in neurodegenerative diseases. Assembly of the PKA holoenzyme, comprising a dimer of heterodimers of regulatory (R) and catalytic (C) subunits, ensures allosteric regulation and functional specificity. Recently, we defined the RIb-L50R variant as a causative mutation that triggers protein aggregation in a rare neurodegenerative disease. However, the mechanism underlying uncontrolled PKA allosteric regulation and its connection to the functional outcomes leading to clinical symptoms remain elusive. In this study, we established an in vitro model using patient-derived cells for a personalized approach and employed direct measurements of purified proteins to investigate disease mechanisms in a controlled environment. Structural analysis and circular dichroism spectroscopy revealed that cellular proteins aggregation resulted from misfolded RIb-subunits, preventing holoenzyme assembly and anchoring through A Kinase Anchoring Proteins (AKAPs). While maintaining high affinity to the C subunit, the resulting RIb-L50R:C heterodimer exhibits reduced cooperativity, requiring lower cAMP concentrations for dissociation. Consequently, there was an increased translocation of C-subunit into the nucleus, impacting gene expression. We successfully controlled C subunit translocation by introducing a mutation that decreased RIb:C dissociation in response to elevated cAMP levels. This research thus sets the stage for developing therapeutic strategies that modulate PKA assembly and allostery, thus exerting control over the unique molecular signatures identified in the disease-associated transcriptome profile.