Aberrant Phase Separation of two PKA RIbeta Neurological Disorder Mutants Leads to Mechanistically Distinct Signaling Deficits

Spatiotemporal regulation of key-node signaling molecules, such as 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), is critical for normal cell physiology and susceptible to dysregulation in disease. Liquid-liquid phase separation (LLPS) is broadly recognized as a fundamental component of signal regulation, yet the connections between physiological and disease-linked biomolecular condensates are not well understood. Here, we show that an understudied, brain-specific PKA regulatory subunit, RIß, forms biomolecular condensates with distinct features from the ubiquitous isoform, RIa. We demonstrate that two RIß mutants linked to neurodegenerative (L50R) or neurodevelopmental (R335W) pathologies produce aberrant condensates which trap the PKA catalytic subunit within a gel-like matrix or cAMP-insensitive holoenzyme complex, respectively. RIßL50R condensates, in particular, lead to disrupted spatiotemporal control of PKA signaling and diminished PKA activity, resulting in phenotypic hallmarks of neurodegeneration. Our work highlights the functional importance of biomolecular condensates and the critical link between dysregulated LLPS and neurological disorders. Overall design: We transiently transfected PKA RIalpha knockout HEK293T cells with RIbeta-mRuby2, the L50R neurodegenerative disease variant, or mRuby2 fluorescent protein alone (as a control), in triplicate. Transfected cultures were harvested to assess transcriptional differences between wild-type and L50R variant RIbeta.