Molecular Determinants of PKA RIα Driven Liquid‐Liquid Phase Separation

Abstract

We recently discovered a regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS), which is critical for effective cAMP compartmentation. PKA RIα LLPS is enhanced by cAMP, which results in phase separated bodies that dynamically sequester cAMP. Interestingly, although cAMP-dependent dissociation of the canonical PKA complex, which consists of regulatory (R) and catalytic (C) subunits, allows RIα to undergo LLPS, the PKA C subunit is co-recruited to RIα phase separated bodies, resulting in high PKA activity. In this study, we performed systematic mutations to the dimerization/docking (D/D) domain, inhibitory sequence (IS), and cyclic nucleotide binding (CNB) domains to elucidate the molecular drivers of RIα LLPS. Mutations of key residues within the CNB domains and within the D/D domain and N3A motif of the CNB-A domain disrupt PKA RIα LLPS, indicating cAMP binding and RIα dimerization are required for LLPS. Conversely, mutations within the IS pseudo-substrate increase RIα LLPS, indicating a disrupted R:C complex enhances LLPS. The IS mutations also prevented recruitment of the catalytic subunit to the RIα LLPS, providing opportunities to further probe this non-canonical recruitment mechanism. Importantly, the loss of RIα LLPS leads to disruption of cAMP compartmentation, and increased cell proliferation, DNA synthesis, and anchorage independent growth, suggesting that dysregulated cAMP signaling plays a role in tumorigenesis.