Phase separation of a PKA type I regulatory subunit regulates β-cell function through cAMP compartmentalization

Cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger, regulates a variety of cellular functions with high specificity. We previously showed that the type I regulatory subunit of cAMP-dependent protein kinase A (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) to facilitate spatial compartmentalization of cAMP. However, how RIα LLPS regulates cellular function is largely unknown. Here, we identify the formation of RIα condensates in MIN6 β cells and reveal key roles for RIα LLPS in regulating β cell function. By combining CRISPR-based RIα knockout with an RIα mutant (Y122A) that exhibits defective cAMP-induced LLPS, we demonstrate that RIα LLPS drives cAMP compartmentalization to tune β cell Ca2+ and cAMP oscillation frequency, control insulin secretion, regulate CREB-mediated gene expression and prevent uncontrolled proliferation. Our data establish the Y122A mutant as a selective molecular tool for studying RIα LLPS and expand our understanding of the functional impact of LLPS-driven protein assemblies.