UC San Diego

The ubiquitous 3’,5’-cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) signaling requires spatiotemporal regulation to specifically execute various cellular functions. However, this spatiotemporal regulation is not well understood. Recently, the PKA regulatory subunit RIα has been shown to undergo cAMP-dependent liquid-liquid phase separation (LLPS), which contributes to cAMP compartmentation and suppresses tumorigenic phenotypes. In Chapter 1, we probe the multi-valency and intrinsic disorder of RIα to determine the molecular drivers of LLPS. Via point mutagenesis, we discover that RIα LLPS requires two separate dimerization interfaces in conjunction with the cAMP-induced release of the PKA-C from the pseudosubstrate inhibitory sequence. Importantly, we found that the RIα pseudosubstrate is essential for the non-canonical, active PKA conformation that enables sequestration of PKA activity to the biomolecular condensates, facilitating low PKA activity in the cytosol. Therefore, RIα LLPS provides distinct spatial organization of PKA activity compared to A-kinase anchoring proteins (AKAPs) recruitment of PKA. In conjunction with the previously established cAMP compartmentation, this spatial constraint of PKA activity underlies the importance of RIα biomolecular condensates in PKA signaling specificity. In Chapter 2, we investigate the role PKA RIα LLPS plays in the functionality of MIN6 β cells, where precise Ca2+ oscillation promotes insulin secretion and cell proliferation controls β-cell expansion. Through a combination of CRISPR-based RIα knockout MIN6 cells and an RIα point mutation (Y122A) to selectively disrupt RIα LLPS, we show that RIα LLPS compartmentalizes cAMP and regulates Ca2+ oscillation frequency and MIN6 β cell proliferation. This study establishes a molecular tool to selectively study RIα LLPS, which may elucidate the functional impact of RIα LLPS in a variety of cellular systems. Together, these studies provide a better understanding of the molecular mechanism and signaling, and functional impact of RIα LLPS. As PKA is implicated in a variety of disease states, including cancer and diabetes, understanding the role of PKA RIα LLPS in disease states is increasingly critical to explore novel therapeutic avenues.