PKA RIα phase separation drives cAMP compartmentation and suppresses tumorigenic signaling

Abstract

The fidelity of intracellular signal transduction hinges on the organization of signaling molecule activities into a dynamic architecture. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G‐protein‐coupled receptors can regulate specific cellular processes despite converging on the ubiquitous second messenger 3’,5’‐cyclic adenosine monophosphate (cAMP). Recent work has challenged the textbook model that cAMP compartmentation is achieved by its local degradation, yet the specific mechanisms responsible for spatially constraining this diffusible messenger remain elusive. We address this longstanding question by identifying the formation of biomolecular condensates of the type I regulatory subunit of cAMP‐dependent kinase (PKA), RIα, as a key driver of cAMP compartmentation. RIα undergoes liquid‐liquid phase‐separation at endogenous levels as a function of dynamic cAMP signaling to form RIα bodies harboring high levels of cAMP and PKA activity. Importantly, we show that this active cAMP sequestration is critical for effective cAMP compartmentation in cells. The pathophysiological relevance of this compartmentation system is illustrated by its key role in the etiology of the atypical liver cancer fibrolamellar carcinoma (FLC). We show that an FLC‐linked oncoprotein fusion between DnaJB1 and the PKA catalytic subunit (PKAcat) potently blocks RIα phase separation and induces aberrant cAMP signaling. Furthermore, loss of RIα phase separation independent of the fusion oncoprotein in normal hepatocytes increased cell proliferation and resulted in cell transformation. Our work reveals liquid‐liquid phase separation as a principle organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.Support or Funding InformationThis work is supported by NIH R35 CA197622, R01 DK073368 (to J.Z). J.Z.Z. is supported by a predoctoral fellowship from the National Science Foundation (DGE‐1650112).In healthy cells, endogenous RIα acts as a dynamic cAMP buffer via phase separation, allowing for PDE‐mediated cAMP compartmentation. For fibrolamellar carcinoma (FLC) patients, DnaJB1‐PKAcat fusion oncogene disrupts RIα phase separation, thus disrupting cAMP compartmentation and leading to tumorigenesis.Figure 1