Julia Hardy (University of California, San Diego), Jason Zhang (University of California, San Diego), Emily Pool (University of California, San Diego), Jaclyn Choi (University of California, San Diego), Susan Taylor (University of California, San Diego, University of California, San Diego), Xin Zhuo (University of California, San Diego), Jin Zhang (University of California, San Diego, University of California, San Diego, University of California, San Diego)
Presenting Author University of California, San Diego
A regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, was recently shown to undergo liquid-liquid phase separation (LLPS), which is critical for cAMP compartmentation. These RIα biomolecular condensates form upon cAMP production, contain high levels of cAMP and active PKA catalytic (PKAcat) subunits, buffer increases in cAMP concentration, and suppress tumorigenic phenotypes. Through mutagenesis studies of key residues within different domains of RIα, we found that cAMP binding to both CNB-A and B domains is required to stimulate RIα LLPS and that the dimerization via both the dimerization/docking (D/D) domain and the N3A motif of the CNB-A is required for RIα LLPS. Our data suggests that the inhibitory sequence (IS), which binds and inhibits the catalytic subunit, is also involved in the regulation of RIα LLPS as well as recruitment of the catalytic subunit to the RIα puncta, likely through a non-canonical interaction. RIα mutants carrying point mutations (G98S and A99S) in the IS showed enhanced RIα LLPS but weakened R:C complex and decreased capability to recruit PKAcat to RIα puncta. Intriguingly, these mutants generated puncta that did not disassemble when cAMP levels decreased, whereas puncta from wildtype RIα showed complete disassembly, suggesting that the IS motif impacts the dynamics of RIα LLPS. Our study led to a further understanding of the stimulation, formation, PKAcat recruitment, and disassembly of PKA biomolecular condensates, which may lead to novel therapeutic approaches for diseases driven by a loss of PKA RIα LLPS, such as fibrolamellar carcinoma.
Support or Funding Information
NIH R35 CA197622 and NIH R01 DK073368 to J.Z.
In low cAMP conditions, the RIα-containing PKA holoenzyme is mostly in its inactive form and is diffuse in the cytosol (left). As the cAMP concentration increases, cAMP binds to the CNB-B domain, which increases the binding affinity of CNB-A (center). Once cAMP binds to both CNB domains, the PKA catalytic activity is unleashed via the transition to a non-canonical PKA conformation. This non-canonical PKA conformation allows recruitment of PKAcat to the RIα biomolecular condensates (right).
