Molecular Determinants and Signaling Effects of PKA RIα Phase Separation

Hardy, J. C.; Pool, E. H.; Bruystens, J. G. H.; Zhou, X.; Li, Q.; Zhou, D. R.; Palay, M.; Tan, G.; Chen, L.; Choi, J. L. C.; Lee, H. N.; Strack, S.; Wang, D.; Taylor, S. S.; Mehta, S.; Zhang, J.

doi:10.1101/2023.12.10.570836

bioRxiv · 10.1101/2023.12.10.570836

Molecular Determinants and Signaling Effects of PKA RIα Phase Separation

Hardy, J. C.·Pool, E. H.·Bruystens, J. G. H.·Zhou, X.·Li, Q.·Zhou, D. R.·Palay, M.·Tan, G.·Chen, L.·Choi, J. L. C.·Lee, H. N.·Strack, S.·Wang, D.·Taylor, S. S.·Mehta, S.·Zhang, J.

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Abstract

Spatiotemporal regulation of intracellular signaling molecules, such as the 3,5-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures the specific execution of various cellular functions. Liquid-liquid phase separation (LLPS) of the ubiquitously expressed PKA regulatory subunit RI was recently identified as a major driver of cAMP compartmentation and signaling specificity. However, the molecular determinants of RI LLPS remain unclear. Here, we reveal that two separate dimerization interfaces combined with the cAMP-induced release of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence are required to drive RI condensate formation in cytosol, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RI pseudosubstrate region is critically involved in the formation of a non-canonical R:C complex, which serves to maintain low basal PKA activity in the cytosol by enabling the recruitment of active PKA-C to RI condensates. Our results suggest that RI LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity.

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Hardy, J. C., Pool, E. H., Bruystens, J. G. H., Zhou, X., Li, Q., Zhou, D. R., Palay, M., Tan, G., Chen, L., Choi, J. L. C., Lee, H. N., Strack, S., Wang, D., Taylor, S. S., Mehta, S., Zhang, J.. 2023-12-11. Molecular Determinants and Signaling Effects of PKA RIα Phase Separation. https://doi.org/10.1101/2023.12.10.570836

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