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Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP

A Corrigendum to this article was published on 20 May 2013

This article has been updated

Abstract

Understanding how specific cyclic AMP (cAMP) signals are organized and relayed to their effectors in different compartments of the cell to achieve functional specificity requires molecular tools that allow precise manipulation of cAMP in these compartments. Here we characterize a new method using bicarbonate-activatable and genetically targetable soluble adenylyl cyclase to control the location, kinetics and magnitude of the cAMP signal. Using this live-cell cAMP manipulation in conjunction with fluorescence imaging and mechanistic modeling, we uncovered the activation of a resident pool of protein kinase A (PKA) holoenzyme in the nuclei of HEK-293 cells, modifying the existing dogma of cAMP-PKA signaling in the nucleus. Furthermore, we show that phosphodiesterases and A-kinase anchoring proteins (AKAPs) are critical in shaping nuclear PKA responses. Collectively, our data suggest a new model in which AKAP-localized phosphodiesterases tune an activation threshold for nuclear PKA holoenzyme, thereby converting spatially distinct second messenger signals to temporally controlled nuclear kinase activity.

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Figure 1: SMICUS in HEK-293 cells.
Figure 2: Spatial manipulation of cAMP production reveals differential PKA activity dynamics in nuclei of HEK-293 cells.
Figure 3: Rapid nuclear PKA responses to local cAMP accumulation require nuclear PKA holoenzyme.
Figure 4: PKA holoenzyme is in the nuclei of HEK-293 cells.
Figure 5: cAMP-PKA signaling in nuclei of HEK-293 cells is tightly regulated by phosphodiesterases and AKAPs.

Change history

  • 19 April 2013

    In the version of this article initially published, the domain schemes in Figure 1a were incorrectly labeled sACt (aa 1–146). The correct text is sACt (aa 1–469). The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank L. Levin (Weill Cornell Medical College, Cornell University) for the gift of sACt cDNA. We thank M. Houslay (Institute of Biomedical and Life Sciences, University of Glasgow) and K. Xiang (University of Illinois, Urbana-Champaign) for the gift of dnPDE4 isoforms. We thank L. Hersh (University of Kentucky College of Medicine) for giving us the A126.1B2 and A126.1B2 Catβ cell line. We also thank S. Mehta, G. Mo, T. Ueno, C. Pohlmeyer and T. Inoue for their technical help. This work was funded by US National Institutes of Health (NIH) grants R01 DK073368, DP1 OD006419 (to J.Z.), F31 DK074381 (to L.M.D.) and R01 HL094476, American Heart Association grant 0830470N (to J.J.S.) and NIH grant GM08715 (to J.H.Y.).

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Authors and Affiliations

Authors

Contributions

Q.N. conceived the original idea for SMICUS. V.S., L.M.D., Q.N. and J.Z. designed the experimental aspects of the project. J.H.Y. and J.J.S. designed the modeling aspects of the project. V.S. and L.M.D. carried out the experiments. J.H.Y. developed the mathematical model and carried out the simulations. V.S., L.M.D., J.H.Y., Q.N., J.J.S. and J.Z. analyzed the data. J.Z., L.M.D., V.S. and J.H.Y. wrote the manuscript.

Corresponding authors

Correspondence to Jeffrey J Saucerman or Jin Zhang.

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Sample, V., DiPilato, L., Yang, J. et al. Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP. Nat Chem Biol 8, 375–382 (2012). https://doi.org/10.1038/nchembio.799

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