Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

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.


  1. Taskén, K. & Aandahl, E.M. Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiol. Rev. 84, 137–167 (2004).

    Article  Google Scholar 

  2. Steinberg, S.F. & Brunton, L.L. Compartmentation of G protein-coupled signaling pathways in cardiac myocytes. Annu. Rev. Pharmacol. Toxicol. 41, 751–773 (2001).

    Article  CAS  Google Scholar 

  3. Houslay, M.D. Underpinning compartmentalised cAMP signalling through targeted cAMP breakdown. Trends Biochem. Sci. 35, 91–100 (2010).

    Article  CAS  Google Scholar 

  4. Carnegie, G.K., Means, C.K. & Scott, J.D. A-kinase anchoring proteins: from protein complexes to physiology and disease. IUBMB Life 61, 394–406 (2009).

    Article  CAS  Google Scholar 

  5. Altarejos, J.Y. & Montminy, M. CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nat. Rev. Mol. Cell Biol. 12, 141–151 (2011).

    Article  CAS  Google Scholar 

  6. Kvissel, A.K. et al. Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing. Exp. Cell Res. 313, 2795–2809 (2007).

    Article  CAS  Google Scholar 

  7. Martin, B.R., Deerinck, T.J., Ellisman, M.H., Taylor, S.S. & Tsien, R.Y. Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1alpha-mediated localization in living cells. Chem. Biol. 14, 1031–1042 (2007).

    Article  CAS  Google Scholar 

  8. Harootunian, A.T. et al. Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion. Mol. Biol. Cell 4, 993–1002 (1993).

    Article  CAS  Google Scholar 

  9. Mayr, B. & Montminy, M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat. Rev. Mol. Cell Biol. 2, 599–609 (2001).

    Article  CAS  Google Scholar 

  10. Meoli, E. et al. Protein kinase A effects of an expressed PRKAR1A mutation associated with aggressive tumors. Cancer Res. 68, 3133–3141 (2008).

    Article  CAS  Google Scholar 

  11. Zippin, J.H. et al. Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain. J. Cell Biol. 164, 527–534 (2004).

    Article  CAS  Google Scholar 

  12. Jarnaess, E. et al. Splicing factor arginine/serine-rich 17A (SFRS17A) is an A-kinase anchoring protein that targets protein kinase A to splicing factor compartments. J. Biol. Chem. 284, 35154–35164 (2009).

    Article  CAS  Google Scholar 

  13. Steegborn, C., Litvin, T.N., Levin, L.R., Buck, J. & Wu, H. Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment. Nat. Struct. Mol. Biol. 12, 32–37 (2005).

    Article  CAS  Google Scholar 

  14. Chen, Y. et al. Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science 289, 625–628 (2000).

    Article  CAS  Google Scholar 

  15. Buck, J., Sinclair, M.L., Schapal, L., Cann, M.J. & Levin, L.R. Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc. Natl. Acad. Sci. USA 96, 79–84 (1999).

    Article  CAS  Google Scholar 

  16. DiPilato, L.M. & Zhang, J. The role of membrane microdomains in shaping β2-adrenergic receptor-mediated cAMP dynamics. Mol. Biosyst. 5, 832–837 (2009).

    Article  CAS  Google Scholar 

  17. Ni, Q. et al. Signaling diversity of PKA achieved via a Ca2+-cAMP-PKA oscillatory circuit. Nat. Chem. Biol. 7, 34–40 (2011).

    Article  CAS  Google Scholar 

  18. Terrin, A. et al. PGE1 stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases. J. Cell Biol. 175, 441–451 (2006).

    Article  CAS  Google Scholar 

  19. DiPilato, L.M., Cheng, X. & Zhang, J. Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc. Natl. Acad. Sci. USA 101, 16513–16518 (2004).

    Article  CAS  Google Scholar 

  20. Allen, M.D. & Zhang, J. Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem. Biophys. Res. Commun. 348, 716–721 (2006).

    Article  CAS  Google Scholar 

  21. Hagiwara, M. et al. Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A. Mol. Cell. Biol. 13, 4852–4859 (1993).

    Article  CAS  Google Scholar 

  22. Burnham, K.P. & Anderson, D.R. Model Selection and Multi-Model Inference: Practical Information-Theoretic Approach Ch. 2, 49–97 (Springer, 2002).

  23. Sugiura, N. Further analysis of data by Akaike's information criterion and finite corrections. Commun. Statist. 7, 13–26 (1978).

    Article  Google Scholar 

  24. Shimojo, M., Paquette, A.J., Anderson, D.J. & Hersh, L.B. Protein kinase A regulates cholinergic gene expression in PC12 cells: REST4 silences the silencing activity of neuron-restrictive silencer factor/REST. Mol. Cell. Biol. 19, 6788–6795 (1999).

    Article  CAS  Google Scholar 

  25. Lynch, M.J. et al. RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with β arrestin to control the protein kinase A/AKAP79-mediated switching of the β2-adrenergic receptor to activation of ERK in HEK293B2 cells. J. Biol. Chem. 280, 33178–33189 (2005).

    Article  CAS  Google Scholar 

  26. McCahill, A. et al. In resting COS1 cells a dominant negative approach shows that specific, anchored PDE4 cAMP phosphodiesterase isoforms gate the activation, by basal cyclic AMP production, of AKAP-tethered protein kinase A type II located in the centrosomal region. Cell. Signal. 17, 1158–1173 (2005).

    Article  CAS  Google Scholar 

  27. de Rooij, J. et al. Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs. J. Biol. Chem. 275, 20829–20836 (2000).

    Article  CAS  Google Scholar 

  28. Beavo, J.A., Bechtel, P.J. & Krebs, E.G. Activation of protein kinase by physiological concentrations of cyclic AMP. Proc. Natl. Acad. Sci. USA 71, 3580–3583 (1974).

    Article  CAS  Google Scholar 

  29. Wong, W. & Scott, J.D. AKAP signalling complexes: focal points in space and time. Nat. Rev. Mol. Cell Biol. 5, 959–970 (2004).

    Article  CAS  Google Scholar 

  30. Dodge-Kafka, K.L. et al. The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways. Nature 437, 574–578 (2005).

    Article  CAS  Google Scholar 

  31. Michel, J.J. & Scott, J.D. AKAP mediated signal transduction. Annu. Rev. Pharmacol. Toxicol. 42, 235–257 (2002).

    Article  CAS  Google Scholar 

  32. Ellis-Davies, G.C. Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat. Methods 4, 619–628 (2007).

    Article  CAS  Google Scholar 

  33. Stierl, M. et al. Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa. J. Biol. Chem. 286, 1181–1188 (2011).

    Article  CAS  Google Scholar 

  34. Schröder-Lang, S. et al. Fast manipulation of cellular cAMP level by light in vivo. Nat. Methods 4, 39–42 (2007).

    Article  Google Scholar 

  35. Kholodenko, B.N., Hancock, J.F. & Kolch, W. Signalling ballet in space and time. Nat. Rev. Mol. Cell Biol. 11, 414–426 (2010).

    Article  CAS  Google Scholar 

  36. Chuang, H.Y., Hofree, M. & Ideker, T. A decade of systems biology. Annu. Rev. Cell Dev. Biol. 26, 721–744 (2010).

    Article  CAS  Google Scholar 

  37. Tay, S. et al. Single-cell NF-κB dynamics reveal digital activation and analogue information processing. Nature 466, 267–271 (2010).

    Article  CAS  Google Scholar 

  38. Paliwal, S. et al. MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast. Nature 446, 46–51 (2007).

    Article  CAS  Google Scholar 

  39. Rich, T.C. et al. Cellular mechanisms underlying prostaglandin-induced transient cAMP signals near the plasma membrane of HEK-293 cells. Am. J. Physiol. Cell Physiol. 292, C319–C331 (2007).

    Article  CAS  Google Scholar 

  40. Xin, W., Tran, T.M., Richter, W., Clark, R.B. & Rich, T.C. Roles of GRK and PDE4 activities in the regulation of β2 adrenergic signaling. J. Gen. Physiol. 131, 349–364 (2008).

    Article  CAS  Google Scholar 

  41. Saucerman, J.J. et al. Systems analysis of PKA-mediated phosphorylation gradients in live cardiac myocytes. Proc. Natl. Acad. Sci. USA 103, 12923–12928 (2006).

    Article  CAS  Google Scholar 

  42. Greenberg, M.E. & Bender, T.P. Identification of newly transcribed RNA. Curr. Protoc. Mol. Biol. 4, 4.10 (2007).

    Google Scholar 

Download references


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.).

Author information

Authors and Affiliations



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.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 3675 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing