Skip to main content

Thank you for visiting nature.com. 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.

Photoswitchable diacylglycerols enable optical control of protein kinase C

Nature Chemical Biology volume 12, pages 755–762 (2016)Cite this article

Subjects

Abstract

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.

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

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Design and synthesis of PhoDAGs.
Figure 2: PhoDAG-1 enables optical control of C1-GFP translocation and intracellular Ca2+ concentration in HeLa cells.
Figure 3: PhoDAG-1 enables optical control of PKC.
Figure 4: Optical control of Ca2+ oscillations in MIN6 and dissociated primary mouse β-cells.
Figure 5: Optical control of insulin secretion in intact mouse pancreatic islets.
Figure 6: PhoDAGs enable optical control of Munc13 and synaptic transmission.

References

  1. Almena, M. & Mérida, I. Shaping up the membrane: diacylglycerol coordinates spatial orientation of signaling. Trends Biochem. Sci. 36, 593–603 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Brose, N. & Rosenmund, C. Move over protein kinase C, you've got company: alternative cellular effectors of diacylglycerol and phorbol esters. J. Cell Sci. 115, 4399–4411 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Newton, A.C. Protein kinase C: structure, function, and regulation. J. Biol. Chem. 270, 28495–28498 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Pessin, M.S. & Raben, D.M. Molecular species analysis of 1,2-diglycerides stimulated by alpha-thrombin in cultured fibroblasts. J. Biol. Chem. 264, 8729–8738 (1989).

    CAS  PubMed  Google Scholar 

  5. Marignani, P.A., Epand, R.M. & Sebaldt, R.J. Acyl chain dependence of diacylglycerol activation of protein kinase C activity in vitro. Biochem. Biophys. Res. Commun. 225, 469–473 (1996).

    Article  CAS  PubMed  Google Scholar 

  6. Hofmann, T. et al. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259–263 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Das, J. & Rahman, G.M. C1 domains: structure and ligand-binding properties. Chem. Rev. 114, 12108–12131 (2014).

    Article  CAS  PubMed  Google Scholar 

  8. Coussens, L. et al. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science 233, 859–866 (1986).

    Article  CAS  PubMed  Google Scholar 

  9. Rhee, J.S. et al. β phorbol ester- and diacylglycerol-induced augmentation of transmitter release is mediated by Munc13s and not by PKCs. Cell 108, 121–133 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Wender, P.A. et al. Modeling of the bryostatins to the phorbol ester pharmacophore on protein kinase C. Proc. Natl. Acad. Sci. USA 85, 7197–7201 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nadler, A. et al. The fatty acid composition of diacylglycerols determines local signaling patterns. Angew. Chem. Int. Edn Engl. 52, 6330–6334 (2013).

    Article  CAS  Google Scholar 

  12. Putyrski, M. & Schultz, C. Protein translocation as a tool: the current rapamycin story. FEBS Lett. 586, 2097–2105 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Feng, S. et al. A rapidly reversible chemical dimerizer system to study lipid signaling in living cells. Angew. Chem. Int. Edn. Engl. 53, 6720–6723 (2014).

    Article  CAS  Google Scholar 

  14. Toettcher, J.E., Voigt, C.A., Weiner, O.D. & Lim, W.A. The promise of optogenetics in cell biology: interrogating molecular circuits in space and time. Nat. Methods 8, 35–38 (2011).

    Article  CAS  PubMed  Google Scholar 

  15. Frank, J.A. et al. Photoswitchable fatty acids enable optical control of TRPV1. Nat. Commun. 6, 7118 (2015).

    Article  PubMed  Google Scholar 

  16. Davis, R.J., Ganong, B.R., Bell, R.M. & Czech, M.P. sn-1,2-dioctanoylglycerol. A cell-permeable diacylglycerol that mimics phorbol diester action on the epidermal growth factor receptor and mitogenesis. J. Biol. Chem. 260, 1562–1566 (1985).

    CAS  PubMed  Google Scholar 

  17. Oancea, E., Teruel, M.N., Quest, A.F.G. & Meyer, T. Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells. J. Cell Biol. 140, 485–498 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wuttke, A., Idevall-Hagren, O. & Tengholm, A. P2Y1 receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion. FASEB J. 27, 1610–1620 (2013).

    Article  CAS  PubMed  Google Scholar 

  19. Nadler, A. et al. Exclusive photorelease of signalling lipids at the plasma membrane. Nat. Commun. 6, 10056 (2015).

    Article  CAS  PubMed  Google Scholar 

  20. Zhao, Y. et al. An expanded palette of genetically encoded Ca2+ indicators. Science 333, 1888–1891 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Plenge-Tellechea, F., Soler, F. & Fernandez-Belda, F. On the inhibition mechanism of sarcoplasmic or endoplasmic reticulum Ca2+-ATPases by cyclopiazonic acid. J. Biol. Chem. 272, 2794–2800 (1997).

    Article  CAS  PubMed  Google Scholar 

  22. Corbalán-García, S. & Gómez-Fernández, J.C. Protein kinase C regulatory domains: the art of decoding many different signals in membranes. Biochim. Biophys. Acta 1761, 633–654 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Kikkawa, U., Matsuzaki, H. & Yamamoto, T. Protein kinase Cδ (PKCδ): activation mechanisms and functions. J. Biochem. 132, 831–839 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Reither, G., Schaefer, M. & Lipp, P. PKCα: a versatile key for decoding the cellular calcium toolkit. J. Cell Biol. 174, 521–533 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Violin, J.D. & Newton, A.C. Pathway illuminated: visualizing protein kinase C signaling. IUBMB Life 55, 653–660 (2003).

    Article  CAS  PubMed  Google Scholar 

  26. Violin, J.D., Zhang, J., Tsien, R.Y. & Newton, A.C. A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J. Cell Biol. 161, 899–909 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Toullec, D. et al. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266, 15771–15781 (1991).

    CAS  PubMed  Google Scholar 

  28. Herbert, J.M., Augereau, J.M., Gleye, J. & Maffrand, J.P. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 172, 993–999 (1990).

    Article  CAS  PubMed  Google Scholar 

  29. Bergsten, P., Grapengiesser, E., Gylfe, E., Tengholm, A. & Hellman, B. Synchronous oscillations of cytoplasmic Ca2+ and insulin release in glucose-stimulated pancreatic islets. J. Biol. Chem. 269, 8749–8753 (1994).

    CAS  PubMed  Google Scholar 

  30. Yang, C. & Kazanietz, M.G. Divergence and complexities in DAG signaling: looking beyond PKC. Trends Pharmacol. Sci. 24, 602–608 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. Kurohane Kaneko, Y. et al. Depression of type I diacylglycerol kinases in pancreatic β-cells from male mice results in impaired insulin secretion. Endocrinology 154, 4089–4098 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Ashcroft, F.M. et al. Stimulus-secretion coupling in pancreatic β cells. J. Cell. Biochem. 55 (Suppl.), 54–65 (1994).

    Article  CAS  PubMed  Google Scholar 

  33. Jacobson, D.A. et al. Kv2.1 ablation alters glucose-induced islet electrical activity, enhancing insulin secretion. Cell Metab. 6, 229–235 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Jacobson, D.A., Weber, C.R., Bao, S., Turk, J. & Philipson, L.H. Modulation of the pancreatic islet β-cell-delayed rectifier potassium channel Kv2.1 by the polyunsaturated fatty acid arachidonate. J. Biol. Chem. 282, 7442–7449 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Rutter, G.A. & Hodson, D.J. Beta cell connectivity in pancreatic islets: a type 2 diabetes target? Cell. Mol. Life Sci. 72, 453–467 (2015).

    Article  CAS  PubMed  Google Scholar 

  36. Lou, X., Korogod, N., Brose, N. & Schneggenburger, R. Phorbol esters modulate spontaneous and Ca2+-evoked transmitter release via acting on both Munc13 and protein kinase C. J. Neurosci. 28, 8257–8267 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Brose, N., Betz, A. & Wegmeyer, H. Divergent and convergent signaling by the diacylglycerol second messenger pathway in mammals. Curr. Opin. Neurobiol. 14, 328–340 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. Basu, J., Betz, A., Brose, N. & Rosenmund, C. Munc13-1 C1 domain activation lowers the energy barrier for synaptic vesicle fusion. J. Neurosci. 27, 1200–1210 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wierda, K.D.B., Toonen, R.F.G., de Wit, H., Brussaard, A.B. & Verhage, M. Interdependence of PKC-dependent and PKC-independent pathways for presynaptic plasticity. Neuron 54, 275–290 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Betz, A. et al. Munc13-1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release. Neuron 21, 123–136 (1998).

    Article  CAS  PubMed  Google Scholar 

  41. Brose, N., Hofmann, K., Hata, Y. & Südhof, T.C. Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins. J. Biol. Chem. 270, 25273–25280 (1995).

    Article  CAS  PubMed  Google Scholar 

  42. Varoqueaux, F. et al. Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming. Proc. Natl. Acad. Sci. USA 99, 9037–9042 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Bargmann, C.I. & Kaplan, J.M. Signal transduction in the Caenorhabditis elegans nervous system. Annu. Rev. Neurosci. 21, 279–308 (1998).

    Article  CAS  PubMed  Google Scholar 

  44. Rand, J.B. in WormBook (ed. The C. elegans Research Community) http://dx.doi.org/10.1895/wormbook.1.131.1 (2007).

  45. Mahoney, T.R., Luo, S. & Nonet, M.L. Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay. Nat. Protoc. 1, 1772–1777 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Fleming, J.T. et al. Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits. J. Neurosci. 17, 5843–5857 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lewis, J.A., Wu, C.H., Berg, H. & Levine, J.H. The genetics of levamisole resistance in the nematode Caenorhabditis elegans. Genetics 95, 905–928 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Fliegl, H., Köhn, A., Hättig, C. & Ahlrichs, R. Ab initio calculation of the vibrational and electronic spectra of trans- and cis-azobenzene. J. Am. Chem. Soc. 125, 9821–9827 (2003).

    Article  CAS  PubMed  Google Scholar 

  49. Nolan, C.J., Madiraju, M.S.R., Delghingaro-Augusto, V., Peyot, M.-L. & Prentki, M. Fatty acid signaling in the β-cell and insulin secretion. Diabetes 55 (Suppl. 2), S16–S23 (2006).

    Article  CAS  PubMed  Google Scholar 

  50. Konrad, D.B., Frank, J.A. & Trauner, D. Synthesis of redshifted azobenzene photoswitches by late-stage functionalization. Chemistry 22, 4364–4368 (2016).

    Article  CAS  PubMed  Google Scholar 

  51. Ishihara, H. et al. Pancreatic β−cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets. Diabetologia 36, 1139–1145 (1993).

    Article  CAS  PubMed  Google Scholar 

  52. Ravier, M.A. & Rutter, Guy.A. in Mouse Cell Cult. 633, 171–184 (2010).

    Article  CAS  Google Scholar 

  53. Ravier, M.A. & Rutter, G.A. Glucose or insulin, but not zinc ions, inhibit glucagon secretion from mouse pancreatic α-cells. Diabetes 54, 1789–1797 (2005).

    Article  CAS  PubMed  Google Scholar 

  54. Dadi, P.K. et al. Inhibition of pancreatic β-cell Ca2+/calmodulin-dependent protein kinase II reduces glucose-stimulated calcium influx and insulin secretion, impairing glucose tolerance. J. Biol. Chem. 289, 12435–12445 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Burgalossi, A. et al. Analysis of neurotransmitter release mechanisms by photolysis of caged Ca2+ in an autaptic neuron culture system. Nat. Protoc. 7, 1351–1365 (2012).

    Article  CAS  PubMed  Google Scholar 

  56. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank M. Sumser, J. Broichhagen and T. Fehrentz for insightful discussions leading to the preparation of the manuscript, and M. Duca, S. Wouters, R. Mitchell and N. Johnston for experimental assistance. We thank F. Stein for providing macros for image analysis. We are grateful for the technical support of EMBL's Advanced Light Microscopy Facility. D.T. and J.A.F. are supported by the Deutsche Forschungsgemeinschaft (DFG) (SFB 1032 project B09, TRR 152) and the European Research Council (ERC Advanced Grant 268795 to D.T.). C.S. is supported by the DFG (TRR 83 and TRR 186). D.A.Y. was funded by the EIPOD Programme at EMBL, EU grant 229597 and an IOCB installation grant. D.J.H. was supported by Diabetes UK RD Lawrence (12/0004431), EFSD/Novo Nordisk Rising Star and Birmingham fellowships. D.J.H. and G.A.R. were supported by MRC project (MR/N00275X/1) and Imperial Confidence in Concept (ICiC) grants. G.A.R. was supported by Wellcome Trust Senior Investigator (WT098424AIA) and Royal Society Wolfson Research Merit awards and grants from the UK Medical Research Council (MR/J0003042/1, MR/L020149/1 and MR/L02036X/1), the Biological and Biotechnology Research Council (BB/J015873/1) and Diabetes UK (11/0004210; 15/0005275). J.N. was supported by DFG grants FOR1279 GO1011/4-1 and GO1011/4-2 and Cluster of Excellence Frankfurt (EXC115) (all grants to A.G.).

Author information

Authors and Affiliations

  1. Department of Chemistry and Center for Integrated Protein Science, Ludwig Maximilians University Munich, Munich, Germany

    James Allen Frank & Dirk Trauner

  2. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    Dmytro A Yushchenko & Carsten Schultz

  3. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic

    Dmytro A Yushchenko

  4. Department of Medicine, Section of Cell Biology and Functional Genomics, Imperial College London, ICTEM, Hammersmith Hospital, London, UK

    David J Hodson & Guy A Rutter

  5. Institute of Metabolism and Systems Research (IMSR) and Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK

    David J Hodson

  6. Centre for Endocrinology, Diabetes and Metabolism (CEDAM), Birmingham Health Partners, Birmingham, UK

    David J Hodson

  7. Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany

    Noa Lipstein, Jeong-Seop Rhee & Nils Brose

  8. Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Germany

    Jatin Nagpal & Alexander Gottschalk

  9. Department for Biochemistry, Institute of Biochemistry, Chemistry and Pharmacy, Goethe University, Frankfurt, Germany

    Jatin Nagpal & Alexander Gottschalk

Authors
  1. James Allen Frank
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dmytro A Yushchenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David J Hodson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Noa Lipstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jatin Nagpal
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Guy A Rutter
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jeong-Seop Rhee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Alexander Gottschalk
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nils Brose
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Carsten Schultz
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Dirk Trauner
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.T. and C.S. coordinated and supervised the study. J.A.F. designed and synthesized the compounds. J.A.F., D.A.Y. and D.J.H. carried out imaging and secretion experiments. J.A.F. and N.L. performed electrophysiological experiments. J.N. performed experiments in C. elegans under the supervision of A.G. J.-S.R. and N.B. coordinated and supervised electrophysiological recordings in hippocampal neurons. G.A.R. provided reagents and assisted with data analysis. All authors contributed to writing the manuscript.

Corresponding authors

Correspondence to Carsten Schultz or Dirk Trauner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–14. (PDF 2306 kb)

Supplementary Note

Supplementary Notes 1–4 (PDF 3060 kb)

PhoDAG-1 enables optical control of PKCδ-RFP translocation. (AVI 5285 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Frank, J., Yushchenko, D., Hodson, D. et al. Photoswitchable diacylglycerols enable optical control of protein kinase C. Nat Chem Biol 12, 755–762 (2016). https://doi.org/10.1038/nchembio.2141

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2141

This article is cited by

Search

Advanced search

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