Article | Published:

Photoswitchable diacylglycerols enable optical control of protein kinase C

Nature Chemical Biology volume 12, pages 755762 (2016) | Download Citation

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.

  • Compound

    (S,E)-2-((4-(4-((4-butylphenyl)diazenyl)phenyl)butanoyl)oxy)-3-hydroxypropyl stearate

  • Compound

    (S,E)-2-((4-(4-((4-butylphenyl)diazenyl)phenyl)butanoyl)oxy)-3-hydroxypropyl octanoate

  • Compound

    (S,E)-3-hydroxy-2-((4-(4-(phenyldiazenyl)phenyl)butanoyl)oxy)propyl octanoate

  • Compound

    (E)-4-(4-(phenyldiazenyl)phenyl)butanoic acid

  • Compound

    (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol

  • Compound

    (S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl stearate

  • Compound

    (R)-2-hydroxy-3-((triethylsilyl)oxy)propyl stearate

  • Compound

    (R,E)-2-((4-(4-((4-butylphenyl)diazenyl)phenyl)butanoyl)oxy)-3-((triethylsilyl)oxy)propyl stearate

  • Compound

    (S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl octanoate

  • Compound

    (R)-2-hydroxy-3-((triethylsilyl)oxy)propyl octanoate

  • Compound

    (R,E)-2-((4-(4-((4-butylphenyl)diazenyl)phenyl)butanoyl)oxy)-3-((triethylsilyl)oxy)propyl octanoate

  • Compound

    (R,E)-2-((4-(4-(phenyldiazenyl)phenyl)butanoyl)oxy)-3-((triethylsilyl)oxy)propyl octanoate

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

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

  2. 2.

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

  3. 3.

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

  4. 4.

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

  5. 5.

    , & Acyl chain dependence of diacylglycerol activation of protein kinase C activity in vitro. Biochem. Biophys. Res. Commun. 225, 469–473 (1996).

  6. 6.

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

  7. 7.

    & C1 domains: structure and ligand-binding properties. Chem. Rev. 114, 12108–12131 (2014).

  8. 8.

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

  9. 9.

    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).

  10. 10.

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

  11. 11.

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

  12. 12.

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

  13. 13.

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

  14. 14.

    , , & The promise of optogenetics in cell biology: interrogating molecular circuits in space and time. Nat. Methods 8, 35–38 (2011).

  15. 15.

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

  16. 16.

    , , & 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).

  17. 17.

    , , & 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).

  18. 18.

    , & P2Y1 receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion. FASEB J. 27, 1610–1620 (2013).

  19. 19.

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

  20. 20.

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

  21. 21.

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

  22. 22.

    & Protein kinase C regulatory domains: the art of decoding many different signals in membranes. Biochim. Biophys. Acta 1761, 633–654 (2006).

  23. 23.

    , & Protein kinase Cδ (PKCδ): activation mechanisms and functions. J. Biochem. 132, 831–839 (2002).

  24. 24.

    , & PKCα: a versatile key for decoding the cellular calcium toolkit. J. Cell Biol. 174, 521–533 (2006).

  25. 25.

    & Pathway illuminated: visualizing protein kinase C signaling. IUBMB Life 55, 653–660 (2003).

  26. 26.

    , , & A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J. Cell Biol. 161, 899–909 (2003).

  27. 27.

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

  28. 28.

    , , & Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 172, 993–999 (1990).

  29. 29.

    , , , & Synchronous oscillations of cytoplasmic Ca2+ and insulin release in glucose-stimulated pancreatic islets. J. Biol. Chem. 269, 8749–8753 (1994).

  30. 30.

    & Divergence and complexities in DAG signaling: looking beyond PKC. Trends Pharmacol. Sci. 24, 602–608 (2003).

  31. 31.

    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).

  32. 32.

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

  33. 33.

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

  34. 34.

    , , , & 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).

  35. 35.

    & Beta cell connectivity in pancreatic islets: a type 2 diabetes target? Cell. Mol. Life Sci. 72, 453–467 (2015).

  36. 36.

    , , & Phorbol esters modulate spontaneous and Ca2+-evoked transmitter release via acting on both Munc13 and protein kinase C. J. Neurosci. 28, 8257–8267 (2008).

  37. 37.

    , & Divergent and convergent signaling by the diacylglycerol second messenger pathway in mammals. Curr. Opin. Neurobiol. 14, 328–340 (2004).

  38. 38.

    , , & Munc13-1 C1 domain activation lowers the energy barrier for synaptic vesicle fusion. J. Neurosci. 27, 1200–1210 (2007).

  39. 39.

    , , , & Interdependence of PKC-dependent and PKC-independent pathways for presynaptic plasticity. Neuron 54, 275–290 (2007).

  40. 40.

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

  41. 41.

    , , & Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins. J. Biol. Chem. 270, 25273–25280 (1995).

  42. 42.

    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).

  43. 43.

    & Signal transduction in the Caenorhabditis elegans nervous system. Annu. Rev. Neurosci. 21, 279–308 (1998).

  44. 44.

    in WormBook (ed. The C. elegans Research Community) (2007).

  45. 45.

    , & Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay. Nat. Protoc. 1, 1772–1777 (2006).

  46. 46.

    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).

  47. 47.

    , , & The genetics of levamisole resistance in the nematode Caenorhabditis elegans. Genetics 95, 905–928 (1980).

  48. 48.

    , , & Ab initio calculation of the vibrational and electronic spectra of trans- and cis-azobenzene. J. Am. Chem. Soc. 125, 9821–9827 (2003).

  49. 49.

    , , , & Fatty acid signaling in the β-cell and insulin secretion. Diabetes 55 (Suppl. 2), S16–S23 (2006).

  50. 50.

    , & Synthesis of redshifted azobenzene photoswitches by late-stage functionalization. Chemistry 22, 4364–4368 (2016).

  51. 51.

    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).

  52. 52.

    & in Mouse Cell Cult. 633, 171–184 (2010).

  53. 53.

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

  54. 54.

    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).

  55. 55.

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

  56. 56.

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

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

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. Section of Cell Biology and Functional Genomics, Department of Medicine, 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. Institute of Biochemistry, Department for Biochemistry, Chemistry and Pharmacy, Goethe University, Frankfurt, Germany.

    • Jatin Nagpal
    •  & Alexander Gottschalk

Authors

  1. Search for James Allen Frank in:

  2. Search for Dmytro A Yushchenko in:

  3. Search for David J Hodson in:

  4. Search for Noa Lipstein in:

  5. Search for Jatin Nagpal in:

  6. Search for Guy A Rutter in:

  7. Search for Jeong-Seop Rhee in:

  8. Search for Alexander Gottschalk in:

  9. Search for Nils Brose in:

  10. Search for Carsten Schultz in:

  11. Search for Dirk Trauner in:

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Carsten Schultz or Dirk Trauner.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–14.

  2. 2.

    Supplementary Note

    Supplementary Notes 1–4

Videos

  1. 1.

    PhoDAG-1 enables optical control of PKCδ-RFP translocation.

About this article

Publication history

Received

Accepted

Published

DOI

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

Further reading