Letter | Published:

GAPDH mediates nitrosylation of nuclear proteins

Nature Cell Biology volume 12, pages 10941100 (2010) | Download Citation

Abstract

S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells1. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly4 or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON6. As the three principal isoforms of NOS—neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) —are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO–GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus7. Here, we show that SNO–GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein–protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.

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.

    , , , & Protein S-nitrosylation: purview and parameters. Nature Rev. Mol. Cell Biol. 6, 150–166 (2005).

  2. 2.

    , , , & S-nitrosylation of histone deacetylase 2 induces chromatin remodelling in neurons. Nature 455, 411–415 (2008).

  3. 3.

    , , & Nitric oxide-dependent negative feedback of PARP-1 trans-activation of the inducible nitric-oxide synthase gene. J. Biol. Chem. 281, 9101–9109 (2006).

  4. 4.

    , & Inducible nitric oxide synthase binds, S-nitrosylates and activates cyclooxygenase-2. Science 310, 1966–1970 (2005).

  5. 5.

    et al. Cysteine regulation of protein function—as exemplified by NMDA-receptor modulation. Trends Neurosci. 25, 474–480 (2002).

  6. 6.

    et al. Dexras1: a G protein specifically coupled to neuronal nitric oxide synthase via CAPON. Neuron 28, 183–193 (2000).

  7. 7.

    et al. S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat. Cell Biol. 7, 665–674 (2005).

  8. 8.

    et al. Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis. Nat. Cell Biol. 10, 866–873 (2008).

  9. 9.

    et al. A protein interaction map of Drosophila melanogaster. Science 302, 1727–1736 (2003).

  10. 10.

    & Mammalian sirtuins—emerging roles in physiology, aging and calorie restriction. Genes Dev. 20, 2913–2921 (2006).

  11. 11.

    , , , & Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat. Cell Biol. 3, 193–197 (2001).

  12. 12.

    , , , & iNOS inactivates Sirt1, a key regulator of stress resistance and metabolism, by S-Nitrosylation. 2006 Annual Meeting American Society of Anesthesiologists conference abstract, A1067 (2006).

  13. 13.

    , , , & Inducible nitric oxide synthase-mediated p53 activation and apoptosis in muscle after burn injury. 2007 Annual Meeting American Society of Anesthesiologists conference abstract, A1856 (2007).

  14. 14.

    et al. Dual role of Zn2+ in maintaining structural integrity and suppressing deacetylase activity of SIRT1. J. Inorg. Biochem. 104, 180–185 (2010).

  15. 15.

    et al. Nutrient control of glucose homeostasis through a complex of PGC-1α and SIRT1. Nature 434, 113–118 (2005).

  16. 16.

    et al. GCN5 acetyltransferase complex controls glucose metabolism through transcriptional repression of PGC-1α. Cell Metab. 3, 429–438 (2006).

  17. 17.

    et al. Regulation of hepatic fasting response by PPARγ coactivator-1α (PGC-1): requirement for hepatocyte nuclear factor 4α in gluconeogenesis. Proc. Natl Acad. Sci. USA 100, 4012–4017 (2003).

  18. 18.

    et al. Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture. Nat. Biotechnol. 27, 557–559 (2009).

  19. 19.

    , & Export by red blood cells of nitric oxide bioactivity. Nature 409, 622–626 (2001).

  20. 20.

    & Thioredoxin catalyzes the S-nitrosylation of the caspase-3 active site cysteine. Nat. Chem. Biol. 1, 154–158 (2005).

  21. 21.

    , , & Thioredoxin is required for S-nitrosation of procaspase-3 and the inhibition of apoptosis in Jurkat cells. Proc. Natl Acad. Sci. USA 104, 11609–11614 (2007).

  22. 22.

    , , & Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins. Science 320, 1050–1054 (2008).

  23. 23.

    et al. Transnitrosylation of XIAP regulates caspase-dependent neuronal cell death. Mol. Cell 39, 184–195 (2010).

  24. 24.

    et al. Evidence for a functional nitric oxide synthase system in brown adipocyte nucleus. FEBS Lett. 514, 135–140 (2002).

  25. 25.

    et al. Nitric oxide signaling via nuclearized endothelial nitric-oxide synthase modulates expression of the immediate early genes iNOS and mPGES-1. J. Biol. Chem. 281, 16058–16067 (2006).

  26. 26.

    et al. Estrogen receptor-α and endothelial nitric oxide synthase nuclear complex regulates transcription of human telomerase. Circ. Res. 103, 34–42 (2008).

  27. 27.

    & Life history of eNOS: partners and pathways. Cardiovasc. Res. 75, 247–260 (2007).

  28. 28.

    , & Intracellular location regulates calcium–calmodulin-dependent activation of organelle-restricted eNOS. Am. J. Physiol. Cell Physiol. 289, C1024–C1033 (2005).

  29. 29.

    , , & Metabolic adaptations through the PGC-1α and SIRT1 pathways. FEBS Lett. 582, 46–53 (2008).

  30. 30.

    et al. Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444, 337–342 (2006).

  31. 31.

    et al. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1α. Cell 127, 1109–1122 (2006).

  32. 32.

    Inflammation and metabolic disorders. Nature 444, 860–867 (2006).

  33. 33.

    & NO at work. Cell 78, 919–925 (1994).

  34. 34.

    & Inhibition of NF-κB by S-nitrosylation. Biochemistry 40, 1688–1693 (2001).

  35. 35.

    & S-nitrosation of Cys 800 of HIF-1α protein activates its interaction with p300 and stimulates its transcriptional activity. FEBS Lett. 549, 105–109 (2003).

  36. 36.

    , & S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component. Cell 114, 255–266 (2003).

  37. 37.

    , , & Proliferative dependent regulation of the glyceraldehyde-3-phosphate dehydrogenase/uracil DNA glycosylase gene in human cells. Carcinogenesis 13, 2127–2132 (1992).

  38. 38.

    & Sequence-specific binding of transfer RNA by glyceraldehydes-3-phosphate dehydrogenase. Science 259, 365–368 (1993).

  39. 39.

    et al. The multifunctional protein glyceraldehyde-3-phosphate dehydrogenase is both regulated and controls colony-stimulating factor-1 messenger RNA stability in ovarian cancer. Mol. Cancer Res. 6, 1375–1384 (2008).

  40. 40.

    , , , & Preparation of enzymatically active recombinant class III protein deacetylases. Methods 36, 338–345 (2005).

  41. 41.

    & The biotin switch method for the detection of S-nitrosylated proteins. Science STKE 86, PL1 (2001).

  42. 42.

    , , & Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex. Proc. Natl Acad. Sci. USA 100, 1609–1614 (2003).

Download references

Acknowledgements

We are grateful to M. Koldobskiy, B. Selvakumar, S.F. Kim, P. Kim, K. Werner and all members of the Snyder laboratory for insight and discussion. We thank P. Puigserver for SIRT1 and PGC1α plasmids. We thank B. Ziegler for organizing the manuscript. This work was supported by USPHS grant DA-000266 and Research Scientist Award DA-00074 to SHS.

Author information

Author notes

    • Makoto R. Hara

    Present address: Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.

Affiliations

  1. Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    • Michael D. Kornberg
    • , Nilkantha Sen
    • , Makoto R. Hara
    • , Krishna R. Juluri
    • , Judy Van K. Nguyen
    • , Adele M. Snowman
    • , Lindsey Law
    • , Lynda D. Hester
    •  & Solomon H. Snyder
  2. Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    • Solomon H. Snyder
  3. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    • Solomon H. Snyder

Authors

  1. Search for Michael D. Kornberg in:

  2. Search for Nilkantha Sen in:

  3. Search for Makoto R. Hara in:

  4. Search for Krishna R. Juluri in:

  5. Search for Judy Van K. Nguyen in:

  6. Search for Adele M. Snowman in:

  7. Search for Lindsey Law in:

  8. Search for Lynda D. Hester in:

  9. Search for Solomon H. Snyder in:

Contributions

M.D.K. designed and performed most of the experiments, analysed the data, prepared the figures, helped write the manuscript and contributed to project design. N.S. performed experiments investigating the effects of GAPDH mutants on SIRT1 nitrosylation in intact cells, performed the GAPDH glycolytic activity assay and the luciferase reporter assay, and analysed the data and prepared the figures from these experiments. M.R.H. identified the physical interaction between GAPDH and SIRT1. K.R.J. identified S-nitrosylation of SIRT1, helped with SIRT1 assay design and prepared constructs. J.V.K.N. performed some in vitro binding and enzyme activity assays. A.M.S. performed site-directed mutagenesis and prepared plasmids. L.L. helped perform some experiments. L.D.H. generated neuronal cultures. S.H.S. designed and supervised the project, wrote the manuscript and provided financial support.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Solomon H. Snyder.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ncb2114

Further reading