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GAPDH mediates nitrosylation of nuclear proteins

Nature Cell Biology volume 12, pages 1094–1100 (2010)Cite this article

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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.

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Figure 1: SNO–GAPDH interacts with SIRT1 near its nitrosylated Cys 150 residue.
Figure 2: Nuclear SNO–GAPDH mediates nitrosylation of SIRT1 through transnitrosylation.
Figure 3: SNO–GAPDH mediates inhibition of SIRT1 enzymatic activity by nitric oxide.
Figure 4: Identification of HDAC2 and DNA-PK as nuclear targets of SNO–GAPDH mediated transnitrosylation.

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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.

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  1. Makoto R. Hara

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

Authors and Affiliations

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

    Solomon H. Snyder

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

    Solomon H. Snyder

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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.

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Correspondence to Solomon H. Snyder.

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Kornberg, M., Sen, N., Hara, M. et al. GAPDH mediates nitrosylation of nuclear proteins. Nat Cell Biol 12, 1094–1100 (2010). https://doi.org/10.1038/ncb2114

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