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Kinase inhibitors modulate huntingtin cell localization and toxicity

Nature Chemical Biology volume 7, pages 453–460 (2011)Cite this article

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Abstract

Two serine residues within the first 17 amino acid residues of huntingtin (N17) are crucial for modulation of mutant huntingtin toxicity in cell and mouse genetic models of Huntington's disease. Here we show that the stress-dependent phosphorylation of huntingtin at Ser13 and Ser16 affects N17 conformation and targets full-length huntingtin to chromatin-dependent subregions of the nucleus, the mitotic spindle and cleavage furrow during cell division. Polyglutamine-expanded mutant huntingtin is hypophosphorylated in N17 in both homozygous and heterozygous cell contexts. By high-content screening in live cells, we identified kinase inhibitors that modulated N17 phosphorylation and hence huntingtin subcellular localization. N17 phosphorylation was reduced by casein kinase-2 inhibitors. Paradoxically, IKKβ kinase inhibition increased N17 phosphorylation, affecting huntingtin nuclear and subnuclear localization. These data indicate that huntingtin phosphorylation at Ser13 and Ser16 can be modulated by small-molecule drugs, which may have therapeutic potential in Huntington's disease.

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Figure 1: Conserved serines in huntingtin N17 can affect cytoplasmic targeting of huntingtin.
Figure 2: Phosphorylation of huntingtin N17 peptides affects α-helical structure.
Figure 3: Serine mutants in huntingtin N17 affect nuclear localization of huntingtin fragments.
Figure 4: The antibody to phospho-N17 huntingtin highlights nuclear puncta.
Figure 5: Anti–phospho-N17 highlights huntingtin at mitotic spindles, chromatin and the cleavage furrow during cell division.
Figure 6: High-content screening with a kinase inhibitor library reveals small molecules that increase or inhibit N17 phosphorylation.
Figure 7: DMAT reduces huntingtin nuclear puncta and increases toxicity.

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Acknowledgements

This work was supported by operating grants from the Canadian Institutes of Health Research (MOP-165174), the Krembil Foundation and the CHDI Foundation (to R.T.). We thank M. Prakesch (Ontario Institute for Cancer Research) for supplying multiple CK2 and IKKβ inhibitors, I. Bezprozvanny (University of Texas Southwestern) and G. Hajnocsky (Thomas Jefferson University) for the gift of huntingtin-null MEF cell lines, M.E. MacDonald (Massachusetts General Hospital) for the gift of mouse striatal STHdh cell lines, and Raquel and Richard Epand for assistance and advice with CD spectroscopy.

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  1. Randy Singh Atwal

    Present address: Present address: Department of Neurology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, USA.,

Authors and Affiliations

  1. Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

    Randy Singh Atwal, Carly R Desmond, Nicholas Caron, Tamara Maiuri, Jianrun Xia & Ray Truant

  2. Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada

    Simonetta Sipione

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Contributions

R.S.A. conceived and performed most experiments. C.R.D., N.C., J.X. and T.M. performed additional experiments. R.T. conceived experiments, prepared final figures and wrote the manuscript with R.S.A., C.R.D., N.C. and T.M. S.S. shared preliminary data to conceive experiments.

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Correspondence to Ray Truant.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results and Supplementary Methods (PDF 3622 kb)

Supplementary Video 1

Huntingtin1-171 Q142-eYFP expression in STHdh cells. 24 hours. This cell population forms inclusions that coalesce into a few large inclusions by a soluble exchange, yet cell death is not evident. (MOV 1003 kb)

Supplementary Video 2

Huntingtin1-171 Q142-eYFP expression in STHdh cells II. 24 hours. This cell population does not form large coalesced inclusions, only small multiple inclusions that rapidly lead to cell death. (MOV 911 kb)

Supplementary Video 3

Huntingtin1-171 (Q142) S13A S16A-eYFP expression in STHdh cells. 24 hours. This cell population rapidly forms multiple inclusions relative to the wild-type N17 constructs, but cell death is delayed. Relative to the wild-type N17 1-171 Q142, this suggests that the two different populations seen with the wild-type protein may correspond to cell signaling in one population versus another. When all huntingtin fragments are phosphomimicked, inclusions are large and often only 1-2 spots, cell death is delayed, suggesting that phospho-N17 inclusions may be innocuous to the cell. (MOV 808 kb)

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Atwal, R., Desmond, C., Caron, N. et al. Kinase inhibitors modulate huntingtin cell localization and toxicity. Nat Chem Biol 7, 453–460 (2011). https://doi.org/10.1038/nchembio.582

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