Kinase inhibitors modulate huntingtin cell localization and toxicity

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    The Huntington's Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell 72, 971–983 (1993).

    Article  Google Scholar 

  2. 2

    Atwal, R.S. et al. Huntingtin has a membrane association signal that can modulate huntingtin aggregation, nuclear entry and toxicity. Hum. Mol. Genet. 16, 2600–2615 (2007).

    CAS  Article  Google Scholar 

  3. 3

    Kim, M.W., Chelliah, Y., Kim, S.W., Otwinowski, Z. & Bezprozvanny, I. Secondary structure of Huntingtin amino-terminal region. Structure 17, 1205–1212 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Tam, S. et al. The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation. Nat. Struct. Mol. Biol. 16, 1279–1285 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Darnell, G., Orgel, J.P., Pahl, R. & Meredith, S.C. Flanking polyproline sequences inhibit beta-sheet structure in polyglutamine segments by inducing PPII-like helix structure. J. Mol. Biol. 374, 688–704 (2007).

    CAS  Article  Google Scholar 

  6. 6

    Mangiarini, L. et al. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87, 493–506 (1996).

    CAS  Article  Google Scholar 

  7. 7

    Martín-Aparicio, E., Avila, J. & Lucas, J.J. Nuclear localization of N-terminal mutant huntingtin is cell cycle dependent. Eur. J. Neurosci. 16, 355–359 (2002).

    Article  Google Scholar 

  8. 8

    Benn, C.L. et al. Huntingtin modulates transcription, occupies gene promoters in vivo, and binds directly to DNA in a polyglutamine-dependent manner. J. Neurosci. 28, 10720–10733 (2008).

    CAS  Article  Google Scholar 

  9. 9

    Seong, I.S. et al. Huntingtin facilitates polycomb repressive complex 2. Hum. Mol. Genet. 19, 573–583 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Godin, J.D. et al. Huntingtin is required for mitotic spindle orientation and mammalian neurogenesis. Neuron 67, 392–406 (2010).

    CAS  Article  Google Scholar 

  11. 11

    Steffan, J.S. et al. SUMO modification of Huntingtin and Huntington's disease pathology. Science 304, 100–104 (2004).

    CAS  Article  Google Scholar 

  12. 12

    Aiken, C.T. et al. Phosphorylation of threonine 3: implications for Huntingtin aggregation and neurotoxicity. J. Biol. Chem. 284, 29427–29436 (2009).

    CAS  Article  Google Scholar 

  13. 13

    Thompson, L.M. et al. IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome. J. Cell Biol. 187, 1083–1099 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Lecerf, J.M. et al. Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease. Proc. Natl. Acad. Sci. USA 98, 4764–4769 (2001).

    CAS  Article  Google Scholar 

  15. 15

    Snyder-Keller, A., McLear, J.A., Hathorn, T. & Messer, A. Early or late-stage anti-N-terminal Huntingtin intrabody gene therapy reduces pathological features in B6.HDR6/1 mice. J. Neuropathol. Exp. Neurol. 69, 1078–1085 (2010).

    CAS  Article  Google Scholar 

  16. 16

    Williamson, T.E., Vitalis, A., Crick, S.L. & Pappu, R.V. Modulation of polyglutamine conformations and dimer formation by the N-terminus of huntingtin. J. Mol. Biol. 396, 1295–1309 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Colby, D.W., Cassady, J.P., Lin, G.C., Ingram, V.M. & Wittrup, K.D. Stochastic kinetics of intracellular huntingtin aggregate formation. Nat. Chem. Biol. 2, 319–323 (2006).

    CAS  Article  Google Scholar 

  18. 18

    Thakur, A.K. et al. Polyglutamine disruption of the huntingtin exon 1 N terminus triggers a complex aggregation mechanism. Nat. Struct. Mol. Biol. 16, 380–389 (2009).

    CAS  Article  Google Scholar 

  19. 19

    Wetzel, M.K. et al. p73 regulates neurodegeneration and phospho-tau accumulation during aging and Alzheimer's disease. Neuron 59, 708–721 (2008).

    CAS  Article  Google Scholar 

  20. 20

    Gu, X. et al. Serines 13 and 16 are critical determinants of full-length human mutant huntingtin induced disease pathogenesis in HD mice. Neuron 64, 828–840 (2009).

    CAS  Article  Google Scholar 

  21. 21

    Greenfield, N.J. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 1, 2876–2890 (2006).

    CAS  Article  Google Scholar 

  22. 22

    Schilling, G. et al. Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum. Mol. Genet. 8, 397–407 (1999).

    CAS  Article  Google Scholar 

  23. 23

    Zhang, H. et al. Elucidating a normal function of huntingtin by functional and microarray analysis of huntingtin-null mouse embryonic fibroblasts. BMC Neurosci. 9, 38 (2008).

    Article  Google Scholar 

  24. 24

    Bordeaux, J. et al. Antibody validation. Biotechniques 48, 197–209 (2010).

    CAS  Article  Google Scholar 

  25. 25

    Hoffner, G., Kahlem, P. & Djian, P. Perinuclear localization of huntingtin as a consequence of its binding to microtubules through an interaction with beta-tubulin: relevance to Huntington's disease. J. Cell Sci. 115, 941–948 (2002).

    CAS  PubMed  Google Scholar 

  26. 26

    Pagano, M.A. et al. 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole: a novel powerful and selective inhibitor of protein kinase CK2. Biochem. Biophys. Res. Commun. 321, 1040–1044 (2004).

    CAS  Article  Google Scholar 

  27. 27

    Cozza, G. et al. Quinalizarin as a potent, selective and cell-permeable inhibitor of protein kinase CK2. Biochem. J. 421, 387–395 (2009).

    CAS  Article  Google Scholar 

  28. 28

    Burke, J.R. et al. BMS-345541 is a highly selective inhibitor of IκB kinase that binds at an allosteric site of the enzyme and blocks NF-κB-dependent transcription in mice. J. Biol. Chem. 278, 1450–1456 (2003).

    CAS  Article  Google Scholar 

  29. 29

    Arrasate, M., Mitra, S., Schweitzer, E.S., Segal, M.R. & Finkbeiner, S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431, 805–810 (2004).

    CAS  Article  Google Scholar 

  30. 30

    Truant, R., Atwal, R.S., Desmond, C., Munsie, L. & Tran, T. Huntington's disease: revisiting the aggregation hypothesis in polyglutamine neurodegenerative diseases. FEBS J. 275, 4252–4262 (2008).

    CAS  Article  Google Scholar 

  31. 31

    Wetzel, R. Nucleation of huntingtin aggregation in cells. Nat. Chem. Biol. 2, 297–298 (2006).

    CAS  Article  Google Scholar 

  32. 32

    Reina, C.P., Zhong, X. & Pittman, R.N. Proteotoxic stress increases nuclear localization of ataxin-3. Hum. Mol. Genet. 19, 235–249 (2009).

    Article  Google Scholar 

  33. 33

    Mueller, T. et al. CK2-dependent phosphorylation determines cellular localization and stability of ataxin-3. Hum. Mol. Genet. 18, 3334–3343 (2009).

    CAS  Article  Google Scholar 

  34. 34

    Fan, M.M., Zhang, H., Hayden, M.R., Pelech, S.L. & Raymond, L.A. Protective up-regulation of CK2 by mutant huntingtin in cells co-expressing NMDA receptors. J. Neurochem. 104, 790–805 (2008).

    CAS  PubMed  Google Scholar 

  35. 35

    Modregger, J., DiProspero, N.A., Charles, V., Tagle, D.A. & Plomann, M. PACSIN 1 interacts with huntingtin and is absent from synaptic varicosities in presymptomatic Huntington's disease brains. Hum. Mol. Genet. 11, 2547–2558 (2002).

    CAS  Article  Google Scholar 

  36. 36

    Plomann, M. et al. PACSIN, a brain protein that is upregulated upon differentiation into neuronal cells. Eur. J. Biochem. 256, 201–211 (1998).

    CAS  Article  Google Scholar 

  37. 37

    Di Maira, G., Brustolon, F., Pinna, L.A. & Ruzzene, M. Dephosphorylation and inactivation of Akt/PKB is counteracted by protein kinase CK2 in HEK 293T cells. Cell. Mol. Life Sci. 66, 3363–3373 (2009).

    Article  Google Scholar 

  38. 38

    Humbert, S. et al. The IGF-1/Akt pathway is neuroprotective in Huntington's disease and involves Huntingtin phosphorylation by Akt. Dev. Cell 2, 831–837 (2002).

    CAS  Article  Google Scholar 

  39. 39

    Kageura, T. et al. Inhibitors from rhubarb on lipopolysaccharide-induced nitric oxide production in macrophages: structural requirements of stilbenes for the activity. Bioorg. Med. Chem. 9, 1887–1893 (2001).

    CAS  Article  Google Scholar 

  40. 40

    Pierce, J.W. et al. Novel inhibitors of cytokine-induced IκBα phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J. Biol. Chem. 272, 21096–21103 (1997).

    CAS  Article  Google Scholar 

  41. 41

    Shaul, J.D., Farina, A. & Huxford, T. The human IKKβ subunit kinase domain displays CK2-like phosphorylation specificity. Biochem. Biophys. Res. Commun. 374, 592–597 (2008).

    CAS  Article  Google Scholar 

  42. 42

    McElhinny, J.A., Trushin, S.A., Bren, G.D., Chester, N. & Paya, C.V. Casein kinase II phosphorylates IκBα at S-283, S-289, S-293, and T-291 and is required for its degradation. Mol. Cell. Biol. 16, 899–906 (1996).

    CAS  Article  Google Scholar 

  43. 43

    Witt, J. et al. Mechanism of PP2A-mediated IKKβ dephosphorylation: a systems biological approach. BMC Syst. Biol. 3, 71 (2009).

    Article  Google Scholar 

  44. 44

    Metzler, M. et al. Phosphorylation of huntingtin at Ser421 in YAC128 neurons is associated with protection of YAC128 neurons from NMDA-mediated excitotoxicity and is modulated by PP1 and PP2A. J. Neurosci. 30, 14318–14329 (2010).

    CAS  Article  Google Scholar 

  45. 45

    Pardo, R. et al. Inhibition of calcineurin by FK506 protects against polyglutamine-huntingtin toxicity through an increase of huntingtin phosphorylation at S421. J. Neurosci. 26, 1635–1645 (2006).

    CAS  Article  Google Scholar 

  46. 46

    Trushin, S.A., Pennington, K.N., Algeciras-Schimnich, A. & Paya, C.V. Protein kinase C and calcineurin synergize to activate IκB kinase and NF-κB in T lymphocytes. J. Biol. Chem. 274, 22923–22931 (1999).

    CAS  Article  Google Scholar 

  47. 47

    Bamborough, P. et al. Progress towards the development of anti-inflammatory inhibitors of IKKβ. Curr. Top. Med. Chem. 9, 623–639 (2009).

    CAS  Article  Google Scholar 

  48. 48

    Trettel, F. et al. Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells. Hum. Mol. Genet. 9, 2799–2809 (2000).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

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.

Corresponding author

Correspondence to Ray Truant.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

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