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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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.
The authors declare no competing financial interests.
Supplementary Results and Supplementary Methods (PDF 3622 kb)
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)
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)
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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