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.

Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin

“…PACSIN1 is a predominantly cytoplasmic neuronal protein that has functions in NMDA receptor recycling (18,19), actin/microtubule reorganization (20), and neuronal spine formation (21). Both N17 and PACSIN1 are substrates of casein kinase 2 (CK2) (12,17).Because both of the flanking regions to the polyglutamine tract are critical in mediating the toxicity of the mutant huntingtin protein in mammalian and yeast models (22), we wanted to determine whether the two domains could be interacting with each other in 3D space using the polyglutamine tract as a flexible region. To test this hypothesis, we developed a Förster resonance energy transfer (FRET) sensor with donor and acceptor fluorophores at the amino and carboxyl-termini of huntingtin fragments, respectively.…”

“…PACSIN1 is a predominantly cytoplasmic neuronal protein that has functions in NMDA receptor recycling (18,19), actin/microtubule reorganization (20), and neuronal spine formation (21). Both N17 and PACSIN1 are substrates of casein kinase 2 (CK2) (12,17).…”

“…The N17 domain can be modified posttranslationally at multiple residues (11)(12)(13)(14). Within N17, phosphorylation of two serines at positions 13 and 16 is critical for modulating huntingtin localization during stress (10, 12), N17 structure (12), and the toxicity of mutant huntingtin in cell biological and mouse models of HD (12)(13)(14)(15).…”

“…Within N17, phosphorylation of two serines at positions 13 and 16 is critical for modulating huntingtin localization during stress (10, 12), N17 structure (12), and the toxicity of mutant huntingtin in cell biological and mouse models of HD (12)(13)(14)(15). We have previously demonstrated that mutant huntingtin is hypophosphorylated at serines 13 and 16; this results in an inability to respond to cell stress (12).…”

“…This could be prevented by treatment of cells with CK2 inhibitors (12). Conversely, Iκβ kinase (IKK) inhibitors paradoxically increased phosphorylation of N17 (12).…”