Steffan, L.M. function promotes accumulation of toxic post-translationally altered mutant Htt. Thus, IKK activation may modulate mutant Htt neurotoxicity depending on the cell’s ability to degrade the altered species. == Introduction == Abnormal accumulation of misfolded and aggregated protein in affected neurons is usually a hallmark of many neurodegenerative diseases associated with aging. The major pathways of protein clearance in the cell are performed by the proteasome and the lysosome, which both become compromised with age (Cuervo et al., 2005;Martinez-Vicente and Cuervo, 2007;Chondrogianni and Gonos, 2008;Tonoki et al., 2009). Parallel with reduced turnover, proteins mutated in familial neurodegenerative diseases accumulate and cause dysfunction and death, and accompanying symptoms. Examples include the polyglutamine (polyQ) disease protein Huntingtin (Htt) in Huntington’s disease (HD), tau in frontotemporal dementias (FTD), -synuclein in Parkinson’s disease (PD), ataxin-1 in spinocerebellar ataxia 1 (SCA1), and SOD1 in amyotrophic lateral sclerosis (ALS). Post-translational modification of target proteins can regulate their clearance from cells. Phosphorylation regulates protein degradation, alters subcellular localization, and/or creates phosphodegrons/binding motifs for interactors that regulate secondary modifications such as ubiquitination, SUMOylation, and acetylation. For instance, phosphorylation of HSF1, MEF2, and GATA-1 activates their SUMOylation (Hietakangas et al., 2006), phosphorylation of FRAX1036 p53 and RelA activates their acetylation (D’Orazi et al., 2002;Hofmann et al., 2002;Chen et al., 2005), and phosphorylation of IB and FOXO3a activates their ubiquitination (Karin and Ben-Neriah, 2000;Karin et al., 2002;Hu et al., 2004). In turn, these modifications may ultimately target the protein for degradation (Hernandez-Hernandez et al., 2006;Hietakangas et al., 2006;Hunter, 2007;Wu et al., 2007;Zuccato et al., 2007;Jeong et al., 2009). As protein clearance mechanisms become impaired upon aging, altered proteins normally targeted for degradation by post-translational modification may accumulate and disease-causing proteins take on toxic functions (Orr and Zoghbi, 2007;Shao and Diamond, 2007). HD is usually a member of a family of polyQ repeat expansion diseases characterized by the accumulation and aggregation of mutant Htt protein in diseased neurons (Orr and Zoghbi, 2007). In HD, when the repeat expands above 40, disease will manifest, typically striking in mid-life (Walker, 2007). Above 65 repeats, a juvenile form of the disease occurs. The polyQ growth exists within the context of a large 350-kD protein; however, expressing just the N-terminal fragment of Htt encoded by exon 1 (Httex1p), which contains a highly expanded polyQ repeat, can precipitate an aggressive HD-like disease in transgenic mice and flies (Mangiarini et al., 1996;Steffan et al., 2001). The first 17 amino acids of Htt can mediate aggregation, subcellular localization and membrane association, stability, and cellular toxicity, each of which are implicated in HD pathogenesis (Steffan et al., 2004;Luo et al., 2005;Warby et al., 2005,2009;Anne et al., 2007;Rockabrand et al., 2007;Atwal and Truant, 2008). The potential for Htt post-translational modification to have a disease-modifying role has recently emerged as a consistent theme, with regulatory functions implicated for other sites within the full-length protein as well, including phosphorylation at S421 by Akt and S434, S1181, and S1201 by Cdk5 (Humbert et al., 2002;Luo et al., 2005;Warby et al., 2005;Anne et al., 2007), SUMOylation and ubiquitination at K6, K9, and K15 (Steffan et al., 2004), palmitoylation at C214 (Yanai et al., 2006), and acetylation at K444 (Jeong et al., 2009). The regulatory properties of post-translational modifications extend to other polyQ repeat diseases, most notably phosphorylation of S776 in expanded ataxin-1, FRAX1036 the mutant protein in SCA1 (Orr and Zoghbi, 2007). We evaluated the effect of phosphorylation within the first 17 amino acids of Htt on its subcellular localization, downstream post-translational modifications, and protein clearance. This domain name FRAX1036 contains two serines at positions 13 and 16, which are adjacent to the lysines found to be altered by SUMO and ubiquitin (Steffan et al., 2004). We demonstrate that this IKK complex, previously shown to directly interact with Htt (Khoshnan et al., 2004), phosphorylates Htt S13 and may activate phosphorylation of S16. Phosphorylation of these residues promotes modification of the adjacent lysine residues and activates Htt clearance in a manner requiring both the proteasome and lysosome. We Rabbit Polyclonal to NMS find that growth of the Htt polyQ repeat may reduce the efficiency of this phosphorylation, potentially contributing to the accumulation of mutant Htt. == Results == == The IKK complex directly phosphorylates Htt == The N-terminal 17 amino acids of Htt contain a number of potentially modifiable residues (Fig. 1 A). In addition to the lysines at 6, 9, and 15, which can be SUMO altered.