Phosphorylation of threonine 3: implications for Huntingtin aggregation and neurotoxicity

Abstract

Huntingtin (Htt) is a widely expressed protein that causes tissue-specific degeneration when mutated to contain an expanded polyglutamine (poly(Q)) domain. Although Htt is large, 350 kDa, the appearance of amino-terminal fragments of Htt in extracts of postmortem brain tissue from patients with Huntington disease (HD), and the fact that an amino-terminal fragment, Htt exon 1 protein (Httex1p), is sufficient to cause disease in models of HD, points to the importance of the amino-terminal region of Htt in the disease process. The first exon of Htt encodes 17 amino acids followed by a poly(Q) repeat of variable length and culminating with a proline-rich domain of 50 amino acids. Because modifications to this fragment have the potential to directly affect pathogenesis in several ways, we have surveyed this fragment for potential post-translational modifications that might affect Htt behavior and detected several modifications of Httex1p. Here we report that the most prevalent modifications of Httex1p are NH(2)-terminal acetylation and phosphorylation of threonine 3 (pThr-3). We demonstrate that pThr-3 occurs on full-length Htt in vivo, and that this modification affects the aggregation and pathogenic properties of Htt. Thus, therapeutic strategies that modulate these events could in turn affect Htt pathogenesis.

FIGURE 1.

Httex1p is post-translationally modified. The following ESI-MS/MS spectra were obtained after chymotryptic digestion and collision induced dissociation (CID) of nickel-purified Httex1p HBH: A, NH₂-terminally acetylated Htt peptide Ac-ATLEKLMKAFESLKSF, [MH₃]³⁺ at m/z 629.09 (MH⁺ = 1885.27 Da); B, NH₂-terminally acetylated and mono-phosphorylated peptide Ac-ApT3LEKLMKAF, [MH₂]²⁺ at m/z 637.32 (MH⁺ = 1273.64 Da). b/y_n* denotes the b/y_n ion with the neutral loss of H₃PO₄.

FIGURE 2.

Thr-3 is phosphorylated on endogenous Htt in vivo. A, specificity of rabbit polyclonal antibody against phospho-T3 Htt (pT3) as determined by dot blot assay. Increasing amounts of phospho-T3 peptide and unmodified peptide were spotted onto a nitrocellulose membrane and probed with the pThr-3 antibody. Corresponding extracts from the striata (B) and cortices (C) of 3 wild-type C57BL/6 mice (1–3) and 5 knock-in CAG140 mice (1–5) were analyzed by immunoblot using the pThr-3 antibody (upper panel), the Htt carboxyl-terminal HF-1 antibody (1981–2580 amino acids) (middle panel), and actin antibody as a loading control (bottom panel).

FIGURE 3.

Htt Thr-3 phosphorylation is affected by polyglutamine length and cell type. Unexpanded and expanded Htt were nickel-purified from ST14A (left two lanes of both panels) or HeLa (right two lanes of both panels) cells and analyzed for relative pThr-3 content by immunoblot (A). Thr-3 phosphorylation was detected using anti-pThr-3 (left panels) and total Htt, including modified and unmodified Htt, was determined using anti-His (right panels). pThr-3 and His signals were detected and quantified using LI-COR. pThr-3:total Htt ratios were calculated for three independent experiments in B. Whole cell extracts from ST14A cells expressing Httex1p with 25Q, 46Q, or 97Q were left untreated (−) or were treated with 100% formic acid (+) to solubilize aggregated material, and then analyzed by immunoblot using pThr-3 (top panel) and His (bottom panel) antibodies (C). pThr-3:total Htt ratios were calculated for 25Q (top bar graph), 46Q (middle bar graph), and 97Q (bottom bar graph), from three independent experiments, to determine the relative amount of pThr-3 in soluble versus total (soluble plus insoluble) Httex1p (D). wt, wild-type; FA, formic acid. Error bars are the S.D. from three independent experiments. *, p ≤ 0.05; **, p ≤ 0.01.

FIGURE 4.

Thr-3 mutant aggregation in striatal progenitor cells. ST14A cells expressing 46Q, 46QT3A, or 46QT3D were immunostained with anti-Htt (S830; green) and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) (A). Images were taken using a ×20 confocal objective (A; top row). The bottom row depicts enlarged regions (×2) of the boxed regions in the top row. The number of transfected cells containing at least 1 aggregate were counted and normalized to total number of transfected cells (B). The scale bar equals 100 μm in A. *, p ≤ 0.05; ***, p ≤ 0.001. Error bars are the S.D. from four independent coverslips.

FIGURE 5.

The effect of Thr-3 mutation on Httex1p aggregation in vivo. Flies expressing 97Q, 97QT3A, or 97QT3D were assayed for soluble Htt (anti-Htt; S830; top panel) and actin (bottom panel) levels using quantitative immunoblot (A). The Htt signal was quantified and normalized to the actin signal (LI-COR) (B). Insoluble material from Htt-expressing flies was measured by filter retardation assay (C). Equal amounts of fly extract were spotted onto a nitrocellulose membrane, and stained with Ponceau for total protein (top panel) and then probed with anti-Htt (S830) to detect the amount of insoluble Htt in 97Q versus 97QT3A versus 97QT3D (bottom panel). The Htt signal was measured by LI-COR and quantified in D. Eye imaginal discs from 3rd instar larvae were stained for Elav (red, first column) and Htt (S830; green, middle column) (E). Boxes drawn in the merged (last) column, in E, identify the area of the Htt signal that was converted to grayscale (F) and analyzed for total aggregate load using Scion Imaging software as described under “Experimental Procedures” (G). The scale bars equal 50 μm in E and F. wt, wild-type. Error bars are the S.D. from four or more imaginal discs for each genotype (E and F) and three independent dot blots (C and D). *, p ≤ 0.05; **, p ≤ 0.01.

FIGURE 6.

The effect of Thr-3 mutation on Httex1p-induced lethality and neurodegeneration. The numbers of male flies expressing no transgene (wild-type, wt; see “Experimental Procedures”), 97Q, 97QT3A, or 97T3D were taken as a percentage of non-expressing siblings carrying a balancer (A). The average rhabdomeres (photoreceptor neurons) per ommatidium (individual eye of a compound eye) were calculated for female flies expressing no transgene (wt), 97Q, 97QT3A, and 97QT3D at 4 days post-eclosion (B). The neurodegeneration was assessed at 11 days post-eclosion for 97QT3A and 97QT3D; there were no 97Q survivors at this time point (C). Error bars are the S.D. from four independent vials (A) or six or more flies per genotype (B and C). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.