title: Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE: Predominant phosphorylation on threonine 3 and regulation by IKKβ.
abstract: Expansion of a CAG triplet repeat within the first exon of the HUNTINGTIN gene encoding for a polyglutamine tract is the cause of a progressive neurodegenerative disorder known as Huntington's disease. N-terminal fragments of mutant huntingtin have a strong propensity to form oligomers and aggregates that have been linked to the Huntington's disease pathology by different mechanisms, including gain of toxic functions. The biological and biophysical properties of the polyglutamine expansion within these huntingtin fragments are influenced by neighboring domains, in particular by the first 17 amino acids of huntingtin (N17), which precede the polyglutamine expansion. It has been suggested that N17 phosphorylation modulate mutant huntingtin aggregation and toxicity, but the study of its functional and pathological relevance requires the capacity to detect this modification in biological samples in a simple, robust way, that ideally provides information on the abundance of a phosphorylated species relative to the total pool of the protein of interest. Using a modified SDS-PAGE protocol (Phos-Tag) followed by Western blotting with specific anti-HUNTINGTIN antibodies, we efficiently resolved huntingtin fragments expressed in cellular contexts based on the presence of phosphorylated residues, we defined threonine 3 as the major site of huntingtin N17 phosphorylation and, finally, we identified IKK-beta as a kinase capable of phosphorylating threonine 3 in N-terminal hungtingtin fragments.
authors.affiliation: Department of Neuroscience IRBM Science Park and IRBM Promidis, Via Pontina km 30, 600, 00071 Pomezia, Rome, Italy.
authors.forename: Maria Blaire
authors.initials: MB
authors.lastname: Bustamante
authors.affiliation: Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Station 19, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Ansaloni
authors.affiliation: Department of Neuroscience IRBM Science Park and IRBM Promidis, Via Pontina km 30, 600, 00071 Pomezia, Rome, Italy.
authors.forename: Jeppe Falsig
authors.initials: JF
authors.lastname: Pedersen
authors.affiliation: Department of Neuroscience IRBM Science Park and IRBM Promidis, Via Pontina km 30, 600, 00071 Pomezia, Rome, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Azzollini
authors.affiliation: Department of Neuroscience IRBM Science Park and IRBM Promidis, Via Pontina km 30, 600, 00071 Pomezia, Rome, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Cariulo
authors.affiliation: Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Station 19, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
authors.forename: Zhe-Ming
authors.initials: ZM
authors.lastname: Wang
authors.affiliation: Department of Neuroscience IRBM Science Park and IRBM Promidis, Via Pontina km 30, 600, 00071 Pomezia, Rome, Italy.
authors.forename: Lara
authors.initials: L
authors.lastname: Petricca
authors.affiliation: Department of Neuroscience IRBM Science Park and IRBM Promidis, Via Pontina km 30, 600, 00071 Pomezia, Rome, Italy.
authors.forename: Margherita
authors.initials: M
authors.lastname: Verani
authors.affiliation: Department of Neuroscience IRBM Science Park and IRBM Promidis, Via Pontina km 30, 600, 00071 Pomezia, Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Puglisi
authors.affiliation: CHDI Foundation/CHDI Management Inc., Los Angeles, CA 90045, USA.
authors.forename: Hyunsun
authors.initials: H
authors.lastname: Park
authors.affiliation: Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Station 19, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
authors.forename: Hilal
authors.initials: H
authors.lastname: Lashuel
authors.affiliation: Department of Neuroscience IRBM Science Park and IRBM Promidis, Via Pontina km 30, 600, 00071 Pomezia, Rome, Italy. Electronic address: a.caricasole@irbm.it.
authors.forename: Andrea
authors.initials: A
authors.lastname: Caricasole
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D057809:HEK293 Cells; D006801:Humans; D000071058:Huntingtin Protein; D020672:I-kappa B Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D013912:Threonine / Q000378:metabolism*
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D009419:Nerve Tissue Proteins; D013912:Threonine
keywords: Huntingtin; Huntington disease; IKKβ; Phos-Tag; Phosphorylation