Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE

Predominant phosphorylation on threonine 3 and regulation by IKKβ

Maria Blaire Bustamante, Annalisa Ansaloni, Jeppe Falsig Pedersen, Lucia Azzollini, Cristina Cariulo, Zhe Ming Wang, Lara Petricca, Margherita Verani, Francesca Puglisi, Hyunsun Park, Hilal Lashuel, Andrea Caricasole

Research output: Contribution to journalArticle

8 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1317-1322
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume463
Issue number4
DOIs
Publication statusPublished - 5 Jun 2015
Externally publishedYes

Fingerprint

Phosphorylation
Threonine
Polyacrylamide Gel Electrophoresis
Exons
Huntington Disease
I-kappa B Kinase
Trinucleotide Repeats
Gene encoding
Poisons
Pathology
Oligomers
Neurodegenerative Diseases
Toxicity
Anti-Idiotypic Antibodies
Phosphotransferases
Agglomeration
Western Blotting
Amino Acids
Genes
1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol

Keywords

  • Huntingtin
  • Huntington disease
  • IKKβ
  • Phos-Tag
  • Phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE : Predominant phosphorylation on threonine 3 and regulation by IKKβ. / Bustamante, Maria Blaire; Ansaloni, Annalisa; Pedersen, Jeppe Falsig; Azzollini, Lucia; Cariulo, Cristina; Wang, Zhe Ming; Petricca, Lara; Verani, Margherita; Puglisi, Francesca; Park, Hyunsun; Lashuel, Hilal; Caricasole, Andrea.

In: Biochemical and Biophysical Research Communications, Vol. 463, No. 4, 05.06.2015, p. 1317-1322.

Research output: Contribution to journalArticle

Bustamante, MB, Ansaloni, A, Pedersen, JF, Azzollini, L, Cariulo, C, Wang, ZM, Petricca, L, Verani, M, Puglisi, F, Park, H, Lashuel, H & Caricasole, A 2015, 'Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE: Predominant phosphorylation on threonine 3 and regulation by IKKβ', Biochemical and Biophysical Research Communications, vol. 463, no. 4, pp. 1317-1322. https://doi.org/10.1016/j.bbrc.2015.06.116
Bustamante, Maria Blaire ; Ansaloni, Annalisa ; Pedersen, Jeppe Falsig ; Azzollini, Lucia ; Cariulo, Cristina ; Wang, Zhe Ming ; Petricca, Lara ; Verani, Margherita ; Puglisi, Francesca ; Park, Hyunsun ; Lashuel, Hilal ; Caricasole, Andrea. / Detection of huntingtin exon 1 phosphorylation by Phos-Tag SDS-PAGE : Predominant phosphorylation on threonine 3 and regulation by IKKβ. In: Biochemical and Biophysical Research Communications. 2015 ; Vol. 463, No. 4. pp. 1317-1322.
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