Identification and characterization of wolframin, the product of the Wolfram syndrome gene (WFS1), as a novel calmodulin-binding protein

Saki Yurimoto, Naoya Hatano, Mitsumasa Tsuchiya, Kiyohito Kato, Tomohito Fujimoto, Tsutomu Masaki, Ryoji Kobayashi, Hiroshi Tokumitsu

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

To search for calmodulin (CaM) targets, we performed affinity chromatography purification of a rat brain extract using CaM fused with GST as the affinity ligand. Proteomic analysis was then carried out to identify CaM-binding proteins. In addition to identifying 36 known CaM-binding proteins, including CaM kinases, calcineurin, nNOS, the IP3 receptor, and Ca 2+-ATPase, we identified an ER transmembrane protein, wolframin [the product of the Wolfram syndrome gene (WFS1)] as interacting. A CaM overlay and an immunoprecipitation assay revealed that wolframin is capable of binding the Ca2+/CaM complex in vitro and in transfected cells. Surface plasmon resonance analysis and zero-length cross-linking showed that the N-terminal cytoplasmic domain (residues 2-285) of wolframin binds to an equimolar unit of CaM in a Ca2+-dependent manner with a KD for CaM of 0.15 μM. Various truncation and deletion mutants showed that the Ca 2+/CaM binding region in wolframin is located from Glu90 to Trp186. Furthermore, we demonstrated that three mutations (Ala127Thr, Ala134Thr, and Arg178Pro) associated with Wolfram syndrome completely abolished CaM binding of wolframin. This observation may indicate that CaM binding is important for wolframin function and that impairment of this interaction by mutation contributes to the pathology seen in Wolfram syndrome.

Original languageEnglish
Pages (from-to)3946-3955
Number of pages10
JournalBiochemistry
Volume48
Issue number18
DOIs
Publication statusPublished - May 12 2009
Externally publishedYes

Fingerprint

Wolfram Syndrome
Calmodulin-Binding Proteins
Tungsten
Calmodulin
Genes
Affinity chromatography
Inositol 1,4,5-Trisphosphate Receptors
Calcium-Calmodulin-Dependent Protein Kinases
Mutation
Surface Plasmon Resonance
Calcineurin
Surface plasmon resonance
Pathology
Affinity Chromatography
Immunoprecipitation
Proteomics
Purification
Adenosine Triphosphatases
Rats
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification and characterization of wolframin, the product of the Wolfram syndrome gene (WFS1), as a novel calmodulin-binding protein. / Yurimoto, Saki; Hatano, Naoya; Tsuchiya, Mitsumasa; Kato, Kiyohito; Fujimoto, Tomohito; Masaki, Tsutomu; Kobayashi, Ryoji; Tokumitsu, Hiroshi.

In: Biochemistry, Vol. 48, No. 18, 12.05.2009, p. 3946-3955.

Research output: Contribution to journalArticle

Yurimoto, Saki ; Hatano, Naoya ; Tsuchiya, Mitsumasa ; Kato, Kiyohito ; Fujimoto, Tomohito ; Masaki, Tsutomu ; Kobayashi, Ryoji ; Tokumitsu, Hiroshi. / Identification and characterization of wolframin, the product of the Wolfram syndrome gene (WFS1), as a novel calmodulin-binding protein. In: Biochemistry. 2009 ; Vol. 48, No. 18. pp. 3946-3955.
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