Identification and characterization of wolframin, the product of the wolfram syndrome gene (WFS1), as a novel calmodulin-binding protein.
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 IP(3) 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 Ca(2+)/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 Ca(2+)-dependent manner with a K(D) for CaM of 0.15 muM. 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.
Yurimoto
Saki
S
Department of Signal Transduction Sciences, Faculty of Medicine, Kagawa University,Kagawa 761-0793, Japan.
Hatano
Naoya
N
Tsuchiya
Mitsumasa
M
Kato
Kiyohito
K
Fujimoto
Tomohito
T
Masaki
Tsutomu
T
Kobayashi
Ryoji
R
Tokumitsu
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics; D051381:Rats; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D008565:Membrane Proteins; C541096:WFS1 protein, rat; D052248:Nitric Oxide Synthase Type I; D000252:Calcium-Transporting ATPases