Characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in Neurospora.
In continuation of our recent observations indicating the presence of a lone calcineurin-dependent response element (CDRE) in the -3730bp upstream region of copper-induced metallothionein (CuMT) gene of Neurospora [K.S. Kumar, S. Dayananda, C. Subramanyam, Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa, FEMS Microbiol. Lett. 242 (2005) 45-50], we isolated and characterized the CDRE-binding protein. The cloned upstream region of CuMT gene was used as the template to specifically amplify CDRE element, which was immobilized on CNBr-activated Sepharose 4B for use as the affinity matrix to purify the CDRE binding protein from nuclear extracts obtained from Neurospora cultures grown in presence of copper. Two-dimensional gel electrophoresis of the affinity purified protein revealed the presence of a single 17kDa protein, which was identified and characterized by MALDI-TOF. Peptide mass finger printing of tryptic digests and analysis of the 17kDa protein matched with the regulatory beta-subunit of calcineurin (Ca(2+)-calmodulin dependent protein phosphatase). Parallel identification of nuclear localization signals in this protein by in silico analysis suggests a putative role for calcineurin in the regulation of CuMT gene expression.
Kumar
Kalari Satish
KS
Department of Biochemistry, Osmania University, Hyderabad 500007, India.
Ravi Kumar
B
B
Siddavattam
Dayananda
D
Subramanyam
Chivukula
C
D000595:Amino Acid Sequence; D019703:Calcineurin / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000737:chemistry / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D008668:Metallothionein / Q000378:metabolism*; D008969:Molecular Sequence Data; D009491:Neurospora / Q000378:metabolism*; D019913:Nuclear Localization Signals; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D020218:Response Elements; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005656:Fungal Proteins; D019913:Nuclear Localization Signals; D010455:Peptides; C007121:copper thionein; D003300:Copper; D008668:Metallothionein; D019703:Calcineurin