Apparent presence of Ser133-phosphorylated cyclic AMP response element binding protein (pCREB) in brain mitochondria is due to cross-reactivity of pCREB antibodies with pyruvate dehydrogenase

Authors

  • Jan Pláteník,

    1. Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan
    2. Nencki Institute, Warsaw, Poland
    3. Institute of Medical Biochemistry, First Faculty of Medicine, Charles University, Prague, Czech Republic
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  • Vladimír J. Balcar,

    1. Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan
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    • 1

      The present address of Vladimír J. Balcar is Department of Anatomy and Histology, Institute for Biomedical Studies, Faculty of Medicine, University of Sydney, Sydney, NSW 2006, Australia.

  • Yukio Yoneda,

    1. Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan
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  • Barbara Mioduszewska,

    1. Nencki Institute, Warsaw, Poland
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  • Richard Buchal,

    1. Institute of Medical Biochemistry, First Faculty of Medicine, Charles University, Prague, Czech Republic
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  • Radovan Hynek,

    1. Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Czech Republic
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    • 2

      The present address of Radovan Hynek is Group of Biochemistry and Nutrition, BioCentrum-DTU, Technical University of Denmark, Lyngby, DK-2800, Denmark.

  • Lukasz Kilianek,

    1. Nencki Institute, Warsaw, Poland
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  • Nobuyuki Kuramoto,

    1. Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan
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    • 3

      The present address of Nobuyuki Kuramoto is Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104, USA.

  • Grzegorz Wilczynski,

    1. Nencki Institute, Warsaw, Poland
    2. Warsaw Medical School, Warsaw, Poland
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  • Kiyokazu Ogita,

    1. Department of Pharmacology, Setsunan University, Hirakata, Osaka, Japan
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  • Yoichi Nakamura,

    1. Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan
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    • 4

      The present address of Yoichi Nakamura is Laboratory of Integrated Physiology, Osaka Prefectural University Graduate School of Agricultural Biology, Sakai, Osaka 599–8531, Japan.

  • Leszek Kaczmarek

    1. Nencki Institute, Warsaw, Poland
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Address correspondence and reprint requests to Jan Pláteník, Institute of Medical Biochemistry, First Faculty of Medicine, Charles University, Kateřinská 32, 121 08 Prague, Czech Republic.
E-mail: jan.platenik@lf1.cuni.cz

Abstract

Cyclic AMP response element binding protein (CREB) is a constitutive transcription factor that activates transcription following stimulus-dependent phosphorylation at Ser133, implicated in synaptic plasticity and neuronal survival pathways. The prevailing view that CREB is exclusively nuclear has been questioned by several studies, and, for example, mitochondrial localization has been reported. Using subcellular fractionation of rat brain cortex coupled with western immunoblotting with Ser133-phospho-CREB (pCREB) antibodies, we found a robust pCREB immunoreactivity (IR) in mitochondria-enriched fractions. The pCREB antibodies also stained the mitochondria, in addition to nuclei, of glial cells in primary cortical cultures. However, two CREB antibodies against different epitopes and gel shift assay detected the CREB protein mainly in the nuclear fraction. The two-dimensional electrophoretic mobility of mitochondrial pCREB IR differed markedly from the nuclear CREB/pCREB IR, indicating that the pCREB antibody cross-reacts with another mitochondrial protein. Immunoprecipitation of the mitochondrial pCREB IR produced three bands on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, which were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as E2, E1 α-subunit, and E1 β-subunit of pyruvate dehydrogenase complex. The cross-reacting epitope was identified as phospho-Ser300 of the α-subunit. In conclusion, this study confirms the presence of pCREB-like IR in brain mitochondria that, after careful scrutiny, turned out to be pyruvate dehydrogenase rather than authentic CREB.

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