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.
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 alpha-subunit, and E1 beta-subunit of pyruvate dehydrogenase complex. The cross-reacting epitope was identified as phospho-Ser300 of the alpha-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.
Pláteník
Jan
J
Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan. jan.platenik@lfl.cuni.cz
Balcar
Vladimír J
VJ
Yoneda
Yukio
Y
Mioduszewska
Barbara
B
Buchal
Richard
R
Hynek
Radovan
R
Kilianek
Lukasz
L
Kuramoto
Nobuyuki
N
Wilczynski
Grzegorz
G
Ogita
Kiyokazu
K
Nakamura
Yoichi
Y
Kaczmarek
Leszek
L
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D050882:CREB-Binding Protein / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D003576:Electron Transport Complex IV / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay; D005455:Fluorescent Antibody Technique / Q000379:methods; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D047468:Immunoprecipitation / Q000379:methods; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods; D008928:Mitochondria / Q000378:metabolism*; D010766:Phosphorylation; D011767:Pyruvate Decarboxylase / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013347:Subcellular Fractions / Q000378:metabolism
D000906:Antibodies; D005904:Glial Fibrillary Acidic Protein; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D012694:Serine; D000255:Adenosine Triphosphate; D003576:Electron Transport Complex IV; D050882:CREB-Binding Protein; D011767:Pyruvate Decarboxylase; D008274:Magnesium