Nuclear condensation of cyclic adenosine monophosphate responsive element-binding protein in discrete murine brain structures.
We have directed a polyclonal antibody against an oligo-peptide (123-136) of the transcription factor cyclic AMP responsive element-binding protein (CREB) including the serine residue at 133. Rabbit sera were purified by ammonium sulfate precipitation, followed by affinity chromatography to homogeneity on one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified antibody not only induced marked supershift of CREB binding, without affecting binding of activator protein-1 on gel retardation electrophoresis, but also differentiated between CREB and CREB phosphorylated at serine133 in brain nuclear fractions on Western blotting. Immunoreactive CREB was detected in both cytosolic and nuclear fractions of discrete murine brain structures but was more highly condensed in cerebellum than in neocortex and hippocampus. Incubation of brain nuclear fractions led to a marked export of immunoreactive CREB in a temperature-dependent manner, whereas the temperature-dependent export activity was significantly lower in cerebellum than in other brain structures. Suppression of general new protein synthesis by cycloheximide (500 mg/kg, i.p.) in vivo resulted in a significant decrease in the nuclear CREB level, with a concomitant increase in the cytosolic level in hippocampus, but not in cerebellum. These results suggest that the nuclear export activity might vary from region to region in murine brains through a hitherto unidentified mechanism other than the nuclear localization signal, to result in different nuclear condensation ratios for subsequent elicitation of differential transcriptional activities by the constitutive transcription factor CREB in the nucleus.
Kuramoto
Nobuyuki
N
Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan.
Kubo
Keita
K
Ogita
Kiyokazu
K
Pláteník
Jan
J
Balcar
Vladimir J
VJ
Takarada
Takeshi
T
Nakamichi
Noritaka
N
Yoneda
Yukio
Y
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D000918:Antibody Specificity; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000276:immunology / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D008297:Male; D051379:Mice; D019579:Neocortex / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011817:Rabbits
D000906:Antibodies; D017362:Cyclic AMP Response Element-Binding Protein; D010446:Peptide Fragments; D016755:Proto-Oncogene Proteins c-jun