Differential expression and phosphorylation of particular fos family members by kainate in nuclear and cytosolic fractions of murine hippocampus.
An i.p. injection of kainate resulted in severe losses of neuronal cells stained by Cresyl Violet in the CA1 and CA3 pyramidal layers of the murine hippocampus within two weeks, without affecting those in the dentate granule layer up to six weeks. Immunohistochemical analysis revealed marked and predominant expression of Fos family members, including c-Fos, Fos-B and Fra-2 proteins, in the dentate granule layer of the hippocampus, but not in the pyramidal layers, 2-18h after administration. Immunoblotting experiments showed that kainate induced more potent expression of c-Fos protein in nuclear fractions obtained 2h after injection than those obtained 18h later, with similar expression between cytosolic fractions obtained 2 and 18h after administration. Both Fos-B and Fra-2 proteins were more potently expressed in nuclear and cytosolic fractions 18h after administration than 2h when determined on immunoblotting analysis. Two-dimensional electrophoresis revealed expression of several proteins immunoreactive to the anti-c-Fos antibody with different isoelectric points in response to kainate in nuclear and cytosolic fractions of the hippocampus for 2-18h after a single injection. Immunoprecipitation analysis using the anti-c-Fos antibody showed phosphorylation of c-Fos protein on serine residues in hippocampal nuclear fractions 2h after administration, with additional phosphorylation on tyrosine residues 18h later. Prior treatment of the protein synthesis inhibitor cycloheximide prevented the expression of immunoreactivities to the anti-c-Fos antibody detected on two-dimensional electrophoresis in hippocampal nuclear fractions obtained 2h after administration.These results suggest that in vivo kainate signals may lead to persistent expression of the transcription factor activator protein-1 that consists of different Fos family members, as well as of c-Fos protein phosphorylated on serine and/or tyrosine residues, at an early stage after administration. Such signal consolidation processes could play a role in mechanisms associated with neuronal survival after kainate in the dentate granular layer of murine hippocampus.
Manabe
T
T
Department of Pharmacology, Setsunan University, Hirakata, 573-0101, Osaka, Japan.
Kitayama
T
T
Ogita
K
K
Yoneda
Y
Y
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D051921:Fos-Related Antigen-2; D006624:Hippocampus / Q000378:metabolism* / Q000473:pathology; D007150:Immunohistochemistry; D007608:Kainic Acid / Q000494:pharmacology*; D008297:Male; D051379:Mice; D010766:Phosphorylation; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D014018:Tissue Distribution; D014157:Transcription Factors / Q000378:metabolism
D004268:DNA-Binding Proteins; D051921:Fos-Related Antigen-2; C497873:Fosl2 protein, mouse; D016760:Proto-Oncogene Proteins c-fos; D014157:Transcription Factors; D007608:Kainic Acid