title: New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.
abstract: Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3'UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future.
authors.affiliation: Institute of Cellular Neurosciences, Medical Faculty, University of Bonn, Bonn, Germany.
authors.forename: Lech
authors.initials: L
authors.lastname: Kaczmarczyk
authors.affiliation: Institut universitaire en santé mentale de Québec, Québec, QC, Canada.
authors.forename: Étienne
authors.initials: É
authors.lastname: Labrie-Dion
authors.affiliation: Institut universitaire en santé mentale de Québec, Québec, QC, Canada.
authors.forename: Kapil
authors.initials: K
authors.lastname: Sehgal
authors.affiliation: Institute of Biochemistry and Molecular Biology, University of Bonn, Bonn, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Sylvester
authors.affiliation: Institute of Cellular Neurosciences, Medical Faculty, University of Bonn, Bonn, Germany.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Skubal
authors.affiliation: Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn, Bonn, Germany.
authors.forename: Michele
authors.initials: M
authors.lastname: Josten
authors.affiliation: Institute of Cellular Neurosciences, Medical Faculty, University of Bonn, Bonn, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Steinhäuser
authors.affiliation: Département de Biochimie, de Microbiologie et de Bio-informatique, Université Laval, Québec, QC, Canada.|Institut universitaire en santé mentale de Québec, Québec, QC, Canada.
authors.forename: Paul
authors.initials: P
authors.lastname: De Koninck
authors.affiliation: Institute of Cellular Neurosciences, Medical Faculty, University of Bonn, Bonn, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Theis
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D057809:HEK293 Cells; D006624:Hippocampus / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: D045163:Antibodies, Phospho-Specific; C513866:CPEB3 protein, human; D020033:Protein Isoforms; D016601:RNA-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2