New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

PLoS One. 2016 Feb 25;11(2):e0150000. doi: 10.1371/journal.pone.0150000. eCollection 2016.


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.

MeSH terms

  • Alternative Splicing
  • Animals
  • Antibodies, Phospho-Specific / metabolism
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism
  • HEK293 Cells
  • Hippocampus / metabolism*
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Neurons / metabolism*
  • Phosphorylation
  • Protein Isoforms / metabolism*
  • RNA-Binding Proteins / metabolism*


  • Antibodies, Phospho-Specific
  • CPEB3 protein, human
  • Protein Isoforms
  • RNA-Binding Proteins
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2

Grants and funding

This work was supported by grants of the German Research Foundation SFB/TR3, N01, C9 (to MT), SFB/TR3, C1, C9 (to CS); STE 552/3 (to CS); the Else-Kröner-Fresenius-Stiftung (to MT); Canadian Institute of Health Research (to PDK) and of the European Community FP7-202167 (to CS and MT).