New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.

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

CPEB3a phosphorylation in cultured hippocampal neurons.

<p>Normalized ratio of phosphorylated CPEB3a (phospho-CPEB3a) fluorescence over total CPEB3a (CPEB3a-EGFP) fluorescence (n = 25–35, 3 experiments). (<b>A</b>) Immunofluorescence of neurons transfected with CPEB3a-mGFP subjected to a 40 mM KCl stimulation for 90 s, which leads to a marked increase in phospho-CPEB3a signal. One hour pretreatment with 10 μM KN93 or 10μM H89 in the stimulation medium reverts this increase. Co-transfection of the natural CaMKII inhibitor (CaMKIIN-HA) prevents the increase in CPEB3a phosphorylation. (<b>B</b>) Quantification of the ratio of phospho-CPEB3a over CPEB3a-mGFP signals in A, which shows a significant increase of CPEB3a phosphorylation following KCl stimulation only, which is not seen in neurons treated with KN93, H89, or those co-transfected with CaMKIIN-HA; n = 10–35, at least 3 experiments per condition. (<b>C</b>) Immunofluorescence of neurons transfected with CPEB3a-mGFP and subjected to either 5 min 50 μM FS stimulation or 1 h 300 nm OA stimulation, which both cause a robust increase in phospho-CPEB3a signal. Pretreatment with 10 μM H89 or 100 μM RP-cAMPS for 1 h partially reverts the effect of FS. (<b>A</b>, <b>C</b>) The scale bar represents 10 μm and applies to all photomicrographs. (<b>D</b>) Quantification of the ratio of phospho-CPEB3a over CPEB3a-mGFP signals in C, which shows a significant increase of CPEB3a phosphorylation following FS, OA and combined FS + OA treatments. Increase in phosphorylation induced by FS is partially reversed by cAMPS-Rp treatment; n = 22–59, 2–5 experiments per condition. (<b>A</b>, <b>B</b>, <b>C</b>, <b>D</b>) *p<0.05, Kruskal-Wallis followed by Tukey’s least-significant difference test.</p

Schematic diagram of CPEB domain structure and sequence comparison of the variable middle region of mouse CPEB3.

<p>Two regions, termed B and C, are alternatively spliced, leading to expression of four isoforms: a (containing both regions), b (lacking the B-region), c (lacking the C-region), and d (lacking both regions) [Uniprot Q7TN99-1, -2, -3, -4 respectively]. Consensus phosphorylation sites for PKA and CaMKII was shown above the alignment. The putative phosphorylated residue was marked with an asterisk.</p

Summary of mass spectrometric evidence for phosphorylation of CPEB3a by PKA, CaMKII and endogenous kinases present in HEK-293 cells.

<p>Summary of mass spectrometric evidence for phosphorylation of CPEB3a by PKA, CaMKII and endogenous kinases present in HEK-293 cells.</p

Multiple sequence alignment of the regions flanking S419/S420 of CPEBs-2-4.

<p>Phosphorylation site was marked with a black asterisk. Residue conservation score was calculated with Jalview software according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150000#pone.0150000.ref061" target="_blank">61</a>]. Color intensity and the score (1–11) in the “conservation index” (below the alignment) reflects the conservation of physicochemical properties of amino acids in the particular column of the alignment. *: conserved column (score 11, highest); +: all the amino acid physical properties conserved (score 10).</p

Non-radioactive phosphorylation assays.

<p>(<b>A</b>) Sequences of the synthetic peptides used for phosphorylation experiments in B, C, D, E. Alternatively spliced exon 7 (the B-region) was highlighted. Putative phosphorylation site is in <b>bold</b>. (<b>B</b>, <b>C</b>, <b>D</b>, <b>E</b>) <i>In vitro</i> phosphorylation of CPEB3 by PKA and CaMKII. (<b>B</b>) MALDI-TOF-MS analysis of CPEB3-derived peptides phosphorylated by PKA-C. A mass shift corresponding to incorporation of 1 or 2 phosphates is observed, as compared with no-kinase control. (<b>C</b>) MALDI-TOF-MS spectra of CPEB3-derived peptides phosphorylated by CaMKII. (<b>B, C</b>) orange trace–spectrum after phosphorylation; black trace–no-kinase control; m/z–mass to charge ratio; AU—arbitrary units. (<b>D</b>) Peptide phosphorylation by PKA-C (PKLight assay). The a/c-isoform peptide, S419A, and S420A mutant peptides are phosphorylated at a significantly higher rate compared to the double mutant, while the b/d-isoform phosphorylation was not significantly different. S420 is phosphorylated preferentially, as shown by decreased phosphorylation rate in case of S420A mutant peptide compared with S419A substitution (p<0.01). Values showing relative levels of unused ATP after phosphorylation reaction were normalized to no-substrate control levels (not shown). Error bars are SEM. *P<0.005, **P<0.001 (student’s t-test). (<b>E</b>) Peptide phosphorylation by CaMKII (ADP-Glo assay). (<b>E1</b>) The a/c-isoform peptide is phosphorylated at significantly higher rate compared with b/d-isoform and S419A/S420A double mutant. (<b>E2</b>) Serine 420 is phosphorylated preferentially, as shown by decreased phosphorylation rate in case of S420A mutant peptide. Values show relative ADP levels after phosphorylation reaction, normalized to no-substrate control levels (not shown). Error bars are SEM. *P<0.01, **P<0.001, ***P<0.05 (student’s t-test, n = 4).</p

qRT-PCR analysis of CPEB3 isoforms of hippocampal mRNA after intraperitoneal kainate injection.

<p>The mRNA levels of isoforms with the B-region increased 30 min after kainate-induced <i>status epilepticus</i>, while the levels of the isoforms lacking the B-region remained unchanged. n = 4 animals = 8 hippocampi (per condition). *p<0.05, student’s t test.</p

CPEB3a phosphorylation in HEK-293 cells.

<p>(<b>A</b>) Immunofluorescence of HEK-293 cells transfected with CPEB3a-EGFP or CPEB3a-EGFP and mRuby-CaMKII, and subjected to different stimulation paradigms. Forskolin (FS) stimulation led to a robust increase of phospho-CPEB3a signal. Ca2+/ionomycin stimulation led to a significant increase in phospho-CPEB3a only when mRuby-CaMKII was co-transfected. The scale bar represents 10 μm and applies to all photomicrographs. (<b>B</b>) Quantification of the immunofluorescence shown in A; n = 4–8 experiments with 20 cells per conditions, *p<0.05, Kruskal-Wallis followed by Tukey’s least-significant difference test. (<b>C</b>) A representative immunoblot showing increased phosphorylation state of CPEB3a protein in transiently transfected HEK-293 cells after FS stimulation (50μM and 200μM). PanCPEB3 and alpha-tubulin antibodies were used to control for equal loading.</p

Correction: High-risk additional chromosomal abnormalities at low blast counts herald death by CML (Leukemia, (2020), 34, 8, (2074-2086), 10.1038/s41375-020-0826-9)

An amendment to this paper has been published and can be accessed via a link at the top of the paper