New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.
New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.
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DOI：
10.1371/journal.pone.0150000
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Theis M
中科院分区：
文献类型：
--
作者：
Kaczmarczyk L;Labrie-Dion É;Sehgal K;Sylvester M;Skubal M;Josten M;Steinhäuser C;De Koninck P;Theis M
胞质多聚腺苷酸化元件结合蛋白（CPEBs）是一类与mRNA的3 'UTR相互作用从而调节基因表达的多聚腺苷酸化因子。CPEB在发育、突触可塑性和细胞衰老中的各种功能已被报道。迄今为止，已经描述了四个部分重叠功能的CPEB家族成员，每个成员包含不同的可变剪接区。该区域在CPEBs-2-4之间高度保守，并且包含推定的磷酸化共识，与CPEB 3的外显子7重叠。我们以前发现CPEBs-2-4剪接异构体含有外显子7主要存在于神经元中，并且异构体表达模式是细胞类型特异性的。在这里，集中在CPEB 3的可变剪接区，我们确定CPEB 3的假定神经元亚型是磷酸化的。使用一种新的磷酸化特异性抗体，我们发现蛋白激酶A和钙/钙调蛋白依赖性蛋白激酶II在体外和原代海马神经元中强烈磷酸化CPEB 3。有趣的是，在小鼠中通过全身性红藻氨酸注射诱导癫痫持续状态导致含有外显子7的CPEB 3亚型的特异性上调。体外CPEB 3磷酸化的广泛分析揭示了另外两个磷酸化位点。此外，我们发现了大量可能靶向CPEB 3的可变剪接激酶共有位点的潜在激酶。由于该位点在CPEB家族成员之间是高度保守的，我们建议存在一种基于剪接的CPEB功能调节机制，并描述了一种强大的磷酸特异性抗体，以在未来对其进行研究。
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.