In vitro embryonic developmental phosphorylation of the cellular nucleic acid binding protein by cAMP-dependent protein kinase, and its relevance for biochemical activities

The zinc-finger cellular nucleic acid binding protein (CNBP) is a strikingly conserved single-stranded nucleic acid binding protein essential for normal forebrain formation during mouse and chick embryogenesis. CNBP cDNAs from a number of vertebrates have been cloned and analysed. CNBP is mainly conformed by seven retroviral Cys-Cys-His-Cys zinc-knuckles and a glycine/arginine rich region box. CNBP amino acid sequences show a putative Pro-Glu-Ser-Thr site of proteolysis and several putative phosphorylation sites. In this study, we analysed CNBP phosphorylation by embryonic kinases and its consequences on CNBP biochemical activities. We report that CNBP is differentially phosphorylated by Danio rerio embryonic extracts. In vitro CNBP phosphorylation is basal and constant at early embryonic developmental stages, it begins to increase after mid-blastula transition stage reaching the highest level at 48 hours postfertilization stage, and decreases thereafter to basal levels at 5 days postfertilization. The cAMP-dependent protein kinase (PKA) was identified as responsible for phosphorylation on the unique CNBP conserved putative phosphorylation site. Site-directed mutagenesis replacing the PKA phospho-acceptor amino acid residue impairs CNBP phosphorylation, suggesting that phosphorylation may not only exist in D. rerio but also in other vertebrates. CNBP phosphorylation does not change single-stranded nucleic acid binding capability. Instead, it promotes in vitro the annealing of complementary oligonucleotides representing the CT element (CCCTCCCC) from the human cellular myelocytomatosis oncogene (c-myc) promoter, an element responsible for c-myc enhancer transcription. Our results suggest that phosphorylation might be a conserved post-translational modification that allows CNBP to perform a fine tune expression regulation of a group of target genes, including c-myc, during vertebrate embryogenesis.