Zebrafish Cellular Nucleic Acid Binding Protein is differentially phosphorylated in the Ser158 residue during early embryogenesis

LOMBARDO, V; CALCATERRA, NORA BEATRIZ

Los Cocos, Córdoba, Argentina.

Workshop; 1st International Workshop “Latest concepts in Developmental Biology; 2006

The zinc-finger cellular nucleic acid binding protein (CNBP) is a single-stranded nucleic acid binding protein essential for normal mouse forebrain (Chen et. al. 2003) and zebrafish craniofacial development (unpublished results). In amphibian and fish, the protein is evenly distributed in the cytosol of embryonic cells until advanced epiboly. At the end of gastrulation, CNBP is translocated to the nucleus (Armas et. al, 2001; Armas et. al. 2004). All the CNBP sequences are highly conserved showing seven tandem CCHC-type zinc knuckle domains and an RGG box between the first and second zinc knuckles. CNBP primary structure shows a nuclear localization signal PKKEREQ, a PEST proteolytic site, and several putative phosphorylation sites. Phosphorylation is a well-documented post-translational modification that affects protein properties, such us the capability of import into the nucleus. Consequently, we analyzed whether CNBP could be phosphorylated by kinases from zebrafish embryo and the developmental behavior of this phosphorylation. In silico analysis revealed the existence of several putative phosphorylation sites in Ser, Thr, and Tyr residues. We determined that CNBP could be phosphorylated in vitro and in vivo, and that this phosphorylation was differential during early development. Phosphorylation was low and uniform during the first stages, reached a maximum at 24 - 48 hours post-fertilisation, and deceased thereafter. Then, we determined by alkaline treatment that CNBP was phosphorylated by Ser/Thr kinases present in zebrafish embryonic extracts. To address which kinase was required to CNBP phosphorylation, we performed phosphorylation assays in presence of specific inhibitors of different kinases. We observed that CNBP phosphorylation was inhibited by the H-89 and PKI inhibitors. Both inhibitors are considered to be selective inhibitors of cAMP-dependent protein kinase (PKA). As in silico analysis showed that the Ser158 residue is part of the PKA kinase-specific site, we generated a site-directed CNBP mutant by changing Ser158 to Ala (zCNBP S158A). This mutant was not able to be phosphorylated by embryonic extracts.  It is noteworthy that the PKA kinase-specific site that involves the Ser158 is conserved in all CNBPs analyzed, suggesting that differential phosphorylation may be important in CNBP biological function.