Differential phosphorylation of CNBP during zebrafish early development.

LOMBARDO, V. A.; WEINER, A.; ARMAS, P.; CALCATERRA, N. B.

Iguazú, Misiones, Argentina.

Congreso; XL Anual Meeting of the Argentine Society for Biochemestry and Molecular Biology.; 2004

Argentine Society for Biochemestry and Molecular Biology.

Cellular nucleic acid binding protein (CNBP) is widespread throughout the animal kingdom. This protein  shows an striking sequence conservation and general structural organisation. Zebrafish CNBP (zCNBP) contains seven retroviral CCHC zinc knuckle motifs, an RGG box, a PEST-type proteolysis sequence and four putative phosphorylation sites in Ser4, Tyr41, Tyr85 and Tyr145. We determined that CNBP is in vitro phosphorylated by zebrafish embryo extracts and that this phosphorylation is differential during early development. Phosphorylation levels were low and uniform during the first stages, reached a maximum between 24 and 48 hours post-fertilisation and deceased later. After incubation with alkali, no phosphorylation was observed in over-exposed SDS-PAGE. Because of this result we presume that the amino acid phosphorylated is Ser4. We have also observed that zCNBP is differentially cleaved during embryonic development and this cleavage starts when the phosphorylation begins to raise, leading us to think that both phenomena could be related. Moreover, in proteolysis experiments, we have observed that the fragment containing the first 29 amino acid residues of zCNBP retains the phosphorylation signal, again suggesting that Ser4 is the phosphorylated residue. The implications of this phosphorylation in nucleic acid binding and oligonucleotide annealing promoting are analysed.