Activation of spleen tyrosine kinase (Syk) at fertilization in Rhinella arenarum eggs

MOUGUELAR, VALERIA S.; COUX, GABRIELA

INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY

U B C PRESS

Año: 2014 vol. 58 p. 811 - 818

0214-6282

Recently, we have provided evidence suggesting the involvement of a cytosolictyrosine?phosphorylatable 70 kDa oocyte protein in Rhinella arenarum (Anura:Bufonidae) fertilization. The aim of this work was to characterize its phosphorylation,determine the identity of this protein and establish a biological role during thefertilization process. Tyrosine phosphorylation of the 70 kDa protein was not observedin eggs activated with calcium ionophore A23187. Pretreatment of oocytes with thetyrosine kinase inhibitor genistein effectively blocked the fertilization-dependentphosphorylation of the 70 kDa protein. In order to identify this protein, we examinedthe presence in amphibian oocytes of non-receptor 70 kDa tyrosine kinase members ofthe Syk/Zap70 and Tec families by RT-PCR using degenerate primers. We found that R.arenarum oocytes contain the transcripts coding for Syk and Tec kinases. Western blotanalysis confirmed the presence of Syk protein in unfertilized oocytes and eggs. Studiesusing phospho-Syk specific antibodies showed that fertilization rapidly (less than 10minutes) induces phosphorylation on Syk tyrosine residues (352 and 525/526) that arenecessary for the activation of the enzyme. Finally, specific inhibition of Syk with theR406 compound provoked a diminished fertilization score, allowing us to confirm afunctional role of the Syk protein during R. arenarum fertilization. To our knowledgethis is the first time that Syk is described as a player in the signaling cascade activatedafter fertilization.