Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I

Abstract

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2.