AKAP3 degradation in sperm capacitation is regulated by its tyrosine phosphorylation

Abstract Background The A-kinase anchoring protein (AKAP) family is essential for sperm motility, capacitation and the acrosome reaction. PKA-dependent protein tyrosine phosphorylation occurs in mammalian sperm capacitation including AKAP3. In a recent study, we showed that AKAP3 undergoes degradation under capacitation conditions. Thus, we tested here whether AKAP3 degradation might be regulated by its tyrosine phosphorylation. Methods The intracellular levels of AKAP3 were determined by western blot (WB) analysis using specific anti-AKAP3 antibodies. Tyrosine phosphorylation of AKAP3 was tested by immunoprecipitation and WB analysis. Acrosome reaction was examined using FITC-pisum sativum agglutinin. Results AKAP3 is degraded and undergoes tyrosine-dephosphorylation during sperm capacitation and the degradation was reduced by inhibition of tyrosine phosphatase and enhanced by inhibition of tyrosine kinase. Sperm starvation or inhibition of mitochondrial respiration, which reduce cellular ATP levels, significantly accelerated AKAP3 degradation. Treatment with vanadate, or Na + or bicarbonate depletion, reduced AKAP3-degradation and the AR rate, while antimycin A or NH 4 Cl elevated both AKAP3-degradation and the AR degree. Treatment of sperm with NH 4 Cl enhanced PKA-dependent phosphorylation of four proteins, further supporting the involvement of AKAP3-degradation in capacitation. To demonstrate more specifically that sperm capacitation requires AKAP3-degradation, we inhibited AKAP3-degradation using anti-AKAP3 antibody in permeabilized cells. The anti-AKAP3-antibody induced significant inhibition of AKAP3-degradation and of the AR rate. Conclusion Sperm capacitation process requires AKAP3-degradation, and the degradation degree is regulated by the level of AKAP3 tyrosine phosphorylation. General significance Better understanding of the molecular mechanisms that mediate sperm capacitation can be used for infertility diagnosis, treatment and the developing of male contraceptives.