AKAP3 degradation in sperm capacitation is regulated by its tyrosine phosphorylation.

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 NH4Cl elevated both AKAP3-degradation and the AR degree. Treatment of sperm with NH4Cl 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.

Dissociation between AKAP3 and PKARII promotes AKAP3 degradation in sperm capacitation.

Ejaculated spermatozoa must undergo a series of biochemical modifications called capacitation, prior to fertilization. Protein-kinase A (PKA) mediates sperm capacitation, although its regulation is not fully understood. Sperm contain several A-kinase anchoring proteins (AKAPs), which are scaffold proteins that anchor PKA. In this study, we show that AKAP3 is degraded in bovine sperm incubated under capacitation conditions. The degradation rate is variable in sperm from different bulls and is correlated with the capacitation ability. The degradation of AKAP3 was significantly inhibited by MG-132, a proteasome inhibitor, indicating that AKAP3 degradation occurs via the proteasomal machinery. Treatment with Ca(2+)-ionophore induced further degradation of AKAP3; however, this effect was found to be enhanced in the absence of Ca(2+) in the medium or when intracellular Ca(2+) was chelated the degradation rate of AKAP3 was significantly enhanced when intracellular space was alkalized using NH4Cl, or when sperm were treated with Ht31, a peptide that contains the PKA-binding domain of AKAPs. Moreover, inhibition of PKA activity by H89, or its activation using 8Br-cAMP, increased AKAP3 degradation rate. This apparent contradiction could be explained by assuming that binding of PKA to AKAP3 protects AKAP3 from degradation. We conclude that AKAP3 degradation is regulated by intracellular alkalization and PKARII anchoring during sperm capacitation.