The two faces of Protein Kinase A regulation during sperm capacitation.

STIVAL, CINTIA; NOVERO, ANALIA G.; GENTILE, IÑAKI; SCHIAVI-EHRENHAUS, LIZA J.; PIGA, ERNESTO; CURCIO, CATALINA; RITAGLIATI, CARLA; XINRAN, XU; KRAPF, DIEGO; BUFFONE, MARIANO G.; KRAPF, DARIO

Congreso; Fertilization and Activation of Development Gordon Research Conference; 2023

Protein Kinase A (PKA) is a central regulator of sperm capacitation and it activates upon association of cAMP. Being a promiscuous enzyme, its specific biological action is fine-tuned by alternative mechanisms that target its subcellular localization as well as its catalytic activity. Our previous study using high-resolution microscopy revealed that PKA positioning is dynamic during capacitation and mediated by interaction with the sperm-specific AKAP4 scaffolding protein. This suggests that altering PKA anchoring may have time-dependent effects. To investigate this, we targeted the PKA-AKAPs interaction in murine sperm using the sHT31 disrupting peptide. Our findings demonstrate that the biological effect of disrupting PKA-AKAP anchoring in mature sperm cells varies greatly depending on the timing of dissociation. When anchoring is impaired from the beginning of the incubation in capacitating medium, capacitation is completely abrogated. The inhibitory mechanism involves changes in in vivo PKA activity and AKAP4 degradation. Considering that AKAP4 is sperm-specific, its interaction with PKA emerges as a promising target for the development of male contraceptives. Interestingly, treatment with sHT31 inhibited acrosomal responsiveness when added from the beginning of capacitation, but potentiated the response to P4 when added after sperm capacitation. In addition to proper localization, the catalytic activity of PKA is also subject to non-canonical modulation during capacitation. Acetylation of PKA seems to be involved in modulating both anchoring and kinase activity. Treatment of sperm with deacetylase inhibitors triggered phosphorylation of PKA substrates, even under non-capacitating conditions. This also stimulated the phosphorylation of PKAcT197 (associated with PKA activation) and increased its interaction with AKAP4. Using a recombinant version of the catalytic subunit of PKA (recPKAc), we conducted NMR experiments and observed spectral shifts in amino acids located at the catalytic site induced by acetylation. Consistent with this, acetylation stimulated the catalytic activity of recPKAc when reconstituted in vitro. Overall, these results suggest that PKA is subjected to different mechanisms other than cAMP binding, that modulate its biological action in the sperm cell. In this sense, interfering with PKA anchoring can lead to opposing effects depending on the timing of the disruption, highlighting a novel binary role for PKA positioning.