PROTEIN KINASE A: BEING IN THE RIGHT PLACE AT THE RIGHT TIME

RITAGLIATI, CARLA; LUQUE, GUILLERMINA M; KRAPF, DIEGO; XU, XINRAN; BARÓ GRAF, CAROLINA; BUFFONE, MARIANO G.; STIVAL, CINTIA; GERVASI, MARIA G; VISCONTI, PABLO E.; KRAPF, DARIO

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

Capacitation is the process by which mammalian sperm acquirethe ability to fertilize and can be mimic in vitro. Once completed,sperm obtain acrosomal responsiveness and a hypeactivated flagellarmovement.When sperm are exposed to HCO3-, there is rapid intracellularincrease in cAMP, that activates PKA, which acts as a key playerduring capacitation. Its regulation has been mainly studied focusingon its catalytic activity. However, biological activity is also dependenton its subcellular localization, which is mediated by interactionwith scaffolding AKAP proteins.Here, we study the role of PKA anchoring during capacitation murinesperm. The binding and anchoring of PKA to AKAP was affectedby the peptide st-HT31, which disrupts PKA-AKAP interaction withoutaffecting catalytic activity.When PKA anchoring was impaired throughout capacitation,sperm neither acquired acrosome responsiveness (assessed byPNA-staining of the acrosome), nor hyperactivated motility. Phosphorylationof PKA substrates as well as tyr phosphorylation werealso blocked, as evaluated by western-blot.Conversely, when PKA de-anchoring was induced in capacitatedsperm, AR was triggered by a mechanism that involved 1) membranedepolarization, and 2) Ca2+ influx, seemingly through Cat-Sper channel. These events were assessed by single-cell microscopyand population fluorimetric measures, using specific-sensitivedyes.Addition of the inhibiting peptide st-HT31 induced AR only in capacitatedsperm but not in non-capacitated cells, showing there arecapacitation-associated events that prime the sperm for this responseto PKA de-anchoring. In addition, AR needed active PKA activity,since inhibition of PKA right before st-HT31 exposure impairedAR. Moreover, an inactive analogue of st-HT31, named st-HT31p,did not show any effect.Overall, we show that PKA anchoring is essential for its biologicalactivity in sperm processes.