Abstract
Protein kinase A (PKA) is a broad-spectrum Ser/Thr kinase involved in the regulation of several cellular activities. Thus, its precise activation relies on being localized at specific subcellular places known as discrete PKA signalosomes. A-Kinase anchoring proteins (AKAPs) form scaffolding assemblies that play a pivotal role in PKA regulation by restricting its activity to specific microdomains. Because one of the first signaling events observed during mammalian sperm capacitation is PKA activation, understanding how PKA activity is restricted in space and time is crucial to decipher the critical steps of sperm capacitation. Here, we demonstrate that the anchoring of PKA to AKAP is not only necessary but also actively regulated during sperm capacitation. However, we find that once capacitated, the release of PKA from AKAP promotes a sudden Ca2+ influx through the sperm-specific Ca2+ channel CatSper, starting a tail-to-head Ca2+ propagation that triggers the acrosome reaction. Three-dimensional super-resolution imaging confirmed a redistribution of PKA within the flagellar structure throughout the capacitation process, which depends on anchoring to AKAP. These results represent a new signaling event that involves CatSper Ca2+ channels in the acrosome reaction, sensitive to PKA stimulation upon release from AKAP.
Highlights
Protein kinase A (PKA) is a broad-spectrum Ser/Thr kinase involved in the regulation of several cellular activities
To study the role of PKA anchoring in the sperm capacitation–associated pathways, we used the peptide sHT31, which inhibits the interaction between PKARII and A-Kinase anchoring proteins (AKAPs) [9]. sHT31 is the permeable stearated form of a short peptide composed of 24 residues (N-stearate-DLIEEAASRIVDAVIEQVKAAGAY) from the PKA-anchoring domain of AKAP
Western blot analysis shows that this preincubation with sHT31 blocked the phosphorylation of PKA substrates associated with sperm capacitation [13], with an IC50 below 1 M (Fig. 1A left panel)
Summary
Protein kinase A (PKA) is a broad-spectrum Ser/Thr kinase involved in the regulation of several cellular activities. Three-dimensional super-resolution imaging confirmed a redistribution of PKA within the flagellar structure throughout the capacitation process, which depends on anchoring to AKAP These results represent a new signaling event that involves CatSper Ca2؉ channels in the acrosome reaction, sensitive to PKA stimulation upon release from AKAP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Its activation appears to be necessary for: 1) membrane potential (Em) hyperpolarization [3, 4], which originates after 15–30 min; 2) development of hyperactivated motility after 30 – 45 min; and 3) triggering of the acrosome reaction, once capacitation is complete, i.e. after 60 min for mouse sperm Even though these processes differ in their kinetics, all of them involve phosphorylation cascades initiated by PKA.
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