Protein kinase A activity leads to the extension of the acrosomal process in starfish sperm

Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction, which is immediately followed by the actin polymerization-dependent extension of an acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome reaction. Asterosap induces a transient elevation of intracellular cGMP and Ca²⁺ levels, and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca²⁺. Yet, the contribution of signaling molecules downstream of cAMP and Ca²⁺ in inducing AV exocytosis and AP extension remain unknown. A modified acrosome reaction assay was used here to differentiate between AV exocytosis and AP extension in starfish sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of target proteins occurs, and these substrates localize at the base of the AP, demonstrating that PKA activation regulates an AP extension step during the acrosome reaction. The major PKA substrate was further identified, from A. amurensis and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin polymerization during the acrosome reaction.