Mussel Spermatozoa Express a Specific Isoform of the Catalytic Subunit of cAMP‐Dependent Protein Kinase

ABSTRACT

cAMP‐dependent protein kinase (PKA) plays a central role in regulating sperm motility in both invertebrates and mammals. The sea mussel Mytilus galloprovincialis provides a valuable model for investigating the molecular mechanisms underlying gamete activation in externally fertilizing organisms. In previous work, several catalytic (C) subunit isoforms of PKA were isolated from this species. Here, we report that one of these isoforms appears to be specifically expressed in spermatozoa, indicating the presence of a sperm‐specific C‐subunit isoform in a non‐mammalian species. The protein was purified from male gonadal tissue and identified by LC‐MS/MS as the isoform corresponding to UniProt entry A0A8B6GIP6. Sequence analysis revealed that this mussel C‐isoform shares strong homology with mammalian sperm‐specific Cα2‐isoform within the catalytic core but possesses a unique N‐terminal region. Whereas mammalian Cα2 contains a short six‐residue N‐terminal extension, the mussel sperm isoform features a longer acidic extension of 17 amino acids. Moreover, phosphoproteomic analysis reveals the presence of two phosphorylation sites (Ser5 and Thr30) in the N‐terminal tail of the mussel C‐subunit that are absent in Cα2. These structural differences suggest specific regulatory mechanisms associated with sperm activation in externally fertilizing bivalves. Immunolocalization studies show that the mussel sperm C‐subunit is distributed in the acrosomal region, mitochondria, and along the flagellum, where it is predominantly associated with R _myt2 , a type II regulatory subunit previously characterized in the mussel.