A novel isoform of human cyclic 3 ',5 '-adenosine monophosphate-dependent protein kinase, C alpha-s, localizes to sperm midpiece

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice vari ant of the human C alpha catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal pa rt of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine C alpha gene. Sequence compa rison revealed similarity to an ovine C alpha variant characterized by prot ein purification and micropeptide sequencing, C alpha-s, identifying the cl oned human cDNA as the C alpha-s isoform. The C alpha-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene sper matocytes was demonstrated. The C alpha-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a C alpha-s-specific anti body indicated that C alpha-s was localized in the midpiece region of the s permatozoon. The majority of C alpha-s was particulate and could not be rel eased from the sperm midpiece by cAMP treatment alone. Furthermore, deterge nt extraction solubilized approximately two-thirds of the C alpha-s pool, i ndicating interaction both with detergent-resistant cytoskeletal and membra ne structures. In addition, we recently identified the regulatory subunit i soforms RI alpha, RII alpha, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target C alpha-s. This novel C alpha-s spl ice variant appeared to have an independent anchor in the human sperm midpi ece as it could not be completely solubilized even in the presence of both detergent and cAMP.