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

Biol Reprod. 2000 Aug;63(2):607-11. doi: 10.1095/biolreprod63.2.607.

Abstract

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human C alpha catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine C alpha gene. Sequence comparison revealed similarity to an ovine C alpha variant characterized by protein purification and micropeptide sequencing, C alpha-s, identifying the cloned 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 spermatocytes was demonstrated. The C alpha-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a C alpha-s-specific antibody indicated that C alpha-s was localized in the midpiece region of the spermatozoon. The majority of C alpha-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the C alpha-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms 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 splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cyclic AMP / pharmacology
  • Cyclic AMP-Dependent Protein Kinases / analysis*
  • Cyclic AMP-Dependent Protein Kinases / chemistry
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Gene Expression
  • Humans
  • Isoenzymes / analysis*
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Male
  • Molecular Sequence Data
  • RNA, Messenger / analysis
  • Sequence Alignment
  • Spermatozoa / enzymology*
  • Spermatozoa / ultrastructure
  • Testis / enzymology

Substances

  • Isoenzymes
  • RNA, Messenger
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases