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 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.
Reinton
N
N
Institute of Medical Biochemistry, University of Oslo, N-0317 Oslo, Norway.
Orstavik
S
S
Haugen
T B
TB
Jahnsen
T
T
Taskén
K
K
Skålhegg
B S
BS
D000595:Amino Acid Sequence; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D013094:Spermatozoa / Q000201:enzymology* / Q000648:ultrastructure; D013737:Testis / Q000201:enzymology
D007527:Isoenzymes; D012333:RNA, Messenger; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases