Identification of tethering domains for protein kinase A type Ialpha regulatory subunits on sperm fibrous sheath protein FSC1.
The fibrous sheath is a unique cytoskeletal structure in the sperm flagellum believed to modulate sperm motility. FSC1 is the major structural protein of the fibrous sheath. The yeast two-hybrid system was used to identify other proteins that contribute to the structure of the fibrous sheath or participate in sperm motility. When FSC1 was used as the bait to screen a mouse testis cDNA library, two clones were isolated encoding the type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase. Deletion analysis using the yeast two-hybrid system and in vitro binding assays with glutathione S-transferase-FSC1 fusion proteins identified two RIalpha tethering domains on FSC1. A domain located at residues 219-232 (termed domain A) corresponds to the reported tethering domain for a type II regulatory subunit (RII) of cAMP-dependent protein kinase, indicating that this binding domain has dual specificity to RI and RII. Another RIalpha tethering site (termed domain B) at residues 335-344 shows specific binding of RIalpha and had no significant sequence homology with known RII tethering domains. However, helical wheel projection analysis indicates that domain B is likely to form an amphipathic helix, the secondary structure of RII tethering domains of protein kinase A anchoring proteins. This was supported by the finding that site-directed mutagenesis to disrupt the amphipathic helix eliminated RIalpha binding. This is apparently the first report of an RIalpha-specific protein kinase A anchoring protein tethering domain.
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Gamete Biology Group, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli; D015723:Gene Library; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029589:Seminal Plasma Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013081:Sperm Motility; D013082:Sperm Tail / Q000378:metabolism*; D013737:Testis / Q000378:metabolism
D017931:DNA Primers; D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins; D029589:Seminal Plasma Proteins; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases