Identification of tethering domains for protein kinase A type Iα regulatory subunits on sperm fibrous sheath protein FSC1

Abstract

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 Iα regulatory subunit (RIα) 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 RIα 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 RIα tethering site (termed domain B) at residues 335-344 shows specific binding of RIα 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 RIα binding. This is apparently the first report of an RIα-specific protein kinase A anchoring protein tethering domain.