ASSOCIATION OF THE REGULATORY SUBUNIT OF A TYPE-II CAMP-DEPENDENT PROTEIN-KINASE AND ITS BINDING-PROTEINS WITH THE FIBROUS SHEATH OF RAT SPERM FLAGELLUM

Demembranated rat sperm flagellar polypeptides capable of binding the regulatory subunit (RII) of a type II cAMP-dependent protein kinase, h aving apparent subunit molecular masses of 120, 80 and 57 kDa were ide ntified by an RII overlay procedure [Horowitz, J. A., Wasco, W., Leise r, M. and Orr, G. A. (1988) J. Biol. Chem. 263, 2098-2104]. In this st udy it is shown that all three polypeptides capable of binding RII on a solid-phase blot are tightly associated with the fibrous sheath. Pur ified fibrous sheath preparations were capable of binding (a) [H-3]cAM P and (b) purified catalytic subunits of cAMP-dependent protein kinase forming a functional holoenzyme. The 57-kDa protein was identified as RII by photoaffinity labeling with 8-azido[P-32]cAMP. This peptide wa s phosphorylated by the catalytic subunit of cAMP-dependent protein ki nase. RII alpha was also shown to form tight, specific complexes with the fibrous sheath demonstrating the presence of functional RII alpha- binding sites. Truncated RII beta fusion proteins were used to identif y the N-terminal amino acids at positions 1-50 as a primary determinan t for RII-binding protein interaction. Differential extraction of adul t testis with buffers containing Triton X-100, urea and sodium dodecyl sulfate revealed the presence of 80-kDa (major) and 120-kDa (minor) R II-binding proteins in particulate extracts. The 80-kDa polypeptide is only expressed at late stages of spermatogenesis, i.e. during spermio genesis, suggesting a developmental role for RU: anchoring to the sper m flagellar fibrous sheath.