CLONING AND CHARACTERIZATION OF A TESTIS-SPECIFIC, DEVELOPMENTALLY-REGULATED A-KINASE-ANCHORING PROTEIN (TAKAP-80) PRESENT ON THE FIBROUS SHEATH OF RAT SPERM

cAMP is important for the initiation of mammalian sperm motility. Prev iously we established that a type II cAMP-dependent protein kinase is tightly associated with the fibrous sheath of rat sperm. This unique c ytoskeletal structure surrounds the 9+2 axonemal network in the princi pal piece of the flagellum. Association of the kinase to the fibrous s heath is mediated via its regulatory subunit, RII. An RII-binding over lay procedure was used to document that RU could specifically associat e with fibrous sheath polypeptides of 120 and 80 kDa. In this study, w e report the cloning of a rat testis-specific, developmentally regulat ed, RII-binding protein (TAKAP-80). A 1.2-kb cDNA clone, isolated by s creening a rat testis expression library with P-32-labeled RII, hybrid ized to a 1.8-kb mRNA transcript present exclusively in testis. This t ranscript appeared at detectable levels at 30 days after birth. Over t he next 10 days the mRNA levels increased greatly. This time interval corresponds to the initiation of spermiogenesis. The complete nucleoti de sequence of TAKAP-80 cDNA was obtained by polymerase chain reaction and contained a continuous open reading frame of 502 amino acids. The deduced amino acid sequence showed a clear demarcation of charged and hydrophobic amino acid residues. Amino acids 1-147 of the protein con tained 45% charged residues, with lysine and arginine predominating. S imilarly, amino acids 268-502 also contained a high percentage of char ged amino acids (35%). In contrast, amino acids 148-267 were mostly hy drophobic and contained clusters of a repeating PXXP motif where X was predominantly valine and alanine or sometimes proline. The 1.2-kb cDN A clone was inserted into the pRSET vector and expressed as a His(6) t ag fusion protein in Escherichia coli. The recombinant protein was sol uble and bound RII alpha, RII beta and type II alpha holoenzyme by the RII-binding overlay procedure. Deletion analysis revealed that the hi gh-affinity interaction site for RII was contained within amino acids 258-378 of TAKAP-80. Antibodies prepared against the fusion protein re cognized an 80-kDa protein present in the urea-insoluble particulate f raction of rat testis and in purified fibrous sheath preparations isol ated from rat epididymal sperm. Levels of the 80-kDa immunoreactive pr otein were significantly higher in mature (60 days old) compared with immature (30 days old) rat testis, correlating with the mRNA levels.