Developmental expression of the protein kinase C substrate/binding protein(clone 72/SSeCKS) in rat testis - Identification as a scaffolding protein containing an A-kinase-anchoring domain which is expressed during late-stage spermatogenesis

The coordinated interaction of kinases, phosphatases and other regulatory m olecules with scaffolding proteins is emerging as a major theme in intracel lular signaling networks. In this report we show that a cDNA isolated from a rat testis expression Library by interactive cloning using the regulatory subunit (R) of a type-II protein kinase A (PKA) is identical with a previo usly characterized protein kinase C (PKC)-binding protein termed either clo ne 72 [Chapline, C., Mousseau, B., Ramsay, K., Duddy, S., Li, Y., Kiley, S. C. & Jaken, S. (1996) J. Biol. Chem. 271, 6417-6422] or SSeCKS [Lin, X., To mbler, E., B., Nelson, P.J., Ross, M. & Gelman, I.H. (1996) J. Biol. Chem. 271, 28430-28438]. Deletion mutagenesis demonstrated that amino acids 1495- 1524 of clone 72/SSeCKS had the ability to interact with RII. Antibodies pr epared against the recombinant protein recognized a 280/290-kDa doublet and a 240-kDa protein on Western blots of rat testis cytosolic and Triton X-10 0 extracts. Expression of clone 72/SSeCKS mRNA and protein levels was devel opmentally regulated in rat testis. Northern-blot analysis showed a dramati c increase in clone 72/SSeCKS-hybridizing mRNA starting 30 days after birth . Immunohistochemical examination showed high expression levels in elongati ng spermatids. Clone 72/SSeCKS was not detected in mature sperm. These stud ies suggest a role for clone 72/SSeCKS, a PKA/PKC scaffolding protein, duri ng the process of spermiogenesis.