Characterization of the flagellar-associated cyclic AMP-dependent protein kinase in mammalian sperm

Abstract

In mammalian sperm, a type II cyclic AMP-dependent protein kinase is associated with the flagellum via its regulatory (RII) subunit. Immunogold labeling has revealed that the majority of RII immunoreactivity is associated with the mitochondria and the fibrous sheath. In order to better understand the role of cyclic AMP-dependent protein kinase in mammalian sperm function, the interaction of RII with these specific cell components has been investigated.;Northern analysis of mouse spermiogenesis mutants has been used to document a high level of expression of a testis-specific RII message that is induced in haploid germ cells. To determine whether multiple forms of RII exist in sperm, polymerase chain reaction has been used to amplify the variable region of the molecule from mouse testis cDNA. Subcloning and sequencing of twenty independent clones containing the amplified fragments has shown them to be identical to the mouse brain RII{dollar}\alpha{dollar} isoform in this variable region.;To characterize the interaction between RII and the fibrous sheath, a sequential extraction procedure has been used to isolate the fibrous sheath from rat sperm. Whole mount and thin section electron microscopy have confirmed that the fibrous sheath is morphologically intact and free of contaminants. An RII overlay procedure has been used to demonstrate that the three RII binding proteins present in intact sperm (120 kDa, 80 kDa and 57 kDa) are all present in the fibrous sheath fraction. Also, RII overlays with truncated RII demonstrate that the amino terminal fifty amino acids of RII{dollar}\beta{dollar} are sufficient for binding to these three proteins. Evidence suggests that the 57 kDa protein is RII and therefore that RII is an integral component of the fibrous sheath.;The nature of the mitochondrial localization of RII, which is probably not mediated via RII binding proteins, has also been investigated. Triton X-114 partitioning behavior of flagellar RII demonstrates the existence of a lipophilic population of RII, and raises the possibility that RII is associated with the mitochondrial membrane via a lipid anchor, as is the case with the yeast mitochondrial cyclic AMP binding protein.