S-AKAP84, a novel developmentally-regulated A kinase anchor protein of the male germ cell cytoskeleton

Abstract

In mammalian spermatozoa, most of the type II{dollar}\alpha{dollar} isoform of cAMP-dependent protein kinase (PKAII{dollar}\alpha{dollar}) is selectively anchored at the cytoplasmic surface of a specialized array of mitochondria in the flagellar cytoskeleton. This mode of organization, which is established by interactions between the regulatory subunits (RII{dollar}\alpha{dollar}) of the kinase and an unknown component of the outer mitochondrial membrane, places the catalytic subunits of PKAII{dollar}\alpha{dollar} in proximity with potential target substrates in the cytoskeleton. Protein phosphorylation catalyzed by anchored PKAII{dollar}\alpha{dollar} may participate in (a) the dynamic re-organization of the cytoskeleton that underlies a classical physiological effect of cAMP--the initiation and maintenance of flagellar motility, and/or (b) the remodelling of the cytoskeleton that generates the flagellum during late stages of sperm development. To date our knowledge of the mechanism by which the bulk of PKAII{dollar}\alpha{dollar} is targeted to and anchored on the outer surface of mitochondria has been extremely limited. In this thesis, I describe the cloning of a cDNA that encodes a novel, germ cell A kinase anchor protein (AKAP) designated S-AKAP84. S-AKAP84 comprises 593 amino acids and it contains a centrally-located sequence that mediates the avid binding of RII{dollar}\alpha{dollar} or RII{dollar}\beta{dollar} subunits. The 3.2 kb S-AKAKP84 mRNA and the cognate S-AKAP84 RII binding protein appear to be expressed principally in the spermatocyte lineage. Moreover, expression of S-AKAP84 is tightly regulated during development. The protein accumulates abruptly as spermatids undergo nuclear condensation and tail elongation. The appearance of S-AKAP84 is preceded by the de novo accumulation of RII subunits. The timing of S-AKAP84 expression is also correlated with the migration of mitochondria from the cytoplasm (round spermatids) to the cytoskeleton (midpiece in elongating spermatids). Residues 1-30 at the N terminus of S-AKAP84 constitute a putative signal/anchor sequence that may target the protein to the outer mitochondrial membrane. Immunofluorescence analysis with antibodies directed against S-AKAP84 documented that this RII binding protein is co-localized with the mitochondria in the flagellum. Therefore, S-AKAP84 represents the first example of a developmentally-regulated, mitochondrially-targeted RII anchor protein.