REGULATION, LOCALIZATION, AND ANCHORING OF PROTEIN-KINASE A SUBUNITS DURING MOUSE SPERM CAPACITATION

The molecular basis of mammalian sperm capacitation, defined as those biochemical and functional changes that render the sperm competent to fertilize the egg, is poorly understood. This extratesticular maturati onal process is accompanied by the activation of a unique signal trans duction pathway involving the cAMP-dependent up-regulation of protein tyrosine phosphorylation presumably through the activation of protein kinase A JFK-A). We demonstrate in this report that capacitation of ca uda epididymal mouse sperm in vitro was accompanied by a time-dependen t increase in PK-A activity. This increase in PK-A activity did not oc cur in a medium that does not support capacitation. While PK-A catalyt ic and RI/RII regulatory subunits, as well as PK-A enzyme activity, we re found in both the Triton X-100-soluble and -insoluble fractions of the sperm, the increase in PK-A activity accompanying capacitation was associated with enzyme activity found in the soluble fraction. Moreov er, the regulatory and catalytic subunits of PK-A were observed by ind irect immunofluorescence to be present throughout the head, midpiece, and principal piece of the sperm. Thus, PK-A appears to be functional in multiple compartments of this highly differentiated cell. A fractio n of the Triton X-100-insoluble PK-A is presumably tethered by AKAP82, the major protein of the fibrous sheath of the sperm flagellum which anchors and compartmentalizes PK-A to the cytoskeleton via the RII sub unit of PK-A. Using various recombinant truncated AKAP82 constructs in a gel overlay assay, the RII subunit-binding domain of this protein w as mapped to a 57-amino-acid residue region at its N-terminus. Compute r analysis revealed a 14-amino-acid region that resembled the RII-bind ing domains of other A Kinase Anchor Proteins. A synthetic peptide cor responding to this domain inhibited AXAP82-RII binding in a gel overla y assay, providing further support that AKAP82 is an anchoring protein for the subcellular localization of PK-A in the mouse sperm fibrous s heath. This work, along with previous findings that cAMP is a key inte rmediary second messenger in regulating protein tyrosine phosphorylati on and capacitation, further supports the importance of PK-A in these processes and necessitates a further understanding of the contribution of both the soluble and insoluble forms of PK-A, as well as AKAP82, t o sperm function. (C) 1997 Academic Press.