Regulation, Localization, and Anchoring of Protein Kinase A Subunits during Mouse Sperm Capacitation

Abstract

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 maturational process is accompanied by the activation of a unique signal transduction pathway involving the cAMP-dependent up-regulation of protein tyrosine phosphorylation presumably through the activation of protein kinase A (PK-A). We demonstrate in this report that capacitation of cauda epididymal mouse sperm in vitro was accompanied by a time-dependent increase in PK-A activity. This increase in PK-A activity did not occur in a medium that does not support capacitation. While PK-A catalytic and RI/RII regulatory subunits, as well as PK-A enzyme activity, were 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. Moreover, the regulatory and catalytic subunits of PK-A were observed by indirect 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 fraction 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 subunit of PK-A. Using various recombinant truncated AKAP82 constructs in a gel overlay assay, the RII subunit-binding domain of this protein was mapped to a 57-amino-acid residue region at its N-terminus. Computer analysis revealed a 14-amino-acid region that resembled the RII-binding domains of other A Kinase Anchor Proteins. A synthetic peptide corresponding to this domain inhibited AKAP82-RII binding in a gel overlay assay, providing further support that AKAP82 is an anchoring protein for the subcellular localization of PK-A in the mouse sperm fibrous sheath. This work, along with previous findings that cAMP is a key intermediary second messenger in regulating protein tyrosine phosphorylation 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, to sperm function.

Haprin, a Novel Haploid Germ Cell-specific RING Finger Protein Involved in the Acrosome Reaction

“…Subcellular Fractionation of Sperm-Subcellular fractionation of sperm was performed according to the previously described method (25,26) with minor modifications. All buffers contained a protease inhibitor mixture (Sigma).…”

Single Amino Acids Determine Specificity of Binding of Protein Kinase A Regulatory Subunits by Protein Kinase A Anchoring Proteins

“…A of FSC1/AKAP82 binds both RI␣ and RII␣ (24,26). This domain was analyzed next to determine the features responsible for the RI␣/RII␣ dual binding capabilities and to relate them to the features of RI␣-specific domain B and the RII␣-specific Ht31 domain.…”

“…4). Domain A contains an Ala, but it is located at the C terminus of the putative RII binding domain (26) and seems unlikely to determine binding specificity (Fig. 3a).…”

Characterization of 3-hydroxyisobutyrate dehydrogenase, HIBADH, as a sperm-motility marker

“…For example, sperm motility is regulated by the cAMP-dependent phosphorylation of flagellar proteins in mammals. One of the downstream targets of cAMP is proteinkinase A (PK-A), a serine/threonine kinase [11]. Mutations of one isoform of PK-A disrupt sperm motility [12].…”