Protein Kinase A-anchoring Inhibitor Peptides Arrest Mammalian Sperm Motility

Abstract

Cyclic AMP-dependent protein kinase (PKA) is anchored at specific subcellular sites through the interaction of the regulatory subunit (R) with protein kinase A-anchoring proteins (AKAPs) via an amphipathic helix binding motif. Synthetic peptides containing this amphipathic helix domain competitively disrupt PKA binding to AKAPs and cause a loss of PKA modulation of cellular responses. In this report we use S-Ht31, a cell-permeant anchoring inhibitor peptide, to study the role of PKA anchoring in sperm. Our analysis of three species of mammalian sperm detected three isoforms of PKA (RIIalpha, RIIbeta, and RIbeta) and one 110-kDa AKAP. The addition of S-Ht31 to bovine caudal epididymal sperm inhibits motility in a time- and concentration-dependent manner. A control peptide, S-Ht31-P, identical to S-Ht31 except for a proline for isoleucine substitution to prevent amphipathic helix formation, had no effect on motility. The inhibition of motility by S-Ht31 is reversible but only if calcium is present in the suspension buffer, suggesting a role for PKA anchoring in regulating cellular calcium homeostasis. Surprisingly, inhibition of PKA catalytic activity had little effect on basal motility or motility stimulated by agents previously thought to work via PKA activation. These data suggest that the interaction of the regulatory subunit of PKA with sperm AKAPs, independent of PKA catalytic activity, is a key regulator of sperm motility and that disruption of this interaction using cell-permeable anchoring inhibitor peptides may form the basis of a sperm-targeted contraceptive.

Targeting of Protein Kinase A by Muscle A Kinase-anchoring Protein (mAKAP) Regulates Phosphorylation and Function of the Skeletal Muscle Ryanodine Receptor

“…Our study is the first to demonstrate that mAKAP plays a key role in regulating PKA-dependent changes in RyR1 phosphorylation and Ca 2ϩ efflux from internal stores, thus indicating an important regulatory role of mAKAP in RyR1 function. These findings are consistent with previous studies showing that disruption of the AKAP-RII interaction by the addition or expression of a competing RII-binding peptide decreases PKA-dependent phosphorylation and/or results in change in PKA-dependent cellular function (2,4,9,10).…”

“…For example, PKA anchoring was required in order to maintain AMPA (␣-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid) responsive glutamate receptor currents in cultured hippocampal neurons (8). Both insulin secretion and increased intracellular Ca 2ϩ are inhibited in pancreatic islet cells following treatment with Ht31, and cell-permeant forms of Ht31 inhibit PKA-dependent sperm motility (9,10). In cardiac myocytes, Ht31 reduces isoproterenol-dependent potentiation of Ca 2ϩ current through the L-type Ca 2ϩ channel.…”

Moving to the beat: a review of mammalian sperm motility regulation

“…In support of the importance of AKAP-based protein anchoring to flagellar function, mice containing a targeted mutation of the AKAP4 gene are infertile, in association with severely reduced sperm motility (Miki et al 2002). In addition, specific inhibition of the anchoring of the type II regulatory subunit of PKA to AKAPs in sperm resulted in an arrest of bovine sperm motility (Vijayaraghavan et al 1997a).…”

Teasing out the role of aromatase in the healthy and diseased testis

“…118 In line with this, studies have shown that it is the cAMP/ PKA pathway that controls aromatase expression in spermatozoa. 44,119,120 In vitro studies have shown that in Leydig tumor cell lines, the aberrant aromatase expression does not occur through this cAMP-dependent pathway, rather other transcription factors specifically expressed by the tumor bind the CRE-like motifs and drive PII aromatase transcription in a cAMP-independent manner. 112 In addition to FSH and LH, two other extracellular regulators have been shown to drive PII aromatase gene expression through regulate testicular PII aromatase transcription in vivo.…”