Protein Kinase A-anchoring Inhibitor Peptides Arrest Mammalian Sperm Motility

doi:10.1074/jbc.272.8.4747

Protein Kinase A-anchoring Inhibitor Peptides Arrest Mammalian Sperm Motility*

^‡ Oregon Regional Primate Research Center, Beaverton, Oregon 97006
the ^§ Promega Corporation, Madison, Wisconsin 53711, and
the ^¶ Veterans Affairs Medical Center and Oregon Health Sciences University, Portland, Oregon 97201

^∥ To whom correspondence should be addressed:
Veterans Affairs Medical Center, Mail Stop 151F, 3710 SW Veterans Hospital Rd., Portland, OR 97201.
Tel.: 503-721-7918; Fax: 503-721-1082; E-mail: carr.daniel_w{at}portland.va.gov

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 (RIIα, RIIβ, and RIβ) 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.

Footnotes

↵** The author is also affiliated with the Dept. of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI 53711.
↵* This research was supported by National Institutes of Health Grants HD30908 (to S. V.) and HD32508 (to D. W. C.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PKA

protein kinase A (cAMP-dependent protein kinase)

R

regulatory

AKAP

protein kinase A-anchoring protein

AIP

anchoring inhibitor peptide

CASMA

computer-automated sperm motility analysis

FMI

forward motility index

CDA

2-chloro-2′-deoxyadenosine

IBMX

isobutylmethylxanthine

H-89

N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide

MOPS

4-morpholinepropanesulfonic acid

HPLC

high pressure liquid chromatography.
↵² S. Vijayaraghavan, K. Trautman, S. A. Goueli, and D. W. Carr, manuscript in preparation.
↵³ S. Vijayaraghan and D. W. Carr, unpublished observations.
↵⁴ S. Vijayaraghavan, G. E. Olson, S. K. NagDas, V. P. Winfrey, and D. W. Carr, manuscript in preparation.
- Received May 23, 1996.
- Revision received September 23, 1996.