Protein kinase A-anchoring inhibitor peptides arrest mammalian sperm motility.

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.

Signal transduction enzymes such as protein kinases and phosphatases play pivotal roles in mediating cellular responses to a wide variety of stimuli. These enzymes are often targeted to specific substrates or cellular compartments through their interaction with cellular "anchoring proteins" (1). This anchoring or compartmentalization is thought to be critical in determining the specificity of response for a particular stimuli (2)(3)(4). The anchoring of PKA 1 is accomplished by the binding of the regulatory subunit (R) to an amphipathic helix binding motif located within A-kinase anchoring proteins (AKAPs) (5). Synthetic peptides containing an amphipathic helix domain are able to competitively disrupt PKA binding to AKAPs (6). Microinjection of these anchoring inhibitor peptides (AIPs) into neurons or skeletal muscle cells disrupts PKA anchoring and PKA modulation of glutamate receptor channels (7) and voltage-gated calcium channels (8). To facilitate studies of PKA anchoring in cells where microinjection is not feasible, we now have developed membrane-permeable AIPs containing an amino-terminal stearic acid moiety. Similar approaches with fatty acid-peptide conjugates have been used to inhibit protein kinase C and tyrosine kinase activities in intact cells (9,10). To our knowledge, this is the first report of the use of a cellpermeable AIP to disrupt a cAMP mediated response.
Sperm are an excellent model in which to test permeant AIPs. Cyclic AMP is known to mediate the motility of sperm and a variety of other ciliated cells (11)(12)(13). Increases in the level of this nucleotide are associated with the development of motility in the epididymis (13,14). Cell-permeant cAMP analogs, cAMP phosphodiesterase inhibitors, and adenylyl cyclase activators all stimulate motility of sperm from several species (15)(16)(17)(18)(19). The kinetic and metabolic responses to cAMP elevation occur within 5-10 min (15,16). Sperm lack nucleic acid and protein synthetic activity, thereby considerably reducing the possible range of targets of cAMP action. The highly polarized sperm cell has distinct subcellular structures easily distinguished at the light microscopic level. Immunogold staining indicates a predominant localization of type II PKA (RII) to the outer membrane of the mitochondria, which spirals around the proximal flagella (20). A developmentally regulated sperm AKAP (S-AKAP84) is also localized to the sperm mitochondria (21). These data suggest that PKA anchoring might be a key factor in the regulation of sperm motility. In this report we characterize sperm PKA isoforms and AKAPs and show that AIPs, but not PKA inhibitors, are able to totally arrest sperm motility. These data suggest that the interaction of RII with AKAPs, but not PKA catalytic activity, is essential for motility.

EXPERIMENTAL PROCEDURES
Sperm Preparation-Testes from mature bulls with intact tunica were obtained from a local slaughterhouse, and sperm from caput or caudal epididymis were isolated and washed as described previously (22). The sperm were resuspended in buffer A (120 mM NaCl, 10 mM KCl, 10 mM Tris, pH 7.4) supplemented with 10 mM glucose and 10 mg/ml bovine serum albumin for motility measurements. Monkey semen was obtained by electroejaculation and processed by procedures previously reported (23). Human semen were obtained from a fertility clinic at the Oregon Health Sciences University.
Sperm Motility Measurement-Head motility parameters were determined as described previously (24,25). A 3-4-l aliquot of sperm suspension (5 ϫ 10 7 /ml) was loaded onto a counting chamber at 37°C. After bulk fluid movement had subsided, six different locations on the slide were recorded. The videotaped segments were analyzed by a computerized image analysis system (CASMA) as described previously (24,25). This computer system measures several parameters of head motion. In this report we have used the forward motility index (FMI) as a measure of motility. FMI is a product of percent motile (%M, percent of sperm moving at velocity greater that 20 mm/s) and average velocity (V a , the five-point smoothed average of the head positions through at least 20 of the 30 frames analyzed). In most cases both components of FMI were found to increase together.
PKA Activity-PKA was assayed as described previously (26) with minor changes. Whole caput or caudal sperm were treated with 2-chloro-2Ј-deoxyadenosine (50 M) or H-89 (50 M), or both, for 30 min. at 37°C. The sperm were then washed 2 times in an ice-cold homogenization buffer supplemented with protease inhibitors, benzamidine (10 mM), leupeptin (4 g/ml), and L-1-tosylamido-2-phenylethyl chloromethyl ketone (100 M) and sonicated for 1 min. Reaction mixtures (20 l total) contained 250 M Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), 250 M [␥-32 P]ATP, 25 mM Na 3 VO 4 , 50 mM MOPS, pH 7.0, 10 mM MgCl 2 , 0.25 mg/ml bovine serum albumin and where indicated, 10 M cAMP. Assays were initiated by the addition of labeled ATP, incubated for 2 min at 30°C, and stopped by addition of 30 l of 1 N HCl. Twenty l of the reaction was then spotted on phosphocellulose paper followed by three washes in 75 mM phosphoric acid. The papers were then analyzed by Cerenkov counting. All determinations were in quadruplicate.
Preparation of Stearated Peptides-The peptides were synthesized on an automated synthesizer using N-(9-fluorenyl)methoxycarbonyl chemistry employing base-mediated coupling. The activator of choice was either benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate or O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate using diisopropylethylamine as solvent. Stearic acid was added together with an activator of attachment to the free NH 2 terminus of the protected peptide. The progress of the stearation reaction was monitored by ninhydrin, a trifluoroacetic acid test cleave, or by mass spectral or HPLC analysis.
The final stearated peptide product was purified by reverse phase HPLC using a C8 column employing a trifluoracetic acid/acetonitrile buffer system. To identify the correct peak and facilitate the recovery of pure material, the molecular weight confirmation of the stearated material was performed using a time of flight mass spectrometry analyzer. Analytical HPLC traces of the pooled fractions confirmed the expected purity. Pooled fractions were lyophilized to a dry powder under nitrogen. The peptides had the following sequences: S-Ht31, N-stearate-DLIEEAASRIVDAVIEQVKAAGAY; S-Ht31-P, N-stearate-DLIEEAAS-RPVDAVPEQVKAAGAY; S-AKAP79, N-stearate-YETLLIETASSL-VKNAIQLSIE; S-CaNBP, N-stearate-SLKRLVTRRKRSESSKQQ-KPLE; and S-PKI, N-stearate-TYADFIASGRTGRRNAI.
Western Blot and Overlay Assays-The overlay procedure is a modified Western blot procedure. Proteins were separated on SDS-polyacrylamide gel electrophoresis and transferred to Immobilon. After treatment with Blotto to prevent nonspecific binding, radiolabeled RII␣ or RII␤ probes were applied (27). Recombinant RII␣ was produced as described previously (5,6). RII␤ and RI␤ were gifts from Dr. John Scott (Oregon Health Sciences University, Portland, OR). Two different variations of the overlay assay were used. In the first procedure, we used recombinant RII that has been radiolabeled by incubating it with the catalytic subunit of PKA and [ 32 P]ATP. After separation from free[ 32 P]ATP, the 32 P-labeled RII (500,000 cpm/10 ml of Blotto) was incubated with the blocked blot for 4 h followed by washing and autoradiography. In the second procedure, we incubated the blot with cold RI␤ (1 g/10 ml of Blotto) and then washed and incubated it with anti-RI␤ antiserum. After washing again, we incubated with secondary antibody conjugated to horseradish peroxidase. A final wash was followed by development with an enhanced chemiluminescence kit (Renaissance, DuPont NEN). The PKA isoform-specific antibodies were affinity-purified antibodies obtained from Triple Point Biologics (Forest Grove, OR).

RESULTS
Identification of AKAPs and PKA Isoforms in Bovine, Human, and Monkey Sperm-Cyclic AMP is known to stimulate sperm motility in a variety of species. To determine if PKA anchoring is involved in regulating motility, we first identified the PKA isoforms and AKAPs present in mammalian sperm. Immunoblot analysis of sperm proteins with affinity-purified isoform-specific antibodies detected three (RII␣, RII␤, and RIb) of the four known PKA isoforms in bovine, human, and monkey sperm (Fig. 1A). RI␣ was not detected in any of the species under these conditions, although in subsequent experiments using concentrated sperm lysates and lower dilutions of antibodies we were able to detect RI␣ in bovine sperm, suggesting that it does exist in sperm, just at lower concentrations than the other isoforms. The lower M r bands detected in bovine and human sperm with anti-RI␣ antibody might be breakdown products of RI␣. However, the other isoforms showed very little apparent proteolysis, suggesting these bands are probably due to the antibody cross-reacting with unrelated proteins. The fuzziness of the bands detected with the RII␣ and RI␤ antibodies suggests that these proteins may be at least partially phosphorylated.
To determine if these isoforms are associated with the solu- Western blotting for regulatory subunits of PKA using isoform-specific antibodies or for AKAPs using 32 P-labeled RII␣ or RII␤ (described under "Experimental Procedures"). A, identification of PKA isoforms in three different species of sperm using four different antibodies. B, bovine caudal epididymal sperm were lysed and separated into supernatant (S) or pellet (P) fractions by centrifugation at 16,000 ϫ g and probed using anti-RII␣, -RII␤, and -RI␤ antibodies. C, a single predominant AKAP was detected in bovine, human, and monkey sperm using either RII␣ or RII␤ as a probe. The bovine and human AKAPs had relative molecular weights of 110,000 and the monkey was approximately 115,000. The overlay assay also detected the endogenous RII isoforms accounting for the bands observed at approximately 55 kDa. D, disruption of RII binding to sperm AKAPs by anchoring inhibitor peptides. RII overlays were performed in the absence (lane 1) or presence of 20 M Ht31 peptide, S-Ht31 peptide, or S-Ht31-P peptide (lane 2, 3, or 4, respectively). Addition of the peptides inhibited RII binding to AKAP 110 by 100, 85, and 0%, respectively, as determined by densitometric scanning analysis. E, treatment of sperm with S-Ht31 produced a significant translocation of RII␤ but not RII␣. Sperm, following treatment with or without 10 M S-Ht31 for 10 min, were lysed and separated by centrifugation at 16,000 ϫ g and analyzed by Western blotting for RII␣ or RII␤. Bands were quantitated by densitometry. S-Ht31 treatment increased the quantity of RII␤ in the supernatant by an average of 24.6 Ϯ 3.0% (n ϭ 4; p Ͻ 0.05) but had no detectable effect on RII␣.
ble or insoluble fractions, bovine sperm were homogenized, centrifuged at 16,000 ϫ g for 30 min, and subjected to Western analysis (Fig. 1B). Greater than 50% of all three R subunit isoforms are present in the pellet fraction of sperm sonicates, and RI␤ is found almost exclusively in this fraction. These results suggest that all of these isoforms are associated with structural or cytoskeletal elements of the sperm.
Overlay analysis of bovine, human, and monkey sperm using 32 P-labeled RII␣ or RII␤ as probes detected a single dominant AKAP in each species (Fig. 1C). The bovine and human AKAP migrated at M r ϭ 110,000, and the monkey AKAP was slightly larger at 115,000. The bands detected at approximately 55 kDa by the RII␤ probe are probably due to dimerization of the probe with endogenous RII, although it is not clear why these proteins are preferentially binding to RII␤ compared with RII␣. Overlay analysis using RI␤ did not detect any binding proteins (data not shown). These data suggest that a single AKAP in sperm may be responsible for the localization of both RII␣ and RII␤. The fact that RI␤ is clearly present in the particulate fraction but does not interact with denatured proteins on the blot suggests that the overlay method may not be appropriate for detecting AKAPs that interact with RI␤.
All AKAPs identified to date contain an amphipathic helix domain responsible for RII binding (5, 6, 28 -30). A peptide, Ht31, containing an amphipathic helix domain binds to RII and competitively inhibits the interaction of RII with other AKAPs (6). The addition of this anchoring inhibitor peptide to the overlay assay blocked RII binding with AKAP 110 (Fig. 1D), suggesting that this sperm AKAP also contains an amphipathic helix binding domain. A cell-permeable stearated Ht31 counterpart, S-Ht31, also inhibited in vitro binding of RII to AKAP 110, albeit at a reduced potency (85% inhibition compared with 100% by the non-stearated Ht31). The control peptide, S-Ht31-P, which has a proline substitution preventing amphipathic helix formation, had no effect on RII binding.
In order to determine if in situ RII/AKAP interactions are disrupted by the S-Ht31, the control and S-Ht31-treated sperm were fractionated to separate RII bound to AKAPs (particulate) from RII that is soluble. The addition of the anchoring inhibitor peptide caused a significant translocation of RII␤ ( Fig. 1E; 24.6 Ϯ 3.0% increase in soluble RII␤, n ϭ 4, p Ͻ 0.05), suggesting the AIP was capable of disrupting RII␤/AKAP interaction. Although we did not detect any S-Ht31-mediated translocation of RII␣, this may be due to the fact the RII␣ is trapped in the flagellar cytoskeletal meshwork after disruption of the RII␣⅐AKAP complex. Previous studies have shown that RII␣ can only be removed from the flagellum by repeated washes with dithiothreitol and that this solubilization of RII by dithiothreitol is not a rapid process but occurs over a period of several hours (31). They also show that sperm RII binding proteins (AKAPs) are not solubilized under these conditions and suggest that the RII␣ is trapped in a highly disulfide cross-linked structure.
Effect of AIPs on Sperm Motility-To determine the effects of anchoring inhibitor peptides on sperm motility, S-Ht31 was added to vigorously motile sperm under a variety of conditions. S-Ht31 inhibits basal motility in a concentration-and time-dependent manner. A complete arrest of motility was observed at concentrations from 5 to 50 M within 3-5 min after treatment ( Fig. 2A). When motility is measured at 5 min post-treatment, the concentration of S-Ht31 needed to produce 50% inhibition is approximately 1 M (Fig. 2B). A control peptide, S-Ht31-P, which is ineffective in disrupting PKA anchoring to AKAPs (see Fig. 1D), had no effect on sperm motility at concentrations of up to 100 M, suggesting that motility inhibition by S-Ht31 was due to a disruption of PKA anchoring.
To test the specificity of the stearated peptides, three other stearated peptides were synthesized. Two of these peptides were based on sequences from AKAP79. The first peptide (S-AKAP79) contained the amphipathic helix RII binding domain (28) and the second (S-CaNBP) contained the domain that binds to protein phosphatase 2B (calcineurin) but should not bind to RII (32). The third peptide (S-PKI) contained the sequence of the protein kinase A inhibitor peptide that should interact with the catalytic subunit of PKA but not the regula-

FIG. 2. Anchoring inhibitor peptides arrest bovine sperm motility in a time-and dose-dependent manner.
A, sperm were incubated in buffer A containing S-Ht31 at 5 (q), 10 (f), or 50 (ࡗ) M or a control peptide, S-Ht31-P at 50 M (ϫ). Motility was assessed at the times indicated by CASMA as described under "Experimental Procedures." B, sperm were incubated for 5 min in buffer A containing increasing concentrations of anchoring inhibitor peptide S-Ht31 (ࡗ) or control peptide S-Ht31-P (å), and motility was assessed. C, sperm were incubated for 5 min in buffer A containing increasing concentrations of anchoring inhibitor peptide S-AKAP79 (circf) or control peptides S-CaNBP (f) or S-PKI (å), and motility was assessed. tory subunit (33). Only the peptide containing the amphipathic helix RII binding motif (S-AKAP79) inhibited sperm motility (Fig. 2C). The S-CaNBP and S-PKI peptides inhibited the activity of calcineurin and PKA, respectively, 2 but had no effect on motility. The reduced potency of S-AKAP79 compared with S-Ht31 might be due to the fact that this peptide was very insoluble in aqueous solution. The peptide would only dissolve in 100% dimethyl sulfoxide, and therefore its final concentration after being diluted into aqueous buffer is uncertain. Equivalent amounts of dimethyl sulfoxide were added to control sperm without effects on motility. These data are consistent with a model where RII/AKAP interaction is required for sperm motility.
To determine if S-Ht31 is effecting the viability or structural integrity of the sperm, the vital dyes SYBR-green and rhodamine 123 were added to sperm before and after treatment with S-Ht31. Only viable, intact cells will take up these dyes. Both treated and control sperm accumulated these dyes to the same extent, suggesting that the stearated peptide did not decrease viability or disrupt the integrity of the sperm plasma membrane. In experiments using sperm loaded with the chromophores 2Ј,7Ј-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein or Fura 2, the addition of digitonin but not S-Ht31 caused the release of these dyes, confirming that peptide treatment did not compromise the integrity of the plasma membrane (data not shown).
Perhaps the best evidence supporting the viability of the S-Ht31-treated sperm is that, under certain conditions, the sperm are able to regain full motility. Monitoring motility beyond 5 min of S-Ht31 treatment showed that sperm kinetic activity recovered spontaneously over 30 -60 min period (Fig.  3A). This recovery occurred only in sperm suspended in calcium containing medium. In medium depleted of external calcium by EGTA, S-Ht31 caused irreversible motility arrest. The motility of untreated sperm was unaffected in the presence or absence of calcium.
Bicarbonate ion has been shown to stimulate sperm adenylyl cyclase (34) and increase intracellular pH (22) and is an essential component of suspension buffers required for optimal sperm function in vitro (35,36). Sperm motility in the presence of bicarbonate is significantly enhanced compared with untreated sperm (Fig. 3B. To determine the effect of AIPs on stimulated sperm, S-Ht31 was added to sperm in both basal and bicarbonate-supplemented media (Fig. 3B). The peptide was equally effective in inhibiting motility in both media. Similar results were obtained when sperm were pretreated with other activators of motility, dibutyryl cAMP, IBMX, and CDA, all thought to increase cAMP content (data not shown). The peptide also caused inhibition of motility when added to undiluted rhesus monkey and human ejaculated semen. 3 Role of PKA in Sperm Motility and S-Ht31 Action-If the effect of anchoring inhibitor peptides on sperm motility is due to the dissociation of the catalytic subunit of PKA from its preferred substrates then this effect should be mimicked by inhibitors of PKA activity. However, the addition of up to 100 M S-PKI to sperm had no effect on motility (Fig. 2C). The addition of high levels of another PKA inhibitor, H-89 (50 M), inhibited basal sperm motility approximately 50% or less and had no effect on sperm motility stimulated by the adenosine analog CDA (Fig. 4A). Essentially identical data were obtained when sperm were stimulated with IBMX or 8-bromo-cAMP instead of CDA. This unexpectedly weak action of H-89 apparently results primarily from a decrease in motility of a sub-population of sperm because a considerable proportion of sperm maintains vigorous motility. This contrasts sharply with the complete arrest of motility seen in S-Ht31-treated sperm. Also, unlike the action of AIPs, H-89 had no effect on CDA-stimulated sperm. The effectiveness of H-89 was also observed with caput sperm, which are immotile unless stimulated with agents such as CDA or IBMX. Motility induction by CDA is unaffected by preincubating sperm with H-89 before treatment (Fig. 4A). Together these observations suggest that AIPs and PKA inhibitors have different mechanisms of action.
In previous reports, initiation or stimulation of sperm motility by CDA was associated with an elevation of cAMP, and an increase in PKA activity was assumed (37). Because H-89 failed to suppress stimulation of motility by CDA, we measured PKA activity from caudal and caput sperm that had been treated with CDA, H-89, or CDA ϩ H-89 (Fig. 4B). All assays were performed in the absence or presence of cAMP to determine the basal and maximal PKA activities. CDA increased basal PKA activity by approximately 60% in both caudal and caput sperm. H-89 treatment, in the presence or absence of CDA, strongly inhibited PKA activity. The addition of cAMP to the assay was not able to overcome this inhibition. To ensure that H-89 was affecting PKA activity in the cells and not just its activity in the in vitro assay, the sperm were washed several times before lysis to remove all exogenous H-89. As a control, the non-permeable PKA inhibitor peptide (50 M) was added to sperm which then were washed, lysed, and assayed for PKA activity in a manner identical to the H-89 treated sperm. PKA inhibitor peptide had no effect on cellular PKA activity, al- though a similar concentration, when added to the PKA assay, will inhibit virtually 100% of activity (data not shown). These data suggest that motility stimulation associated with treatments that elevate cAMP does not require increases in the catalytic activity of PKA and can occur even if PKA activity is substantially inhibited.

DISCUSSION
As an initial step in studying the role of PKA anchoring in regulation of sperm function, we first identified PKA isoforms and AKAPs that are present in mammalian sperm. Three of the four isoforms of the regulatory subunit of PKA were detected in bovine, human, and monkey sperm. RI␣ was only detected when higher concentrations of antibody were used, suggesting that this isoform may be less prevalent than the other isoforms. Consistent with previous reports (31), the major proportion of all isoforms remained in the particulate fraction, presumably due to their interaction with AKAPs. Bovine, human, and monkey sperm all contain one predominant AKAP with a relative molecular weight of 110,000 -115,000. The AKAPs predominantly localize to the particulate fraction and bind both RII␣ and RII␤, suggesting that a single AKAP is responsible for the localization of both these RII isoforms. Overlay assays using RI␤ as a probe did not detect any binding protein in sperm. Preliminary analysis at the electron microscope level detects RII␤ in both the head and tail while RII␣ is found almost exclusively associated with the flagellum. 4 In concordance with our observations, Orr and colleagues (31) reported one predominant AKAP at M r of approximately 120,000 in bovine sperm (31), and Rubin and colleagues (21) found a single RII␤ AKAP (ϳ120 kDa) in mature mouse sperm. Others, however, have reported RII␣ AKAPs of 82 kDa in mouse (38), 80 kDa in rat (31), and 72 kDa in human (39). The reason for these differences is not clear, although proteolysis is one possibility. We find that prolonged storage of SDS-solubilized extracts leads to the loss of the 110-kDa band and the appearance of an 80-kDa band. The 72-kDa RII␣ binding band reported in human sperm (39) is distinct from all other known AKAPs due to the fact that it was detected using a peptide from RII␣ (45-75) that does not contain the AKAP binding domain (40 -43). Finally, a recent report has described the cloning of a sperm AKAP of 84 kDa uniquely expressed during spermatogenesis (21). Intriguingly, this AKAP is not found in mature sperm and thus its relationship, if any, to AKAP 110 in mature sperm is unknown. To date, only sperm cells have been shown to contain an AKAP of 110 kDa. If it turns out that AKAP 110 is unique to sperm, this protein could possibly be used as a target for a male contraceptive.
Several previous reports have suggested that flagellar activity in sperm is regulated by cAMP (12, 44 -46). Both PKA and AKAPs have been shown to be located at the same subcellular site in sperm in the outer mitochondrial membrane located on the proximal flagella, the sperm "midpiece" (20,21). Our study is the first to investigate the physiological role of PKA anchoring in sperm function. The addition of cell-permeable anchoring inhibitor peptides dramatically inhibits sperm motility in a dose-and time-dependent manner. This inhibition is reversible, but only in the presence of external calcium, suggesting that the regulatory mechanism being disrupted may involve the maintenance of calcium homeostasis or a calcium-regulated function that recovers only in the presence of exogenous calcium. Since the mitochondria plays an important role in sperm calcium homeostasis (47)(48)(49)(50)(51), it is possible that disruption of RII anchoring in this region could be responsible for the changes in sperm Ca 2ϩ regulation. The control peptide, S-Ht31-P, had no effect on motility, suggesting that motility inhibition by S-Ht31 and S-AKAP79 was not due to the stearate moiety but due to the disruption of PKA anchoring. Although we cannot prove that the anchoring inhibitor peptides arrest motility by disrupting RII/AKAP interaction instead of another protein/protein interaction, evidence supporting this hypothesis include the following: 1) S-Ht31 disrupts RII/AKAP interaction in vitro, 2) the addition of S-Ht31 to sperm causes a translocation of RII␤ from the particulate to the soluble fraction, and 3) two different PKA-anchoring inhibitor peptides with totally unique sequences arrest motility while three other stearated peptides, including the S-Ht31-P control peptide, had no effect on motility. Reversibility of the motility inhibition and the observations that S-Ht31-treated sperm take up vital dyes provide evidence that the motility inhibition is not due to disruption of sperm plasma membrane integrity. The simplest model consistent with these data is that the interaction of the regulatory subunit of PKA with AKAP 110 is essential for sperm movement.
It is usually assumed that the main function of PKA/AKAP interaction is to anchor the catalytic subunit at a preferential subcellular site for specific phosphorylation of protein substrates in the vicinity. This model is supported by studies showing that microinjection of AIPs into neuronal or muscle cells mimics the effect of PKA inhibitors, causing a loss of cAMP modulation of the glutamate receptor and voltage-gated Ca 2ϩ channels (7,8). In sperm, however, inhibition of the catalytic subunit of PKA does not mimic the effect of AIPs. Our studies now document that under conditions where sperm PKA is clearly inhibited, compounds that elevate cAMP such as CDA, IBMX, and 8-bromo-cAMP still induce or stimulate sperm motility. The lack of effect of S-PKI and H-89, even at concentrations as high as 100 M, on cAMP-mediated induction and stimulation of motility is quite unequivocal. What then is the role of cAMP, independent of PKA activity, in sperm motility? Our data using anchoring inhibitor peptides provides a possible answer: that the interaction of the regulatory subunit with sperm AKAPs has regulatory actions independent of the catalytic subunit. The interaction between RII and other sperm proteins may also be cAMP-dependent, since cAMP is known to produce a conformational change in the regulatory subunit. Others have also suggested that the regulatory subunit acts independently of the catalytic subunit (52). It is known that RII can inhibit phosphatases (53), and preliminary data from our laboratory show that RII will inhibit sperm PP1. 2 An independent role of RII in the regulation of sperm motility has also been suggested by reports showing that the addition of axokinin (later shown to be RII (54,55)) to demembraned sperm was sufficient to induce motility (56 -58).
The regulation of sperm motility may involve interactions between the RII⅐AKAP complex and other sperm proteins. The demonstration that phosphatase PP2B, PKA, and protein kinase C are all anchored to the same AKAP in neurons (59) opens up several possible, previously unidentified roles for the individual members of this multimeric complex. Clearly, the isolation and characterization of the sperm AKAP and its associated proteins are required for further insights into their possible functions. Whatever the biochemical pathways controlled by the sperm RII anchoring complex may be, our data suggest that RII anchoring, independent of PKA catalytic activity, is essential for sperm motility and that cell-permeable AIPs are a useful tool for studying PKA anchoring in cellular function.