|
|
|
|||||||
|
|||||||||
(Received for publication, May 23, 1996, and in revised form, September 23, 1996)
From the 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 (RII INTRODUCTION 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 (2-4). The anchoring
of PKA1 is accomplished by the binding of
the regulatory subunit (R) to an amphipathic helix binding motif
located within 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 (11-13). 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 (15-19). 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 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.
Head motility parameters were
determined as described previously (24, 25). A 3-4-µl aliquot of
sperm suspension (5 × 107/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 (Va, 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 was assayed as described previously (26)
with minor changes. Whole caput or caudal sperm were treated with
2-chloro-2 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
NH2 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-DLIEEAASRPVDAVPEQVKAAGAY; S-AKAP79,
N-stearate-YETLLIETASSLVKNAIQLSIE; S-CaNBP,
N-stearate-SLKRLVTRRKRSESSKQQKPLE; and S-PKI,
N-stearate-TYADFIASGRTGRRNAI.
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 RESULTS 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
To determine if these isoforms are associated with the soluble or
insoluble fractions, bovine sperm were homogenized, centrifuged at
16,000 × 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 Overlay analysis of bovine, human, and monkey sperm using
32P-labeled RII 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 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 µM 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 µM (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 µM, 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 regulatory
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 2 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
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 µM S-PKI to
sperm had no effect on motility (Fig. 2C). The addition of high levels
of another PKA inhibitor, H-89 (50 µM), 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 subpopulation 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 + 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 µM) 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, although 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 In concordance with our observations, Orr and colleagues (31) reported
one predominant AKAP at Mr of approximately
120,000 in bovine sperm (31), and Rubin and colleagues (21) found a
single RII 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
(47-51), it is possible that disruption of RII anchoring in this
region could be responsible for the changes in sperm Ca2+
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 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
Ca2+ 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 µM, 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
RII·AKAP 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.
FOOTNOTES Acknowledgments We acknowledge the excellent technical
expertise of Kevin Trautman and Greg Liberty. We would also like to
thank Dr. Donner Babcock for insightful discussions. This publication
is No. 2022 from the Oregon Regional Primate Research Center.
REFERENCES
Volume 272, Number 8,
Issue of February 21, 1997
pp. 4747-4752
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,
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
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
, RII
, and RI) 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.
-
inase 
nchoring
roteins (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 cell-permeable AIP to disrupt a cAMP mediated response.
-deoxyadenosine (50 µM) or H-89 (50 µM), 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 µg/ml), and L-1-tosylamido-2-phenylethyl chloromethyl
ketone (100 µM) and sonicated for 1 min. Reaction
mixtures (20 µl total) contained 250 µM
Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), 250 µM [
-32P]ATP, 25 mM
Na3VO4, 50 mM MOPS, pH 7.0, 10 mM MgCl2, 0.25 mg/ml bovine serum albumin and
where indicated, 10 µM cAMP. Assays were initiated by the
addition of labeled ATP, incubated for 2 min at 30 °C, and stopped
by addition of 30 µl of 1 N HCl. Twenty µl 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.
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
[32P]ATP. After separation from
free[32P]ATP, the 32P-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 µg/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).
, 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 Mr 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.
Fig. 1.
Identification of PKA subunits and AKAPs in
mammalian sperm. Sperm from bovine (B), human
(h), and monkey (M) were lysed, and the proteins
were separated by SDS-PAGE and analyzed by Western blotting for
regulatory subunits of PKA using isoform-specific antibodies or for
AKAPs using 32P-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 × 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 µM 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 µM S-Ht31
for 10 min, were lysed and separated by centrifugation at 16,000 × 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 ± 3.0% (n = 4; p < 0.05) but had no
detectable effect on RII.
[View Larger Version of this Image (77K GIF file)]
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.
or RII as probes detected a single
dominant AKAP in each species (Fig. 1C). The bovine and
human AKAP migrated at Mr = 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.
(Fig. 1E; 24.6 ± 3.0% increase in soluble RII, n = 4, p < 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·AKAP 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.
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 (
), 10 (
), or 50 (
) µM or a control peptide,
S-Ht31-P at 50 µM (×). 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 () or control peptide S-Ht31-P (
), 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 () or S-PKI (), and
motility was assessed.
[View Larger Version of this Image (17K GIF file)]
,7-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).
Fig. 3.
Reactivation of S-Ht31 inhibited motility in
the presence of calcium and bicarbonate. A, bovine caudal
epididymal sperm were suspended in buffer A supplemented with 2 mM calcium (+ Calcium) or 2 mM EGTA
(
Calcium). Motility was assessed by CASMA (as described under "Experimental Procedures") at 5, 30, and 60 min following the
addition of either water (control) or 10 µM S-Ht31.
B, sperm were incubated in buffer A containing 2 mM calcium and 50 mM bicarbonate (where
indicated). Motility was assessed at 5, 30, and 60 min following the
addition of either water (control) or 10 µM S-Ht31.
[View Larger Version of this Image (44K GIF file)]
Fig. 4.
Effect of the protein kinase inhibitor H-89
on sperm motility and PKA activity from control and CDA activated
sperm. A, bovine caudal and caput sperm were incubated in
buffer A in the presence or absence of 50 µM CDA and/or
50 µM H-89. When sperm were treated with both CDA and
H-89, the H-89 was added 15 min prior to CDA. Motility was assessed by
CASMA (as described under "Experimental Procedures") at 15 min
following treatment. B, bovine sperm were incubated as
described for A and then assessed for PKA activity as
described under "Experimental Procedures." Where indicated, 10 µM cAMP was added to the PKA assay.
[View Larger Version of this Image (25K GIF file)]
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
AKAP (~120 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.
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.
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.
**
The author is also affiliated with the Dept. of Pathology and
Laboratory Medicine, University of Wisconsin Medical School, Madison,
WI 53711.
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.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  O. M. Faruque, D. Le-Nguyen, A.-D. Lajoix, E. Vives, P. Petit, D. Bataille, and E.-H. Hani Cell-permeable peptide-based disruption of endogenous PKA-AKAP complexes: a tool for studying the molecular roles of AKAP-mediated PKA subcellular anchoring Am J Physiol Cell Physiol, February 1, 2009; 296(2): C306 - C316. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Puri, K. Myers, D. Kline, and S. Vijayaraghavan Proteomic Analysis of Bovine Sperm YWHA Binding Partners Identify Proteins Involved in Signaling and Metabolism Biol Reprod, December 1, 2008; 79(6): 1183 - 1191. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Wang, Y. Yang, and P. A. Friedman Na/H Exchange Regulatory Factor 1, a Novel AKT-associating Protein, Regulates Extracellular Signal-regulated Kinase Signaling through a B-Raf-Mediated Pathway Mol. Biol. Cell, April 1, 2008; 19(4): 1637 - 1645. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E Fiedler, M. Bajpai, and D. W Carr Identification and Characterization of RHOA-Interacting Proteins in Bovine Spermatozoa Biol Reprod, January 1, 2008; 78(1): 184 - 192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nie, C. B. McDonough, T. Huang, P. V. Nguyen, and T. Abel Genetic Disruption of Protein Kinase A Anchoring Reveals a Role for Compartmentalized Kinase Signaling in Theta-Burst Long-Term Potentiation and Spatial Memory J. Neurosci., September 19, 2007; 27(38): 10278 - 10288. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Burton and G. S. McKnight PKA, Germ Cells, and Fertility Physiology, February 1, 2007; 22(1): 40 - 46. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Kaidanovich-Beilin and H. Eldar-Finkelman Peptides Targeting Protein Kinases: Strategies and Implications. Physiology, December 1, 2006; 21(6): 411 - 418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Gardner, S. J. Tavalin, A. S. Goehring, J. D. Scott, and S. W. Bahouth AKAP79-mediated Targeting of the Cyclic AMP-dependent Protein Kinase to the beta1-Adrenergic Receptor Promotes Recycling and Functional Resensitization of the Receptor J. Biol. Chem., November 3, 2006; 281(44): 33537 - 33553. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lavialle-Defaix, H. Gautier, A. Defaix, B. Lapied, and F. Grolleau Differential Regulation of Two Distinct Voltage-Dependent Sodium Currents by Group III Metabotropic Glutamate Receptor Activation in Insect Pacemaker Neurons J Neurophysiol, November 1, 2006; 96(5): 2437 - 2450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Warwar, S. Efendic, C.-G. Ostenson, E. P. Haber, E. Cerasi, and R. Nesher Dynamics of Glucose-Induced Localization of PKC Isoenzymes in Pancreatic {beta}-Cells: Diabetes-Related Changes in the GK Rat Diabetes, March 1, 2006; 55(3): 590 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bajpai, S. E. Fiedler, Z. Huang, S. Vijayaraghavan, G. E. Olson, G. Livera, M. Conti, and D. W. Carr AKAP3 Selectively Binds PDE4A Isoforms in Bovine Spermatozoa Biol Reprod, January 1, 2006; 74(1): 109 - 118. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Yang and P. Yang The Flagellar Motility of Chlamydomonas pf25 Mutant Lacking an AKAP-binding Protein Is Overtly Sensitive to Medium Conditions Mol. Biol. Cell, January 1, 2006; 17(1): 227 - 238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Lynch, G. S. Baillie, A. Mohamed, X. Li, C. Maisonneuve, E. Klussmann, G. van Heeke, and M. D. Houslay RNA Silencing Identifies PDE4D5 as the Functionally Relevant cAMP Phosphodiesterase Interacting with {beta}Arrestin to Control the Protein Kinase A/AKAP79-mediated Switching of the {beta}2-Adrenergic Receptor to Activation of ERK in HEK293B2 Cells J. Biol. Chem., September 30, 2005; 280(39): 33178 - 33189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Jones and Y. Sharief Asymmetrical protein kinase A activity establishes neutrophil cytoskeletal polarity and enables chemotaxis J. Leukoc. Biol., July 1, 2005; 78(1): 248 - 258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Jungmann and O. Kiryukhina Cyclic AMP and AKAP-mediated Targeting of Protein Kinase A Regulates Lactate Dehydrogenase Subunit A mRNA Stability J. Biol. Chem., July 1, 2005; 280(26): 25170 - 25177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G Buffone, J. C Calamera, S. V Verstraeten, and G. F Doncel Capacitation-associated protein tyrosine phosphorylation and membrane fluidity changes are impaired in the spermatozoa of asthenozoospermic patients Reproduction, June 1, 2005; 129(6): 697 - 705. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Luconi, I. Porazzi, P. Ferruzzi, S. Marchiani, G. Forti, and E. Baldi Tyrosine Phosphorylation of the A Kinase Anchoring Protein 3 (AKAP3) and Soluble Adenylate Cyclase Are Involved in the Increase of Human Sperm Motility by Bicarbonate Biol Reprod, January 1, 2005; 72(1): 22 - 32. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. NagDas, V. P. Winfrey, and G. E. Olson Tyrosine Phosphorylation Generates Multiple Isoforms of the Mitochondrial Capsule Protein, Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx), During Hamster Sperm Capacitation Biol Reprod, January 1, 2005; 72(1): 164 - 171. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Y. Rawe, J. Ramalho-Santos, C. Payne, H. E. Chemes, and G. Schatten WAVE1, an A-kinase anchoring protein, during mammalian spermatogenesis Hum. Reprod., November 1, 2004; 19(11): 2594 - 2604. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Stokes, L. M.N. Shimoda, M. Koblan-Huberson, C. N. Adra, and H. Turner A TRPV2-PKA Signaling Module for Transduction of Physical Stimuli in Mast Cells J. Exp. Med., July 19, 2004; 200(2): 137 - 147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Huang, K. Myers, B. Khatra, and S. Vijayaraghavan Protein 14-3-3{zeta} Binds to Protein Phosphatase PP1{gamma}2 in Bovine Epididymal Spermatozoa Biol Reprod, July 1, 2004; 71(1): 177 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Liu, J.-Y. Hu, S. Schacher, and J. H. Schwartz The Two Regulatory Subunits of Aplysia cAMP-Dependent Protein Kinase Mediate Distinct Functions in Producing Synaptic Plasticity J. Neurosci., March 10, 2004; 24(10): 2465 - 2474. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Luconi, V. Carloni, F. Marra, P. Ferruzzi, G. Forti, and E. Baldi Increased phosphorylation of AKAP by inhibition of phosphatidylinositol 3-kinase enhances human sperm motility through tail recruitment of protein kinase A J. Cell Sci., March 1, 2004; 117(7): 1235 - 1246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. TASKEN and E. M. AANDAHL Localized Effects of cAMP Mediated by Distinct Routes of Protein Kinase A Physiol Rev, January 1, 2004; 84(1): 137 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Turner Tales From the Tail: What Do We Really Know About Sperm Motility? J Androl, November 1, 2003; 24(6): 790 - 803. [Full Text] [PDF]
|
|
|
|
|
|
S. Aquila, D. Sisci, M. Gentile, A. Carpino, E. Middea, S. Catalano, V. Rago, and S. Ando Towards a physiological role for cytochrome P450 aromatase in ejaculated human sperm Hum. Reprod., August 1, 2003; 18(8): 1650 - 1659. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. K. Carnegie and J. D. Scott A-kinase anchoring proteins and neuronal signaling mechanisms Genes & Dev., July 1, 2003; 17(13): 1557 - 1568. [Full Text] [PDF]
|
|
|
|
 |
|
 N. M. Alto, S. H. Soderling, N. Hoshi, L. K. Langeberg, R. Fayos, P. A. Jennings, and J. D. Scott Bioinformatic design of A-kinase anchoring protein-in silico: A potent and selective peptide antagonist of type II protein kinase A anchoring PNAS, April 15, 2003; 100(8): 4445 - 4450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Burns-Hamuro, Y. Ma, S. Kammerer, U. Reineke, C. Self, C. Cook, G. L. Olson, C. R. Cantor, A. Braun, and S. S. Taylor Designing isoform-specific peptide disruptors of protein kinase A localization PNAS, April 1, 2003; 100(7): 4072 - 4077. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Huang, B. Khatra, M. Bollen, D. W. Carr, and S. Vijayaraghavan Sperm PP1{gamma}2 Is Regulated by a Homologue of the Yeast Protein Phosphatase Binding Protein sds221 Biol Reprod, December 1, 2002; 67(6): 1936 - 1942. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Aquila, D. Sisci, M. Gentile, E. Middea, L. Siciliano, and S. Ando Human Ejaculated Spermatozoa Contain Active P450 Aromatase J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3385 - 3390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Jones Protein kinase A regulates {beta}2 integrin avidity in neutrophils J. Leukoc. Biol., June 1, 2002; 71(6): 1042 - 1048. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. K. Rathee, C. Distler, O. Obreja, W. Neuhuber, G. K. Wang, S.-Y. Wang, C. Nau, and M. Kress PKA/AKAP/VR-1 Module: A Common Link of Gs-Mediated Signaling to Thermal Hyperalgesia J. Neurosci., June 1, 2002; 22(11): 4740 - 4745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Anway, N. Ravindranath, M. Dym, and M. D. Griswold Identification of a Murine Testis Complementary DNA Encoding a Homolog to Human A-Kinase Anchoring Protein-Associated Sperm Protein Biol Reprod, June 1, 2002; 66(6): 1755 - 1761. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. O. Williams Cutting Edge: A-Kinase Anchor Proteins Are Involved in Maintaining Resting T Cells in an Inactive State J. Immunol., June 1, 2002; 168(11): 5392 - 5396. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K Cho, M W Brown, and Z I Bashir Mechanisms and physiological role of enhancement of mGlu5 receptor function by group II mGlu receptor activation in rat perirhinal cortex J. Physiol., May 1, 2002; 540(3): 895 - 906. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. R. Kelemen, K. Hsiao, and S. A. Goueli Selective in Vivo Inhibition of Mitogen-activated Protein Kinase Activation Using Cell-permeable Peptides J. Biol. Chem., March 1, 2002; 277(10): 8741 - 8748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Chaudhry, C. Zhang, and J. G. Granneman Characterization of RIIbeta and D-AKAP1 in differentiated adipocytes Am J Physiol Cell Physiol, January 1, 2002; 282(1): C205 - C212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y Hayabuchi, C Dart, and N B Standen Evidence for involvement of A-kinase anchoring protein in activation of rat arterial KATP channels by protein kinase A J. Physiol., October 15, 2001; 536(2): 421 - 427. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Xie and J.-P. Raufman Association of protein kinase A with AKAP150 facilitates pepsinogen secretion from gastric chief cells Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G1051 - G1058. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Belyamani, E. A. Gangolli, and R. L. Idzerda Reproductive Function in Protein Kinase Inhibitor-Deficient Mice Mol. Cell. Biol., June 15, 2001; 21(12): 3959 - 3963. [Abstract] [Full Text]
|
|
|
|
 |
|
 A. R. Gaillard, D. R. Diener, J. L. Rosenbaum, and W. S. Sale Flagellar Radial Spoke Protein 3 Is an A-Kinase Anchoring Protein (AKAP) J. Cell Biol., April 16, 2001; 153(2): 443 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Reinton, S. Ørstavik, T. B. Haugen, T. Jahnsen, K. Taskén, and B. S. Skålhegg A Novel Isoform of Human Cyclic 3',5'-Adenosine Monophosphate-Dependent Protein Kinase, C{alpha}-s, Localizes to Sperm Midpiece Biol Reprod, August 1, 2000; 63(2): 607 - 611. [Abstract] [Full Text]
|
|
|
|
|
|
S. Vijayaraghavan, J. Mohan, H. Gray, B. Khatra, and D. W. Carr A Role for Phosphorylation of Glycogen Synthase Kinase-3{alpha} in Bovine Sperm Motility Regulation Biol Reprod, June 1, 2000; 62(6): 1647 - 1654. [Abstract] [Full Text]
|
|
|
|
|
|
F. Sun, M. J. Hug, N. A. Bradbury, and R. A. Frizzell Protein Kinase A Associates with Cystic Fibrosis Transmembrane Conductance Regulator via an Interaction with Ezrin J. Biol. Chem., May 5, 2000; 275(19): 14360 - 14366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.A. Harrison, D.W. Carr, and S. Meizel Involvement of Protein Kinase A and A Kinase Anchoring Protein in the Progesterone-Initiated Human Sperm Acrosome Reaction Biol Reprod, March 1, 2000; 62(3): 811 - 820. [Abstract] [Full Text]
|
|
|
|
|
|
K. Yuasa, K. Omori, and N. Yanaka Binding and Phosphorylation of a Novel Male Germ Cell-specific cGMP-dependent Protein Kinase-anchoring Protein by cGMP-dependent Protein Kinase Ialpha J. Biol. Chem., February 18, 2000; 275(7): 4897 - 4905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A Fujita, K Nakamura, T Kato, N Watanabe, T Ishizaki, K Kimura, A Mizoguchi, and S Narumiya Ropporin, a sperm-specific binding protein of rhophilin, that is localized in the fibrous sheath of sperm flagella J. Cell Sci., January 1, 2000; 113(1): 103 - 112. [Abstract] [PDF]
|
|
|
|
 |
|
 K. L. Dodge, D. W. Carr, C. Yue, and B. M. Sanborn A Role for AKAP (A Kinase Anchoring Protein) Scaffolding in the Loss of a Cyclic Adenosine 3',5'-Monophosphate Inhibitory Response in Late Pregnant Rat Myometrium Mol. Endocrinol., December 1, 1999; 13(12): 1977 - 1987. [Abstract] [Full Text]
|
|
|
|
 |
|
 K. L. Dodge, D. W. Carr, and B. M. Sanborn Protein Kinase A Anchoring to the Myometrial Plasma Membrane Is Required for Cyclic Adenosine 3',5'-Monophosphate Regulation of Phosphatidylinositide Turnover Endocrinology, November 1, 1999; 140(11): 5165 - 5170. [Abstract] [Full Text]
|
|
|
|
|
|
A. Mandal, S. Naaby-Hansen, M. J. Wolkowicz, K. Klotz, J. Shetty, J. D. Retief, S. A. Coonrod, M. Kinter, N. Sherman, F. Cesar, et al. FSP95, A Testis-Specific 95-Kilodalton Fibrous Sheath Antigen That Undergoes Tyrosine Phosphorylation in Capacitated Human Spermatozoa Biol Reprod, November 1, 1999; 61(5): 1184 - 1197. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. M.O. Turner, R. L.M. Eriksson, G. L. Gerton, and S. B. Moss Relationship between sperm motility and the processing and tyrosine phosphorylation of two human sperm fibrous sheath proteins, pro-hAKAP82 and hAKAP82 Mol. Hum. Reprod., September 1, 1999; 5(9): 816 - 824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Burton, B. Treash-Osio, C. H. Muller, E. L. Dunphy, and G. S. McKnight Deletion of Type IIalpha Regulatory Subunit Delocalizes Protein Kinase A in Mouse Sperm without Affecting Motility or Fertilization J. Biol. Chem., August 20, 1999; 274(34): 24131 - 24136. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Moss, R. M.O. Turner, K. L. Burkert, H. VanScoy Butt, and G. L. Gerton Conservation and Function of a Bovine Sperm A-Kinase Anchor Protein Homologous to Mouse AKAP82 Biol Reprod, August 1, 1999; 61(2): 335 - 342. [Abstract] [Full Text]
|
|
|
|
|
|
Y. Si and M. Okuno Role of Tyrosine Phosphorylation of Flagellar Proteins in Hamster Sperm Hyperactivation Biol Reprod, July 1, 1999; 61(1): 240 - 246. [Abstract] [Full Text]
|
|
|
|
|
|
S. Vijayaraghavan, G. A. Liberty, J. Mohan, V. P. Winfrey, G. E. Olson, and D. W. Carr Isolation and Molecular Characterization of AKAP110, a Novel, Sperm-Specific Protein Kinase A-Anchoring Protein Mol. Endocrinol., May 1, 1999; 13(5): 705 - 717. [Abstract] [Full Text]
|
|
|
|
|
|
M. Salanova, S.-Y. Chun, S. Iona, C. Puri, M. Stefanini, and M. Conti Type 4 Cyclic Adenosine Monophosphate-Specific Phosphodiesterases Are Expressed in Discrete Subcellular Compartments during Rat Spermiogenesis Endocrinology, May 1, 1999; 140(5): 2297 - 2306. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. S. TASH and G. E. BRACHO Microgravity alters protein phosphorylation changes during initiation of sea urchin sperm motility FASEB J, May 1, 1999; 13(9001): 43 - 54. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Klussmann, K. Maric, B. Wiesner, M. Beyermann, and W. Rosenthal Protein Kinase A Anchoring Proteins Are Required for Vasopressin-mediated Translocation of Aquaporin-2 into Cell Membranes of Renal Principal Cells J. Biol. Chem., February 19, 1999; 274(8): 4934 - 4938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. O. Turner, L. R. Johnson, L. Haig-Ladewig, G. L. Gerton, and S. B. Moss An X-linked Gene Encodes a Major Human Sperm Fibrous Sheath Protein, hAKAP82. GENOMIC ORGANIZATION, PROTEIN KINASE A-RII BINDING, AND DISTRIBUTION OF THE PRECURSOR IN THE SPERM TAIL J. Biol. Chem., November 27, 1998; 273(48): 32135 - 32141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Feliciello, C. S. Rubin, E. V. Avvedimento, and M. E. Gottesman Expression of A Kinase Anchor Protein 121 Is Regulated by Hormones in Thyroid and Testicular Germ Cells J. Biol. Chem., September 4, 1998; 273(36): 23361 - 23366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Dell'Acqua and J. D. Scott Protein Kinase A Anchoring J. Biol. Chem., May 16, 1997; 272(20): 12881 - 12884. [Full Text] [PDF]
|
|
|
|
|
|
K. Yuasa, J. Kotera, K. Fujishige, H. Michibata, T. Sasaki, and K. Omori Isolation and Characterization of Two Novel Phosphodiesterase PDE11A Variants Showing Unique Structure and Tissue-specific Expression J. Biol. Chem., September 29, 2000; 275(40): 31469 - 31479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. W. Carr, A. Fujita, C. L. Stentz, G. A. Liberty, G. E. Olson, and S. Narumiya Identification of Sperm-specific Proteins That Interact with A-kinase Anchoring Proteins in a Manner Similar to the Type II Regulatory Subunit of PKA J. Biol. Chem., May 11, 2001; 276(20): 17332 - 17338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Cho, M.W. Brown, and Z. I. Bashir Mechanisms and physiological role of enhancement of mGlu5 receptor function by group II mGlu receptor activation in rat perirhinal cortex J. Physiol., March 8, 2002; (2002) 200101392. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |