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
Kimberly L. Dodge,
Daniel W. Carr,
Caiping Yue and
Barbara M. Sanborn
Department of Biochemistry and Molecular Biology (K.L.D., C.Y.,
B.M.S.) University of Texas Medical School at Houston Houston,
Texas 77030
Department of Medicine (D.W.C.) Veterans
Affairs Medical Center and Oregon Health Science University
Portland, Oregon 97201
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ABSTRACT
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During pregnancy in the rat, there is a change in
the ability of chlorophenylthio (CPT)-cAMP to inhibit myometrial
phosphatidylinositide turnover. This is accompanied by a change in the
association of proteins with a plasma membrane A kinase anchoring
protein (AKAP). Both CPT-cAMP and isoproterenol inhibited
oxytocin-stimulated phosphatidylinositide turnover on days 12 through
20 of gestation, whereas neither agent had an effect on day 21.
Accompanying this change was a dramatic decrease in the concentration
and activity of cAMP-dependent protein kinase [protein kinase A
(PKA)] and an increase in the concentration of protein phosphatase 2B
(PP2B) in plasma membranes from day 21 compared with day 19 pregnant
rats. In contrast, both PKA and PP2B concentrations and activities
increased in total myometrial homogenates. Both PKA and PP2B
coimmunoprecipitated with an antibody against the 150-kDa AKAP found in
rat myometrial plasma membranes. More PKA was associated with AKAP150
on day 19 than on day 21, while the reverse was true for PP2B.
Disruption of PKA/AKAP association in day 19 pregnant rat myometrial
cells with the specific interaction inhibitor peptide S-Ht31 resulted
in the loss of the cAMP-inhibitory effect on phosphatidylinositide
turnover. PP2B activity in myometrial homogenates dephosphorylated
PLCß3, a PKA substrate targeted in the
inhibition of G
q-stimulated
phosphatidylinositide turnover. The dramatic loss of the
cAMP-inhibitory effect on day 21 of pregnancy may alter the balance
between uterine contraction and relaxation near parturition. The
changes in the relative concentrations of PKA and PP2B associated with
AKAP150 are consistent with a functional role for AKAP150 scaffolding
in the alteration of cellular signaling.
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INTRODUCTION
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The colocalization of enzyme and substrate is often important for
the specificity of enzyme action. The cAMP-dependent protein kinase
[protein kinase A (PKA)] is localized to subcellular organelles
through association with A kinase anchoring proteins (AKAPs), and this
association is important for PKA action in specific cellular events
(1). AKAPs have now been shown to bind other proteins as well as PKA.
For example, AKAP79 binds not only PKA, but also protein kinase C,
calcineurin, calmodulin, and phosphatidylinositide-4,5-bisphosphate
(2, 3, 4). The association of these signaling proteins with AKAP79
suggests that this AKAP may act as a scaffolding protein, integrating
multiple signaling pathways to act on specific cellular events.
However, the physiological implications of the subcelluar localization
of these proteins have not been demonstrated to date.
In the uterus, elevation of cAMP concentration has been correlated with
relaxation of the myometrium, and the cAMP pathway has been targeted by
some tocolytics to control preterm labor (5). We have recently shown
that the cAMP- dependent protein kinase (PKA) inhibits the
G
q-stimulated activation of PLCß3 (6) and
is localized at the plasma membrane through association with a 86-kDa
AKAP in human myometrial cells (6A ).
During pregnancy, the ability of ß-adrenergic agonists to inhibit
contraction and to generate cAMP decreases with gestational age in both
human and rat myometrium (7, 8). It has been suggested that this
decrease is the result of changes in the signaling pathways involved in
cAMP generation. ß-Adrenergic receptors, G
s and
adenylyl cyclase expression, and G
s-stimulated adenylyl
cyclase activity decrease in term rat and human myometrium (8, 9, 10, 11, 12).
However, in the day 21 pregnant rat myometrium, forskolin did not
inhibit oxytocin-induced phosphatidylinositide turnover, although it
did increase cAMP concentration (13). These data suggested that the
cAMP-mediated inhibitory mechanism itself may also decline at the end
of pregnancy.
In the present study, we report a dramatic decline in the ability of
chlorophenylthio (CPT)-cAMP to inhibit oxytocin-stimulated
phosphatidylinositide turnover on day 21 of pregnancy near term. This
decline in the cAMP-inhibitory mechanism was accompanied by a
reciprocal change in the concentrations of PKA and protein phosphatase
2B (PP2B) associated with a plasma membrane AKAP of 150 kDa. These
changes indicate that AKAP150 can act as a scaffolding protein and that
the gestation-associated shift in the enzymes associated with AKAP150
may help determine the physiological state of the uterus.
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RESULTS
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The cAMP-Mediated Inhibition of Phosphatidylinositide Turnover Is
Absent in the Myometrium of the Day 21 Pregnant Rat
The concentration of oxytocin receptor protein changes in the
pregnant rat myometrium, and this might affect maximal response of the
oxytocin-stimulated increase in phosphatidylinositide turnover (14).
For the purpose of comparison, we elected to measure the effect of cAMP
at the same relative stimulation point. To reduce any ambiguity that
might arise from binding of oxytocin to vasopressin receptors, the
oxytocin agonist Thr4, Glu7-oxytocin (TGOT) was
used to determine the EC50 for phosphatidylinositide
turnover on the various days of pregnancy (15). Although the
EC50 did not change, the phosphatidylinositide turnover
elicited by TGOT was significantly higher on day 21 than on prior days
of pregnancy (Fig. 1
). A TGOT
concentration of 200 nM, close to the EC50, was
used in all subsequent experiments.

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Figure 1. A Summary of the Dose Response of Myometrial
Phosphatidylinositide Turnover to TGOT on Days 1221 of Gestation in
the Rat
Data are from a single experiment (mean, n = 3) and are
representative of three different experiments. The data were analyzed
by a four-parameter logistics curve-fitting program (M. L. Jaffe,
Silver Spring, MD). The mean EC50 values ±
SE are as follows: day 12, 100 ± 13; day 15, 95
± 28; day 19, 135 ± 20; day 20, 90 ± 8; and day 21,
150 ± 21 (nsd).
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To test the hypothesis that the cAMP-inhibitory effect may be
diminished near term, myometrial strips from rats at various days of
pregnancy were treated with CPT-cAMP and isoproterenol, and the effect
on TGOT-stimulated phosphatidylinositide turnover was determined.
CPT-cAMP decreased the TGOT-stimulated increase on day 19 by more than
90%, as did isoproterenol (Fig. 2A
). The
cAMP-inhibitory effect was also demonstrated on days 12, 15, and 20, as
summarized in Fig. 2C
. However, on day 21, the results were strikingly
different (Fig. 2B
). Neither CPT-cAMP nor isoproterenol inhibited
TGOT-stimulated phosphatidylinositide turnover. The loss of the
cAMP-inhibitory mechanism occurred sharply between days 20 and 21 of
gestation (Fig. 2C
). These rats normally deliver on the afternoon of
day 21.

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Figure 2. The Effect of CPT-cAMP and Isoproterenol on the
TGOT-Stimulated Increase in [3H]IP3 in
Pregnant Rat Myometrium
Rat myometrial strips from day 19 (A) or day 21 (B) of pregnancy were
incubated with CPT-cAMP (CPT) for 5 min or isoproterenol (ISO) for 15
min before being stimulated with 200 nM TGOT (OT) for 3
min. Data are expressed as mean ± SE (n = 3) and
are representative of three different experiments. Significant
differences at P < 0.05 between groups are
designated by different lowercase letters. C, The mean
percent inhibition by CPT-cAMP of the TGOT-stimulated increase in
phosphatidylinositide turnover for days 1221 of pregnancy (mean
± SE, n = 3).
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The Decrease in the cAMP-Inhibitory Effect Correlates with
Membrane-Associated Changes in PKA and PP2B
There are several explanations for the lack of CPT-cAMP-mediated
inhibition of agonist-stimulated phosphatidylinositide turnover in day
21 pregnant rat myometrium. Since an equivalent concentration of TGOT
elicits a greater response on day 21 than day 19, it is possible that
the concentration of CPT-cAMP used was not adequate to counteract the
response. However, 1.5 mM CPT-cAMP completely inhibited
phosphatidylinositide turnover stimulated by 2 µM TGOT on
day 20 of pregnancy (control, 735 ± 135; 2 µM TGOT,
1858 ± 82; CPT+TGOT, 747 ± 61, n = 3), even though
this concentration of TGOT elicited a similar effect as did 200
nM TGOT on day 21 (control, 791 ± 51; 200
nM TGOT, 1825 ± 121; CPT+TGOT, 1862 ± 103,
n = 3). These results indicate that the absence of cAMP-mediated
inhibition on day 21 is not due to the inability of cAMP to compensate
for the increased amount of TGOT-stimulated phosphatidylinositide
turnover.
Another explanation might be that the target protein for PKA has
decreased in concentration in the plasma membrane. We have found that
PKA inhibited the coupling of G
q to PLCß3
as a result of phosphorylation of PLCß3 on
Ser1105 (6). Therefore, a significant decrease in
PLCß3 in the plasma membrane could influence the effect
of cAMP on oxytocin receptor/G
q/PLCß coupling. Figure 3A
shows that PLCß1 and
PLCß3 concentrations did not change significantly between
days 19 and 21, as detected by Western blot, whereas G
q
concentration increased. PLCß2 was not detected by
Western blot for either day (data not shown). These data suggest that
the loss of the cAMP-inhibitory mechanism is not due to a significant
decrease in the target PLC.

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Figure 3. Detection of PLCß3, PKA, and Protein
Phosphatases in Rat Myometrial Plasma Membranes from Days 19 and 21 of
Pregnancy
Immunoblots were probed with antibodies specifically directed against
PLCß1, PLCß3, G q, PKA
catalytic subunit, PKA regulatory subunit type II, PP1, or PP2B (1:500
each), respectively. The molecular weights of the proteins identified
are indicated by arrows. D, Coomassie blue stain of
protein from purified myometrial plasma membranes from equivalent
samples and the same gel as that shown for PKA catalytic subunit in
panel B, indicating that sample loading was equivalent. Also, an
immunoblot probed simultaneously with antibodies against both PKA
catalytic subunit and PLCß3 (1:500) is shown. In a number
of cases, blots were reprobed with different antibodies, which also
reproduced the changes illustrated in this figure.
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The concentration or activity of PKA could decrease in the plasma
membrane during late pregnancy, and this would decrease the
effectiveness of PKA. Figure 3B
shows that the expression of both the
PKA catalytic and regulatory type II subunits decreased dramatically
between days 19 and 21 of pregnancy in purified myometrial plasma
membrane preparations. The changes in protein concentration have been
confirmed in tissue from three separate rats in each group. The
cAMP-stimulated kinase activity was also significantly higher in
pregnant rat myometrial plasma membranes on day 19 of gestation
compared with day 21 (Fig. 4A
).

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Figure 4. PKA and PP2B Activities in Both Purified Myometrial
Plasma Membranes and Total Myometrial Homogenates on Days 19 and 21 of
Pregnancy in the Rat
PKA activity in plasma membrane (A) and total homogenates (C) and PP2B
in plasma membrane (B) and total homogenates (D) are expressed as means
of duplicates from a single experiment, with individual data points
represented by dots. Data are representative of results
from two experiments. Differences in panels A, C, and D were
statistically significant (P > 0.05) as determined
by Students t test.
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We have recently demonstrated that PKA is localized to the plasma
membrane through association with AKAPs of 86 kDa in an immortalized
pregnant human myometrial cell line and 150 kDa in the nonpregnant rat
myometrium (6A ). One possible explanation for the decrease in
PKA levels at the plasma membrane could be that the AKAP150
concentration decreased. However, as seen in Fig. 3B
, no decrease in
AKAP150 was detected by overlay analysis.
An increase in phosphatases at the plasma membrane could
dephosphorylate PKA target proteins and oppose PKA action. Figure 3C
shows changes in concentrations of phosphatases known to associate with
the plasma membrane (16). Although protein phosphatase 1 (PP1)
concentration did not change between days 19 and 21 of pregnancy, PP2B
concentration significantly increased in the plasma membrane on day 21
compared with day 19. Calcium-stimulated PP2B activity in plasma
membranes prepared from myometrium on days 19 and 21 of pregnancy did
not increase significantly under the experimental conditions (Fig. 4B
).
Changes in the concentrations of these proteins in total tissue
homogenates are shown in Fig. 5
. In
contrast to the decline in PKA concentration in the plasma membrane
between days 19 and 21, total homogenate PKA concentrations increased
over this time interval (Fig. 5B
), as did cAMP-stimulated PKA activity
(Fig. 4C
). PP2B concentration increased in total tissue homogenates
between days 19 and 21 of pregnancy (Fig. 5C
), similar to the increase
in PP2B concentration seen in the plasma membrane (Fig. 5C
). The
activity of PP2B in the total homogenates increased between days 19 and
21 as well (Fig. 4D
). In contrast, PP1 expression was unchanged in
total homogenates (Fig. 5C
).

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Figure 5. Detection of PLCß3, PKA, and Protein
Phosphatases in Total Tissue Homogenates from 19- and 21-Day Pregnant
Rat Myometrium
Immunoblots were probed with antibodies specifically directed against
PLCß1, PLCß3, G q, PKA
catalytic subunit, PKA regulatory subunit type II, PP1, or PP2B (1:500
each), respectively. The molecular weights of the proteins identified
are indicated by arrows. D, Coomassie blue stain of
protein from myometrial cellular homogenates. An immunoblot probed
simultaneously with antibodies against both PKA catalytic subunit and
PLCß3 (1:500) is also shown.
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Figure 6
summarizes the average changes
in protein expression on day 21 relative to day 19 of gestation in rat
myometrial plasma membranes (A) and total myometrial homogenates (B),
observed in tissue from three separate rats in each group. The most
striking contrast is between the decrease in plasma membrane PKA in the
face of an increase in total PKA and no change in the concentration of
AKAP150. On the other hand, the concentration of PP2B associated with
the plasma membrane paralleled the increase in total cellular
enzyme.

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Figure 6. Changes in the Distribution of PKA and Other
Proteins between Day 19 and Day 21 of Gestation in the Rat Myometrium
Densitometric scans were performed on immunoblots from either purified
myometrial plasma membranes (A) or total myometrial homogenates (B)
from three separate rats. Data are expressed as mean ±
SE of the changes on day 21 relative to day 19.
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The Association of PKA with AKAP 150 Is Important for Its
Inhibitory Effect
We have recently demonstrated a functional role for PKA
association with AKAP86 in the cAMP-mediated inhibition of
phosphatidylinositide turnover in human PHM141 cells (6A ). If
PKA associated with an AKAP in the plasma membrane in day 19 pregnant
rat myometrium is similarly responsible for the inhibitory effect of
cAMP, it should be possible to eliminate the cAMP response by
interfering with the PKA/AKAP interaction. We have recently found that
the AKAP interaction inhibitory peptide S-Ht31 effectively decreases
the association of PKA with the plasma membrane in human myometrial
cells (6A ). Furthermore, in crude membrane from the nonpregnant rat
uterus, S-Ht31 reduced the association of both PKA catalytic and
regulatory subunits with the particulate fraction and increased the
concentration of PKA in the supernatant. Consistent with these
observations, S-Ht31 completely reversed the inhibition by
CPT-cAMP of TGOT-stimulated phosphatidylinositide turnover in day 19
pregnant rat myometrial cells, whereas the control peptide P-S-Ht31 had
no effect (Fig. 7
). The disruption of
PKA/AKAP interaction by S-Ht31 in day 19 cells mimics the lack of
repression by PKA seen in day 21 pregnant rat myometrium, where the
concentration of PKA in the plasma membrane is decreased.

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Figure 7. S-Ht31 Reversed the cAMP Inhibition of
TGOT-Stimulated Phosphatidylinositide Turnover in Day 19 Pregnant
Myometrial Cells
Myometrial cells were incubated with 1 µM S-Ht31 for 10
min and 1.5 mM CPT-cAMP for 5 min before being stimulated
with 200 nM TGOT (OT) for 3 min. Data are expressed as
mean ± SE (n = 3) in a single experiment.
Lowercase letters denote statistical differences at
P < 0.05.
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To explore the role of AKAP150 in the pregnancy-related changes in the
action of PKA, the ability of PKA to coimmunoprecipitate with AKAP150
in detergent extracts of purified myometrial plasma membranes was
tested. Figure 8A
shows that the amount
of PKA coimmunoprecipitated with AKAP150 dramatically decreased on day
21 of pregnancy compared with day 19. These data correlate with the
decrease in PKA concentration at the plasma membrane that occurred at
this time.

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Figure 8. PKA and PP2B Coimmunoprecipitate with AKAP150 in
Different Relative Proportions on Day 19 and Day 21 of Pregnancy
Plasma membrane protein (50 µg) was incubated with 5 µg of antibody
directed against AKAP150 or control IgG, designated as C. The
immunoprecipitates were subjected to SDS-PAGE, transferred to
nitrocellulose, and probed first with an antibody against PKA (A) and
then stripped and probed with an antibody against PP2B (B) (1:500
each).
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AKAP 150 Also Binds PP2B
Human brain AKAP79 binds not only PKA, but also PP2B, protein
kinase C, phosphatidyl-4,5-bisphosphate, and calmodulin (2, 3, 4). Rat
brain AKAP150 is considered the rat homolog of AKAP79 (17). We have
found that rat brain and myometrial AKAP150 react with a common
antibody (Dodge et al., submitted). Both brain and
myometrial RNA produce an 800-bp RT-PCR product using primers spanning
bases 233-1033 in the brain sequence (data not shown). These data
indicate that the brain and myometrial proteins are probably identical.
To test whether AKAP150 can also associate with PP2B, the
nitrocellulose membrane used in Fig. 8A
was stripped and probed with an
antibody against PP2B. Figure 8B
shows that PP2B coimmunoprecipitated
with AKAP150. In contrast to PKA, the binding of PP2B to AKAP150
increased on day 21 of pregnancy, in parallel with the increase in PP2B
protein in the membrane. These data suggest that PP2B/AKAP150
association plays a role in the localization of PP2B to the plasma
membrane in the rat myometrium.
Previous data from our laboratory showed that the cAMP-inhibitory
effect on G
q-stimulated PLCß3 activity was
mediated via PKA phosphorylation of PLCß3 (6). Figure 9
shows that day 21 pregnant rat
myometrial extracts dephosphorylated recombinant
32P-labeled PLCß3 by 82% in 15 min at 30
C. In the presence of the specific PP2B autoinhibitory peptide,
32P-PLCß3 was only dephosphorylated 36%,
while it was dephosphorylated 74% in the presence of okadaic acid, a
PP1 and PP2A inhibitor. These data indicate that
32P-PLCß3 is a substrate for PP2B and that
PP2B is the major phosphatase in the myometrial extract
dephosphorylating this protein.

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Figure 9. 32P-Labeled PLCß3
Is Dephosphorylated by PP2B in 21-Day Pregnant Rat Myometrial Cellular
Extracts.
A, As described in Materials and Methods, okadaic acid (15
nM) (OA) and the PP2B autoinhibitory peptide (PP2B-I) (4.4
nM) were added to the incubation mixture where indicated.
B, Coomassie blue stain of PLCß3(His)6 blot
used in A.
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DISCUSSION
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The initiation of labor may be influenced by a decrease in the
signaling pathways that stimulate relaxation and an increase in the
pathways that stimulate contraction. The mechanisms proposed for the
changes in these signaling pathways to date have involved aspects of
receptor-G protein-effector coupling related to cAMP production (5, 7, 8, 9, 10, 11, 12). The data presented here provide insight into a new mechanism
that exists beyond cAMP production itself. We demonstrate here a
striking decrease in the cAMP-inhibitory response at the end of
pregnancy in the rat, which could affect contractant-induced inositol
trisphosphate production and calcium mobilization. We present
evidence that changes in the concentrations of PKA and PP2B associated
with AKAP150 in the plasma membrane are integrally related to the loss
of the cAMP-inhibitory response.
Both G
s concentration and the ability of
G
s to stimulate adenylyl cyclase activity decrease in
the laboring human uterus (10). Furthermore, there is a decrease in
adenylyl cyclase concentration in the day 21 pregnant rat uterus (11).
Therefore, decreased ability of uterine relaxants to generate cAMP
would affect their ability to inhibit phosphatidylinositide turnover.
However, this explanation does not explain the loss of effect of
CPT-cAMP itself on phosphatidylinositide turnover, since this treatment
bypasses the need for the generation of cAMP.
We had found previously that CPT-cAMP inhibited the oxytocin-stimulated
increase in phosphatidylinositide turnover in the nonpregnant
estrogen-primed rat myometrium (18, 19). In contrast to this finding,
Khac et al. (13) found that forskolin did not attenuate the
oxytocin-stimulated increase in phosphatidylinositide turnover in the
day 21 pregnant rat uterus although it increased cAMP. These apparent
discrepancies could be reconciled if the cAMP- inhibitory mechanism
decreased near the end of pregnancy. This indeed appears to be the
case. Although CPT-cAMP and isoproterenol inhibited the
oxytocin-stimulated increase in phosphatidylinositide turnover on days
1220 of pregnancy in the rat, neither agent inhibited the
oxytocin-stimulated increase on day 21.
In the rat myometrium, the predominant PLCß subtypes are
PLCß1 and PLCß3 (20). Phosphorylation of
PLCß3 by PKA inhibits stimulation of this enzyme by
G
q (6). A decrease in the concentration of
PLCß3 would decrease the sensitivity of
phosphatidylinositide turnover to cAMP. Indeed, PLCß3
mRNA has been reported to decrease in the 21 day pregnant rat
myometrium (21). However, no significant changes were seen in
PLCß3 protein concentration at the level of the
myometrial plasma membrane or in total cellular protein between days 19
and 21 of gestation. The concentration of G
q increased,
consistent with previous reports (22). These data suggest that the lack
of cAMP inhibition of phosphatidylinositide turnover on day 21 is not
the result of a decrease in PLCß3 concentration. It is
unlikely that a shift in the relative coupling of the oxytocin receptor
from G
q to G
i (23) could contribute to
the loss of the effect of cAMP, since Gß
-stimulated
PLCß3 is also inhibited by PKA (24).
A decrease in PKA activity could also contribute to a decrease in the
cAMP-inhibitory response. Consistent with this hypothesis, PKA
concentration and activity decreased in myometrial plasma membranes on
day 21 of gestation compared with day 19. This change in PKA
concentration was not the result of a decrease in cellular PKA
concentration, but rather a decrease in the localization of PKA to the
plasma membrane. The amount of PKA coimmunoprecipitated with plasma
membrane-associated AKAP150 was greater on day 19 of gestation than on
day 21. Disruption of PKA/AKAP association reversed the cAMP-mediated
inhibitory effect in the day 19 pregnant rat myometrium, mimicking the
lack of cAMP responsiveness seen on day 21. These data suggest that PKA
localized to the plasma membrane through the association with AKAP150
is necessary for the cAMP-inhibitory mechanism and indicate that
pregnancy-related mechanisms regulate the localization of PKA in the
myometrium.
Dephosphorylation of proteins is of equal importance as phosphorylation
in the regulation of protein activity. Western blot analysis of
myometrial plasma membrane demonstrated a significant increase in PP2B
expression on day 21 of pregnancy compared with day 19 in both plasma
membrane fractions and total homogenates. PP2B was also shown to
associate with AKAP150, suggesting PP2B localization to the plasma
membrane is accomplished via this association. This demonstration that
AKAP150 can associate with proteins other than PKA extends its homology
to AKAP79. In contrast to PKA, more PP2B was associated with AKAP150 on
day 21 than on day 19 of pregnancy. In vitro experiments
demonstrated 32P-labeled PLCß3 could be
dephosphorylated by PP2B, suggesting that PLCß3 could be
a target of PP2B action in vivo. By decreasing the
effectiveness of PKA on the regulation of PLCß3 activity,
PP2B could enhance G
q/PLCß3 coupling,
favoring contraction of the myometrium. The association of PP2B with
AKAP150 could contribute to the lack of a significant change in PP2B
activity associated with the plasma membrane fractions between day 19
and day 21. Interaction of PP2B with AKAP79 inhibits enzyme activity
in vitro (3). However, it has been hypothesized that PP2B
could be activated when locally released from AKAPs under specific
physiological conditions, and therefore the increased concentration
could have functional significance (3).
The association of PKA and PP2B with AKAP150 showed a reciprocal
binding pattern, which, in the case of PKA, was not a reflection of
changes in tissue PKA expression. Calmodulin binding to AKAP79
decreases PKC binding (4). By analogy, it is possible that the binding
affinity of PKA for AKAP150 may decrease as a consequence of PP2B
binding, and this possibility is being explored. Understanding the
regulation of the association of PKA and PP2B with AKAP150 could be
helpful in understanding the control of preterm labor and
parturition.
The association of PKA and PP2B with AKAP150 suggests that AKAP150 acts
as a scaffolding protein in the myometrium. The gestation-dependent
association of these enzymes with AKAP150 in the plasma membrane may
help to regulate events controlling the physiological state of the
uterus. The higher concentration of PKA associated with AKAP150 on day
19 of pregnancy could favor inhibition of the action of
PLCß3, promoting uterine quiescence. On day 21 of
pregnancy, the decreased association of PKA and the increased
association of PP2B with AKAP150 could diminish PKA action at the
plasma membrane, facilitating uterine contraction. These data suggest
that AKAPs, functioning as scaffolding proteins, play an important
physiological role in events associated with parturition.
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MATERIALS AND METHODS
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Materials
8-(4-Chlorophenylthio)-cAMP (CPT-cAMP), Kemptide (synthetic PKA
substrate), isoproterenol, and collagenase (type II,
500 U/mg) were
obtained from Sigma (St. Louis, MO). Antibodies against
PLCß1, PLCß2, PLCß3, PP1, PKA
regulatory subunit II
(will detect all RII subunits per supplier),
PKA catalytic subunit
(cross-reactive with ß and
subunits),
G
q/ll, normal mouse IgG, and Protein A- and A/G-agarose
conjugates were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antiprotein phosphatase 2B (PP2B)
antibody was obtained from Transduction Laboratories (Lexington, KY).
The PKA RII subunit and PP2B autoinhibitory peptides were obtained from
BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA).
Recombinant purified PLCß3 was prepared as described
previously (6). Goat antirabbit IgG horseradish peroxidase conjugate
was obtained from Bio-Rad Laboratories, Inc. (Hercules,
CA). Cell culture reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). The AKAP interaction inhibitor peptide
S-Ht31 and the control peptide P-S-Ht31 have been characterized
previously (25). Timed pregnant Sprague Dawley rats were obtained from
Harlan Sprague Dawley, Inc. (Houston, TX) and normally
delivered on the afternoon of day 21 of pregnancy. Thr4,
Gly7-Oxytocin (TGOT) was obtained from Peninsula Laboratories, Inc. (Belmont, CA).
-P32-ATP (3000
Ci/mol) and [3H]myoinositol (22.3 Ci/mmol) were obtained
from Dupont-NEN (Boston, MA).
Phosphatidylinositide Turnover
Rats on various days of pregnancy were killed between 0600 and
0800 h by ether excess according to institutional guidelines.
Uteri were cut open, the fetuses and placentae removed, and the
endometrium removed by gentle scraping. The myometrium was cut into
approximately 50-mg strips and labeled with 0.4 µM
myo-[3H]-inositol (8.3 µCi/ml) in 1 ml Kreb-Ringers
(118 mM NaCl, 4.7 mM KCl, 25 mM
NaHCO3, 1.2 mM KH2PO4,
1.2 mM MgSO4, 10 mM glucose, 10
µM myoinositol, 1.2 mM CaCl2, pH
7.4) buffer at 37 C for 3 h in the presence of 5%
CO2/95% O2. The tissue was incubated with 10
mM LiCl in Kreb-Ringers buffer for 10 min. Isoproterenol
and CPT-cAMP were added 15 min before stimulation with various amounts
of TGOT for 3 min. Reactions were terminated by freezing strips in
liquid N2. [3H]Inositol phosphates were
isolated and counted essentially as described previously (19). Data are
expressed as mean ± SE and were analyzed by one-way
ANOVA and Duncans modified multiple range test.
Isolation and Culture of Pregnant Rat Myometrial Cells
Isolation of myometrial cells from a day 19 pregnant rat was
performed by the method of Arnaudeau et al. (26) with a few
modifications. The myometrium, isolated as described above, was placed
in HBSS plus 4000 mg/liter glucose, 200 mg/liter CaCl2 and
98 mg/liter MgCl2, minced finely, and shaken at 37 C for 5
min in HBSS containing 0.1% BSA. The tissue was then digested with
0.15% collagenase in HBSS with shaking for 40 min at 37 C. After
centrifugation at 1200 x g for 5 min, the supernatant
was removed and the pellet digested with fresh collagenase for an
additional 40 min. The supernatants were pooled, and the cells washed
with HBSS. The cells were plated in two 150-mm plates, replated 24
h later at 1.8 x 105 in 35-mm plates, and used for
phosphatidylinositide turnover 24 h later.
Immunoblot Analysis
Purified myometrial plasma membranes were prepared as previously
described (27). Protein from either total pregnant myometrium
homogenates (10 µg) or purified myometrial plasma membrane (5 µg)
was subjected to SDS-PAGE in 10% gels and transferred to
nitrocellulose membranes (Millipore Corp., Bedford, MA).
Blots were probed with antibodies, and bands were visualized by
enhanced chemiluminescence (DuPont-NEN).
Immunoprecipitation
Plasma membranes (50 µg) from pregnant rat myometrium on days
19 and 21 of pregnancy were incubated on ice in RIPA buffer (1x
phosphate buffer solution, 1% NP40, 0.5% sodium deoxycholate, 0.1%
SDS, and 1% Triton X-100) with 1 µg normal mouse IgG/ml buffer.
After 30 min, Protein A/G-agarose conjugate was added and the mixture
was centrifuged in a microfuge for 5 min at 4 C. AKAP150 antibody (5
µg) was added to the supernatant and incubated at 4 C with shaking in
RIPA buffer for 18 h. The mixture was incubated for 4 h with
Protein A/G-agarose beads (20 µl) and centrifuged at 5000 x
g. The Protein A/G-agarose bead pellet was washed four times
with RIPA buffer, suspended in electrophoresis loading buffer, and
subjected to SDS-PAGE in 10% gels and the proteins transferred to
nitrocellulose membranes. The blots were probed with antibodies against
PKA catalytic subunit and PP2B.
AKAP Overlay Assay
The AKAP overlay assay is a modified Western blot procedure
(28). Purified plasma membrane protein (15 µg) from pregnant rat
myometrium was subjected to SDS-PAGE in 10% gels and transferred to
nitrocellulose membranes. Blots were probed with radiolabeled
recombinant PKA regulatory subunit type II
produced as previously
described (24), and bands were visualized by autoradiography.
PKA Activity
PKA activity was assayed by the method of Roskoski (29) with
minor modifications. Either plasma membrane protein (5 µg) or total
myometrial extracts (10 µg) from myometrium on different days of
pregnancy were incubated in 50 µl of reaction buffer [10
mM Mg acetate, 20 mM Tris-Cl (pH 7.4), 0.5
mM 3-isobutyl-1-methylxanthine, 10 mM
dithiothreitol, 5 mM NaF] containing 2 µM
CPT-cAMP, 30 µM Kemptide (PKA substrate), 100
µM ATP, and 5 µM 32P-
-ATP.
After 5 min at 30 C, 20 µl of the reaction were spotted onto
phosphocellulose strips and washed five times in 75 mM
phosphoric acid and once in 95% ethanol. Filters were air dried and
counted by liquid scintillation.
PP2B Activity
Substrates were labeled in reaction buffer containing 100
µM ATP, 5 µM 32P-
-ATP, 20
mM 4-morpholinepropanesulfonic acid, pH 7.0, 2
mM magnesium acetate, 15 mM
ß-mercaptoethanol, and either 94 mM PKA RII peptide or
100 mM recombinant PLCß3(His)6
with PKA catalytic subunit at a molar ratio of 20:1. The mixture was
incubated at 30 C for 30 min.
32P-PLCß3(His)6 was bound to
nickel-nitrilotriacetic acid (Ni-NTA) resin and separated from
free 32P-
-ATP via centrifugation as described previously
(6).
PP2B activity was measured using either 32P-labeled PKA RII
peptide or 32P-labeled
PLCß3(His)6. Five micromoles of each
labeled substrate were added to 30 µl reaction buffer (40
mM Tris, pH 7.5, 0.1 M KCl, 0.5 mM
CaCl2, 6 mM magnesium acetate, 2.5
µM calmodulin, 0.5 mM dithiothreitol, 10
µM IP20) containing either 5 µg total
plasma membrane protein or 10 µg total myometrial extracts from the
different days of pregnancy. The reactions were incubated for 15 min at
30 C. For experiments using the RII peptide, 20 µl of the reaction
mixture were spotted onto phosphocellulose strips and washed five times
in 75 mM phosphoric acid and once in 95% ethanol. Filters
were air-dried and counted by liquid scintillation.
For experiments using PLCß3(His)6, okadaic
acid (15 nM) and PP2B autoinhibitory peptide (4.4
nM) were also added to the reaction mixture where
specified. After termination of the reaction,
PLCß3(His)6 was bound to Ni-NTA resin and
washed, and the pellet was extracted with gel loading buffer. Samples
were electrophoresed on 10% SDS-PAGE gels and subjected to
autoradiography. Quantitation was accomplished by densitometry.
 |
ACKNOWLEDGMENTS
|
---|
The authors would like to thank Dr. J. D. Scott for
supplying the antibody against rat brain AKAP150.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Barbara M. Sanborn, Ph.D., Department of Biochemistry and Molecular Biology, University of Texas Medical School, PO Box 20708, Houston, Texas 77225.
This work was supported in part by NIH Grants HD-09618 (B.M.S.),
HD36408 (D.W.C.), and T32-HD07325 (K.L.D.)
Received for publication March 18, 1999.
Revision received July 23, 1999.
Accepted for publication August 11, 1999.
 |
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