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Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030; and the Department of Medicine, Veterans Affairs Medical Center and Oregon Health Science University (D.W.C.), Portland, Oregon 97201
Address all correspondence and requests for reprints to: Barbara M. Sanborn, Ph.D., Department of Biochemistry and Molecular Biology, University of Texas Medical School, P.O. Box 20708, Houston, Texas 77225. E-mail: bsanborn{at}bmb.med.uth.tmc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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q/phospholipase C (PLC) coupling (1). PLC
hydrolyzes phosphatidylinositol bisphosphate to form inositol
1,3,4-triphosphate (IP3) and diacylglycerol.
IP3 binds to receptors on the endoplasmic reticulum,
resulting in the release of calcium from intracellular stores (2).
Smooth muscle relaxation is facilitated by relaxants, but the
mechanisms used are not well defined. Sutherland and Rall first
suggested the involvement of cAMP in relaxation of smooth muscle in
1960 on the basis of a correlation between epinephrine-induced
relaxation of smooth muscle and an increase in cAMP (3). However, the
relationship has since been challenged (4). The major function of cAMP
is to activate the cAMP-dependent protein kinase (PKA). PKA is composed
of dimeric regulatory subunits, each binding a catalytic
subunit (5). When cAMP concentrations rise in the cell, two cAMP
molecules bind to each regulatory subunit, resulting in a
conformational change that releases the catalytic subunits. The
catalytic subunit catalyzes the transfer of the 
-phosphate of ATP to
a serine or threonine on a specific protein, often resulting in
modification of protein function.
cAMP affects many physiological events in the myometrium, such
as ion movement and phosphatidylinositide turnover (6). Previous work
in PHM1–41 cells demonstrated cAMP-mediated inhibition of
phosphatidylinositide turnover via the action of PKA (7). Further
examination of the targets of PKA action revealed that although
Gq and PLCß1 were not substrates, PKA
phosphorylation of PLCß3 inhibited Gq
stimulation of this enzyme (8).
PKA is a multifunctional enzyme with many substrates. The localization of PKA near a particular substrate appears to be highly important for the regulation of specific physiological events (9). PKA is targeted to specific subcellular locations through the association with A kinase-anchoring protein (AKAPs). AKAPs bind the RII regulatory subunits of PKA through a common amphipathic helix motif (10). Anchoring inhibitor peptides designed to mimic the amphipathic helix motif on AKAP Ht31 have been demonstrated to disrupt PKA localization in neurons when microinjected into the cells (11). Cell-permeable anchoring inhibitors containing an amino-terminal stearic acid moiety (S-Ht31) have been synthesized and used to study the effect of disruption of PKA binding to AKAPs in bovine sperm (12).
There is no information on the characteristics of the PKA
involved in cAMP-mediated inhibition of phosphatidylinositide turnover
in the myometrium. In previous experiments, the addition of
chlorophenylthio-cAMP (CPT-cAMP) to rat myometrial plasma membranes
attenuated GTPS-stimulated PLC activity (13). These data suggest
that PKA activity already associated with the plasma membrane could be
involved in the cAMP inhibitory mechanism.
The present study was designed to investigate the possible importance of PKA localization to the plasma membrane in mediating the inhibitory effect of cAMP on phosphatidylinositide turnover in the myometrium. The ability of the cell-permeable anchoring peptides to prevent the inhibitory effect of both endogenous and exogenous increases in cAMP on the oxytocin-stimulated increase in phosphatidylinositide turnover in an immortalized pregnant human myometrial cell line (PHM1–41) was tested. The putative AKAP involved in the cAMP inhibitory mechanism was identified by an overlay assay of purified plasma membrane of the PHM1–41 cells, and the putative rat homolog AKAP was also discovered in the plasma membrane of the nonpregnant rat uterus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP (3000 Ci/mmol) were
obtained from Amersham Pharmacia Biotech (Arlington
Heights, IL). H-89 was obtained from Seikagaku Corp.
(Rockville, MD). Antibodies against AKAP79 and AKAP150 were obtained
from Dr. John D. Scott (Vollum Institute, Oregon Health Science
University, Portland, OR). The antibody against PKA catalytic subunit
was obtained from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). The anchoring inhibitor peptide S-Ht31 and the control
peptide P-S-Ht31 were synthesized as previously described (12).
Cell culture
The immortalized pregnant human myometrial cell line PHM1–41
has been characterized previously (15). Cells were cultured at 37 C and
5% CO2 in DMEM containing 4 g/liter glucose, 0.1 mg/ml
geneticin, 10% FBS, 2 mM L-glutamine, 50 U/ml
penicillin, and 50 µg/ml streptomycin and used at passages
15–19.
Phosphatidylinositide turnover
Cultured cells were incubated for 18 h in culture medium
containing 0.4 µM [3H]myoinositol. The
cells were incubated with H-89, S-HT31, or P-S-HT31 for the time and
concentrations indicated, followed by 10 mM LiCl for 10
min. Agents that elevate cAMP were added 15 min (relaxin and forskolin)
or 5 min (CPT-cAMP) before stimulation with 100 nM oxytocin
for 10 min. The reactions were terminated by aspiration and the
addition of 0.6 ml cold 10% trichloroacetic acid.
[3H]Inositol phosphates were isolated and counted
essentially as described by Anwer et al. (16). Data
represent the mean ± SE of three determinations and
were analyzed by one-way ANOVA and Duncan’s modified multiple range
test.
AKAP overlay assay
The overlay procedure is a modified Western blot procedure and
has been characterized previously (17). Protein (30 µg) from whole
cell lysates of PHM1–41 cells or protein (15 µg) from PHM1–41 cell
purified plasma membranes was subjected to SDS-PAGE in 10% gels and
transferred to nitrocellulose membranes (Millipore Corp.,
Bedford, MA). Blots were probed with radiolabeled PKA regulatory
subunits II, and bands were visualized by autoradiography.
cAMP-dependent protein kinase activity
cAMP-dependent protein kinase activity was assayed by the method
of Roskoski with minor modifications (18). Five micrograms of plasma
membrane were incubated in 50 µl reaction buffer [10 mM
magnesium acetate, 20 mM Tris-Cl (pH 7.4), 0.5
mM 3-isobutyl-1-methylaxanthine, 10 mM
dithiothreitol, and 5 mM NaF] with either 1
µM S-Ht31 or 1 µM P-S-Ht31 at 30 C for 5
min. The mixture was then spun at 40,000 x g for 15
min. Reaction buffer (40 µl) containing 2 µM CPT-cAMP,
30 µM Kemptide, 100 µM ATP, and 5
µM [-32]PATP was added to the pellet.
After 5-min incubation 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
then counted by liquid scintillation.
Immunoblot analysis
Purified plasma membrane protein (10 µg) or crude membrane
protein (20 µg) from PHM1–41 cells was incubated with either 1
µM S-Ht31 or 1 µM control P-S-Ht31 for 5
min at 30 C. The mixture was then spun at 40,000 x g
for 15 min. The pellet was subjected to SDS-PAGE in 10% gels and
transferred to nitrocellulose membranes (Millipore Corp.).
Purified plasma membranes (15 µg) from either PHM1–41 cells or the
nonpregnant rat myometrium were also subjected to SDS-PAGE in 10% gels
and transferred to nitrocellulose membrane filters. Blots were probed
with antibodies, and bands were visualized by enhanced
chemiluminescence (DuPont NEN Life Science Products).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To determine whether disruption of
PKA anchoring inhibits the ability of CPT-cAMP to inhibit the
oxytocin-stimulated increase in phosphatidylinositide turnover,
PHM1–41 cells were preincubated with either the cell-permeable
anchoring inhibitor peptide S-Ht31 or a control peptide that contains a
proline introduced into the amphipathic helix (P-S-Ht31). This
insertion disrupts the helical structure of the peptide and eliminates
the interaction determinants (10). Pretreatment with S-HT31 blocked the
ability of CPT-cAMP to inhibit the oxytocin-stimulated increase in
phosphatidylinositide turnover by 94%. The control peptide P-S-Ht31
had no effect on the ability of CPT-cAMP to inhibit the
oxytocin-stimulated increase in phosphatidylinositide turnover.
Consistent with previous results (7), reversal of the inhibitory effect
of CPT-cAMP was also seen when PHM1–41 cells were pretreated with
H-89, a protein kinase inhibitor specific for PKA.
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). The addition of 15
µM forskolin inhibited the oxytocin-stimulated increase
in phosphatidylinositide turnover by 90%. Pretreatment with S-HT31
reversed the inhibition of phosphatidylinositide turnover by forskolin
by 97%. The control peptide P-S-Ht31 had no effect. The data in Fig. 1
suggest that disruption of PKA anchoring inhibits the ability of cAMP
to inhibit the oxytocin-stimulated increase in phosphatidylinositide
turnover.
The uterine relaxant relaxin both increased intracellular cAMP
concentration in rat myometrial cells and inhibited the
oxytocin-stimulated increase in phosphatidylinositide turnover (7, 14, 16). However, the increase in cAMP was relatively slow and small
compared with that elicited by isoproterenol, a ß-adrenergic agonist
(14), and may not be involved in the cellular action of this hormone.
The inhibitory effects of relaxin could be blocked by the addition of
protein kinase inhibitors with a high degree of specificity for PKA,
suggesting that relaxin exerts its effects on phosphatidylinositide
turnover via the action of PKA (7, 19). In light of the effect of
S-Ht31 on PKA function, the effect of this peptide on the inhibitory
effect of relaxin was tested in PHM1–41 cells. As shown in Fig. 2, treatment of PHM1–41 cells with 100
nM oxytocin resulted in a 6.5-fold increase in
[3H]IP3, and 1 µg/ml relaxin inhibited the
oxytocin-stimulated increase by 100%. Pretreatment with 10
µM S-HT31 inhibited the action of relaxin by 100%,
whereas the control peptide P-S-Ht31 was ineffective. These data
suggest that the ability of relaxin to inhibit the
oxytocin-stimulated increase in phosphatidylinositide turnover is also
dependent on PKA anchoring to an AKAP.
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shows a time course of S-Ht31
incubation in PHM1–41 cells. Significant effects of S-Ht31 on
relaxin-mediated inhibition of phosphatidylinositide turnover were
observed as early as 1 min after incubation with S-Ht31, with maximal
effects reached by 10 min. The effect of S-Ht31 was dose dependent,
with a significant effect at 100 nM (Fig. 4) and complete reversal of the
inhibition of the effect of relaxin at 1 µM. Notably,
incubation with 10 µM S-Ht31 alone did not affect the
oxytocin-stimulated increase in phosphatidylinositide turnover.
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examines the effect of S-Ht31 on PKA
activity in PHM1–41 plasma membrane, using the synthetic Kemptide
substrate. The addition of 1 µM S-Ht31 inhibited the
ability of cAMP to stimulate PKA activity by 95%, whereas P-S-Ht31 had
no effect on cAMP-stimulated PKA activity.
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). In a similar experiment, both PKA catalytic and
regulatory proteins were decreased in crude PHM1–41 membranes after
incubation with S-Ht31 (data not shown). These data demonstrate
directly that PKA is found in myometrial plasma membrane and that
S-Ht31 is effective in disrupting this localization.
Identification of the AKAP involved in the cAMP inhibitory
mechanism
A number of different AKAPs have been reported to be located in
different subcellular locations in cells. We employed an overlay
binding assay using radiolabeled murine RII protein as a probe to
determine identity of AKAPs associated with PHM1–41 cell plasma
membranes. Figure 6 shows that although
PHM1–41 cells contain many AKAPs, the plasma membrane contains only
one predominant AKAP with an apparent molecular mass of 86 kDa.
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shows that
an AKAP of 150 kDa is associated with rat myometrial plasma membrane.
These data suggest that AKAP86 and AKAP150 could be responsible for
localization of the PKA regulating phosphatidylinositide turnover
in the human and rat, respectively.
The molecular weights of AKAP86 and AKAP150 are very similar to those
of two homologous AKAPs previously characterized in human and rat brain
plasma membranes, AKAP79 and AKAP150, respectively (20, 21). AKAP79 has
been shown to localize to the plasma membrane in postsynaptic densities
(20, 22). To determine whether the proteins we observed were related to
these AKAPs, Western blot analysis was performed on protein from
PHM1–41 cell plasma membranes and rat myometrial plasma membranes with
monoclonal antibodies directed against AKAP79 and AKAP150,
respectively. Figure 7 shows that
antibodies against brain human AKAP79 and rat AKAP150 reacted with
AKAPs found in PHM1–41 cell plasma membranes and rat myometrial plasma
membranes, respectively. These proteins corresponded in size to AKAP86
and AKAP150, respectively. These data suggest that in the same species,
myometrial and brain AKAPs have similar epitopes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S-stimulated phosphatidylinositide turnover (13).
This paper demonstrates the importance of PKA anchoring, presumably on
the plasma membrane, for cAMP-mediated regulation of
phosphatidylinositide turnover. The cell-permeable anchoring inhibitor
peptide S-Ht31 was able to block the ability of both CPT-cAMP and
forskolin to inhibit oxytocin-stimulated phosphatidylinositide
turnover. This is the first demonstration of the importance of PKA
anchoring in myometrial signaling and the second study to demonstrate
the influences of the cell-permeable anchoring inhibitor S-Ht31 on PKA
action (12). Relaxin is a relaxant of uterine smooth muscle, but many details of the signal transduction cascades used by relaxin to exerts its effects are unknown at present. Relaxin has been shown to increase cAMP concentration and inhibit the oxytocin-stimulated increase in phosphatidylinositide turnover (7, 14, 16). The inhibition of phosphatidylinositide turnover by relaxin has been shown to be reversed by PKA inhibitors, suggesting that the action of PKA is involved (7, 19). Consistent with this hypothesis, S-Ht31 blocked the ability of relaxin to inhibit the oxytocin-stimulated increase in phosphatidylinositide turnover in PHM1–41 cells. Therefore, it is most likely that PKA associated with the plasma membrane is involved in the inhibitory effect of relaxin on phosphatidylinositide turnover.
The pharmacology of the anchoring inhibitor peptide S-Ht31 with respect to specific cellular events is not well characterized. The data presented here demonstrate that a 10-min incubation of S-Ht31 at a dose of 1 µM is sufficient to reverse PKA-mediated effects on phosphatidylinositide turnover in PHM1–41 cells. Similar to these findings, a previous study using S-Ht31 found that a 5-min incubation with 1 µM S-Ht31 arrested sperm mobility (12). As the peptide has a stearic linkage, it may remain closely associated with the plasma membrane. It remains to be demonstrated whether this peptide can also be used to disrupt intracellular AKAP/PKA association at other locations within the myometrial cells.
Wen et al. demonstrated that cAMP stimulated inhibition of
phospholipase C action in purified myometrial membranes, suggesting
that the cAMP inhibitory effect is a membrane-associated event (13).
Overlay analysis of PHM1–41 cell purified plasma membrane found only
one protein, an AKAP of 86 kDa, associated with this fraction.
Therefore, PKA localized to the plasma membrane, probably through the
association with AKAP86, could be responsible for cAMP-mediated
inhibition of Gq stimulation of PLCß3 via
phosphorylation of the enzyme (8).
A previously characterized 79-kDa human AKAP was located at the plasma membrane in postsynaptic densities (20, 22). The similarity in molecular weights and cellular location between AKAP79 and AKAP86 suggested that myometrial AKAP86 may be similar or identical to AKAP79. A monoclonal antibody generated against AKAP79 detected an AKAP in PHM1–4l plasma membranes that migrated at 86 kDa in SDS-PAGE, presumably AKAP86. The basis for the differences in molecular mass in SDS-PAGE is not known at present, but could represent slight differences in sequence or covalent modification. Thus, AKAP79 and AKAP86 contain similar epitopes recognized by the monoclonal antibody and could represent members of a family of AKAP proteins.
The cAMP inhibitory mechanism has also been demonstrated in the nonpregnant rat myometrium (16, 19), suggesting that mechanisms similar to those found in PHM1–41 cells exist in this tissue. Overlay analysis detected an AKAP of 150 kDa in purified rat myometrial plasma membrane. This AKAP could be the rat homolog of AKAP86, although the effect of S-Ht31 on PKA function in this tissue is not known at present. Similarly, a monoclonal antibody against the rat brain AKAP150 recognized an AKAP of 150 kDa in nonpregnant rat myometrial plasma membranes.
The data presented here provide strong support for a growing theme in cell biology, that colocalization of enzyme and substrate is very important for cellular signaling. For example, five proteins involved in the yeast pheromone mating complex become localized together through their association with the scaffolding protein Sterile 5 (23). Similarly, many tyrosine kinases are coupled to their downstream cytoplasmic enzymes through adapter proteins that contain SH2 and SH3 domains (24).
There is evidence that AKAP proteins are also scaffolding proteins. AKAP79 has been shown to bind not only PKA, but also protein kinase C, calcineurin, calmodulin, and phosphatidylinositide bisphosphate (22, 25, 26). However, the physiological importance of the close association of these proteins has not been demonstrated to date. AKAP86 may also bind multiple proteins. Although this hypothesis has not been explored, association with other enzymes could affect the physiology of the myometrium. Protein kinase C stimulated by diacylglycerol released in phosphatidyl-1,4-bisphosphate hydrolysis by PLC has been shown to inhibit PLC activity (27). The binding of phosphatidylinositide bisphosphate to AKAP86 may also localize PKA close to its substrate, PLCß3 (8). Also, calcineurin could be potentially important in turning off PKA signaling to facilitate uterine contraction.
Ion movement is important for regulation of the contractile state of the uterus. Voltage-gated calcium channels are important for the influx of calcium during contraction, and calcium-stimulated potassium channels are important for hyperpolarization of the membrane potential after contraction (28). One of the more characterized functions of AKAPs is to regulate ion channel activity (29). Association of PKA with AKAP15 in the heart has been shown to be important for PKA stimulation of calcium influx via L-type calcium channels (30). PKA anchoring is also important for mediating the effect of PKA on the ROMK1 potassium channel in the kidney and calcium-stimulated potassium channels in the trachea (31, 32). Both of the channels have been shown to be affected by PKA in smooth muscle (33, 34). The effects of S-Ht31 on ion channel activity and regulation in the myometrium are unknown at present.
In summary, the data presented here demonstrate the importance of PKA anchoring through AKAP86 to the plasma membrane for cAMP-mediated regulation of phosphatidylinositide turnover in PHM1–41 myometrial cells. The association of PKA with the plasma membrane would bring PKA into close proximity with its substrate protein PLCß3, allowing for the regulation of PLCß3 activity by uterine relaxants. This regulation could be important for the promotion of uterine quiescence and may be particularly important during pregnancy.
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Acknowledgments |
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Footnotes |
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Received February 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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q/11.
Endocrinology 136:1509–1515[Abstract]
q/phospholipase C(PLC)
coupling by a mechanism not involving PLCß2.
Endocrinology 139:2265–2271q.
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