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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}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{alpha}s and adenylyl cyclase expression, and G{alpha}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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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 12–21 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).

 
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. 2AGo). The cAMP-inhibitory effect was also demonstrated on days 12, 15, and 20, as summarized in Fig. 2CGo. However, on day 21, the results were strikingly different (Fig. 2BGo). 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. 2CGo). 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 12–21 of pregnancy (mean ± SE, n = 3).

 
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{alpha}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{alpha}q/PLCß coupling. Figure 3AGo shows that PLCß1 and PLCß3 concentrations did not change significantly between days 19 and 21, as detected by Western blot, whereas G{alpha}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{alpha}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.

 
The concentration or activity of PKA could decrease in the plasma membrane during late pregnancy, and this would decrease the effectiveness of PKA. Figure 3BGo 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. 4AGo).



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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 Student’s t test.

 
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. 3BGo, 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 3CGo 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. 4BGo).

Changes in the concentrations of these proteins in total tissue homogenates are shown in Fig. 5Go. 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. 5BGo), as did cAMP-stimulated PKA activity (Fig. 4CGo). PP2B concentration increased in total tissue homogenates between days 19 and 21 of pregnancy (Fig. 5CGo), similar to the increase in PP2B concentration seen in the plasma membrane (Fig. 5CGo). The activity of PP2B in the total homogenates increased between days 19 and 21 as well (Fig. 4DGo). In contrast, PP1 expression was unchanged in total homogenates (Fig. 5CGo).



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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{alpha}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.

 
Figure 6Go 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.

 
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 PHM1–41 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. 7Go). 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.

 
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 8AGo 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).

 
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. 8AGo was stripped and probed with an antibody against PP2B. Figure 8BGo 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{alpha}q-stimulated PLCß3 activity was mediated via PKA phosphorylation of PLCß3 (6). Figure 9Go 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}s concentration and the ability of G{alpha}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 12–20 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{alpha}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{alpha}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{alpha}q to G{alpha}i (23) could contribute to the loss of the effect of cAMP, since Gß{gamma}-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{alpha}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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} (will detect all RII subunits per supplier), PKA catalytic subunit {alpha} (cross-reactive with ß and {gamma} subunits), G{alpha}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). {gamma}-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 Duncan’s 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 {alpha} 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-{gamma}-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-{gamma}-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-{gamma}-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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