Phosphorylation of the salivary Na+-K+-2Clminus cotransporter

Kinji Kurihara1,3, Nobuo Nakanishi2, Marilyn L. Moore-Hoon3, and R. James Turner3

Departments of 1 Oral Physiology and 2 Biochemistry, Meikai University, School of Dentistry, Sakada-shi, Saitama 350-0283, Japan; and 3 Membrane Biology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda MD 20892


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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We studied the phosphorylation of the secretory Na+-K+-2Cl- cotransporter (NKCC1) in rat parotid acinar cells. We have previously shown that NKCC1 activity in these cells is dramatically upregulated in response to beta -adrenergic stimulation and that this upregulation correlates with NKCC1 phosphorylation, possibly due to protein kinase A (PKA). We show here that when ATP is added to purified acinar basolateral membranes (BLM), NKCC1 is phosphorylated as a result of membrane-associated protein kinase activity. Additional NKCC1 phosphorylation is seen when PKA is added to BLMs, but our data indicate that this is due to an effect of PKA on endogenous membrane kinase or phosphatase activities, rather than its direct phosphorylation of NKCC1. Also, phosphopeptide mapping demonstrates that these phosphorylations do not take place at the site associated with the upregulation of NKCC1 by beta -adrenergic stimulation. However, this upregulatory phosphorylation can be mimicked by the addition of cAMP to permeabilized acini, and this effect can be blocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatory phosphorylation of NKCC1 and suggest that an additional factor present in the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.

exocrine glands; fluid secretion; stimulus-secretion coupling; cation-chloride cotransporter


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE "SECRETORY" ISOFORM of the Na+-K+-2Cl- cotransporter, NKCC1 (also known as BSC2) plays a central role in the secretion of salt and water by many exocrine epithelia (5, 8). In these tissues NKCC1 is localized to the basolateral membrane of the secretory cell, where it concentrates Cl- in the cytoplasm above electrochemical equilibrium. The apical membrane typically contains a secretagogue-activated Cl- channel. During stimulation, Cl- thus enters the cell via NKCC1 and exits via the apical Cl- channel. The resulting transepithelial Cl- secretion then leads to the secretion of salt and osmotically obliged water. There is now good evidence from a number of these tissues that NKCC1 fluxes also are dramatically increased as a result of secretagogue action and that this effect is not simply due to the changes in electrochemical driving forces for Na+, K+, and Cl- that accompany stimulation (3, 4, 6, 15, 18). Rather, this upregulation appears to arise directly from the increased activity of individual cotransporters.

In previous studies from our laboratories we have examined this phenomenon in some detail in rat parotid acinar cells (3, 9, 17-19). In particular, we have characterized the upregulation of NKCC1 activity induced by both muscarinic and beta -adrenergic stimulation (Ca2+-mobilizing and cAMP-generating stimuli, respectively, in these cells). In the case of beta -adrenergic stimulation, which mainly concerns us here, we have shown that there is a close correlation between isoproterenol-induced increases in NKCC1 transport activity and NKCC1 phosphorylation (18, 19). More specifically, we found that the isoproterenol dose responses for both of these phenomena were essentially identical, with a half-maximal effect at ~20 nM agonist in each case, and that isoproterenol stimulation was accompanied by the increased phosphorylation of a 17-kDa peptide resulting from NKCC1 digestion by V8 protease. In addition, these effects are paralleled by an increase in the number of high-affinity binding sites for the NKCC1 inhibitor bumetanide in membranes prepared from stimulated acini (9). These results are consistent with the hypothesis that beta -adrenergic stimulation and the accompanying phosphorylation result in the activation of previously quiescent, or near-quiescent, transporters.

In contrast to many other exocrine epithelia, salivary glands secrete fluid in response to Ca2+-mobilizing rather than cAMP-generating agonists (the increased NKCC1 activity resulting from the latter is thought to account for the increased fluid secretion observed when beta -adrenergic stimulation is superimposed on muscarinic stimulation in these tissues; see, for example, Ref. 18). Thus beta -adrenergic stimulation of salivary acini does not result in the intracellular Cl- loss and cell shrinkage that typically accompany fluid secretion by exocrine cells (14, 18), and the involvement of these effects in the isoproterenol-induced upregulation of the acinar NKCC1 can therefore be excluded. In addition, we have shown that the effect of isoproterenol on NKCC1 can be prevented by protein kinase inhibitors and mimicked by permeant cAMP analogs as well as maneuvers known to increase cytosolic cAMP levels (18, 19), suggesting the involvement of protein kinase A (PKA). However, somewhat surprisingly we also have found that the site of isoproterenol-induced phosphorylation is found in the NH2-terminal cytosolic tail of NKCC1 (9) which does not contain the sole PKA consensus site in this cotransporter.

Here we continue our studies of the phosphorylation events associated with the rat parotid NKCC1. Briefly stated, we show that NKCC1 is phosphorylated by endogenous kinases associated with isolated rat parotid acinar basolateral membranes (BLMs). Additional NKCC1 phosphorylation is seen in the presence of added PKA, but our data suggest that this is due to an effect of PKA on endogenous membrane kinase or phosphatase activities rather than its direct phosphorylation of NKCC1. Moreover, these phosphorylations do not take place at the site associated with the upregulation of NKCC1 by isoproterenol stimulation. Finally, we show that the phosphorylation of NKCC1 induced by isoproterenol treatment can be mimicked by the addition of cAMP to permeabilized acini and that this effect can be blocked by a specific peptide inhibitor of PKA. Taken together, these results provide strong evidence that PKA is indeed involved in the phosphorylation of NKCC1 induced by isoproterenol treatment but that an additional, non-membrane associated factor (for example, a cytosolic kinase activated by PKA) is required to effect the phosphorylation event.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Materials. [gamma -32P]ATP (3,000 Ci/mmol) was obtained from NEN. Molecular weight standards and prepoured SDS-PAGE and tricine gels were from Novex. Protein G/Sepharose beads were from Pierce. L-Tosylamido-2-phenylethyl chloromethyl ketone, phenylmethylsulfonyl fluoride, ovalbumin, and the PKA inhibitor PKI(5-24) were from Sigma. The catalytic subunit of PKA (cPKA) also was obtained from Sigma (P8289) and was reconstituted in deionized water containing 5 mM dithiothreitol (DTT) at a concentration of 2 U/µl. Pepstatin, leupeptin, and V8 protease were from Boehringer Mannheim. Calyculin A, K252a, okadaic acid, and staurosporine were from Calbiochem. Bisindolylmaleimide I HCl, lavendustin A, microcystin-LR, and H-89 were from LC Laboratories.

Protein was measured by using the Bio-Rad Protein Assay Kit with bovine IgG as the standard.

The stop solution (SS) for the phosphorylation reactions contained 20 mM HEPES (pH 7.4 with NaOH), 10 mM Na2ATP, 50 mM NaF, 15 mM sodium pyrophosphate, 0.1 mM sodium orthovanadate, 0.1 µM calyculin A, 10 mM EDTA, 300 µM phenylmethylsulfonyl fluoride, 100 µM L-tosylamido-2-phenylethyl chloromethyl ketone, 1.5 µM pepstatin, 1.5 µM leupeptin, and 0.3% Triton X-100.

Membrane preparation. Rat parotid BLMs were prepared by isopycnic centrifugation as previously described (12). Briefly, rat parotid glands were minced and vigorously homogenized in an isotonic sucrose buffer. The resulting homogenate was centrifuged (2,500 g, 5 min) to remove cellular debris, and the supernatant was recentrifuged (22,000 g, 20 min) to recover a crude membrane pellet. This pellet was spun on a Percoll gradient from which a fraction highly enriched in BLMs was recovered. The basolateral membrane fraction was then washed twice in buffer A (10 mM HEPES buffered with Tris to pH 7.4 plus 100 mM mannitol) containing 100 mM KCl and 1 mM EDTA, resuspended at a protein concentration of 5-10 mg/ml, snap frozen in aliquots, and stored above liquid nitrogen. Relative to the starting tissue homogenate, the activity of the basolateral membrane marker K-stimulated p-nitrophenyl phosphatase is enriched 15-20 times in this final basolateral membrane preparation (12). On the day of an experiment a suitable number of aliquots of BLMs were thawed at room temperature for 30 min, diluted in buffer A containing 1 mM EGTA, centrifuged at 100,000 g for 60 min, and resuspended in buffer A containing 1 mM EGTA at a protein concentration of 1 mg/ml.

Antibody preparation and conjugation. The antibodies used in these studies (9), alpha -wCT(m) and alpha -wCT(r), were raised (in mice and rabbits, respectively) against a 6× His fusion protein corresponding to the COOH-terminal 454 amino acids of rat NKCC1. In Western blots of rat parotid BLMs (not shown), these antisera recognize a single 170-kDa protein, the molecular mass we previously established as that of (fully glycosylated) rat NKCC1 in this tissue. For immunoprecipitation studies, antibodies were preconjugated to protein G beads as previously described (9) and suspended in SS containing 1% ovalbumin as a slurry of 50% beads and 50% buffer. In control experiments (not shown), we established that both alpha -wCT(m) and alpha -wCT(r) yield quantitative immunoprecipitations of rat NKCC1 under the experimental conditions described here, and we have not detected any differences in their behavior. alpha -wCT(m) was used in the experiments shown in Figs. 1-5 and alpha -wCT(r) was used in the experiment shown in Fig. 6.


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Fig. 1.   Phosphorylation of rat parotid basolateral membrane (BLM) proteins and the Na+-K+-2Cl- cotransporter (NKCC1) following addition of [gamma -32P]ATP to isolated membranes. Rat parotid BLMs were incubated for 30 s or 30 min (as indicated) with 40 µM [gamma -32P]ATP. After the phosphorylation reaction was terminated, total BLM proteins (left lanes) or immunoprecipitated NKCC1 (IPP; right lanes) were analyzed by SDS-PAGE and autoradiography. All procedures are described in METHODS.



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Fig. 2.   Effects of protein kinase inhibitors on the phosphorylation of NKCC1 by membrane-associated kinase(s). All procedures are described in METHODS. The addition of protein kinase inhibitors briefly preceded that of [gamma -32P]ATP. The autoradiograph shows typical NKCC1 immunoprecipitates. The kinase inhibitors tested were staurosporine (10 nM; Ss), K252a (100 nM; K), H-89 (1 µM), lavendustin A (3 µM; Lav), and bisindolylmaleimide I (2 µM; Bis). The bar graph shows the combined results of densitometric analysis of 4 independent experiments for each kinase inhibitor; phosphorylation of NKCC1 in the presence of the various kinase inhibitors is expressed as a percentage of that seen in an untreated control (C).



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Fig. 3.   Effects of protein phosphatase inhibitors on the dephosphorylation of NKCC1 by membrane-associated protein phosphatase(s). All procedures are described in METHODS. Reactions were stopped 30 min after the addition of [gamma -32P]ATP. The autoradiographs show typical NKCC1 immunoprecipitates (the exposure of the strip at right is shorter than that at left). The inhibitors tested were as follows: none (C), calyculin A (100 nM; CA), okadaic acid (100 nM; OA), microcystin-LR (100 nM; Mic), and sodium orthovanadate (0.1 and 1.0 mM; VO4). In the case of CA, OA, and Mic, addition of the inhibitor briefly preceded that of [gamma -32P]ATP; in all cases NKCC1 phosphorylation 30 s later was not significantly affected by the presence of inhibitor (data not shown). In the case of VO4, the inhibitor was added 60 s after the addition of [gamma -32P]ATP. The data shown in the bar graph represent the combined results of densitometric analysis of 3-5 independent experiments for each experimental condition; phosphorylation of NKCC1 in the presence of the various inhibitors is expressed as a percentage of that seen 30 s after the addition of [gamma -32P]ATP.



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Fig. 4.   Effects of cAMP and the catalytic subunit of protein kinase A (cPKA) on the phosphorylation of NKCC1 in isolated rat parotid BLMs. Rat parotid BLMs were incubated for 30 s in the presence of 40 µM [gamma -32P]ATP and 1.0 mM dithiothreitol (DTT) (C), 1.0 mM DTT plus 10 µM cAMP, or 1.0 mM DTT plus 16 U of cPKA as indicated. After the phosphorylation reaction was terminated, immunoprecipitated NKCC1 (top left) and immunoprecipitated NKCC1 digested with V8 protease (top right) were examined by autoradiography after SDS-PAGE or tricine-SDS electrophoresis, respectively. Combined results are shown (bottom) of densitometric analyses of 4 independent experiments after V8 protease digestion (see text for details). All experimental procedures are described in METHODS.



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Fig. 5.   Evidence that cPKA does not phosphorylate NKCC1 directly in rat parotid BLMs. The experiment was carried out in 2 steps. First, rat parotid BLMs in buffer I containing 2 mM DTT and 80 µM unlabeled ATP were preincubated for 15 s at 30°C in the presence (+cPKA) or absence (-cPKA) of 16 U cPKA (final volume 20 µl, containing 0.4 mg BLM/ml; this step was initiated by the addition of unlabeled ATP). Next, the following reagents (final concentrations) were added in the order specified: 1 µM H89 to both reactions, 16 U of cPKA to "-cPKA" and an equivalent volume of buffer to "+cPKA," and 40 µM [gamma -32P]ATP to both reactions (final volume 40 µl). Incubation at 30°C was continued for an additional 30 s, and then both reactions were stopped and analyzed for NKCC1 phosphorylation by immunoprecipitation, SDS-PAGE, and autoradiography as usual. A representative autoradiograph and the densitometric analysis of 4 independent experiments are shown. Preinc, preincubation.



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Fig. 6.   Direct evidence for the involvement of PKA in the phosphorylation of NKCC1 associated with beta -adrenergic stimulation. Digitonin-permeabilized rat parotid acini were incubated with [gamma -32P]ATP in the absence of 10 µM cAMP (C), in the presence of 10 µM cAMP, or in the presence of 10 µM cAMP plus 10 µM PKI(5-24) as indicated (all additions were made with [gamma -32P]ATP). The reaction was stopped after 1 min of incubation and analyzed for NKCC1 phosphorylation by immunoprecipitation, gel purification, V8 protease digestion, tricine-SDS electrophoresis, and autoradiography as described in METHODS. A representative autoradiograph (top) and the densitometric analysis (bottom) of the phosphorylation of the 17-kDa peptide from 4 independent experiments are shown. Measured relative to control conditions (C; 100%), the phosphorylations observed in the presence of cAMP and cAMP plus PKI were 184 ± 22 and 112 ± 13% for the 17-kDa phosphopeptide, 108 ± 11 and 85 ± 5% for the 10-kDa phosphopeptide, 111 ± 4 and 92 ± 3% for the 7-kDa phosphopeptide, and 115 ± 3 and 98 ± 2% for total undigested NKCC1, respectively.

Phosphorylation studies using intact rat parotid BLMs. Phosphorylation reactions were carried out at 30°C in buffer I (buffer A plus 20 mM sodium acetate, 60 mM potassium acetate, 60 mM KCl, 10 mM magnesium acetate, and 1 mM EGTA) containing 0.2 mg/ml rat parotid BLMs and other additions as indicated. The reaction was initiated by the addition of 40 µM [gamma -32P]ATP and stopped by the addition of an appropriate solution (see below). Unless otherwise stated, phosphorylation reactions were stopped 30 s after the addition of [gamma -32P]ATP. Under these experimental conditions we found that the half-time of [gamma -32P]ATP hydrolysis was ~10 s (data not shown; release of 32Pi from ATP was monitored by using the method described in Ref. 13).

In experiments in which the phosphorylation of all basolateral membrane proteins was examined, the phosphorylation reaction was stopped by the addition of 160 µl of ice-cold 18.85% TCA to 40 µl of reaction mixture (8 µg of membrane protein), and proteins were collected by centrifugation at 10,000 g for 10 min at 4°C. The tube was then rinsed with 200 µl of distilled water without disturbing the protein pellet, and the pellet was solubilized in SDS-PAGE sample buffer.

In experiments in which the phosphorylation of immunoprecipitated NKCC1 was examined, an ice-cold solution containing twice the concentrations of the ingredients of SS was added to an equal volume of reaction mixture. A 120-µl aliquot of the stopped phosphorylation reaction mixture (12 µg of membrane protein) was then combined with 40 µl of preconjugated protein G beads (slurry) and incubated for 4 h at 4°C. The beads were then collected by centrifugation, washed six times with 500 µl of SS (changing the tube on the last wash), and extracted with SDS-PAGE sample buffer.

Phosphorylation studies using permeabilized rat parotid acini. Rat parotid acini were prepared as previously described (19). Phosphorylation reactions were carried out at 37°C in buffer I containing a 20% suspension of acini, 0.1 mM sodium orthovanadate, and other additions as indicated (final volume 100 µl). The reaction was initiated by the addition of 40 µM [gamma -32P]ATP and 0.1 mM digitonin and was stopped by the addition of 500 µl of SS. After the addition of SS, insoluble debris was removed by centrifugation at 10,000 g for 10 min, and the resulting supernatant was combined with 30 µl (slurry) of preconjugated protein G beads for immunoprecipitation of NKCC1 as described in Phosphorylation studies using intact rat parotid BLMs.

Digestion with V8 protease. Digestion with V8 protease was carried out as previously described (9, 19). Briefly, bands corresponding to immunoprecipitated rat NKCC1 were cut from dried SDS-PAGE gels, rehydrated in 50 mM NH4HCO3 (pH 8.0) plus 1 mM DTT, and incubated with V8 protease (20 U/ml) for 6 h. The digested material recovered from the gel fragments was then dried, taken up in sample buffer, and subjected to tricine-SDS electrophoresis.

Gel electrophoresis and autoradiography. SDS-PAGE, tricine-SDS electrophoresis, autoradiography, and densitometry were carried out as previously described (9). Densitometric results are given as means ± SE for the number of replicates indicated. For SDS-PAGE we employed 4-20% gradient gels. For tricine-SDS electrophoresis we used 16% gels. Unless otherwise noted, the results shown are from SDS-PAGE gels.


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ABSTRACT
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DISCUSSION
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The two left-hand lanes of Fig. 1 show the results of an experiment in which rat parotid BLMs were incubated with [gamma -32P]ATP for 30 s or 30 min, as described in METHODS, and then analyzed by SDS-PAGE and autoradiography. After 30 s of incubation, a number of proteins, particularly in the molecular mass range 50-200 kDa, were labeled with 32P. Because the BLMs used in these experiments were extensively washed during their preparation (see METHODS), this result indicates the presence of one or more protein kinases tightly associated with the rat parotid BLM. In control experiments (not shown) we have demonstrated that overall BLM protein phosphorylation reaches a maximum ~30 s after the addition of [gamma -32P]ATP under our experimental conditions (as indicated in METHODS, the half-time of [gamma -32P]ATP hydrolysis was ~10 s under these conditions). After 30 min of incubation, much of the membrane phosphorylation is lost (Fig. 1), likewise demonstrating the presence of significant membrane-associated protein phosphatase activity (as indicated below, this loss of phosphorylation can be blocked by inhibition of protein phosphatases, confirming that it is not due to nonspecific effects such as proteolysis). As illustrated in the two right-hand lanes of Fig. 1, when NKCC1 was immunoprecipitated from these membranes, we found that it was one of the phosphoproteins detected. Analysis by scanning densitometry indicated that phosphorylation of NKCC1 at 30 s was 3.40 ± 0.08 times that observed at 30 min (n = 7).

Figure 2 illustrates the effects of various protein kinase inhibitors on the ATP-induced phosphorylation of NKCC1 in rat parotid BLMs. The effects of 10 nM staurosporine and 100 nM K252a (general serine/threonine kinase inhibitors with IC50 values in the range 1-10 nM and 20-200 nM, respectively), 1 µM H-89 (a relatively specific inhibitor of PKA with IC50 ~0.05 µM), 3 µM lavendustin A (a relatively specific inhibitor of tyrosine kinases with IC50 ~0.01 µM), and 2 µM bisindolylmaleimide I (a relatively specific inhibitor of protein kinase C with IC50 <0.1 µM for most isotypes) are shown. Marked inhibition of phosphorylation is seen with staurosporine and K252a, which were used in their usual effective range, but there is little or no effect of the other kinase inhibitors, all of which were used at concentrations at least an order of magnitude above their IC50 values. When we examined the effects of these protein kinase inhibitors on the ATP-induced phosphorylation of all BLM proteins (cf., 2 left-hand lanes of Fig. 1), qualitatively similar results were found (not shown).

Figure 3 shows the effects of various protein phosphatase inhibitors on the dephosphorylation of NKCC1 by membrane-associated protein phosphatase(s). The effects of calyculin A, okadaic acid, and microcystin-LR, all used at 100 nM, are shown in Fig. 3, left. Both calyculin A and microcystin-LR are known to inhibit serine/threonine protein phosphatase type 1 at concentrations of ~1 nM, and all three compounds inhibit serine/threonine protein phosphatase type 2A at concentrations of ~1 nM. However, even at 100 nM concentration these compounds have only modest effects on the dephosphorylation of NKCC1 by membrane-associated phosphatases; we suspect that this is because there are relatively small quantities of these predominantly cytosolic phosphatases that copurify with the BLM. In contrast, the more general phosphatase inhibitor, sodium orthovanadate, whose effects are shown in Fig. 3, right, markedly inhibits the dephosphorylation of NKCC1 at 0.1 mM concentration. Qualitatively similar results were found for the effects of these protein phosphatase inhibitors on overall dephosphorylation of all BLM proteins by membrane-associated phosphatases (not shown).

As already discussed, our previous studies demonstrate that the activity of NKCC1 is dramatically upregulated in response to beta -adrenergic stimulation of rat parotid acini and indicate that this effect is the result of NKCC1 phosphorylation. Our results also suggest that PKA may be involved in this effect. To explore this possibility further, we tested the effects of adding cAMP or cPKA to rat parotid BLMs in the presence of [gamma -32P]ATP. The results of such an experiment are shown in Fig. 4. The top, left-hand panel of Fig. 4 shows an autoradiograph of immunoprecipitated NKCC1 showing apparent increases in phosphorylation by both treatments relative to an untreated control. The top, right-hand panel of Fig. 4 shows an autoradiograph of V8 protease digests of these samples run on a tricine-SDS gel; in these digests we see a single phosphopeptide with molecular mass ~7 kDa. The bottom panel of Fig. 4 presents a densitometric analysis confirming that the phosphorylation of this 7-kDa peptide is increased by the presence of cAMP or cPKA. The effect of cAMP in this experiment is presumably due to the presence of PKA bound to A-kinase anchoring proteins (AKAPs) (1, 16) in the BLM and thereby copurified with them. We have, in fact, previously presented evidence for the presence of endogenous PKA and its cAMP-dependent phosphorylation of Na+-K+-ATPase in this BLM preparation (10).

However, as also stated earlier, the phosphorylation of NKCC1 that accompanies isoproterenol stimulation of intact acini and correlates with functional NKCC1 upregulation is associated with a 17-kDa peptide resulting from V8 protease digestion. Although an ~7-kDa phosphopeptide also was seen in these previous V8 protease digests (9, 19), its phosphorylation was not significantly affected by isoproterenol treatment. To better understand the effect of cPKA on the 7-kDa phosphopeptide shown in Fig. 4, we carried out the experiment shown in Fig. 5. Here BLMs were preincubated with unlabeled ATP in the presence or absence of cPKA, and then additions were made to both reactions to bring them to the same levels of cPKA and the PKA inhibitor H-89 (final concentration 1 µM), before adding [gamma -32P]ATP and measuring NKCC1 phosphorylation. As can be readily appreciated from Fig. 5, significantly more incorporation of 32P into NKCC1 is seen in the membranes preincubated with cPKA. However, because cPKA was present in both reactions with [gamma -32P]ATP, this increase in phosphorylation could not be due to a direct phosphorylation of NKCC1 by cPKA. Instead, the results of this experiment are consistent with the hypothesis that cPKA treatment of acinar BLMs results in the activation of endogenous membrane-associated kinase(s) or the inhibition of membrane phosphatases, which in turn leads to increased phosphorylation of NKCC1.

To be able to evaluate this result in the context of our earlier experiments with intact acini, we carried out the experiment illustrated in Fig. 6. Here digitonin-permeabilized acini were incubated with [gamma -32P]ATP alone, with [gamma -32P]ATP in the presence of cAMP, or with [gamma -32P]ATP plus cAMP and PKI(5-24), a specific peptide inhibitor of PKA. After incubation, the phosphorylation of NKCC1 was analyzed by V8 protease digestion. In this digest, three phosphopeptides are shown (Fig. 6, top), the 17-kDa and 7-kDa phosphopeptides previously observed in NKCC1 digests from intact acini (9, 19) and a 10-kDa phosphopeptide only seen with permeabilized acini (a 5.7-kDa phosphopeptide reported in Ref. 19 was not seen in our more recent experiments described here and in Ref. 9, where NKCC1 was immunopurified before gel purification and V8 protease digestion; in the experiments reported in Ref. 19 we digested gel slices containing all BLM proteins with Mr ~170 kDa, so the 5.7-kDa phosphopeptide is presumably from another ~170-kDa membrane protein). As shown in Fig. 6, the addition of cAMP to permeabilized acini results in the characteristic increase in phosphorylation of the 17-kDa peptide observed in our previous studies with intact acini stimulated with isoproterenol (9, 19). This result is consistent with our earlier observations that the effects of isoproterenol on NKCC1 can be mimicked by maneuvers that increase intracellular cAMP levels (18, 19). Moreover, as also shown in Fig. 6, the effect of cAMP on phosphorylation of the 17-kDa peptide can be blocked by PKI(5-24), providing strong evidence that PKA itself is indeed involved in this phosphorylation event in permeabilized acini, and thus presumably in intact acini as well. The fact that cPKA is unable to phosphorylate the 17-kDa peptide when it is added to isolated acinar BLM (Fig. 4) suggests that there is a participant in this phosphorylation reaction that is present in the acinar cell but absent from isolated membranes.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated here that isolated rat parotid BLMs possess significant membrane-associated protein kinase and protein phosphatase activities and that one of the proteins phosphorylated and dephosphorylated by these enzymes is NKCC1 (Figs. 1-3). The membrane-associated protein kinase activity appears to be of the serine/threonine type, because its effect on NKCC1 was blocked by the general serine/threonine kinase inhibitors staurosporine and K252a; however, specific inhibitors of PKA, tyrosine kinase, and protein kinase C had little or no effect on this kinase activity even at high concentrations (Fig. 2). The membrane-associated phosphatase activity was inhibited by the nonspecific phosphatase inhibitor sodium orthovanadate, but more specific inhibitors of protein phosphatases 1 and 2A were relatively ineffective (Fig. 3). Moreover, because our experiments were carried out in essentially Ca2+-free medium (1 mM EGTA), the involvement of protein phosphatase 2B (calcineurin) also can be excluded. Further experiments designed to identify the specific protein kinases and phosphatases involved in these effects were not attempted.

The phosphorylation of NKCC1 produced by the membrane-associated protein kinase activity was on a 7-kDa peptide resulting from V8 protease digestion (Fig. 4). Additional phosphorylation of this peptide was seen when isolated BLMs were incubated with ATP in the presence of cAMP (Fig. 4). This effect is presumably due to the presence of endogenous PKA in parotid BLMs. It has been shown that membrane-bound AKAPs bind PKA via its regulatory subunit RII and thereby anchor it near specific target proteins (1, 16). These target proteins are then rapidly phosphorylated in response to increased cellular cAMP, which releases cPKA from RII. Phosphorylation of Na+-K+-ATPase in response to cAMP addition has, in fact, previously been demonstrated in this same parotid BLM preparation and the involvement of AKAPs confirmed (10). As already emphasized, however, the upregulation of NKCC1 function associated with isoproterenol treatment and increased acinar cAMP levels correlates with the phosphorylation of a 17-kDa peptide resulting from V8 protease digestion. Initially we wondered if PKA was actually anchored near NKCC1 via AKAPs but had been stripped off during the BLM preparation, thus accounting for the absence of phosphorylation of the 17-kDa peptide with cAMP treatment. However, attempts to reload membrane-bound AKAPs with PKA, followed by centrifugation and cAMP plus [gamma -32P]ATP treatment, failed to yield phosphorylation of the 17-kDa peptide (not shown). Likewise, direct addition of cPKA plus [gamma -32P]ATP to BLM did not result in phosphorylation of the 17-kDa peptide, although NKCC1 phosphorylation of the 7-kDa peptide was increased (Fig. 4).

We did find, however, that preincubation of BLMs with cPKA before the addition of [gamma -32P]ATP resulted in markedly higher levels of NKCC1 phosphorylation than observed in untreated controls (Fig. 5). This result suggests that the effects of cAMP and cPKA on phosphorylation of NKCC1 are due to activation of the endogenous membrane-associated kinase activity or inhibition of membrane phosphatases, rather than a direct phosphorylation of NKCC1 by cPKA. Thus it appears as if PKA may be unable to directly phosphorylate NKCC1 at any site in isolated BLMs.

We previously observed a 7-kDa phosphopeptide in V8 protease digests of immunoprecipitated NKCC1 from intact rat parotid acini (9, 19). Because the endogenous protein kinase activity of the rat parotid BLM appears to be quite high and is clearly able to overpower the endogenous protein phosphatase activity (Fig. 1), we speculate that phosphorylation of NKCC1 on the 7-kDa peptide site is maintained near saturation levels in the intact acinar cell. This could account for the observation that no increase in phosphorylation at this site is observed with isoproterenol stimulation despite the resulting activation of PKA. The significance of this phosphorylation of NKCC1 in rat parotid acinar cells remains to be determined.

The fact that the phosphorylation of NKCC1 induced by isoproterenol treatment can be mimicked by the addition of cAMP to permeabilized acini and that this effect can blocked by a specific peptide inhibitor of PKA (Fig. 6) provides strong evidence for the involvement of PKA in this effect. Because this phosphorylation cannot be produced by the addition of PKA to BLM, we conclude that some factor present in intact cells but missing in isolated BLMs may be involved. Further experiments are required to identify this (putative) participant in the phosphorylation reaction; however, an obvious candidate would be a cytosolic kinase that is activated by PKA. In this regard, Forbush and colleagues have presented evidence that NKCC1 can be upregulated as a result of phosphorylation by an as yet unidentified Cl--dependent kinase (7, 11) and that this phosphorylation can be reversed by protein phosphatase type 1, which is directly targeted to NKCC1 (2). Whether these same enzymes are involved in beta -adrenergic stimulation-dependent NKCC1 phosphorylation in salivary glands remains to be determined. If this is the case, however, then this kinase must be activated by a non-Cl--dependent mechanism in these tissues because, as already stressed, intracellular Cl- levels in salivary acinar cells are not affected by beta -adrenergic stimulation.


    ACKNOWLEDGEMENTS

We thank Dr. Bruce J. Baum for helpful discussions and encouragement during the course of this work.


    FOOTNOTES

Address for reprint requests and other correspondence: R. J. Turner, Bldg. 10, Rm. 1A06, 10 Center Dr. MSC 1190, National Institutes of Health, Bethesda MD 20892-1190 (E-mail: rjturner{at}nih.gov).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00352.2001

Received 26 July 2001; accepted in final form 27 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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