Regulation and Identification of Na,K-ATPase α1 Subunit Phosphorylation in Rat Parotid Acinar Cells*

The stimulation of fluid and electrolyte secretion in salivary cells results in ionic changes that promote rapid increases in the activity of the Na,K-ATPase. In many cell systems, there are conflicting findings concerning the regulation of the phosphorylation of the Na,K-ATPase α subunit, which is the catalytic moiety. Initially, we investigated the phosphorylation sites on the α1 subunit in native rat parotid acinar cells using tandem mass spectrometry and identified two new phosphorylation sites (Ser222, Ser407), three sites (Ser217, Tyr260, Ser47) previously found from large scale proteomic screens, and two sites (Ser23, Ser16) known to be phosphorylated by PKC. Subsequently, we used phospho-specific antibodies to examine the regulation of phosphorylation on Ser23 and Ser16 and measured changes in ERK phosphorylation in parallel. The G-protein-coupled muscarinic receptor mimetic carbachol, the phorbol ester phorbol 12-myristate 13-acetate, the Ca2+ ionophore ionomycin, and the serine/threonine phosphatase inhibitor calyculin A increased Ser23 α1 phosphorylation. Inhibition of classical PKC proteins blocked carbachol-stimulated Ser23 α1 subunit phosphorylation but not ERK phosphorylation, which was blocked by an inhibitor of novel PKC proteins. The carbachol-initiated phosphorylation of Ser23 α1 subunit was not modified by ERK or PKA activity. The Na,K-ATPase inhibitor ouabain reduced and enhanced the carbachol-promoted phosphorylation of Ser23 and Ser16, respectively, the latter because ouabain itself increased Ser16 phosphorylation; thus, both sites display conformational-dependent phosphorylation changes. Ouabain-initiated phosphorylation of Ser16 α1 was not blocked by PKC inhibitors, unlike carbachol- or phorbol 12-myristate 13-acetate-initiated phosphorylations, suggesting that this site was also a substrate for a kinase other than PKC.

activity due to its insertion into the plasma membrane (18). In contrast, ERK contributed positively to Na,K-ATPase activity in intact cells and in vitro in a manner that did not rely on changes in Na,K-ATPase insertion into the plasma membrane (19). Relative increases of Na,K-ATPase activity in intact cells by PKA/cAMP were linearly related to the relative increases in the ␣ subunit phosphorylation (20). In comparison, the phosphorylation of the ␣ subunit on Ser 943 by PKA increased the PKC-dependent inhibition of Na,K-ATPase activity in isolated membranes (21). In addition to the sites mentioned above, additional sites on serine, tyrosine, and threonine residues have been identified, and a total of at least 20 phosphorylation sites are reported at databases such as PhosphoSitePlus.
Phosphorylation of the ␣ subunit on the C and N termini may be differentially regulated by the conformation of the ␣ subunit, although the particular regulation may vary for in vitro and in situ findings. In studies with purified rat kidney Na,K-ATPase, the phosphorylation of the ␣ subunit by PKC was increased and the phosphorylation by PKA was decreased by the presence of the Na,K-ATPase inhibitor ouabain (22). In contrast, in intact rat kidney cells, ouabain blocked the phosphorylation of the ␣ subunit by PKC (23).
The Na,K-ATPase plays an important role in regulating Na ϩ and K ϩ transport during the initiation of fluid secretion by neurotransmitters in salivary glands, including parotid, submandibular, and sublingual glands. Activation of the G-proteincoupled M3 muscarinic acetylcholine receptor (M3R) produces the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, which release intracellular Ca 2ϩ stores and increase PKC activity, respectively. Activation of the M3R by acetylcholine or the muscarinic mimetic carbachol promotes the elevation of [Ca 2ϩ ] i and the opening of Ca 2ϩ -sensitive K ϩ and Cl Ϫ channels and increases Na ϩ entry via the Na-K-2Cl cotransporter and other ion transporters (for review, see Ref. 24). Consequently, carbachol increases the Na,K-ATPase activity as much as 8-fold in parotid acinar cells (25). Inhibition of the Na,K-ATPase with ouabain blocks the increases in fluid secretion. In an early study, carbachol promoted a transient increase in the phosphorylation of the Na,K-ATPase ␣ subunit in metabolically labeled rat submandibular gland cells (26). PKA and cAMP increased the ␣ subunit phosphorylation in basolateral membrane vesicles purified from rat parotid glands, and it was suggested that protein kinase A anchoring protein 150 (27) plays an important role in promoting the phosphorylation of the ␣ subunit by PKA (28). In addition to the phosphorylation of the ␣ subunit by PKA, recent studies indicate that modulation of the Na,K-ATPase activity by PKA in cardiac cells involved glutathionylation of the ␤ subunit (29).
In the present studies, we examine the regulation of the ␣1 subunit phosphorylation in intact rat parotid acinar cells. In view of some of the conflicting results from previous studies of the ␣ subunit phosphorylation, we focused on identifying sites of phosphorylation on ␣1, determining the role of PKC in ␣1 phosphorylation, examining whether other kinases affect ␣1 phosphorylation by PKC, and determining whether conformational changes in ␣1 affect its phosphorylation.
LC/MS/MS Tandem Mass Spectrometry-For all mass spectrometry (MS) experiments, Na,K-ATPase ␣1 subunit immunoprecipitates were separated using SDS-PAGE, the gel was stained with Coomassie Blue and destained, and the ␣1 subunit band was excised. Samples were subjected to reduction with 10 mM dithiothreitol (DTT) for 30 min, alkylation with 55 mM iodoacetamide for 45 min, and in-gel digestion with L-1-tosylamido-2-phenylethyl chloromethyl ketone-modified trypsin or chymotrypsin overnight at pH 8.3 (50 mM ammonium bicarbonate) followed by reversed-phase microcapillary/tandem mass spectrometry (LC/MS/MS). LC/MS/MS was performed using an EASY-nLC splitless nanoflow HPLC (Proxeon Biosciences) with a self-packed 75-m inner diameter ϫ 15-cm C 18 column coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo Scientific) in the data-dependent acquisition and positive ion mode at 300 nl/min with one full MS-Fourier transform scan followed by six MS/MS spectra. MS/MS spectra collected via collision-induced dissociation in the ion trap were searched against the concatenated target and decoy (reversed) single entry ␣1-Na,K-ATPase and full Swiss-Prot protein databases using Sequest (Proteomics Browser software, Thermo Scientific) with differential modifications for Ser/Thr/Tyr phosphorylation (ϩ79.97) and the sample processing artifacts Met oxidation (ϩ15.99), deamidation of Asn and Gln (ϩ0.984), and Cys alkylation (ϩ57.02). Phosphorylated and unphosphorylated peptide sequences were identified whether they initially passed the following Sequest scoring thresholds against the target database: 1ϩ ions, Xcorr Ն 2.0 Sf Ն 0.4, p Ն 5; 2ϩ ions, Xcorr Ն 2.0, Sf Ն 0.4, p Ն 5; 3ϩ ions, Xcorr Ն 2.60, Sf Ն 0.4, p Ն 5 against the target protein database, where Xcorr represents Sequest cross-correlation score, and Sf represents Sequest final score. Passing MS/MS spectra were manually inspected to make sure that all b-and y-fragment ions aligned with the assigned sequence and modification sites. Determination of the exact sites of phosphorylation was aided using FuzzyIons and GraphMod, and phosphorylation site maps were created using the ProteinReport software (Proteomics Browser software suite, Thermo Scientific). False discovery rates of peptide hits (phosphorylated and unphosphorylated) were estimated below 1.50% based on reversed database hits.
Titanium Dioxide (TiO 2 ) Phosphopeptide Enrichment-Half of the digested peptide pool was reserved for enrichment with the Phos-trap phosphopeptide Enrichment Kit (PerkinElmer Life Sciences) containing TiO 2 -coated magnetic beads according to the vendor's protocol. Briefly, peptide mixtures containing phosphopeptides were acidified with Binding Buffer and incubated with 20 l of 20ϫ TiO 2 magnetic beads diluted in 180 l of HPLC grade water for 1 h at room temperature with continuous shaking in a room temperature incubator followed by washing three times with Binding Buffer and one time with Washing Buffer. Phosphopeptides were then incubated with 35 l of Basic Elution buffer with continuous shaking. Elution Buffer was then transferred to a 12 ϫ 32-mm autosampler vial with 50 l of HPLC A Buffer (0.1% formic acid), and the final solution was concentrated to 5 l using a SpeedVac prior to injection via LC/MS/MS.
Western Blot Analysis and Immunoprecipitation-At the end of the treatment period, native rat parotid acinar cells suspended at ϳ0.5-1 mg/ml were lysed and cleared of insoluble proteins by sedimentation at 15,000 ϫ g for 15 min at 4°C. For samples subjected to Western blot analysis without immunoprecipitation, cells were lysed in ice-cold Lysis buffer (137 mM NaCl, 20 mM Tris base, pH 7.5, 1 mM EGTA, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) IGEPAL) containing the following reagents: 1 mM vanadate, 4.5 mM sodium pyrophosphate, 47.6 mM NaF, 9.26 mM ␤-glycerophosphate, 0.5 mM dithiothreitol, 2 mg/ml leupeptin, 2 mg/ml pepstatin, 2 mg/ml aprotinin, and 2 mg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. The cleared supernatants were diluted with 5ϫ Laemmli sample buffer, heated for 30 min at 37°C, and stored at Ϫ20°C prior to electrophoresis. For rat parotid acinar cell and Par-C10 cell samples subjected to ␣1 immunoprecipitation for Western blot analysis, cells were lysed in the following Immunoprecipitation buffer: 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% IGEPAL, 0.5% SDS, 0.5 mM dithiothreitol, 20 mM NaF, 1 mM sodium vanadate, and the same mix of phosphatase inhibitors in the Lysis buffer. The lysate was cleared as above, and a6F antibody (ϳ20 g/ml) and a mixture of protein A-and protein G-Sepharose beads was added overnight (unless stated otherwise). The beads were col-lected by sedimentation, washed three times in PBS, 1% IGE-PAL solution, and then heated for 30 min at 37°C in Laemmli sample buffer. All samples were separated using SDS-polyacrylamide gel electrophoresis with an 8% separating gel and a 3% stacking gel. Proteins were transferred to nitrocellulose, and immunoblots were probed overnight with various antibodies according to the supplier's specifications. When probing immunoblots for ␣1 subunit, lysates were probed using either mouse monoclonal ␣1 (SC-21712) or rabbit polyclonal ␣1 (3010); immunoprecipitates (and lysates on the same blot) were probed using rabbit ␣1. Proteins were visualized using chemiluminescence reagents and x-ray film. Alternatively, in some experiments, proteins were visualized and quantified by direct infrared fluorescence using an Odyssey imaging system (LI-COR Biosciences) as reported previously (31).
For ␣1 subunit immunoprecipitates subjected to mass spectrometry analysis, cells were suspended at ϳ3-6 mg/ml and lysed in Immunoprecipitation buffer, and a6F antibody (ϳ40 g/ml) and a mixture of protein A-and protein G-Sepharose beads were added overnight. The collected beads were washed and treated as above and subjected to SDS-PAGE as described above for mass spectrometry.
Quantification of Protein Phosphorylation-The phosphorylation status of proteins visualized on film was quantified by densitometry using the NIH ImageJ software program. For each sample, the phosphoproteins were normalized to total protein level (ERK or ␣1 subunit) to account for gel loading/transfer variations. Blots were probed for phosphoproteins, stripped, and reprobed for total proteins. The phosphorylations for the various conditions were normalized to the phosphorylation under basal control (non-stimulated, no inhibitors) conditions as indicated. In experiments using the Odyssey imaging system, blots were simultaneously probed for phosphoproteins and total protein levels using polyclonal antibodies and mouse monoclonal antibodies, respectively, and fluorescent anti-rabbit and anti-mouse antibodies were used for visualization and subsequent quantification.
Data Analysis-Values were calculated as the mean Ϯ S.E. of n number of independent experiments (each n from a different cell preparation). The differences between the basal/control and the experimental samples were evaluated using a Student's t test. All Western blot experiments were performed at least three different times. Representative blots are shown in each figure. For each experiment to be analyzed using Western blotting techniques and/or the Odyssey system, multiple (duplicate or triplicate) cell samples were collected for each condition, and the average of the values obtained within each individual experiment was treated as n ϭ 1.

Identification of Sites of ␣1 Subunit Phosphorylation by Mass
Spectrometry-The main isoform of the ␣ subunit of the Na,K-ATPase in rat parotid acinar cells is ␣1 (31,32). Initially, we used LC/MS/MS to identify phosphorylation sites on ␣1 obtained from immunoprecipitating the protein from rat parotid cells, and we also attempted to determine differences in phosphorylation status in cells exposed to different stimuli. We identified seven different phosphorylation sites, of which six were on serine residues and one was on tyrosine. These are listed in Table 1, along with the surrounding sequence, database scoring information about the identification, and putative kinases responsible for phosphorylation. Two sites (Ser 222 , Ser 407 ) appear to be novel ones that have not yet been reported. Two sites (Ser 47 , Ser 217 ) were previously identified by mass spectrometry in several published proteomic screens, and the tyrosine phosphorylation site (Tyr 260 ) was identified in many proteomic studies, most of them internal studies by Cell Signaling Technologies. Two sites (Ser 16 , Ser 23 ) have been identified as sites phosphorylated by PKC and are the subject of various studies (5,7,8,11,14,42).
In addition to trypsin, we used chymotrypsin for digestion of the ␣ subunit protein, and in some studies, we also utilized TiO 2 to enrich digestions for phosphopeptides (33,34). In several experiments, after not finding any phosphorylation sites on the ␣1 subunit, we pooled samples obtained from cells treated with different agents to increase the amount of protein subjected to analysis. Ultimately, using LC/MS/MS, we were unable to clearly demonstrate quantitative changes in phosphorylation due to stimulation of the cells with various agents, including carbachol, isoproterenol, and PMA, because phosphorylation levels were generally of low stoichiometry and peptide signals are sometimes suppressed by the addition of phosphate groups (35). Consequently, we decided to use commercially available phospho-antibodies to evaluate the regulation of ␣1 subunit phosphorylation.
Effects of Various Stimuli on ␣1 Subunit Phosphorylation-Because M3R stimulation produces a rapid and large increase in Na,K-ATPase activity in parotid acinar cells (25), we examined the effects of the muscarinic ligand carbachol on ␣1 phosphorylation. We also measured changes in ERK phosphorylation as a representative signaling molecule because carbachol also rapidly activates ERK in parotid acinar cells. Carbachol increased Ser 23 ␣1 subunit phosphorylation within 2 min, and the phosphorylation declined after peaking at 2-3 min (Fig. 1). The carbachol-induced phosphorylation of ERK also displayed a similarly transient time course, as we recently reported (36).
We also examined changes in Ser 23 ␣1 subunit and ERK phosphorylation by other agents that affect the Na,K-ATPase activity and fluid and protein secretion. Not surprisingly, the phorbol ester PMA, an activator of PKC, increased Ser 23 ␣1 subunit and ERK phosphorylations ( Fig. 2A), consistent with the phosphorylation of both proteins being dependent on PKC. Ionomycin, a divalent ionophore that elevates intracellular Ca 2ϩ and stimulates fluid secretion and Na,K-ATPase activity by opening Ca 2ϩ -sensitive K ϩ and Cl Ϫ channels, increased Ser 23 ␣1 subunit but not ERK phosphorylation. In contrast, nystatin, a monovalent cationophore that increases the activity of the Na,K-ATPase in parotid acinar cells by increasing the intracellular Na ϩ /K ϩ ratio (25), did not increase Ser 23 ␣1 subunit or ERK phosphorylation. Ouabain, a cardiac glycoside that binds to the ␣ subunits of the Na,K-ATPase and inhibits its activity, also did not increase Ser 23 ␣1 subunit or ERK phosphorylation. Neither Ser 23 ␣ subunit nor ERK phosphorylation was increased by isoproterenol (2-5 min), which activates the adenylyl cyclase-coupled parotid ␤-adrenergic receptor, or by forskolin (2-12 min), which directly activates adenylyl cyclase. Isoproterenol and forskolin stimulate PKA downstream of adenylyl cyclase, promoting protein exocytosis in parotid acinar cells. The relative effects of all of these agents on Ser 23 ␣1 subunit phosphorylation are shown in Fig. 2B, along with that of calyculin A, a serine/threonine phosphatase inhibitor that promoted the largest increase. Notably, immunoblots obtained using cell lysates were similar to those found using ␣1 subunit immunoprecipitations (Fig. 2C), confirming that the protein recognized by the Ser 23 phospho-specific antibody was indeed ␣1.
cPKC Inhibition Blocks Phosphorylation of Ser 23 ␣1 Subunit but Not ERK-Because the ␣1 subunit is directly phosphorylated on Ser 23 by PKC in various cells, we examined the effects of PKC inhibitors on ␣1 phosphorylation promoted by carbachol and PMA in parotid acinar cells. We compared the inhib-

TABLE 1
Tandem mass spectrometry identification of phosphorylation sites on Na,K-ATPase ␣1 subunit in rat parotid acinar cells Samples were obtained prepared using ␣1-immunopreciptations followed by proteolysis and LC/MS/MS analyses. Shown are conditions for which each site was identified and putative kinases that are known or predicted to phosphorylate specific sites. The abbreviations used are: CK2, casein kinase 2; IR, insulin receptor; IGF1R, IGF1 receptor; ATMK, ataxia telangiectasia mutated kinase; B, basal; I, isoproterenol; C, carbachol; CA, calyculin A; P, PMA; pool, BϩCϩI combined; Charge, charge state of peptide ion; ⌬mass, difference between experimental peptide mass and known sequence; Sf, Sequest final score; MHϩ, protonated molecular mass; Xcorr, Sequest cross-correlation score; ⌬Cn, Xcorr difference between the top ranked and next best sequence; Sp, Sequest preliminary score; rank, top hit from protein database. Accession number was AAB81285 for all sequences. All sequences were ranked No. 1 as the top hit from the protein database. itory effects of Go6976, an inhibitor that blocks cPKC family members, with those of GF109203X, a PKC inhibitor that blocks both cPKC and nPKC family members. In parallel, we examined the phosphorylation of ERK, which is also dependent on PKC in rat parotid acinar cells (37). Both PKC inhibitors substantially blocked Ser 23 ␣1 subunit phosphorylation by carbachol (Fig. 3A) and PMA (Fig. 3B), suggesting that a cPKC protein was largely responsible for phosphorylating ␣1 subunit on this site. In contrast, Go6976 was ineffective at blocking ERK phosphorylation, indicating that ERK phosphorylation is dependent on nPKC proteins in parotid acinar cells. The differ-ent relative effects of the two PKC inhibitors on the ␣1 subunit (Fig. 3C) and ERK (Fig. 3D) phosphorylation indicate that different PKC family members are upstream of ERK phosphorylation relative to those responsible for ␣1 phosphorylation. We also examined changes in ␣1 subunit phosphorylation in Par-C10 cells, an immortalized rat parotid acinar cell line that also expresses ␣1, although at levels much less than those found in native parotid acinar cells (31). Both PKC inhibitors also blocked ␣1 phosphorylation on Ser 23 in carbachol-treated Par-C10 cells (Fig. 3E), similar to the effects noted in freshly isolated rat parotid acinar cells.

Sequence
Although the data indicated that the stimulation of ERK and ␣1 subunit phosphorylation was due to distinct PKC family members, we examined the possibility that ERK activity contributed to ␣1 phosphorylation on Ser 23 . Treatment of cells with U0126, an inhibitor of MEK, the kinase immediately upstream of ERK, blocked both the basal and the carbacholstimulated ERK phosphorylation but did not affect the carbachol-promoted ␣1 subunit phosphorylation on Ser 23 (Fig. 4), consistent with the lack of a contribution of ERK to this phosphorylation in parotid acinar cells.
Protein Kinase A Does not Affect Ser 23 ␣1 Subunit Phosphorylation-It was reported that phosphorylation of ␣ on Ser 943 by PKA increased the PKC-promoted inhibition of Na,K-ATPase activity in vitro, perhaps by enhancing the PKC-dependent phosphorylation of ␣1 (21). Therefore, we examined whether PKA affected Ser 23 phosphorylation by pretreating cells with isoproterenol and forskolin prior to activating PKC with carbachol and PMA (Fig. 5). Isoproterenol and forskolin, which both promote cAMP production, increased PKA activity, as indicated in immunoblots probed with an antibody that recognizes substrate proteins phosphorylated by PKA. Neither of the PKA activators reduced the carbachol-or PMA-promoted phosphorylation of ␣1 subunit on Ser 23 . In contrast, the PKA activators blocked the increases in ERK phosphorylation promoted by either carbachol or PMA, similar to our recent report that PKA inhibits PKC-dependent ERK activation by these and other stimuli by Ͼ60% (36). The different effects of PKA on ERK (inhibition) and Ser 23 ␣1 subunit (no effect) phosphorylation is also consistent with the phosphorylation of these two proteins being downstream of different PKC families (see "Discussion").
Ouabain Blocks Ser 23 and Increases Ser 16 ␣1 Subunit Phosphorylation-Because the phosphorylation of the ␣ subunit by different kinases is affected by its conformation, we examined the effect of ouabain on the phosphorylation of ␣1 on Ser 23 promoted by various agents. In these experiments, the Na,K-ATPase activity of intact parotid acinar cells was inhibited by ouabain, and then cells were exposed to other phosphorylationpromoting agents. Notably, ouabain substantially blocked the increases in Ser 23 ␣1 subunit phosphorylation initiated by exposure of the cells to carbachol, PMA, and calyculin (Fig. 6, A  and C). This suggests that the availability of Ser 23 for PKC phosphorylation is reduced when ouabain induces a change in the conformation of ␣1 subunit. In contrast, ouabain did not block increases in ERK phosphorylation by various stimuli; moreover, we reported previously that ouabain increased the phosphorylation of ERK by submaximal (e.g. 10 Ϫ6 M) concentrations of carbachol (38). Although ouabain did not affect the basal phosphorylation of ␣1 subunit on Ser 23 , the exposure of cells to ouabain produced an increase in Ser 16 phosphorylation (Fig. 6B). Carbachol and PMA produced much more modest relative increases in the phosphorylation of the ␣1 subunit on Ser 16 (Fig. 6D) than on Ser 23 (Fig. 6C). Pretreatment of cells with ouabain for 1 min enhanced the increase in Ser 16 phosphorylation promoted by carbachol, increasing it to the level stimulated by PMA, which was not enhanced further by ouabain (Fig. 6D). Because ouabain depolarizes rat parotid acinar cells and elevates [Ca 2ϩ ] i in some cells (although not in parotid acinar cells (38)), we compared the stimulatory effect of ouabain on ␣1 subunit Ser 16 phosphorylation with that of increasing the extracellular KCl concentration by 20 mM, which depolarizes parotid acinar cells as much as does 1 mM ouabain 3 , and with that of ionomycin, a Ca 2ϩ ionophore that increases the [Ca 2ϩ ] i . The depolarizing effects of KCl did not produce an increase in ␣1 subunit Ser 16 phosphorylation, and ionomycin promoted a decrease, not an increase, in this phosphorylation (Fig. 6D). Not surprisingly, a 20 mM increase in the extracellular NaCl concentration, an experimental control to mimic the extracellular osmotic change of KCl, also did not affect Ser 16 phosphorylation.
We also examined the PKC dependence of changes in the Ser 16 phosphorylation of ␣1 subunit by various stimuli. The increases promoted by carbachol and PMA were significantly blocked by GF109203X but not by Go6976 (Fig. 6E). These results are different from the similar inhibitory effects of both PKC inhibitors on the increases in Ser 23 phosphorylation promoted by carbachol and PMA (Fig. 3C). Moreover, the increases in Ser 16 phosphorylation in cells exposed to ouabain were not blocked by either PKC inhibitor. These results suggest that there are multiple mechanisms by which the ␣1 subunit is phosphorylated on Ser 16 .

DISCUSSION
The results of this study demonstrate the following characteristics of the phosphorylation of the ␣1 subunit of the Na,K-ATPase in rat parotid acinar cells. 1) Carbachol, PMA, and calyculin A increase its phosphorylation on Ser 23 ; 2) in contrast to the phosphorylation of ERK by nPKC proteins, the carbacholinitiated phosphorylation of ␣1 subunit on Ser 23 is largely dependent on cPKC proteins; 3) PKA and ERK do not affect the carbachol-initiated phosphorylation of ␣1 subunit on Ser 23 ; and 4) the Na,K-ATPase inhibitor ouabain reduces the stimulus-dependent phosphorylation on Ser 23 and promotes the stimulus-independent phosphorylation on Ser 16 .
Using mass spectrometry, we identified several novel phosphorylation sites (Ser 222 and Ser 407 ) on the ␣1 subunit in addition to other known phosphorylated sites that have been found in large proteomic screens (see Table 1), which can be accessed from the PhosphoSite database. Due to the abundance of lysine residues around Ser 23 , we did not see this site phosphorylated until we used chymotrypsin for digestion and consolidated the phosphorylated peptides using TiO 2 . Consequently, we focused our studies on using phospho-specific ␣1 antibodies for Ser 23 and Ser 16 , the two sites previously demonstrated to be phosphorylated directly by PKC.
Carbachol and PMA produced a rapid increase in the phosphorylation of the ␣1 subunit on Ser 23 . By comparing the effects of two PKC inhibitors (Go6976, G203109X) that had different 3 S. P. Soltoff, unpublished studies. inhibitory selectivities, we demonstrated that a member(s) of the cPKC family (␣, ␤, ␥) was responsible for the majority of phosphorylation on Ser 23 . The contrasting effects of ionomycin (increase) and nystatin (no effect) on ␣1 subunit Ser 23 phosphorylation indicate that increases in the phosphorylation on this site are not secondary to increases in Na,K-ATPase activity, which these agents produce via changes in monovalent (nystatin) and divalent (ionomycin) cations. Although ERK phosphorylation induced by carbachol is largely PKC-dependent, it was not blocked by Go6976, which is selective for cPKC proteins. The same was true for PMA-promoted ERK phosphorylation. These data clearly demonstrate that carbachol activates different PKC families and that these have different functional roles downstream of the M3R activation in parotid cells. The parotid ␤-adrenergic receptor largely does not increase PKC activity, so it was not surprising that the ␤-receptor ligand isoproterenol did not increase ␣1 subunit phosphorylation on Ser 23 .
Because there is literature regarding cross-talk between ERK-and PKC-dependent ␣ subunit phosphorylation (8) (see the Introduction), we examined the effect of UO126, a MEK inhibitor that blocks ERK phosphorylation, on the carbacholinitiated Ser 23 ␣1 phosphorylation. The ineffectiveness of UO126 in blocking this phosphorylation indicates that ERK does not have an impact on the PKC-dependent phosphorylation of ␣1 subunit on Ser 23 in parotid acinar cells. Similarly, we did not find any cross-talk between PKA and Ser 23 ␣1 phosphorylation. Because isoproterenol and forskolin block PKC-dependent ERK phosphorylation (36) but not PKC-dependent Ser 23 ␣1 phosphorylation, these results are a second demonstration (in addition to the PKC inhibitor studies) that different PKC families promote ERK and ␣1 phosphorylation. These findings also indicate that PKA blocks the phosphorylation of a protein downstream of nPKC activation (ERK) but not downstream of cPKC activation (␣1 subunit).
Although ouabain increased the PKC-dependent phosphorylation of purified rat kidney ␣ in vitro (22), it was not entirely surprising that ouabain reduced the stimulus (carbachol, PMA, calyculin A)-dependent phosphorylation of ␣1 subunit on Ser 23 in parotid acinar cells because ouabain blocked the phorbol ester-initiated phosphorylation of ␣1 in intact rat kidney cells (23). One can imagine that the binding of the Na,K-ATPase inhibitor and the subsequent change in the conformation of the  ␣1 subunit alters the accessibility of a PKC phosphorylation site. However, it was surprising that ouabain increased the phosphorylation of ␣1 on Ser 16 in a PKC-independent fashion. These effects of ouabain were not secondary to [Ca 2ϩ ] i elevation or depolarization of the cells. This suggests that Ser 16 becomes more accessible for phosphorylation, perhaps involving the inhibition of a phosphatase, when the Na,K-ATPase is inhibited in the absence of a stimulus that increases PKC activity. This still begs the question of the identity of the kinase that is upstream of the ouabain-promoted phosphorylation of Ser 16 . Notably, the increases of the phosphorylation of ␣1 on Ser 16 by carbachol and PMA were PKC-dependent; however, these phosphorylations appeared to be due to nPKCs, a different family from that which promoted the phosphorylation of ␣1 subunit on Ser 23 by these stimuli.
The effects of PKC on Na,K-ATPase activity are varied, and this may be due to the fact that there are four ␣ isoforms that may be differentially regulated (reviewed in Refs. 1 and 3). Sottejeau et al. (39) recently reported that an isoformspecific region in ␣1, a dileucine motif that is conserved among mammalian ␣1 proteins but not other ␣ isoforms, affected the trafficking and PKC-dependent activation of Na,K-ATPase activity. They suggest that the two leucines may be part of a recognition motif for adapter proteins that affects the internalization of the Na,K-ATPase, thereby acting as an additional regulatory site that, along with the PKC phosphorylation sites on the ␣1 subunit, affect the expression and functional activity of the Na,K-ATPase. Because some agents produce multiple changes in ␣1 subunit phosphorylation, in some cases, it can be difficult to conclude that specific phosphorylation sites are causal for changes in Na,K-ATPase activity. The PKC inhibitor GF109203X blocked insulin-promoted increases in Na,K-ATPase activity and decreases in Ser 23 ␣1 subunit phosphorylation in mouse corneal endothelial cells (40). These observations led investigators to suggest that the insulin receptor tyrosine kinase promoted a PKCmediated activation of Ser/Thr phosphatase, although contributions from tyrosine phosphorylation were not assessed in these studies. In contrast, in response to a protein tyrosine phosphatase inhibitor, human renal carcinoma cells displayed a decrease in Na,K-ATPase activity that was attributed to decreases in ␣1 subunit Ser/Thr phosphorylation and/or increases in its tyrosine phosphorylation (41).
In studies examining the phosphorylation of human ␣l, which has a glycine instead of a serine at position 23, the phosphorylation on Ser 16 promoted either stimulation or inhibition of Na,K-ATPase activity depending on the PKC family member that mediated this phosphorylation. In addition, stimuli that increased the phosphorylation of both Ser 16 and Ser 23 on rat ␣1 subunit promoted the recruitment of the Na,K-ATPase to the plasma membrane, but this enzyme was internalized by endocytosis when it was phosphorylated only on Ser 23 (42). Our findings demonstrated that several conditions (ouabain; PMA in the presence of Go6976) imposed on rat parotid acinar cells FIGURE 6. Effects of ouabain on ␣1 subunit phosphorylation on Ser 23 and Ser 16 . In A-D, rat parotid acinar cells were treated for 1 min with vehicle (Ϫ) or 1 mM ouabain and subsequently treated with carbachol (CCh, 10 Ϫ5 M, 2 min), PMA (100 nM, 2 min), or calyculin A (CalyA,100 nM, 10 min). Ouabain treatment without other agents was for 3 min, except that cells also were exposed for 15 min (ϩ*) in D. In D, cells were exposed to 20 mM KCl, 20 mM NaCl, and 10 Ϫ6 M ionomycin (Iono) (all for 3 min). A and B, cell lysates were analyzed by Western blot as indicated. P-S23-␣1, ␣1 subunit Ser 23 phosphorylation; P-S16-␣1, ␣1 subunit Ser 16 phosphorylation; P-ERK, ERK phosphorylation. C and D, the phosphorylation of ␣ subunit on Ser 23 (C, S23-Alpha1) and Ser 16 (D, S16-Alpha1) was quantified relative to basal conditions (no ouabain). n ϭ 3-11. *, p Ͻ 0.01, and **, p Ͻ 0.05 versus basal (no additions) or for paired (ϩ ouabain) as indicated. In E, rat parotid acinar cells were treated for 10 min with vehicle (Ϫ), Go6976 (Go, 1 M), and GF109203X (10 M) followed by ouabain (1 mM, 3 min), carbachol (10 Ϫ5 M, 2 min), or PMA (100 nM, 2 min). n ϭ 5-7. *, p Ͻ 0.01 and **, p Ͻ 0.05 versus basal (no additions) or for paired (ϩ GF109203X (GF)) as indicated.
would establish yet a different end point: the phosphorylation of ␣1 mainly on Ser 16 but not Ser 23 .
In summary, we have identified multiple phosphorylation sites on the ␣1 subunit of the Na,K-ATPase in native rat parotid acinar cells, which initiate fluid and electrolyte secretion leading to saliva formation. These sites include two that appear to be novel and others previously detected mainly by large proteomic studies. Activation of the M3R and PKC rapidly increases the phosphorylation of the ␣1 subunit on Ser 23 and Ser 16 , and these phosphorylations are differentially modified by inhibition of the Na,K-ATPase with ouabain. It remains to be determined whether the phosphorylation of multiple N-terminal sites on the parotid ␣1 subunit promotes its insertion into or removal from the basolateral membrane, events that are among the fates of the phosphorylated Na,K-ATPase in other cellular systems, and to determine the localization changes of ␣1 in response to modulating these phosphorylations.