©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Basis for Species-specific Differences in the Phosphorylation of Na,K-ATPase by Protein Kinase C (*)

Marina S. Feschenko (1), Kathleen J. Sweadner (1)(§)

From the (1) Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
FOOTNOTES
REFERENCES

ABSTRACT

There is considerable evidence that protein kinases play a role in regulation of the activity of the Na,K-ATPase, but the characteristics of direct kinase phosphorylation of Na,K-ATPase subunits are still not well understood. There are 36 sites that could qualify as protein kinase C motifs in rat 1. Here we have used protein fragmentation with trypsin to localize the site of phosphorylation of the rat Na,K-ATPase 1 subunit to within the first 32 amino acids of the N terminus and then used direct sequencing of the phosphorylated protein to determine which of two candidate serine residues was modified. The result was that at most 25% of the P was found on Ser-11, a site that is well conserved in Na,K-ATPase 1 subunits. The remaining 75% or more of the P was found on Ser-18, a site that is absent in many Na,K-ATPase subunit sequences. This accounts for the observation that dog and pig 1 subunits can be phosphorylated by protein kinase C only to much lower levels than can rat 1. It is also likely to be relevant to other known species-specific effects of protein kinase C on Na,K-ATPase.


INTRODUCTION

Na,K-ATPase() is an integral membrane protein that maintains the gradients of sodium and potassium ions, using the energy of ATP hydrolysis. It is composed of two subunits in equimolar amounts: , the catalytic subunit, and , a glycoprotein. Both subunits have multiple isoforms (1, 2) . Three isoforms of the subunit, 1, 2, and 3, share 85-86% amino acid sequence identity. A major region of divergence, both among isoforms and among species, is the N-terminal 60-70 amino acids. There is evidence that the N terminus plays a role in regulating the K deocclusion pathway and the Na affinity of the Na,K-ATPase (3, 4), making this region of particular interest. A recent study has demonstrated protein kinase C-mediated phosphorylation of serine and threonine residues in the corresponding region of the Bufo marinus 1 subunit (5) .

A physiological role for protein kinase C regulation of Na,K-ATPase is controversial. Although there are reports of inhibition of enzyme activity as a direct result of phosphorylation in the dogfish shark and duck salt gland enzymes (6, 7) , our own attempts to detect inhibition of rat kidney enzyme have been negative (8) . Other studies performed on rat nerve have shown puzzling inconsistencies, nonetheless consistent with a role of protein kinase C in physiological regulation of the Na,K-ATPase (see Ref. 9 and references therein). The discrepancies might be better understood if the sites of phosphorylation were known with certainty.

Previously we showed that (in contrast to phosphorylation by protein kinase A) phosphorylation of Na,K-ATPase by protein kinase C was not equal in stoichiometry in different species. A stoichiometry of 0.9 mol/mol was found for rat 1, while only 0.15 mol/mol was found for either dog or pig 1 (8) . Here we investigated the underlying basis for the different levels of phosphorylation.


EXPERIMENTAL PROCEDURES

Na,K-ATPase and Protein Kinase C

Purified rat, pig and dog kidney Na,K-ATPases were prepared by the method of J(10) using frozen kidneys obtained from PelFreeze Biologicals (Rogers, AR). Rat axolemma Na,K-ATPase was purified from frozen rat brains (PelFreeze Biologicals) according to Sweadner (11) . All enzyme preparations were stored in buffer containing 20 mM Tris (pH 7.2), 320 mM sucrose, 1 mM EDTA. Specific activities of Na,K-ATPase from kidneys and axolemma were 1200-1700 and 850-1000 µmol/h/mg, respectively. Protein concentration was determined by the method of Lowry using bovine serum albumin as a standard (12) . PKC from rat brain was purchased from CalBiochem, La Jolla, CA, or Boehringer Mannheim GmbH Biochemica, Germany. The PKC preparations used consists of , , and isoforms (information provided by the supplier).

Phosphorylation of Na,K-ATPase by PKC

Purified Na,K-ATPase at a concentration of 50 µg/ml was preincubated with PKC (1.5 µg/ml) for 3 min at 30 °C in buffer containing 10 mM Tris phosphate (pH 7.4), 5 mM magnesium acetate, 500 µM CaCl, 100 nM phorbol 12-myristate 13-acetate (Sigma), 80 µg/ml phosphatidyl serine (Sigma, P7769) in a sonicated suspension. The reaction was started by the addition of [-P]ATP (4000-6000 cpm/pmol) to a final concentration 40-50 µM. Phosphorylation proceeded for 30 min at 30 °C and was stopped with an equal volume of electrophoresis sample buffer containing 125 mM Tris (pH 6.8), 4% SDS, 20% glycerol, and bromphenol blue (Laemmli sample buffer).

Gel Electrophoresis, Electrophoretic Transfer, and Immunostaining

Electrophoresis was performed as described previously (13) , using 7.5, 10, or 12.5% polyacrylamide gels. Electrophoretic transfer of proteins was performed in buffer containing 20 mM Tris, 150 mM glycine, and 20% methanol at 0.15 A overnight. Nitrocellulose blots were stained with 0.1% Amido Black, and phosphoproteins were visualized by autoradiography of the dried nitrocellulose on Kodak XAR-5 film. Densitometry was performed with a Molecular Dynamics laser densitometer. Film exposures were chosen in which the darkest bands were not overexposed.

For immunostaining, nitrocellulose membranes were immersed in 20 mM Tris-HCl buffer (pH 7.5), containing 150 mM NaCl and 0.5% Tween 20 (TBS-T) for 30-40 min at room temperature. Incubations with the primary and horseradish peroxidase-conjugated secondary antibodies were performed in the same buffer for 1.5 and 1 h, respectively. Membranes were extensively washed with TBS-T after each step, and finally stained with luminescent ECL reagents (Amersham Corp.). Four different Na,K-ATPase antibodies were used in this study: peptide-directed antibodies 754 (anti N terminus) and 845 (anti C terminus) (the generous gift of W. J. Ball, Jr., University of Cincinnati) (14) ; monoclonal antibody McK1, which binds near the N terminus of the 1-subunit (13) ; and polyclonal antiserum K1, which was raised against purified rat kidney Na,K-ATPase 1 subunit and is known to recognize polypeptide fragments from different parts of the molecule (N terminus, C terminus, and central loop) (15, 16) .

Trypsinolysis of Na,K-ATPase

Phosphorylated rat kidney Na,K-ATPase was incubated with trypsin at a protease/ATPase ratio of 1:100 either directly in phosphorylation medium or after centrifugation (40,000 rpm, 4 °C for 1 h) and resuspension in 25 mM imidazole, 1 mM EDTA (pH 7.4), 15 mM NaCl. Tryptic hydrolysis was allowed to proceed for the times indicated and then stopped by the addition of 1 mM diisopropyl fluorophosphate. Tryptic hydrolyzates were precipitated with cold methanol (9 volumes of methanol/1 volume of protein solution), and then resuspended in Laemmli sample buffer. Some aggregation occurred after precipitation (see Fig. 4D). L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (type XIII) was from Sigma.


Figure 4: Mapping of PKC phosphorylation site by limited tryptic hydrolysis of Na,K-ATPase in the E1 conformation. After phosphorylation by PKC, purified Na,K-ATPase was transferred to medium containing 25 mM imidazole, 1 mM EDTA (pH 7.4), and 15 mM NaCl. Trypsin was added at a protease/ATPase ratio of 1:100. Lane1, no trypsin; lane2, 1 min of incubation with trypsin. A, autoradiograms of P-labeled polypeptides after SDS-gel electrophoresis and electrophoretic transfer to nitrocellulose; B, staining of the blot with the antibody against the N terminus (754); C, restaining with monoclonal antibody McK1; D, restaining with polyclonal antibody K1. Except for a smudge of low molecular mass material, the pattern of P signal closely followed the presence of intact N terminus, and no smaller fragments were found to have incorporated phosphate.



Proteolytic Fingerprinting

Tryptic digestion of Na,K-ATPase was also performed during gel electrophoresis (17) . Phosphorylation of Na,K-ATPase was stopped with electrophoresis sample buffer, and then soybean trypsin inhibitor (Sigma) and trypsin were added to concentrations indicated in figure legends. Samples were loaded on a 1.5 mm thick 12.5% gel and run at constant current of 30 mA.

N-terminal Sequence Analysis

Purified rat kidney Na,K-ATPase was phosphorylated by PKC with [-P]ATP as described above. After 30 min at 30 °C, proteins were precipitated with 3 volumes of cold chloroform/methanol (2:1 (v/v)), vortexed, and centrifuged at 3000 rpm for 15 min, at 4 °C. The separated aqueous and organic phases were carefully removed. The protein extract located at the interface was saved, dried under a stream of nitrogen, and dissolved in Laemmli sample buffer. Labeled Na,K-ATPase (M 95,000) and autophosphorylated PKC (M 80,000) were separated on a 7.5% Laemmli gel and then electroblotted onto polyvinylidine difluoride membranes (ProBlott, Applied Biosystems Inc.) in 10 mM CAPS, pH 11, containing 20% methanol. Membranes were stained with 0.1% Coomassie Blue R-250, and the band of Na,K-ATPase was cut out, washed with deionized water, and air dried. N-terminal sequence analysis by Edman degradation was performed at the Biopolymer Core Facility, Endocrine Unit, Massachusetts General Hospital, with an Applied Biosystems 477A pulsed liquid sequencer in conditions developed to permit elution and quantification of phosphorylated serines.() In two separate experiments, 50 and 25 pmol of subunit were applied for sequencing, and initial yields of phenylthiohydantoin-derivatives were 20-25 and 14-15 pmol, respectively.


RESULTS

Phosphorylation of Na,K-ATPase from Different Species by Protein Kinase C

Fig. 1 illustrates phosphorylation of purified Na,K-ATPase from rat, dog, and pig kidneys. In conditions where phosphorylation of rat kidney -subunit was close to 1 mol of P/mol of Na,K-ATPase, pig and dog Na,K-ATPases gave only 10-19% of this level (data of four experiments). In all three cases, the Na,K-ATPase isoform present is 1. Attempts to increase phosphate incorporation in pig and dog Na,K-ATPases by changing the phosphorylation conditions (time, concentrations of ATPases and PKC, medium constituents) were unsuccessful. The possibility that there is contaminating phosphatase activity in dog and pig Na,K-ATPase preparations but not in that from the rat was previously tested and ruled out (8) .


Figure 1: Phosphorylation of Na,K-ATPase by PKC from different species. Autoradiogram of P-labeled proteins after SDS-gel electrophoresis and electrophoretic transfer of the samples to nitrocellulose. Purified Na,K-ATPase (0.5 µg) was incubated with 15 ng of PKC for 30 min at 30 °C in a final volume of 10 µl. PK, pig kidney Na,K-ATPase; RK, rat kidney Na,K-ATPase; DK, dog kidney Na,K-ATPase. The -subunits and PKC are marked. It is notable that the level of autophosphorylation of PKC itself is similar in the different lanes, despite the differences in the level of phosphorylation of Na,K-ATPase subunit. This confirms our prior report that the substrate-dependent activation of PKC was similar despite the different susceptibilities to incorporation of P (8).



Mapping of Phosphorylation Site with Tryptic Fragments

The simplest hypothesis to explain the low level of phosphorylation of dog and pig kidney Na,K-ATPases is that a key phosphorylation site is missing from their primary structures. When the sequences of rat, pig, and dog 1 are compared, there are only three locations where serines or threonines present in rat 1 are missing from both dog and pig 1 (Ser-18, Ser-114, and Thr-884). We mapped the phosphorylation site on fragments obtained by limited tryptic hydrolysis and identified the fragments with appropriate antibodies. Fig. 2diagrams the fragments predicted to be found after three different digestion procedures; the reproducible fragmentation patterns have been described before (17, 18) .


Figure 2: Diagram of the fragments predicted to be found after different digestion procedures. The topline represents full-length 1. Cleavage by trypsin in the E2 conformation (the K form) rapidly results in two major fragments as shown, plus the slower loss of 32 or more amino acids at the N terminus. Cleavage in the E1 conformation (the Na form) results in rapid removal of the first 32 amino acids, followed much more slowly by generation of a fragment of about 80 kDa. Fingerprinting (digestion by trypsin/soybean trypsin inhibitor complex) during gel electrophoresis results in many fragments (as seen in Fig. 5), but only the smallest fragment containing the intact N terminus (25 kDa) is shown in this diagram. At the bottom of the figure, the first 42 amino acids of rat 1 are shown (with numbering corresponding to the protein as found after post-translational cleavage of the first five amino acids). The tryptic cleavage site known as T2 is shown, as well as the epitopes for two antibodies used in these studies, the peptide-directed polyclonal antibody 754 and monoclonal antibody McK1.



Digestion in the Na,K-ATPase conformation known as E2 (the K form) would remove Ser-18, leave Ser-118 in a 40-kDa fragment, and leave Thr-884 in a 60-kDa fragment. The E2 conformation should be adopted in our phosphorylation conditions (Mg and P, plus low ATP, Ca and other constituents necessary for the kinase), and so digestion immediately following phosphorylation was tried first (Fig. 3). Rapid disappearance of the P-labeled subunit band was observed under these conditions (Fig. 3A). Immunostaining with a peptide-directed antibody against the N terminus gave a picture similar to the autoradiogram (Fig. 3B). No phosphorylated tryptic fragments were detected. The same blot was restained with antibody against a peptide at the C terminus. This antibody recognized a 64-kDa fragment, as expected ( Fig. 2and Fig. 3C). The appearance of the fragment of about 40 kDa at the 4-min time point (also stained by the C-terminal antibody) is due to secondary cleavage at a site named T4, observed earlier in the E2 conformation of Na,K-ATPase (19) . The result clearly indicates that the PKC phosphorylation site or sites are located in the N-terminal part of the molecule and not in the C-terminal half.


Figure 3: Mapping of PKC phosphorylation site by limited tryptic hydrolysis of Na,K-ATPase in the E2 conformation. Purified Na,K-ATPase at a concentration of 50 µg/ml was incubated with PKC (1.5 µg/ml) for 30 min at 30 °C in phosphorylation buffer, and then trypsin was added at a protease/ATPase ratio of 1:100. Samples were collected after 0, 1, 2, 4, 8, 16, and 30 min of incubation (lanes1-7). A, autoradiogram of P-labeled protein after SDS-gel electrophoresis and electrophoretic transfer to nitrocellulose; B, staining of the blot with peptide-directed antibody against the N terminus (754). The absence of stain of a band of approximately 40 kDa indicates that cleavage had occurred both in the middle of the protein (the site known as T1) and near the N terminus (T2); C, the same blot was restained with the antibody against the C terminus (845), demonstrating the expected product of cleavage at site T1, which however is seen to not be phosphorylated in A. Although the original blot was still radioactive, the time required to expose x-ray film for visualization of the ECL signal is too short (1-10 min) for autoradiographic detection of P (which required 5-16 h) (data not shown).



The failure to see any phosphorylated tryptic fragments suggested that the site may lie in a short region at the N terminus that is clipped off by digestion at a site known as T2. This site can be cleaved preferentially by trypsinolysis in the E1 conformation (the Na form) (Fig. 2). After phosphorylation, the Na,K-ATPase was pelleted and resuspended in Na-containing buffer and then digested. After 1 min with trypsin, a significant decrease in radioactivity was seen (Fig. 4A). Staining with the peptide-directed antiserum against the N terminus or with the monoclonal antibody McK1 (Fig. 4, B and C) marked a pattern of bands indistinguishable from that detected on the autoradiogram. The epitope of McK1 is known to be located between the T2 site and the epitope of the peptide-directed antibody (13) (Fig. 2). Polyclonal antiserum K1 recognized additional fragments (Fig. 4D). One of the abundant fragments had a molecular weight slightly lower than the -subunit, but it was recognized by neither N terminus antibody, and it was not radioactively labeled. We can conclude that the PKC phosphorylation site is located within the first 32 amino acids before the T2 cleavage site.

To demonstrate retention of P label in a fragment including the N terminus, proteolytic fingerprinting was used (Fig. 5). Tryptic cleavage was performed directly in the Laemmli gel in the presence of soybean trypsin inhibitor, which paradoxically stabilizes trypsin against SDS inactivation (17) . This approach permitted us to get a completely different tryptic pattern and thus to visualize fragments bearing the P label. The most highly radioactive polypeptide was about 25 kDa (Fig. 5A); other labeled fragments represent sequential degradation of the Na,K-ATPase. Exactly the same fragments were stained by antibody against the N terminus (Fig. 5B). Staining with polyclonal antibodies revealed additional nonradioactive fragments (Fig. 5C). Taken together, the results of the three experiments suggest that the PKC phosphorylation site is located close to the N terminus. When digestion was performed in E1 or E2 conditions ( Fig. 3and 4), extensive digestion at the numerous Lys and Arg residues there (Fig. 2) apparently precluded recovery of a labeled fragment.


Figure 5: Proteolytic fingerprinting of Na,K-ATPase phosphorylated by PKC. Purified Na,K-ATPase was phosphorylated by PKC, and the reaction was stopped by the addition of an equal volume of Laemmli sample buffer. Then soybean trypsin inhibitor was added to a final concentration of 25 µg/ml, and aliquots of 40 µl containing 2 µg of ATPase each were removed. Trypsin was added shortly before loading samples on a 12.5% Laemmli gel. The protease/ATPase ratios in each sample were as follows: 1, no trypsin; 2, 1:100; 3, 1:50; 4, 1:10; 5, 1:5. A, autoradiograms of P-labeled polypeptides after SDS-gel electrophoresis and electrophoretic transfer to nitrocellulose; B, staining of the blot with the antibody against N terminus (754); C, restaining with polyclonal antibody K1 to reveal additional fragments.



Identification of Amino Acid(s) Phosphorylated by PKC in Rat Kidney Na,K-ATPase

When the N-terminal sequence of rat kidney 1-subunit is examined, only two serines (11 and 18) and no threonines are found. Although only Ser-18 is within a consensus motif for PKC-dependent phosphorylation, we could not rule out that Ser-11 might be phosphorylated as well. To identify the amino acid(s) phosphorylated, we used direct Edman degradation of P-labeled protein. Purified rat kidney Na,K-ATPase was phosphorylated by PKC in conditions shown to result in phosphate incorporation of close to 1 mol of P/mol of -subunit (8). Labeled Na,K-ATPase was electrophoresed and transferred to ProBlott membrane for N-terminal amino acid sequence analysis. At each sequencing cycle, one-third of the eluted phenylthiohydantoin-derivative was identified by high performance liquid chromatography, and two-thirds was collected and subjected to liquid scintillation counting. Fig. 6displays the amount of P radioactivity recovered at each sequence cycle along with the identified amino acid and its yield.


Figure 6: Detection of P-serines by N-terminal sequencing and liquid scintillation counting. 20 µg of purified Na,K-ATPase was phosphorylated by PKC, subjected to SDS-electrophoresis and electrophoretic transfer to ProBlott membrane, and then used for N-terminal sequence analysis. The radioactivity (bars) and yield of amino acid/cycle (dots) was determined, and the expected protein sequence was confirmed as indicated over each bar. The same result was obtained in two separate sequencing attempts.



Some nonphosphorylated serine was recovered at both cycles 11 and 18. The recovery of unphosphorylated serine of course cannot be used to estimate the yield for that cycle. The average repetitive yield in this analysis was 94.88%.() Actual yields appeared to be even lower toward the end, possibly due to the elution of proteins noncovalently adsorbed to membrane, and the typically poor recovery of lysines, which are clustered at the end surrounding Ser-18. To estimate the ratio of label in Ser-18 to Ser-11, we normalized the counts obtained against the calculated repetitive yield of 94.88%. This most conservative estimate suggests that there was at least 3 times as much P in Ser-18 as in Ser-11. Since the real yields at the end of sequencing run were probably lower, it is likely that the ratio is even higher.


DISCUSSION

There is a large body of evidence that Na,K-ATPase can be a substrate for PKC-dependent phosphorylation in vitro(5, 6, 20, 21, 22) and in intact cells (5, 9, 23, 37) . Phosphorylatability of Na,K-ATPase appears to vary among species. During in vitro experiments, we found that phosphate incorporation in rat kidney Na,K-ATPase was 6-8-fold higher than in that from pig and dog. Low stoichiometries (0.15-0.18 mol of phosphate/mol of subunit) for PKC phosphorylation of dog (20) and B. marinus(5) Na,K-ATPase were reported by other investigators, while in shark and rat, higher phosphate incorporation (1-2 mol of phosphate/mol of ) was found (6, 8) . An interesting observation was made by Middleton et al.(23) , who studied phosphorylation of Na,K-ATPase by PKC in two different kidney cell lines. Stimulation of endogenous PKC increased phosphate incorporation in Na,K-ATPase -subunit from opossum kidney cells (OK cells), but not from porcine kidney cells (LLC-PK1). The data not only demonstrate that phosphorylation does take place in living cells, but they also are in line with our finding that PKC phosphorylation of Na,K-ATPase from different species is not equivalent. We would predict that the opossum -subunit should contain an additional site analogous to that in the rat.

In contrast to protein kinase A phosphorylation (for which only one consensus motif exists in rat 1) there are more than 30 consensus sequences for PKC-dependent phosphorylation. It was shown by different methods that the protein kinase A site is actually used for phosphorylation both in vitro and in vivo (5, 8, 24). Recently Beguin et al.(5) identified Thr-10 and Ser-11() by site-directed mutagenesis as PKC phosphorylation sites in B. marinus Na,K-ATPase (Fig. 7). Total phosphate incorporation was only 0.18 mol of phosphate/mol of -subunit, however, and neither of the phosphorylated amino acids was within a conventional phosphorylation motif for PKC, which is known to be (K/R)X(S/T) (25) . Mutations of a number of other serine and threonine residues that were in phosphorylation motifs did not alter phosphate incorporation into the 1-subunit from B. marinus. Thus the authors concluded that a novel motif for PKC-mediated phosphorylation was found. A similar motif exists in no other known PKC substrates, except for the autophosphorylation sites of PKC itself (26) , where lysine or arginine residues flanking the phosphorylated amino acid are not necessary.


Figure 7: N-terminal sequence alignment of Na,K-ATPase subunit from different species. The N-terminal sequences, starting at the initiator methionine, are shown for the indicated species and isoforms, as derived from cDNA or DNA sequences available in GenBank. Gaps were allowed to optimize the alignment of homologous sites. All serines and threonines are underlined. The numbering begins at amino acid 6 because for rat, pig, and dog 1 as well as for rat 2, the first five amino acids are known to be cleaved during biosynthesis. The arrows indicate Ser-11 and Ser-18 and its homologs.



In the present study, we identified two PKC phosphorylation sites with different phosphorylatability in rat kidney 1 subunit, Ser-11 and Ser-18. Phosphorylation was found exclusively in the N terminus of the molecule, similar to that found in B. marinus (Fig. 7). Since the majority of P label (75% or more) was found in Ser-18, and this amino acid is within a consensus motif for PKC phosphorylation, we would hypothesize that Ser-18 is the main site of phosphorylation in rat 1. Although Ser-11 was observed to be phosphorylated to a lower extent, we cannot rule out the possibility that the two sites are equivalent in their phosphorylatability, but some preexisting, stable phosphorylation of Ser-11 interfered with the incorporation of additional P. In this case, the level of phosphorylation in rat Na,K-ATPase still would be higher than in pig and dog Na,K-ATPases, and the theoretical stoichiometries would be 2 and 1 mol of phosphate/mol of Na,K-ATPase, respectively. It is of interest that phosphorylation of Na,K-ATPase by PKC is highly specific, as it occurs only at the N terminus of the -subunit in spite of the many consensus sequences.

There is general interest in the role of the N terminus because of its isoform and species divergence in a family of highly homologous proteins. Although cutting the N terminus off (3, 27, 28) or deleting it before expression (4, 29, 30, 31) leaves the enzyme partially active and capable of carrying out its basic functions, changes in Na affinity (28) , K deocclusion kinetics (3, 4) , dephosphorylation kinetics (32) , voltage dependence of K binding and transport (33) , as well as a shift toward E1 conformation (34), and a change in sodium/potassium transport ratio (35) have been reported. The most prominent feature of the N terminus is that the region contains a high proportion of positively charged amino acids, and thus the introduction of the negative phosphate groups may alter the structure with functional consequences similar to deletion. Our attempts to find an influence of PKC phosphorylation on the rat Na,K-ATPase activity assessed in maximal turnover conditions were unsuccessful (8) , although recently it was reported that phosphorylation of duck salt gland subunit by PKC decreased its affinity for Na 2.4-fold (7) .

Interestingly, a serine analogous to Ser-11 is conserved in all vertebrate 1s and all vertebrate 2s, while it is missing in all arthropods, Hydra, and vertebrate 3s (). In contrast, a serine similar to Ser-18 of rat 1 is found only in rat 3 (KKSKAK), and in Catostomus (KKKSKNK), Torpedo (KNSKKSKSK), and Hydra (KKSAP) 1s. Nothing like it is found in any 2; human or chicken 3; the H,K-ATPase of the colon; or any of these 1s: dog, pig, human, mouse, horse, chicken, Bufo, Xenopus, Anguilla (European eel), Drosophila, or Artemia.() A few other s have candidate sites within the first 30 amino acids that are consensus sequences for PKC but which could not be called homologous: rat, human, and chicken 2 (REYS); rat, human, and chicken 3 (KSS, KDS, KES, respectively); a different Artemia subunit() (KQLS); cat flea 1 (RSDS, RVAT, RRKT, and KTPT); and gastric H,K-ATPase of rat, human, and pig (KMSKKK).

This analysis of PKC consensus sites suggests that rat 3 should be a good candidate for phosphorylation. Thus we tested this hypothesis using a purified axolemmal Na,K-ATPase preparation, which is known to consist of 2 and 3 isoforms predominantly (16) . The preparation we used contained no more than 5% of 1 isoform as detected with the help of a monoclonal antibody (McK1) specific for the rat 1. Phosphorylation of rat axolemma Na,K-ATPase by PKC was 16-20% of the maximal level seen with rat kidney enzyme (data not shown). This is more than can be accounted for by the 1 present. The low level of phosphorylation of Na,K-ATPase from rat axolemma suggests, like for pig and dog 1, that the other isoforms are either isolated in an already partly phosphorylated state or that phosphorylatability of the relevant residue(s) is intrinsically low.


CONCLUSION

The identification of PKC-mediated phosphorylation at sites close to the N terminus of the subunit of the Na,K-ATPase focuses interest on this relatively divergent segment of the protein. No other sites were detectably phosphorylated, including the site near the C terminus, RRNSV, that is known to be phosphorylated by protein kinase A. The relatively poorly phosphorylated PKC site is well conserved among various Na,K-ATPases, while the better phosphorylated site found in the rat has only a few homologs among all of the known sequences. This suggests that species-specific adaptations, like those affecting cardiac glycoside sensitivity, may extend to mechanisms of kinase-mediated regulation as well.

  
Table: 0p4in Other serines or threonines found before the cannonical T2 tryptic cleavage site (ELKK) (28).(119)


FOOTNOTES

*
This work was supported by National Institutes of Health Grant NS 27653. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. 149-6118, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02114. Tel.: 617-726-8579; Fax: 617-726-7526.

The abbreviations used are: Na,K-ATPase, sodium and potassium ion-activated adenosine triphosphatase; PKC, protein kinase C (calcium- and phospholipid-dependent protein kinase); CAPS, 3-(cyclohexylamino)propanesulfonic acid.

A. Khatri, T. R. Ostrea, M. S. Feschenko, and K. J. Sweadner, manuscript in preparation.

To calculate the average repetitive yield, we used the repetitive yields of the most stable amino acids, Gly, Asp, and Ala.

The Artemia Na,K-ATPase sequence found in GenBank, accession number X56650.

The distinct Artemia Na,K-ATPase sequence found in GenBank, accession number Y07513.

These amino acids are called Thr-15 and Ser-16 by Beguin et al. (5) because they did not assume, as we do, that the first five amino acids are removed during biosynthesis.


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