Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129
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ABSTRACT |
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Regulation of Na-K-ATPase by cAMP-dependent protein kinase occurs in a variety of tissues. Phosphorylation of the enzyme's catalytic subunit at a classical phosphorylation consensus motif has been observed with purified enzyme. Demonstration of phosphorylation at the same site in normal living cells or tissues has been more difficult, however, making it uncertain that the Na-K-ATPase is a direct physiological substrate of the kinase. Recently, the structure of the homologous sarco(endo)plasmic reticulum Ca-ATPase (SERCA1a) has been determined at 2.6 Å resolution (Toyoshima C, Nakasako M, Nomura H, and Ogawa H. Nature 405: 647-655, 2000.), and the Na-K- ATPase should have the same fold. Here, the Na-K-ATPase sequence has been aligned with the Ca-ATPase structure to examine the predicted disposition of the phosphorylation site. The location is close to the membrane and partially buried by adjacent loops, and the site is unlikely to be accessible to the kinase in this conformation. Conditions that may expose the site or further bury it are discussed to highlight the issues facing future research on regulation of Na-K-ATPase by cAMP-dependent pathways.
adenosine 3',5'-cyclic monophosphate; crystal structure
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INTRODUCTION |
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THE ION GRADIENTS that are necessary for a wide range of physiological processes are maintained by Na-K- ATPase, and regulation of the Na-K-ATPase plays a role in the hormonal and homeostatic responses of many cells (recently reviewed in Ref. 55). Regulation occurs on several levels: biosynthesis and degradation; reversible recruitment to and internalization from the plasma membrane; alteration of affinity for Na; and either stimulation or inhibition of activity. The involvement of cAMP-dependent protein kinase (PKA) in acute sodium pump regulation has been documented in 20 different mammalian tissues and in lower vertebrates (55). The result can be either pump stimulation or inhibition, however, and in no case is the pathway completely understood.
The first demonstration of phosphorylation of the Na-K-ATPase catalytic
subunit () by the catalytic subunit of PKA was by Mardh in 1979 (38), but little more was done until 1991, when phosphorylation was observed at stoichiometries of 0.5-1.0
phosphate per
(8, 19). Soon it was shown that
the site was within 12 kDa of the COOH terminus (20),
specifically at Ser-938, the only serine in a consensus motif (RRNSF)
for PKA (6, 25, 27). The earliest in vitro phosphorylation
studies were with enzyme treated with a mild detergent like Triton
X-100, and it was observed that the detergent was actually required to
obtain phosphorylation in the test tube (6, 18, 20, 25).
Since then, phosphorylation by PKA in the test tube in the absence of detergent has been detected only in preparations that were mildly heat
denatured (4) or in preparations that were completely solubilized with C12E8 or
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate detergent and then reconstituted with lipids (21).
It normally should be a straightforward matter to demonstrate
phosphorylation in intact cells at a site like RRNSF on an abundant protein like the Na-K-ATPase, but, in fact, there is not a lot evidence
for it. One lab has reported dibutyryl cAMP-stimulated or
forskolin-stimulated incorporation of 32P in in renal
cortical or medullary tubule preparations (14, 35),
although the site of phosphorylation was not defined. In several cases,
experiments designed to detect it have given negative results: rat
sciatic nerve (12), choroid plexus (28), lung cells (10), striatal neurons (45), C6 glioma,
and normal rat kidney (NRK-52E) epithelial cells (26).
Evidence for the phosphorylation of Ser-938 on normally expressed (not
transfected) Na-K-ATPase by endogenously activated PKA has been
presented in only one paper to our knowledge. In this case,
untransfected COS cells stimulated with a
-adrenergic agonist,
isoproterenol, showed increased phosphorylation of the endogenous
monkey
1-subunit detected with an antibody directed
against the phosphorylated Ser-938 site (16). There was a
70% increase in immunoreactivity of a band at 100 kDa over a
substantial background of stain in unstimulated cells. In the most
recent attempt (26), an antibody prepared identically was observed to cross-react with many other phosphoproteins in cells that
had been treated with phosphatase inhibitors. Although forskolin-IBMX treatment of the cells raised cAMP to very high levels, phosphorylation of endogenous Na-K-ATPase was not detected in immunoprecipitates. In
cell extracts, however, addition of Triton X-100-treated exogenous Na-K-ATPase resulted in its phosphorylation, showing that PKA was
present and active. The detergent seemed to be required for access to
the site.
Two groups investigated the effects of mutation of Ser-938 to alanine
(S938A) in transfected cells. Beguin and colleagues (5, 6)
found evidence for phosphorylation in exogenous Bufo enzyme
expressed in COS cells that was reduced in mutants expressing Ala-938.
In studies of activity, Cheng and Fisone (16, 27) and
their associates demonstrated that forskolin treatment to raise cAMP
levels caused a 19% reduction in Na-K-ATPase activity in COS cells
expressing wild-type rat 1 Na-K-ATPase, whereas cells
expressing S938A lacked forskolin-induced inhibition. Similar results,
a 25% reduction in activity, were found for isoproterenol, a
-adrenergic agonist (16). In the latter paper, the
phosphorylation site-specific antibody detected an increase in Ser-938
phosphorylation in wild-type rat
1 but not in S938A,
both in stable transfectants and in transient transfectants that were
overexpressing rat
1 Na-K-ATPase by 5- to 10-fold.
Oddly, however, treatment with ouabain, a specific inhibitor of the
Na-K-ATPase, caused the same increase in phosphorylation of Ser-938 in
the host cell
-subunit, and no additional effect was seen with
isoproterenol. Mutation of Ser-938 to aspartic acid was apparently not
inhibitory (17).
Inhibition of Na-K-ATPase activity by PKA has been reported in crude
membrane preparations (22) and purified enzyme (8; reviewed in Ref. 15). An inhibitory effect of detergent
was shown to be a complication that precluded assessment of inhibition in some systems (21, 25). Most recently, phosphorylation
by PKA was observed to be dissociable from inhibition in two reports. In both cases, phosphorylation of Ser-938 was constant, while its
functional consequence varied with either intracellular Ca level
(15) or oxygen tension (35). In enzyme
expressed in insect cells, treatment of intact cells with a cAMP analog
resulted in inhibition of 1 and
2 by
~30%, whereas
3 was stimulated by ~35%, all
without detergent treatment (11). In this case, phosphorylation was detected, but not correlated with modulatory effects.
A simple, unified scheme of sodium pump inhibition by direct
phosphorylation at the PKA site has thus not been obtained. The only
consistent trend is that phosphorylation of at the PKA site is
usually detected in conditions where there might be some denatured
enzyme: in detergent, after reconstitution, after mild heating, and in
cells expressing exogenous
-subunit from transfected DNA. Here we
examine the hypothesis that the PKA site might be inaccessible to the kinase.
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DATA ANALYSIS |
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Homology of Ca-ATPase and Na-K-ATPase.
The sarco(endo)plasmic reticulum Ca-ATPase (SERCA) is in the same gene
family as the Na-K-ATPase and is highly homologous, showing ~30%
identity, 65% similarity, and a similar total length (41). For years, it has been believed that the similarity
between Ca-ATPase and Na-K-ATPase breaks down in the COOH-terminal
third of the protein, where six of the transmembrane spans are found, and the predicted topology of that portion of the protein has been
quite controversial. The perception that the COOH-terminal ends were
different was based on the premise that hydropathy plots should show
aligned blocks of largely hydrophobic transmembrane spans, while
hydropathy comparisons between Ca-ATPase and Na-K-ATPase (and even
between Na-K-ATPase and H-K-ATPase) showed major inconsistencies. With
the 2.6 Å crystal structure of the Ca-ATPase, however, the actual
transmembrane helices can be seen, as well as key structural features
such as the locations of residues involved in ion binding and
disruptions of helical structure to create an ion-binding pocket. It
can now be seen that the hydropathy predictions failed because of the
relatively high content of polar amino acids in the transmembrane spans
and a relatively high content of nonpolar amino acids in the connecting
loops. The underlying homology between Ca-ATPase and Na-K-ATPase is
strong and is completely consistent with a shared fold.
Topology studies did not always give consistent results for the
Na-K-ATPase, but most biochemical and immunological evidence obtained
with native enzyme supports the corresponding transmembrane spans
(2, 3, 24, 29, 37, 40, 44, 53, 56). This means that
overall organization of the Ca-ATPase and Na-K-ATPase -subunits
should be essentially the same except for scattered loops at the
surface and the NH2- and COOH-terminal tails, where the
sequences diverge the most.
Structure of the homologous site.
Figure 1 shows the sequence surrounding
the PKA site (RRNSF) in the rat 1 Na-K-ATPase aligned
with the corresponding segment (ENQSL) of rabbit SERCA1a. The site lies
on an intracellular loop between M8 and M9, and the serine itself is
conserved. Under the alignment, identical amino acids are marked with
asterisks, conservative substitutions with lines, and less conservative
substitutions with dots. The degree of similarity, conservatively
defined, is 35%, and with the broader definition it is ~70%. In the
Ca-ATPase, the Arg-Arg sequence of the PKA motif is replaced with other
polar amino acids, glutamic acid and asparagine.
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Structure of a PKA autophosphorylation site.
For comparison, the structure of a known PKA site is shown in Fig.
6. This is the autophosphorylation site
on PKA itself (MMDB ID 4194, PDB ID 1CDK), which is located on the
activation loop near but not in the active site (31). The
structure is the same as that pictured in Fig. 2B, but now
the sequence RTWT*L is highlighted. The phosphate on the second
threonine is shown in red. The underlying -carbon backbone is
stretched out (Fig. 6A), and the site is highly exposed on
the surface (6B). In Fig. 6, C and D,
the structure has been rotated 90° to display the inhibitory peptide
(pink) that lies in the active site. It, too, adopts a stretched-out conformation.
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DISCUSSION |
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Predicted inaccessibility of the PKA site.
The structure of the Ca-ATPase loop homologous to the Na-K-ATPase PKA
site suggests that the PKA site should be inaccessible to kinase. This
is for several reasons: its relatively sunken position, proximity to
the membrane, crowding by the nearby COOH-terminal tail, the partially
overhanging N domain, and the potential for - (and
-) subunits to
lie nearby. The true structure of the Na-K-ATPase is, of course, not
yet known, and it is possible that there are enough adjustments to the
M8-M9 loop to make the site locally more prominent and to move away the
residues that crowd it and the COOH terminus. The site should remain
close to the membrane and tucked under the P domain, however, which
would make the approach of a kinase difficult. It is notable that the
arginine residues have not been observed to be digested by trypsin in
membrane-bound Na-K-ATPase, although digestion did occur in denatured
or chemically modified enzyme (39, 42, 48), and digestion
at (K)ILIF (the last residue before the beginning of M9) has been
reported in enzyme destabilized by Ca ions (52).
Experimental increases in accessibility. It is clear that the Na-K-ATPase can be phosphorylated at the PKA site in the presence of Triton X-100. At the very least, the detergent should eliminate the planar lipid bilayer, leaving an annulus of detergent and, perhaps, allowing closer approach of the kinase from the side.
There is evidence that detergents can cause a more profound structural alteration of the Na-K-ATPase. Covalent oxidative cross-linking of cysteine residues in trypsin-digested Na-K-ATPase has been shown to result in a cross-link between theThe implications of conformation differences. The discouraging analysis above might be incorrect if the Na-K-ATPase PKA site is exposed specifically in different enzyme conformations or by interaction with other cellular components. Interestingly, enzyme conformation appeared to play a role in the extent of Na-K-ATPase phosphorylation in the test tube, even though detergent was required (25). The Na-K-ATPase, like other P-type ATPases, is thought to cycle through conformation changes that alternately expose Na and K binding sites on opposite sides of the membrane. There is an abundance of physical and kinetic evidence for two major conformations termed E1 and E2 (33). The conformation changes are thought to entail large interdomain movements (46). These conformation changes have been long known to affect the accessibility of certain proteolytic cleavage sites (34). Experimentally, despite the presence of enough Triton X-100 to inactivate ATP hydrolysis, we observed that conditions favoring the E1 conformation resulted in higher levels of PKA phosphorylation than conditions favoring E2 (25).
The conformation of the Ca-ATPase in the crystal structure is not certain, but it is quite different from that observed by cryoelectron microscopy in the same enzyme crystallized without Ca and with decavanadate in the active site (46, 57, 60). The 2.6 Å structure has wide-spread domains, whereas the 8 Å structure is more condensed, so the 2.6 Å structure may represent E1 or a conformation related to it. The extent of PKA phosphorylation in different ligands suggests that the site should be even less exposed in E2 (25). In fact, in our hands, ouabain, which induces an E2-like conformation, completely blocked phosphorylation of pig kidney Na-K-ATPase. One could argue, however, that the rotation of the N domain closer to the A domain in E2 could conceivably expose the PKA site better, and the site could emerge more from the lipid bilayer. A salient example of a specific change in accessibility of sites is the demonstration by Lutsenko et al. (37) of changes in the accessibility of an extracellularly exposed cysteine residue, Cys-964 of sheepPossible physiological regulatory mechanisms. It should be noted that despite all the negative evidence discussed earlier in this paper, Cheng et al. (16) and Fisone et al. (27) have reported blockage of Na-K-ATPase regulation by PKA in transfectants expressing S938A, in the latter case modulated by the intracellular concentration of Ca. They also reported the involvement of phosphorylation of Ser-938 in modulation of phosphorylation by protein kinase C in similar transfectants (17). Difficulties in demonstrating phosphorylation in purified enzyme and isolated microsomes might be caused by separation of the enzyme from important regulatory components. Protein kinase A is now known to associate with anchoring proteins (23). A long time ago, Lingham and Sen (36) reported that an intermediary protein was required in the regulation of the Na-K-ATPase by PKA.
There is evidence for physical interaction of the Na-K-ATPaseConclusions. On the whole, evidence for direct phosphorylation of the Na-K-ATPase at Ser-938 in the native state is weak. Examination of the location of the homologous sequence in the atomic structure of the Ca-ATPase suggests that the site should be inaccessible to the kinase. The most conservative interpretation is that much of the cAMP-dependent Na-K-ATPase regulation known to occur in cells is carried out without direct covalent phosphorylation of the enzyme. It is possible that E1-E2 conformation changes control accessibility of the site, but the likelihood is diminished in view of the rapid cycle of conformation changes at Vmax. It remains possible that other cellular proteins interact with the Na-K-ATPase in vivo to improve the accessibility of the site to kinase. In this case, regulation should be complex in the sense that two regulatory pathways may have to converge to achieve phosphorylation of the Na-K-ATPase.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants RO1NS-27653 and RO1HL-36271 (to K. J. Sweadner).
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. J. Sweadner, Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, Charlestown, MA 02129 (E-mail: sweadner{at}helix.mgh.harvard.edu).
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.
Received 19 September 2000; accepted in final form 8 November 2000.
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