From the
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
Na,K-ATPase
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
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
To demonstrate retention of
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
In contrast to protein kinase A phosphorylation (for which
only one consensus motif exists in rat
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
Interestingly, a serine analogous to Ser-11 is conserved in all
vertebrate
This analysis of PKC consensus sites suggests
that rat
The identification of PKC-mediated phosphorylation at sites
close to the N terminus of the
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.
(
)
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) .
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.
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.
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.
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.
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.
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.
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.
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) .
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).
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.
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)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.