(Received for publication, December 9, 1995; and in revised form, December 25, 1995)
From the
Antidiuretic hormone modulates the water permeability (P) of epithelial cells in the rat kidney
by vesicle-mediated insertion and removal of the aquaporin-2 (AQP-2)
water channel. AQP-2 possesses a single consensus cAMP-dependent
protein kinase A (PKA) phosphorylation site (Ser-256) hypothesized to
regulate channel P
(Kuwahara, M.,
Fushimi, K., Terada, Y., Bai, L., Sasaki, S., and Marumo, F.(1995) J. Biol. Chem. 270, 10384-10387). To test whether PKA
phosphorylation of AQP-2 alters channel P
, we compared the P
values of purified AQP-2 endosomes after incubation with
either PKA or alkaline phosphatase. Studies using
[
-
P]ATP reveal that AQP-2 endosomes contain
endogenous PKA and phosphatase activities that add and remove
P label from AQP-2. However, the P
(0.16 ± 0.06 cm/s) of endosomes containing
phosphorylated AQP-2 (0.7 ± 0.3 mol of PO
/mol of
protein) is not significantly different from the same AQP-2 endosomes
where 95 ± 8% of the phosphate has been removed (P
0.14 ± 0.06 cm/s). These data
do not support a role for PKA phosphorylation in alteration of
AQP-2's P
. Instead, AQP-2
phosphorylation by PKA may modulate AQP-2's distribution between
plasma membrane and intracellular vesicle compartments.
ADH ()stimulation of rat kidney IMCD causes a rapid
increase in its apical membrane osmotic water permeability (P
)(1, 2, 3) .
This large ADH-elicited increase in P
occurs by the fusion of cytoplasmic vesicles containing
water channels with the apical
membrane(1, 4, 5) . Withdrawal of ADH
stimulation induces retrieval of apical membrane water channels by
endocytosis and returns apical membrane P
to its low base-line
value(6, 7, 8) . In the rat kidney IMCD, this
insertion and removal process is mediated by an increase in
intracellular cAMP and activation of cAMP-dependent
PKA(9, 10) .
Data from many laboratories have now established the central role of aquaporin water channel proteins as water-selective pores in the plasma membranes of various renal epithelial cells (reviewed in Refs. 2, 3, and 11). Reconstitution studies of AQP-1 in liposomes suggest that AQP water channel proteins exist as homotetramers in plasma membranes where each AQP polypeptide forms a narrow water-selective channel(12) . A total of four AQPs expressed in the mammalian kidney have been cloned and characterized. All four AQPs possess six transmembrane domains composed of highly conserved sequences, while the structures of their carboxyl-terminal regions are divergent(2, 3) . Antisera specific for the respective carboxyl-terminal sequences of various AQPs have been utilized to localize these proteins to either the apical or basolateral plasma membranes and/or vesicles within individual cell types within specific nephron segments(13, 14, 15, 16) .
A large body of data demonstrates that AQP-2 is the ADH-elicited water channel. AQP-2 is expressed exclusively by ADH-responsive cells in rat collecting duct(14, 17, 18) , where it is prominently located in the apical membrane as well as a population of subapical vesicles(13, 19) . Recent ultrastructural studies have demonstrated that ADH stimulation and withdrawal redistributes AQP-2 from cytoplasmic vesicles to the apical membrane followed its retrieval into endosomes(19, 20, 21) . Purified endosomes originating from the apical membrane of rat IMCD are highly enriched for AQP-2(22) . Finally, structural alterations of the human AQP-2 gene are associated with the disease nephrogenic diabetes insipidus, where affected individuals lack the ability to produce hypertonic urine despite high serum ADH levels(23) .
The
cDNA sequence of AQP-2 predicts a 271-amino acid protein with one N-linked glycosylation site(17, 18) . This
corresponds to a 29-kDa protein and its 35-40-kDa glycosylated
form as identified by multiple anti-AQP-2
antisera(13, 18) . In addition, the cDNA sequence
corresponding to the carboxyl-terminal domain of AQP-2 reveals several
putative phosphorylation sites including a cAMP-dependent protein
kinase A (PKA) site (Ser-256), a site for protein kinase C (Ser-226),
and two potential sites of casein kinase phosphorylation (Ser-229 and
Thr-244)(17) . The presence of these potential phosphorylation
sites has raised the possibility that the P of individual AQP-2s may be altered by phosphorylation in a
manner similar to that described for other ion
channels(24, 25, 26, 27) . This
includes alteration of channel function by PKA phosphorylation (28) of the major intrinsic protein of the lens (29) that possesses a 59% sequence homology with
AQP-2(30) .
Expression studies of AQP-2 in Xenopus oocytes demonstrate that preincubation with cAMP or its analogs
increases the P of oocytes injected with
either total medullary RNA (31) or cRNA from wild type
AQP-2(17, 32) . In recent studies, Kuwahara and
co-workers (32) have utilized site-directed mutagenesis
techniques in an attempt to identify a potential role for cAMP-mediated
PKA phosphorylation of Ser-256 in modulation of AQP-2 function. Their
data demonstrate that alteration of AQP-2 Ser-256 results in a loss of
the 2-fold increase in P
induced by
preincubation of oocytes expressing AQP-2 with cAMP. To distinguish
between the cAMP-mediated increases in either 1) the P
of individual AQP-2 proteins resident
in the oocyte plasma membrane or 2) alterations in the number of AQP-2
proteins present in the oocyte membrane by insertion and retrieval of
AQP-2-containing membrane, these studies quantified binding of an
anti-AQP-2 antiserum to the external surface of the oocyte membrane.
Since no differences in anti-AQP-2 binding to oocytes were observed
after cAMP stimulation, these authors suggested that cAMP-mediated
phosphorylation of Ser-256 alters the P
of individual AQP-2 proteins.
To further test the
conclusions from these data, we have utilized a homogeneous population
of purified endosomes derived from the apical membrane of rat IMCD that
are highly enriched for AQP-2(22) . We tested whether AQP-2 is
phosphorylated or dephosphorylated by endogenous membrane-bound enzymes
present in these purified endosomes and if phosphorylation of AQP-2
alters endosomal membrane P. Our data
show that purified IMCD endosomes possess endogenous PKA and
phosphatase activities that phosphorylate and dephosphorylate AQP-2.
However, paired measurements of membrane P
show no significant differences between endosomes containing
phosphorylated AQP-2 after incubation with exogenous PKA as compared to
the same endosomes where AQP-2 is dephosphorylated by alkaline
phosphatase treatment. These data do not support a role for
cAMP-mediated PKA phosphorylation in regulating the permeability of the
AQP-2 water channel.
Purified P-labeled AQP-2 was then transferred
to a polyvinylidene difluoride membrane (Immobilon, DuPont NEN) in 10
mM CAPS, pH 11.0, and its location determined by
autoradiography and staining with 0.1% Amido Black. Equal portions of
the same
P-labeled AQP-2 band was used to determine 1) its
molar phosphate content by scintillation counting using appropriate
calculations and determination of both
P isotope specific
activity and scintillation counting efficiency, as well as 2) molar
protein content by quantitative amino acid hydrolysis using published
data of the molar ratios of individual amino acids of
AQP-2(17) .
To insure that phosphorylation did not alter the mean diameters of AQP-2 endosomes, paired samples were fixed and sectioned and mean diameters of endosomes visualized under identical magnifications were determined as described previously(22) .
In previous work, we have employed a protocol consisting of five separate differential centrifugation steps combined with Percoll gradient sedimentation to purify a homogeneous population of apically derived endosomes from homogenates of rat kidney inner medulla and papilla(22) . These endosomes average 144 ± 5 nm in diameter, contain functional water channels, and are highly enriched for AQP-2 protein as determined by immunoblotting using a specific rabbit anti-AQP-2 antiserum(14, 22) . In experiments described below, these AQP-2 endosomes were utilized to determine the functional consequences of AQP-2 phosphorylation by PKA.
As shown in Fig. 1, incubation of AQP-2 endosomes with Mg [
-
P]ATP alone (lane 1)
results in the appearance of 7 major
P-labeled SDS-PAGE
protein bands of approximately 100-150, 94, 52, 40-43,
30-34, and 22-24 kDa as well as a band of very large
molecular mass greater than 200 kDa. Their respective locations in
autoradiograms of total endosomal proteins shown in Fig. 1(lanes 1 and 3) are indicated by small arrows or brackets. However, Triton X-100
solubilization and immunoprecipitation of the endosomal proteins shown
in lane 1 with anti-AQP-2 Sepharose shows AQP-2 protein is not
phosphorylated (Fig. 1, lane 2). In contrast, addition
of purified catalytic PKA subunit to these endosomes results in an
overall increase in
P labeling of endosomal protein bands (Fig. 1, lane 3) and prominent
P labeling
of AQP-2 protein in anti-AQP-2 immunoprecipitates (Fig. 1, lane 4). This includes protein bands of 28 and 35-45 kDa
corresponding to the nonglycosylated and glycosylated forms of AQP-2 as
described previously(18) . The locations of AQP-2 bands present
in autoradiograms of whole endosomal proteins are indicated by stars of Fig. 1(lane 3). Under these
conditions, phosphorylation of AQP-2 could be attributed directly to
PKA since
P labeling of AQP-2 was not observed after
addition of either an excess of purified PKA regulatory subunit (lanes 5 and 6) or IP
, a 20-mer peptide
that is a highly specific competitive inhibitor of PKA catalytic
activity (Fig. 1, lanes 7 and 8)(35) .
As expected, addition of excess regulatory subunit (molecular mass 55
kDa) to endosomes results in the appearance of a prominent 55-kDa
P-labeled band shown in lane 5 that is not
present in lane 7. A star in lane 3 denotes
the location of a fainter 55-kDa band that appears upon addition of
both [
-
P]ATP and PKA catalytic subunit that
likely represents phosphorylation of endogenous PKA regulatory subunit
by exogenous PKA.
Figure 1:
Patterns of protein phosphorylation
exhibited by AQP-2 endosomes after incubation with
[-
P]ATP under various conditions. Identical
aliquots of purified AQP-2 endosomes were suspended in Buffer B
containing 2 mM MgCl
, 5% glycerol and 0.05%
2-mercaptoethanol and incubated under assay conditions (see
``Experimental Procedures'') for 5 min with various additions
listed below.
P-Labeled proteins were identified by
SDS-PAGE and autoradiography after solubilization of individual
aliquots either in SDS directly (lanes 1, 3, 5, and 7) or after immunoprecipitation of AQP-2 by
anti-AQP-2 Sepharose 4B-linked antiserum (lanes 2, 4, 6, and 8). Incubation conditions included: 0.1 mM [
P]ATP only (lanes 1 and 2), in combination with (20 units/ml) PKA (lanes 3 and 4), together with either the regulatory subunit of
PKA (lanes 5 and 6) or a 20-mer peptide IP
(35) a specific inhibitor of PKA phosphorylation (lanes 7 and 8). The representative autoradiogram was
developed after 24 h and is representative of eight separate
experiments. The position of proteins of known molecular mass
10
are indicated by the arrowheads, while
the top (T) and dye front (D) of the gel are shown by
the small arrowheads. In lanes 1 and 3,
proteins
P-labeled by addition of ATP only are indicated
by small arrowheads or brackets. Stars indicate bands corresponding to the 28- and 35-45-kDa AQP-2
bands shown in lane 4 as well as a fainter band of 55 kDa
corresponding to the regulatory subunit of PKA as shown in lane 5 but not lane 6.
The ratio of P label between the
35-45- and 28-kDa AQP-2 bands after 5 min of
P
incorporation by PKA and purification by immunoprecipitation was 3.75
± 0.57 (n = 12) as determined by densitometry of
autoradiograms of anti-AQP-2 immunoprecipitates. This value is
comparable to the ratio of these AQP-2 protein bands as detectable by
immunoblotting(14, 18, 22) . These data
demonstrate that under conditions described in Fig. 1, both the
28- and 35-45-kDa forms of AQP-2 present in these purified
endosomes are specific substrates for PKA phosphorylation.
Previous
work in brain (reviewed in (37) and (38) ),
erythrocytes (39) , epithelial(40) , and neuroendocrine
cells (41) has demonstrated that hormone activation causes cAMP
accumulation in distinct cellular compartments where PKA subunits are
differentially localized and often bound to membranes through their
associations with a family of PKA-anchoring proteins. In the kidney
medulla and papilla, a significant portion of total cAMP-dependent PKA
activity is present in the particulate fractions of
homogenates(42) . To determine if bound endogenous
cAMP-dependent PKA activity is present in endosomes where it could
phosphorylate AQP-2, purified endosomes were incubated in the presence
of 100 µM cAMP and [-
P]ATP
only (Fig. 2, lanes 1 and 4). The patterns of
phosphorylation obtained in SDS-PAGE analysis of both whole endosomal
proteins (lane 1) and anti-AQP-2 immunoprecipitates (lane
4) were then compared to their respective counterparts after
incubation of AQP-2 endosomes with either
[
-
P]ATP alone (Fig. 2, lanes 2 and 5) or after addition of a combination of
[
-
P]ATP and exogenous PKA catalytic subunit (Fig. 2, lanes 3 and 6). Addition of 100
µM cAMP consistently resulted in
P labeling
of AQP-2 (lane 4) that was similar to that present after
addition of both [
-
P]ATP and exogenous PKA (lane 6).
P labeling of AQP-2 was absent after
incubation of endosomes with [
-
P]ATP only (lane 5). These data demonstrate the presence of endogenous
PKA activity that is activated by 100 µM cAMP and
phosphorylates AQP-2 protein in these endosomes.
Figure 2:
Purified AQP-2 endosomes contain
endogenous cAMP-dependent protein kinase A activity. Aliquots of
endosomes were incubated under conditions described in Fig. 1with [-
P]ATP and 100
µM cAMP (lanes 1 and 4),
[
-
P]ATP only (lanes 2 and 5) or [
-
P]ATP and 20 units/ml PKA
catalytic subunit (lanes 3 and 6). After 5 min of
incubation,
P-labeled vesicle proteins were either
analyzed directly by SDS-PAGE (lanes 1-3) or following
immunoprecipitation using anti-AQP-2 Sepharose (lanes
4-6). The autoradiogram shown (exposed for 72 h) is from a
single experiment representative of a total of four. The relative
migration of proteins of known molecular mass are indicated as
described in Fig. 1.
To obtain
conditions where maximal phosphorylation of AQP-2 was achieved after
addition of exogenous PKA catalytic subunit, the time course of P labeling of AQP-2 was determined as shown in Fig. 3A. Maximal incorporation of
P into
AQP-2 occurred within 3 min (Fig. 3A, lanes 2 and 6) and was followed by progressive loss of label over
an interval of 20 min (Fig. 3A, lanes 3, 4, 7, and 8). Quantitation of autoradiograms
of immunoprecipitates using anti-AQP-2 antiserum (lanes
1-4) showed loss of 47 ± 23% (n = 4)
of AQP-2
P label after 10 min as compared to that present
after 3 min of phosphorylation by PKA. After 20 min, only 25 ±
13% (n = 4) of
P label remained in AQP-2.
There were no significant differences observed in ratios of
P content of 45-35-kDa/28-kDa AQP-2 bands at 3 min
(3.4 ± 0.43; n = 4), 10 min (3.71 ± 0.29; n = 4), or 20 min (3.82 ± 0.30; n = 4).
Figure 3:
Purified AQP-2 endosomes contain
endogenous phosphatase activity. Panel A, identical aliquots
of endosomes were incubated for 0 min (lanes 1 and 5), 3 min (lanes 2 and 6), 10 min (lanes
3 and 7), and 20 min (lanes 4 and 8)
with [-
P]ATP and PKA. The
P-labeled protein content of samples were then analyzed by
SDS-PAGE and autoradiography as described in Fig. 1, either
after addition of SDS solubilization buffer to endosomes (lanes
5-8) or following immunoprecipitation using anti-AQP-2
Sepharose (lanes 1-4). The autoradiogram (48-h exposure)
shown is from a single experiment representative of a total of four.
The relative migration of proteins of known molecular mass is displayed
in a fashion identical to that described in Fig. 1. Panel
B, to determine if the reduction of
P-labeled AQP-2
in the immunoprecipitates shown in panel A was the result of
proteolysis, a single aliquot of endosomes was divided equally and one
subaliquot (lane 1) exposed to phosphorylation conditions (see lanes 1 and 2 in Fig. 1) for 20 min at 37
°C, while the other (lane 2) was incubated on ice.
Subsequently, both aliquots received additions of PKA, were incubated
for 5 min at 37 °C and solubilized and their
P-labeled
AQP-2 content determined by immunoprecipitation as shown in panel
A, which displays the 28- and 35-45-kDa
P-labeled AQP-2 bands. The resulting autoradiogram from
this single aliquot of endosomes was exposed for 48 h and is
representative of a total of two
experiments.
The progressive reduction of P-labeled AQP-2 present in immunoprecipitates shown in Fig. 3A could result from either dephosphorylation by
endogenous phosphatase activity or proteolysis of AQP-2 during the
interval that endosome proteins are phosphorylated. To distinguish
between these possibilities, identical aliquots of endosomes were
either preincubated under phosphorylation conditions for 20 min at 37
°C (Fig. 3B, lane 1) or held on ice (Fig. 3B, lane 2). After subsequent
phosphorylation of endosomes for 5 min, the content of
P-labeled immunoprecipitable AQP-2 in each aliquot was
compared by autoradiography. The
P-labeled AQP-2 content
of endosomes preincubated under phosphorylation conditions (panel
B, lane 1) was not significantly different (0.98 ±
0.05; n = 3) as compared to control samples (panel
B, lane 2). These data suggest that the progressive loss
of
P label from AQP-2 in purified endosomes results from
endogenous phosphatase activity. Under these conditions, neither the
35-45-kDa nor the 28-kDa AQP-2 band appears to be preferential
substrates for dephosphorylation.
As shown in Fig. 4,
incubation of endosomes with 150 µg/ml alkaline phosphatase for 20
min at 37 °C resulted in a loss of greater than 95 ± 8% (n = 7) of P label from purified
immunoprecipitates of AQP-2. Prior to quantitation of the
P-labeled phosphate content of AQP-2, the
P
label resulting from AQP-2 in endosomes subjected to preincubation with
alkaline phosphatase was compared to that displayed by control
endosomes. This experiment was performed to determine if AQP-2 present
in endosomes already possesses a significant content of endogenous
nonradioactive phosphate prior to its phosphorylation in vitro by exogenous PKA and [
-
P]ATP.
Preincubation with alkaline phosphatase would remove any endogenous
phosphate and thus be expected to increase incorporation of
P label into AQP-2 in subsequent exogenous PKA
phosphorylation. However, as shown in Fig. 5, paired experiments
revealed no significant difference (0.91 ± 0.1; p >
0.05; n = 4) in the
P label of AQP-2
derived from either control (lane 2) or alkaline
phosphatase-treated (lane 1) endosomes. These data suggest that AQP-2
present in purified endosomes does not possess a significant content of
phosphate prior to its phosphorylation with exogenous PKA.
Figure 4:
Incubation of AQP-2 endosomes with
alkaline phosphatase dephosphorylates P-labeled AQP-2.
AQP-2 in purified endosomes was
P-labeled by 5 min of
incubation with PKA and [
-
P]ATP under
conditions described in Fig. 1and the reaction terminated by
addition of EDTA to final concentration of 2 mM. One half of
these endosomes were then incubated with 150 µg/ml alkaline
phosphatase for 20 min at 37 °C, and both subaliquots were then
analyzed after immunoprecipitation with anti-AQP-2 Sepharose 4B as
described in Fig. 1and Fig. 3, where lane 1 is
AQP-2 protein derived from alkaline phosphatase-treated endosomes and lane 2 from control endosomes. The autoradiogram (exposed 48
h) displayed is from a single experiment and is representative of a
total of seven. The relative migration of proteins of known molecular
mass are displayed in manner identical to that described in Fig. 1.
Figure 5:
Preincubation of AQP-2 endosomes with
alkaline phosphatase does not alter its content of P label
after subsequent phosphorylation with exogenous PKA. Identical aliquots
of AQP-2 endosomes were preincubated in the absence (Cont) or
presence (Alk. P.) of alkaline phosphatase as described in Fig. 4. AQP-2 endosomes were then repeatedly centrifuged in
ice-cold Buffer B to remove soluble alkaline phosphatase enzyme. Both
endosome aliquots were then phosphorylated and analyzed as described
for lane 4 of Fig. 1. The autoradiogram exposed for 36
h displays a representative experiment performed a total of four times. Arrows located on the right denote the 28- and
35-45-kDa
P-labeled AQP-2
bands.
To
quantify the P phosphate content of AQP-2,
P-labeled AQP-2 protein was purified by
immunoprecipitation and SDS-PAGE. As described under
``Experimental Procedures,'' the 35-40-kDa region of
individual 35-45-kDa AQP-2 protein bands was used for analyses
since prior studies had demonstrated that it was free of contamination
by IgG protein present in AQP-2 immunoprecipitates (data not shown).
Quantitation of
P content by scintillation counting and
protein by amino acid analysis showed AQP-2 protein possessed 0.7
± 0.3 (n = 3) mol of
P
phosphate/mol of AQP-2 protein.
Having established conditions where
AQP-2 in endosomes contains an average of 0.7 mol of phosphate/mol of
protein (Fig. 5) or greater than 95 ± 8% of the P phosphate is removed by alkaline phosphatase (Fig. 4), the membrane water flux (J
) of
these respective endosomes was compared in a series of paired
experiments. AQP-2 endosomes containing entrapped F-dextran were
prepared from rats receiving intravenous F-dextran injections and the J
of aliquots of individual preparation of
endosomes determined using stopped-flow
fluorimetery(22, 36) . There was no significant
difference between the magnitude of entrapped fluorescence as well as
the J
of endosomes phosphorylated by PKA and ATP (Fig. 6, upper panel) as compared to those displayed
when these endosomes were subjected to dephosphorylation by alkaline
phosphatase treatment (Fig. 6, lower panel).
Ultrastructural analyses also revealed no significant difference (p > 0.5) in the mean diameter of phosphorylated AQP-2 endosomes
(144 ± 15 nm; n = 50) as compared to
dephosphorylated endosomes (146 ± 9 nm; n = 50).
Hence, the P
of endosomes containing
phosphorylated AQP-2 (0.16 ± 0.06 cm/s; n = 3)
was not significantly different from the P
of
endosomes where AQP-2 had been dephosphorylated by alkaline phosphatase
(0.14 ± 0.06 cm/s; n = 3). These data
demonstrate that alterations in the phosphorylation state of AQP-2 do
not result in significant changes in the P
of
these endosomes.
Figure 6:
Comparison of the membrane water flux (J) of endosomes containing AQP-2 present
in phosphorylated and dephosphorylated states. AQP-2 endosomes
containing entrapped F-dextran were isolated from rats as described in (22) . A single aliquot of endosomes was first phosphorylated
for 3 min using ATP and PKA under conditions described in Fig. 4(lanes 1 and 2). While the membrane J
of one half of these endosomes (upper tracing) was being quantified using stopped-flow
fluorimetry as described under ``Experimental Procedures''
and in (22) , the remainder were incubated with alkaline
phosphatase in a fashion identical to that described in Fig. 4(lane 1). After dephosphorylation of AQP-2, the J
of these endosomes was determined (lower tracing) under identical conditions. These data display
individual paired recordings from a single experiment performed a total
of three times.
The data contained in Fig. 1and Fig. 2demonstrate that AQP-2 protein present in purified
endosomes can be phosphorylated by both endogenous PKA or exogenous PKA
catalytic subunit in vitro. Although addition of only
[-
P]ATP to endosomes results in
phosphorylation of several proteins, both the 28- and 35-45-kDa
bands of AQP-2 are phosphorylated exclusively after addition of cAMP or
exogenous PKA catalytic subunit. Thus, under these in vitro conditions, phosphorylation of AQP-2 by other kinase activities
does not occur.
Kinetic studies of P incorporation into
AQP-2 reveals its rapid phosphorylation by PKA followed by a net loss
of
P label (Fig. 3, panel A). Present data
suggest that the loss of
P label from AQP-2 results from
endogenous phosphatase activity rather than proteolysis. First,
preincubation of endosomes under these conditions prior to AQP-2
phosphorylation results in no detectable loss of immunoprecipitable
P-labeled AQP-2 as compared to control (Fig. 3, panel B). Had significant proteolysis of AQP-2 occurred during
the preincubation interval, we would have anticipated a reduction in
the
P-content of AQP-2 as compared to control. Second, our
anti-AQP-2 antiserum specifically recognizes the amino acid sequence
(residues 258-271) located immediately adjacent to the consensus
PKA phosphorylation site at Ser-256 of AQP-2(14) . If limited
proteolysis of AQP-2 had occurred, then additional
P-labeled AQP-2 bands would likely be present in
autoradiograms of AQP-2 immunoprecipitates. However, we have observed
no such additional
P bands in our AQP-2
immunoprecipitates.
The presence of bound PKA and phosphatase activities in purified AQP-2 endosomes is intriguing because recent reports have demonstrated a specific role for both membrane-bound PKA (43) and phosphatase enzymes (44) in modulation of functional aspects of various ion channels(45) . Alternatively, it is possible that the presence of both PKA and phosphatase activities in purified AQP-2 endosomes results from the nonspecific binding of these enzymes to vesicles. A detailed characterization of both enzymatic activities that is beyond the scope of this report will be necessary to distinguish between these possibilities.
These data
shown in Fig. 4and Fig. 5demonstrate that covalent
phosphate can be removed from AQP-2 protein by alkaline phosphatase
treatment and that AQP-2 present in purified endosomes is present in a
relatively dephosphorylated state. In contrast, quantitation of the
AQP-2 P content after its incubation with exogenous PKA
shows it is efficiently phosphorylated in vitro. Although our
analysis of AQP-2 phosphate content was limited to the 35-40-kDa
region of the 35-45-kDa band, densitometry data showing that both
the 28- and 35-45-kDa AQP-2 bands possess
P label
proportional to their protein content suggest that both glycosylated
and nonglycosylated forms of AQP-2 possess similar
P
contents.
Paired experiments shown in Fig. 6reveal no
significant differences between the P values
exhibited by endosomes possessing AQP-2 phosphorylated by PKA and ATP
or dephosphorylated by alkaline phosphatase. Having quantitated the
phosphate content of AQP-2 using
P label, these
experiments were designed to detect alterations in the P
exhibited by endosomes possessing AQP-2 in phosphorylated versus dephosphorylated states. Under our conditions, where a
30% difference in vesicle P
could be measured and
endosome size did not change, no significant differences in either the
magnitude of the entrapped vesicular fluorescence or its time course
were noted. Thus, it is difficult to attribute the nearly 5-fold
increase in apical membrane P
observed after
incubation of rat inner medullary collecting duct with cAMP (46) to changes in P
resulting from AQP-2
phosphorylation. These data do not support the speculation that
phosphorylation of AQP-2 by PKA alters the P
of
AQP-2 proteins(18, 32) . However, our data cannot
exclude entirely this possibility since more complex speculations such
as interactions between AQP-2 protein and other proteins present in
purified AQP-2 endosomes that may modulate AQP-2 protein function were
not tested. Further study is necessary to reconcile the apparent
differences between the role of phosphorylation of AQP-2 in modulation
of plasma membrane P
in Xenopus oocytes (18, 32) as compared to AQP-2 endosomes purified from
rat kidney as reported here.