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INTRODUCTION |
Plasma membrane Ca2+/calmodulin-dependent
calcium pumps (PMCAs,1 also
known as plasma membrane Ca2+-ATPases) are abundantly
expressed in eukaryotic cells (for review, see Refs. 1 and 2) and
regulate intracellular Ca2+ homeostasis. PMCAs along with
sarco-/endoplasmatic reticulum Ca2+-ATPases, belong to
the P-type family of ATPases, which form an aspartyl phosphate
intermediate during their reaction cycle (for review, see Ref. 3). The
biochemical properties and structure of PMCAs have been well
characterized and were recently reviewed in Strehler and Zacharias (1).
Four different genes have been identified (4-10), coding for the four
known isoforms and their multiple splice variants (for review, see Ref.
1). The complex variation generated by alternative splicing raised the
question of different functions of the variants produced. In addition, PMCA activity is regulated by various signaling events, e.g.
Ca2+/calmodulin dependence, phosphorylation of the enzyme,
activation by phospholipids, and others (summarized, with nomenclature
explained in Carafoli (11).
Especially in excitable cells the function of PMCAs remains enigmatic.
Recently, a role of the carboxyeosin-sensitive Ca2+-ATPase
PMCA in controlling resting [Ca2+]i and the
reduction in [Ca2+]i after an increase in
[Ca2+]i in rat ventricular myocytes has been
demonstrated (12). In contrast to these observations, overexpression of
PMCA4b in the myocardium of transgenic rats did not result in an
altered regulation of contraction/relaxation cycle but in an enhanced growth response of cardiac myocytes to different stimuli (13). Additionally, it has been shown that the C termini of several PMCA
variants, especially variants PMCA4b and PMCA2b, bind to PDZ domains of
proteins of the MAGUK (membrane-associated guanylate kinases) family
(14, 15), to the PDZ domain of nitric-oxide synthase I (nNOS, NOS-I
(16)) and to Na+/H+ exchanger regulatory factor
(NHERF (23)). We observed that isoform PMCA4b (PMCA4CI) not only
interacted physically with nitric-oxide synthase I, but also
down-regulated its activity in a dose-dependent manner
through local Ca2+ depletion (16). A compilation of
interaction partners currently known is given in Table IA.
Gene knock-out studies of PMCA2 have revealed balance and hearing
deficits and slower growth of PMCA2-deficient animals compared with
wild-type littermates. Histological analysis of the cerebellum and
inner ear showed that null mutants had slightly increased numbers of
Purkinje neurons, an absence of otoconia in the vestibular system, and
a range of abnormalities of the organ of Corti (17), underlining a
potential role of PMCAs in differentiation and developmental processes.
Taken together, these results suggest an involvement of specific PMCAb
isoforms in signaling events via binding to PDZ domains and regulation
of local calcium concentrations in the proximity of
Ca2+-dependent enzymes.
CASK (calcium/calmodulin-dependent serine protein kinase),
a MAGUK family member, originally identified as an interaction partner
of neurexins (18), contains several modular protein domains. Each
domain mediates a specialized function of the protein; a
calmodulin-dependent protein kinase-like domain is followed by a PDZ-, an SH3-, and a guanylate kinase-like domain (18). The
protein binding PDZ domains of MAGUKs are thought to be responsible for
assembly of multiprotein complexes in distinct cellular compartments responsible for signaling pathways (19, 20). Binding to syndecans, protein 4.1 (21), neurexin (18), and a junctional adhesion molecule
(22) has also been demonstrated. A recently described novel type of
protein domain in CASK (L27 domain) mediates interaction with the MAGUK
family member SAP97 (23), belonging to the growing group of proteins
interacting with members of the PMCA family (14, 15, 24).
Despite its role as an organizer of protein complex assembly, it has
been shown that CASK interacts via its guanylate kinase-like domain
with the T-box transcription factor Tbr-1 (25). In conjunction with
Tbr-1 it enters the nucleus and binds to the palindromic T-element
(AATTTCACACCTAGGTGTGAAATT), thereby acting as a co-activator of
transcription of T-element-containing promoters, e.g. the
reelin promoter (25).
Here we show that the C-terminal amino acids of PMCA4b interact with
CASK/LIN-2. The results were verified by conventional immunoprecipitation experiments using kidney and brain extracts. Co-expression of both proteins in distal tubules was demonstrated in
rat kidney sections. Additionally, using a T-element-containing reporter vector, we have shown that increased PMCA activity
down-regulates T-element-dependent reporter activity,
providing a direct link from the cell membrane to the nucleus and to
transcriptional regulation of T-element-containing promoters.
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EXPERIMENTAL PROCEDURES |
Peptide Affinity Chromatography and Immunoprecipitations--
To
identify potential interaction partners of the PMCA C terminus a
peptide consisting of the last 15 C-terminal amino acids of the PMCA4b
(LPQSDSSLQSLETSV) was immobilized to streptavidin beads (Amersham
Biosciences) via an N-terminal biotinylation of the peptide
(MWG-Biotech). Streptavidin beads without the PMCA C-terminal peptide
served as controls. 2.5 ml of 50% slurry streptavidin beads were
washed with PBS and incubated with 1 mg of biotinylated C-terminal
peptide for 5 h at 4 °C, washed several times in NET buffer
(350 mM NaCl, 1 mM EDTA, 50 mM
Tris, pH 7.5, without detergents), and resuspended in 2.5 ml of NET
buffer. Rat organ lysates were prepared in NET buffer by Dounce
homogenization. The homogenate was centrifuged (20 min, 15,000 × g, 4 °C), and the pellet was dissolved in NET, 1% Triton
X-100 by vortexing for 30 min at 4 °C. After the second
centrifugation (30 min, 15,000 × g, 4 °C) protein
content of the supernatant was estimated using Bio-Rad protein assay
following the manufacturer's instructions. 150 µl of peptide-loaded
or unloaded beads were incubated with ~1-2 mg of tissue lysate and
rotated for 16 h at 4 °C. Subsequently, beads were washed
extensively with NET, 1% Triton X-100 and boiled in 100 µl of SDS
sample buffer, and 30 µl of each sample was separated in a 4-20%
gradient gel.
Immunoprecipitations were made using a standard protocol. In brief,
tissues were lysed in radioimmune precipitation assay buffer (1× PBS,
1% IGEPAL CA-630, Sigma, 0.5% sodium deoxycholate, Sigma/CompleteTM
EDTA-free protease inhibitor mixture, Roche Diagnostics). After an
initial preclearing step of 1 h at 4 °C (100-µl mix of a 50%
slurry of protein A and G-Sepharose beads, Amersham Biosciences, added
to ~1 mg of tissue lysate) antigens were coupled to the following
antibodies (5 µg of purified antibodies or 5 µl of serum): anti-CASK polyclonal rabbit anti-CASK, Zymed Laboratories
Inc., catalog no. 71-5000; anti-CASK monoclonal,
Transduction Laboratories, catalog no. C63120; anti-PMCA polyclonal
rabbit anti-hPMCAb, generated against the last 15 amino acid residues
of the human PMCA4b; anti-PMCA monoclonal clone 5F10, Sigma, catalog
no. A-7952. As an irrelevant antibody control, an estrogen
receptor-specific antiserum was employed. Samples containing 500 µg
of tissue lysate and antibodies were rotated for 1 h at 4 °C.
Protein-antibody complexes were precipitated using a mix of 50 µl of
protein A and 50 µl of protein G (50% slurry each)-Sepharose beads
for 1 h at 4 °C. After 4 washing steps with radioimmune
precipitation assay buffer and 1 with 50 mM Tris, pH 8.0, 100 µl of sample buffer was added to the washed beads, and 30 µl of
each sample was loaded and separated in an 8% SDS-PAGE.
Western Blotting and Detection of Proteins--
Samples were
boiled (5 min, 95 °C) in SDS-loading buffer (Bio-Rad), and the
proteins were separated on an 8% SDS-polyacrylamide gel and
transferred to nitrocellulose membranes (Amersham Biosciences). Membranes were blocked for 1 h in PBS, 5% nonfat milk, 0.05%
Tween 20. The following antibodies were used: anti-PMCA clone 5F10, Sigma, catalog no. A-7952; anti-CASK, Transduction Laboratories, catalog no. C63120; anti-SAP97 monoclonal antibody directed against
N-terminal amino acids 1-163 of SAP97 (26) were a kind gift from Craig
C. Garner, University of Alabama. Horseradish peroxidase-coupled
secondary antibodies and ECLTM Western blotting detection reagents (all
Amersham Biosciences) were used according to the manufacturer's protocols.
Immunocytochemistry--
After cannulation of the rat aorta
below the level of kidneys, kidneys were perfused with 4%
paraformaldehyde in PBS and used for frozen tissue sections (27).
5-µm sections were fixed in 2% paraformaldehyde in PBS (15 min,
4 °C), permeabilized in 0.1% Triton X-100 (15 min, 21 °C), and
blocked in PBS, 10% goat serum (3 h, 21 °C).
The following antibodies were used in fluorescence stainings: anti-PMCA
monoclonal antibody clone 5F10 (Sigma, 1:100), anti-CASK rabbit
polyclonal antibody (Zymed Laboratories Inc., 1:100),
and anti-calbindin-D-28K (Sigma, catalog 9848, 1:200). Secondary antibodies were fluorescein isothiocyanate-labeled
goat anti-mouse IgG and Cy3-labeled goat anti-rabbit IgG (Jackson Laboratories).
Vectors and Co-transfection Assays--
PMCA4b expression vector
has been described previously (16). Insertion of the point mutation
Asp672
Glu in the hPMCA4b expression vector was
achieved according to Adamo et al. (28) and is described
elsewhere (16). The constitutively active form of PMCA4b (PMCAct120
(29)) was a generous gift from Dr. E. E. Strehler;
expression vector design was described before (16). To
investigate the transactivation activity of CASK, two repeats of T
element (2× AATTTCACACCTAGGTGTGAAATT) were cloned into the
pGL3-Promoter vector (Promega) containing a SV40 minimal promotor.
Co-transfection assays were performed in HEK293 cells cultured in
6-well plates using the LipofectAMINE PLUSTM reagent (Invitrogen)
according to the manufacturer's protocol. 16 h after transfection
cells were stimulated with 0.5 µM ionomycin (Sigma) for
3 h at 37 °C. Relative luciferase activity was estimated using
the luciferase assay system (Promega) in comparison to control cells
co-transfected with the pGL3 Promoter vector.
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RESULTS |
Comparison of C Termini of PMCA4b Isoforms--
Similarities
between the potential CASK binding partner PMCA4b and the previously
described interaction partner neurexin were identified by comparing
amino acid sequences. Comparison of C termini of human PMCA4b (ETSV)
and neurexin (EYYV) revealed identity in the last amino acid position
(V) and in
3 position (E) of both proteins. Position of these amino
acid residues are also conserved in PMCA4b proteins of different
species (Table IB), suggesting
physiological relevance for these residues.
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Table I
Compilation of PDZ domain-containing proteins interacting with PMCA
isoforms and comparison of PMCA4b C termini with C terminus of
neurexin
A, PMCA2b and -4b have been shown to interact with different proteins
containing PDZ interaction domains. Corresponding references are given
in the last column. B, three PMCA4b isoforms of different species and
the CASK binding neurexin show amino acid sequence similarities in
their C terminus. They contain a Val at their final position and a Glu
E at 3 (numbering according to Sheng and Sala (43)) position,
suggesting a conserved motif.
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CASK Interacts with the C Terminus of the PMCA4b--
Peptide
affinity chromatography was performed to test if the PDZ
domain-containing protein CASK/LIN-2 interacts with the C terminus of
the PMCA4b. Use of the last 15 C-terminal amino acid residues of the
human PMCA4b coupled to Sepharose beads revealed interaction of the
PMCA4b C-terminal peptide with CASK/LIN-2 and SAP97 (Fig.
1). Binding of both proteins to the
C-terminal peptide has been demonstrated in extracts from various
organs. Interaction of PMCA4b with SAP97 has been previously shown (14,
15); therefore, it was regarded as the positive control.

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Fig. 1.
SAP97 and CASK interact with the C terminus
of PMCA4b. The last 15 amino acids of the hPMCA4b were immobilized
to Sepharose beads (+) and used to pull down interacting
proteins from various indicated organs. Subsequent Western blots
revealed interaction of SAP97 and CASK with the C terminus of the
PMCA4b. Sepharose beads without peptide ( ) served as the negative
control. Note that interaction with SAP97 is not a prerequisite for
binding of CASK to the PMCA C terminus (e.g. independent
binding in kidney, spleen, heart, and skeletal (Sk.)
muscle).
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CASK Co-precipitates with PMCA in Extracts of Brain and
Kidney--
Co-immunoprecipitations were made to test for interaction
of the full-length proteins in brain and kidney, which express both PMCA4b and CASK (1, 30). Both CASK-specific antibodies (mono- and
polyclonal) precipitated CASK (Fig. 2,
A and B; lanes 3 and 4,
respectively) as did the calmodulin positive control (Fig. 2,
A and B, lanes 2). Polyclonal and
monoclonal PMCA-specific antibodies precipitated a CASK-containing
complex from rat brain and kidney extracts (Fig. 2, A and
B, lanes 5 and 6) as did (expectedly) calmodulin-coated Sepharose beads (lanes 2 in Fig. 2,
A and B). Use of an irrelevant antibody
(polyclonal anti-estrogen receptor
) in immunoprecipitation assays
did not result in CASK precipitation (Fig. 2, lanes 1). In
both organ extracts a comparable pattern of precipitation was observed;
however, in kidney the interaction seemed to be more prominent, and the
following experiments were, therefore, performed with kidney sections
or HEK293 cells.

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Fig. 2.
CASK co-immunoprecipitates using CASK- and
PMCA-specific antibodies. Lysates of brain (A) and
kidney (B) were incubated with poly (p)- and
monoclonal (m) antibodies specific for CASK and PMCA (for
details see "Experimental Procedures"), and subsequently protein
complexes were precipitated with protein A/G beads. Western blots of
precipitated proteins were probed with an antibody specific for CASK
(A and B), and co-precipitation was observed in
both organs tested using different CASK- (lanes 3 and
4) or PMCA-specific antibodies (lanes 5 and
6). Irrelevant antibodies (irrel. ab, lanes
1, polyclonal anti-estrogen receptor) were used as negative
controls, and brain lysates (lane 7) were used as the
positive control. As another positive control CASK binding calmodulin
(CaM) beads were used instead of protein G beads
(lanes 2). C, HEK293 cells were transfected with
either the wild-type PMCA4b or the Asp672 Glu PMCA
mutant. Equal expression levels of CASK in both HEK293 lysates used is
shown in the first through fifth lanes. Irrelevant
antibodies do not precipitate CASK (second and sixth
lanes), whereas PMCA- and CASK-specific antibodies do
(third and seventh, fourth, and
last lanes).
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To test if the point mutation Asp672
Glu in the PMCA
may influence protein interaction (e.g. by inducing
conformational changes in the PMCA) we made co-immunoprecipitations
with lysates of HEK293 cells previously transfected with the wild-type
PMCA4b or point-mutated PMCA4b. We did not observe a difference in
binding behavior of isoforms. Both isoforms were capable of
precipitating CASK in a similar amount (Fig. 2C).
Co-expression of CASK and PMCA in the Distal Nephron of the Rat
Kidney--
Immunofluorescence staining of rat kidney sections
revealed congruent distribution of PMCA and CASK in this organ (Fig.
3, A-C). To identify the
nephron segments in kidney cortex where both proteins are co-expressed,
kidney sections were stained for calbindin distribution (Fig. 3,
D-F), a marker of distal convoluted tubule
(31). As in human kidney, PMCA (41) and CASK were strongly expressed in
the distal convoluted tubules (co-expression with calbindin, Fig. 3,
D-F). In kidney medulla, PMCA and CASK are co-expressed in the medullary thick ascending limb, identified by the
expression of Tamm-Horsfall protein (32) (data not shown).

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Fig. 3.
Co-expression of PMCA and CASK in
distal convoluted tubules of rat kidney. Double immunofluorescent
staining of PMCA and CASK (A-C), and calbindin, and CASK
(D-F) of rat kidney cortex. Kidney sections
were incubated with mouse anti-PMCA (A), rabbit anti-CASK
(B and E), or rabbit anti-calbindin
(D) antibodies followed by anti-rabbit Cy3 conjugated or
anti-mouse fluorescein isothiocyanate-conjugated antibodies.
C and F, merger of the images. PMCA and CASK are
co-expressed in the distal convoluted tubules (identified by the
expression of calbindin). Glomeruli (G) and proximal
convoluted tubules (PC) were not stained. Bar, 50 µm.
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PMCA Inhibits Activity of a T-element-regulated Reporter
Vector--
Not only is CASK a member of the MAGUK family, thought to
be involved in clustering proteins to the membrane, it is also a transcriptional co-factor involved in regulation of
T-element-containing promoters. We therefore determined whether the
interaction of the Ca2+-transporting PMCA and the
Ca2+/calmodulin-binding protein CASK may have an effect on
transcriptional activation of a T-element-containing reporter vector.
This was tested using a reporter vector containing two
CASK-dependent T-elements in tandem orientation (Fig.
4A). Insertion of
the tandem resulted in a 10-fold increase in reporter activity compared
with ("empty") control vector in HEK293 cells after stimulation
with a Ca2+ ionophore (Fig. 4B, first
and second columns). Co-transfection with PMCA4b expression
vector inhibited ionophore-induced reporter activity to ~20% of that
obtained with the reporter vector alone (Fig. 4B,
second and third columns). To test if this
inhibition was a result of the Ca2+-transporting activity
of the PMCA (and not an inhibition resulting from interaction of both
proteins or unspecific effects due to co-transfection) we
co-transfected a mutated form of the PMCA4b (Asp672
Glu, thereby replacing an amino acid residue in the ATP binding site
(28)). This resulted in a loss of inhibition of the
CASK-dependent reporter (Fig. 4B, fourth
column). Loss of inhibition was not complete; this mutation has
previously been demonstrated to retain ~10%
Ca2+-transporting activity (29). CASK protein expression in
these cells was not altered by co-transfection (Fig. 4C),
and expression of PMCA4b and mutated PMCA4b was increased as expected
and gave similar protein levels after co-transfection (example in Fig. 4D).

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Fig. 4.
PMCA regulates CASK-mediated transcription of
T-element containing reporter vector. A, schematic overview
of CASK reporter vector construct used in co-transfection assays. A
repeat of the Tbr-1·CASK complex binding T-element was cloned in
front of an SV40 minimal promoter (SV40MP) and a luciferase reporter
gene. B, relative luciferase units (rlu) after
co-transfection of CASK-reporter vector (p2×T-Luci) and PMCA4b
expression constructs (pCMV-PMCA and pCMV-PMCAmut). Insertion of 2×
T-element in the reporter vector enhanced reporter activity more than
10-fold compared with empty pGL3 promoter vector (B,
first and second columns). Co-transfection of
wild-type PMCA expression vector resulted in an ~80% decrease in
reporter activity (third column), which was nearly brought
back to a normal level when a point-mutated PMCA (Asp672
Glu) was co-transfected with CASK reporter vector (fourth
column). C, representative Western blot showing
constitutive expression of CASK protein level despite co-transfection
with reporter and expression vectors. D, representative
Western blot showing strong expression of wild-type (wt)
hPMCA4b (lane 3) and mutated (mut) hPMCA4bmut
(lane 4) after co-transfection of HEK293 cells.
E, use of calcium ionophore enhances activity of reporter vector and C-terminal truncation of PMCA4b
disrupts PMCA-mediated down-regulation of transcriptional activity. 0.5 µM ionomycin enhanced transcriptional activity
~1.5-fold (second column, compared with activity without
addition of ionophore (first column)) and was, therefore,
used to ensure availability of free Ca2+ in co-transfection
assays shown in B. Overexpression of wild-type PMCA4b
reduced reporter activity dramatically (0.5 µg of expression vector,
third column). The addition of ionomycin (0.5 µM) did not enhance reporter activity significantly in
the presence of overexpressed PMCA4b (fourth column).
Co-transfection with a constitutively active PMCA lacking the
regulatory domain (PMCAct120, fifth column) restored
reporter activity even in the presence of calcium ionophore.
p < 0.05 for ionomycin/ PMCA versus
+ionomycin/ PMCA; +ionomycin/ PMCA versus
+ionomycin/+PMCA and +ionomycin/+PMCA versus
+ionomycin/+PMCAct120, n = 10.
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To make sure that the observed down-regulation of promoter activity was
caused by the Ca2+-pumping activity and not by binding to
CASK, we tested a constitutively active form of the pump (PMCAct120)
lacking the regulatory domain and the C terminus normally mediating
interaction. Compared with the PMCA-mediated reduction of luciferase
activity (even in the presence of ionomycin) the PMCAct120 did not
reduce reporter luciferase activity, suggesting necessity of direct
interaction of CASK and PMCA to mediate function (Fig.
4E).
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DISCUSSION |
In our attempts to gain insight into the molecular function of
PMCAs, we have identified the PDZ domain containing CASK as a physical
and functional interaction partner of PMCA4b.
Interaction of PMCA4b with PDZ Domain-containing Proteins--
The
majority of the hitherto described interaction partners of the PMCA4b
belong to the family of MAGUKs (14, 15); additionally, nitric-oxide
synthase I and Na+/H+ exchanger regulatory
factor 2 (NHERF2) have recently been identified as partners interacting
with PMCA C termini (16, 24). The C terminus of the PMCA splice variant
4b differs from other b variants not only in the last amino acid
(valine instead of leucine) but also in amino acid positions 1200-1204
(comparison of different PMCA C termini in Strehler and Zacharias (1)),
suggesting specificity of interaction of the various b forms with other
proteins (24). The C terminus of the human PMCA4b (ETSV) resembles the
C terminus of neurexin (EYYV), a known interacting partner of CASK
(18). Therefore, the last four amino acids of human PMCA4b have the same level of identity (two of four amino acids) as the previously described CASK PDZ-interacting protein syndecan (EFYA). Consensus sequences of PDZ domain binding C termini are often described as
tripeptide ((T/S)XV, X is any amino acid
(44)) or tetrapeptide ((E/Q)(T/S)XV), but there are
several indications that six (33, 34) or even eight amino acid residues
mediate proper binding to PDZ domains.
In general, PDZ domains appear to display different levels of
specificity, some interacting promiscuously with a variety of proteins
harboring different C-terminal amino acid sequences (35); others
require a higher level of specificity for binding (24). Although the
PDZ domain of CASK is classified as a class II PDZ domain (for review,
see Ref. 30), it has been shown to interact with different C-terminal
sequences (EYYV from neurexin and EFYA from syndecan (18, 36)).
Additionally, it has been demonstrated for the PDZ domain of PICK-1
that it falls into both class I and II PDZ domains in terms of
preference for the
2 amino acid (37, 38). Our results demonstrate
that this may also be true for the PDZ domain of CASK/LIN-2 since the
PMCA4b C terminus is, strictly speaking, a class I PDZ domain ligand.
Recently, it was shown that CASK and SAP97 may also interact directly
via a novel protein-protein interaction domain called L27 domain, which
is present twice in CASK (23). One could assume that interaction of
PMCA and CASK is indirect and mediated by naturally occurring SAP97,
but this hypothesis seems to be not tenable since interaction of PMCA4b
C terminus and CASK also occurs in organs not displaying binding of
SAP97 (Fig. 1). Of course, indirect interaction or formation of larger
multiprotein complexes cannot be completely excluded.
Localization of CASK and PMCA in Distal Tubuli of Kidney--
CASK
is expressed in different parts of the brain and in liver, kidney,
uterus, and lung (18). In adult rat brain CASK is localized to the
plasma membrane of neuronal synapses (18, 39). PMCAs are also located
in this cellular compartment, and interaction of PMCAs with other PDZ
domain-containing proteins has been demonstrated in these structures
(for review, see Ref. 1).
Localization of PMCA4b in distal tubuli of human kidney has been shown
previously (40). Our results extend these findings to rat kidney and
showed the co-expression of PMCA and CASK in this nephron segment.
Also, differences in Ca2+ transport in proximal and distal
convoluted tubuli have been reported, originating from the observation
that Ca2+ ATPase activity was highest in distal tubuli
(41). Here we show that PMCA interacts with CASK and, therefore,
provides a clue for the function of PMCA by interaction with CASK.
PMCA4b may not be only a simple Ca2+ pump but also a
regulator of formation of intracellular PDZ domain-mediated complexes
and/or signal transduction.
Regulation of CASK Function by PMCA4b--
CASK contains a
Ca2+/calmodulin-dependent protein kinase II
domain. The calmodulin binding domain and the threonine of the
autophosphorylation site are highly conserved (18). Although no kinase
activity was observed for CASK, a recombinant CASK calmodulin binding
site bound calmodulin efficiently in a
Ca2+-dependent manner (18). These data
suggested that recently observed nuclear translocation and
transcriptional regulation by CASK (25) may at least in part depend on
Ca2+ concentration in close proximity of the protein.
Ca2+/calmodulin binding to CASK may induce a conformational
change of CASK and, thereby, allow binding to the
transcription factor Tbr-1 and translocation of CASK or the
CASK·Tbr-1 complex from the membrane to the nucleus. Depletion
of local Ca2+ by PMCA4b in close proximity to CASK may
inhibit Ca2+/calmodulin binding and subsequently inhibit
binding to Tbr-1 and/or translocation of CASK or the CASK·Tbr-1
complex to the nucleus. By also translocating to the nucleus, CASK
invites analogies with the MAGUK family member ZO-1, which translocates
from tight junctions to the nucleus in a cell contact-regulated manner,
but the mechanisms governing this process are unclear (42).
Our results, obtained in co-transfection studies, support the
hypothesis of Ca2+-dependent nuclear
translocation and are in line with the observed regulation of
nitric-oxide synthase I, also a
Ca2+/calmodulin-dependent protein. Nitric-oxide
synthase I activity was negatively regulated by PMCA4b overexpression
in a dose-dependent manner. This negative regulation was
not dependent on conformational change due to PDZ ligand binding but on
Ca2+ depletion in close proximity of the enzyme (16). This
also seems to be true for the regulation of CASK. Co-transfection of mutated or truncated PMCA4b resulted in restoration of
CASK-dependent transcriptional activity to expected levels.
In summary, our results demonstrate a novel role for the plasma
membrane calcium pump; it is likely to participate in transcriptional
regulation through a highly specific (PDZ domain-mediated) physical and
functional interaction.