Interaction of the Plasma Membrane Ca2+ Pump 4b/CI with the Ca2+/Calmodulin-dependent Membrane-associated Kinase CASK*

Kai SchuhDagger , Stjepan Uldrijan§, Stepan GambaryanDagger , Nicola RoethleinDagger , and Ludwig Neyses||

From the Dagger  Department of Medicine, University of Wuerzburg, D-97080 Wuerzburg, Germany, § Masaryk Memorial Cancer Center, University of Brno, CZ-65653 Brno, Czech Republic, and  University of Manchester, Department of Medicine, Manchester Royal Infirmary, Manchester M13 9WL, Great Britain

Received for publication, December 9, 2002, and in revised form, December 23, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Spatial and temporal regulation of intracellular Ca2+ is a key event in many signaling pathways. Plasma membrane Ca2+-ATPases (PMCAs) are major regulators of Ca2+ homeostasis and bind to PDZ (PSD-95/Dlg/ZO-1) domains via their C termini. Various membrane-associated guanylate kinase family members have been identified as interaction partners of PMCAs. In particular, SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bind to the C-terminal tails of PMCA "b" splice variants. Additionally, it has been demonstrated that PMCA4b interacts with neuronal nitric-oxide synthase and that isoform 2b interacts with Na+/H+ exchanger regulatory factor 2, both via a PDZ domain. CASK (calcium/calmodulin-dependent serine protein kinase) contains a calmodulin-dependent protein kinase-like domain followed by PDZ, SH3, and guanylate kinase-like domains. In adult brain CASK is located at neuronal synapses and interacts with various proteins, e.g. neurexin and Veli/LIN-7. In kidney it is localized to renal epithelia. Surprisingly, interaction with the Tbr-1 transcription factor, nuclear transport, binding to DNA T-elements (in a complex with Tbr-1), and transcriptional competence has been shown. Here we show that the C terminus of PMCA4b binds to CASK and that both proteins co-precipitate from brain and kidney tissue lysates. Immunofluorescence staining revealed co-expression of PMCA, CASK, and calbindin-D-28K in distal tubuli of rat kidney sections. To test if physical interaction of both proteins results in functional consequences we constructed a T-element-dependent reporter vector and investigated luciferase activity in HEK293 lysates, previously co-transfected with PMCA4b expression and control vectors. Expression of wild-type PMCA resulted in an 80% decrease in T-element-dependent transcriptional activity, whereas co-expression of a point-mutated PMCA, with nearly eliminated Ca2+ pumping activity, had only a small influence on regulation of transcriptional activity. These results provide evidence of a new direct Ca2+-dependent link from the plasma membrane to the nucleus.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 right-arrow 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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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).

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 beta ) 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 right-arrow 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).

To test if the point mutation Asp672 right-arrow 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.

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 right-arrow 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 right-arrow 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.

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).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    FOOTNOTES

* 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.

|| To whom correspondence should be addressed. Tel.: 44-161-276-5738; Fax: 44-161-276-8904; E-mail: ludwig.neyses@mhc.cmht.nwest.nhs.uk.

Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M212507200

    ABBREVIATIONS

The abbreviations used are: PMCA, plasma membrane Ca2+-ATPase; CASK, calcium/calmodulin-dependent serine protein kinase; MAGUK, membrane-associated guanylate kinases; PBS, phosphate-buffered saline.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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