Publications by authors named "Ágnes Enyedi"

37 Publications

The Plasma Membrane Ca Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton.

Cancers (Basel) 2021 Mar 17;13(6). Epub 2021 Mar 17.

Department of Transfusiology, Semmelweis University, H-1089 Budapest, Hungary.

We demonstrated that the plasma membrane Ca ATPase PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells. Actin dynamics are essential for cells to move, invade and metastasize, therefore, we hypothesized that PMCA4b affected cell migration through remodeling of the actin cytoskeleton. We found that expression of PMCA4b in A375 BRAF mutant melanoma cells induced a profound change in cell shape, cell culture morphology, and displayed a polarized migratory character. Along with these changes the cells became more rounded with increased cell-cell connections, lamellipodia and stress fiber formation. Silencing PMCA4b in MCF-7 breast cancer cells had a similar effect, resulting in a dramatic loss of stress fibers. In addition, the PMCA4b expressing A375 cells maintained front-to-rear Ca concentration gradient with the actin severing protein cofilin localizing to the lamellipodia, and preserved the integrity of the actin cytoskeleton from a destructive Ca overload. We showed that both PMCA4b activity and trafficking were essential for the observed morphology and motility changes. In conclusion, our data suggest that PMCA4b plays a critical role in adopting front-to-rear polarity in a normally spindle-shaped cell type through F-actin rearrangement resulting in a less aggressive melanoma cell phenotype.
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http://dx.doi.org/10.3390/cancers13061354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002435PMC
March 2021

P38 MAPK Promotes Migration and Metastatic Activity of BRAF Mutant Melanoma Cells by Inducing Degradation of PMCA4b.

Cells 2020 05 13;9(5). Epub 2020 May 13.

2nd Institute of Pathology, Semmelweis University, H-1091 Budapest, Hungary.

Metastatic melanoma is the most aggressive type of skin cancer. Previously, we identified the plasma membrane Ca pump isoform 4b (PMCA4b or ) as a putative metastasis suppressor in BRAF mutant melanoma cells. Metastasis suppressors are often downregulated in cancer, therefore, it is important to identify the pathways involved in their degradation. Here, we studied the role of p38 MAPK in PMCA4b degradation and its effect on melanoma metastasis. We found that activation of p38 MAPK induces internalization and subsequent degradation of PMCA4b through the endo/lysosomal system that contributes to the low PMCA4b steady-state protein level of BRAF mutant melanoma cells. Moreover, BRAF wild type cell models including a doxycycline-inducible HEK cell system revealed that p38 MAPK is a universal modulator of PMCA4b endocytosis. Inhibition of the p38 MAPK pathway markedly reduced migration, colony formation and metastatic activity of BRAF mutant cells in vitro partially through an increase in PMCA4b and a decrease in β4 integrin abundance. In conclusion, our data suggest that the p38 MAPK pathway plays a key role in PMCA4b degradation and inhibition of this pathway-by increasing the stability of PMCA4b-may provide a potential therapeutic target for inhibition of melanoma progression and metastasis.
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http://dx.doi.org/10.3390/cells9051209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290426PMC
May 2020

Endoplasmic Reticulum Calcium Pumps and Tumor Cell Differentiation.

Int J Mol Sci 2020 May 9;21(9). Epub 2020 May 9.

AP-HP, Service d'Anatomie et Cytologie Pathologiques, Hôpital Lariboisière, 75010 Paris, France.

Endoplasmic reticulum (ER) calcium homeostasis plays an essential role in cellular calcium signaling, intra-ER protein chaperoning and maturation, as well as in the interaction of the ER with other organelles. Calcium is accumulated in the ER by sarco/endoplasmic reticulum calcium ATPases (SERCA enzymes) that generate by active, ATP-dependent transport, a several thousand-fold calcium ion concentration gradient between the cytosol (low nanomolar) and the ER lumen (high micromolar). SERCA enzymes are coded by three genes that by alternative splicing give rise to several isoforms, which can display isoform-specific calcium transport characteristics. SERCA expression levels and isoenzyme composition vary according to cell type, and this constitutes a mechanism whereby ER calcium homeostasis is adapted to the signaling and metabolic needs of the cell, depending on its phenotype, its state of activation and differentiation. As reviewed here, in several normal epithelial cell types including bronchial, mammary, gastric, colonic and choroid plexus epithelium, as well as in mature cells of hematopoietic origin such as pumps are simultaneously expressed, whereas in corresponding tumors and leukemias SERCA3 expression is selectively down-regulated. SERCA3 expression is restored during the pharmacologically induced differentiation of various cancer and leukemia cell types. SERCA3 is a useful marker for the study of cell differentiation, and the loss of SERCA3 expression constitutes a previously unrecognized example of the remodeling of calcium homeostasis in tumors.
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http://dx.doi.org/10.3390/ijms21093351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247589PMC
May 2020

Molecular Diversity of Plasma Membrane Ca Transporting ATPases: Their Function Under Normal and Pathological Conditions.

Adv Exp Med Biol 2020 ;1131:93-129

2nd Department of Pathology, Semmelweis University, Budapest, Hungary.

Plasma membrane Ca transport ATPases (PMCA1-4, ATP2B1-4) are responsible for removing excess Ca from the cell in order to keep the cytosolic Ca ion concentration at the low level essential for normal cell function. While these pumps take care of cellular Ca homeostasis they also change the duration and amplitude of the Ca signal and can create Ca gradients across the cell. This is accomplished by generating more than twenty PMCA variants each having the character - fast or slow response, long or short memory, distinct interaction partners and localization signals - that meets the specific needs of the particular cell-type in which they are expressed. It has become apparent that these pumps are essential to normal tissue development and their malfunctioning can be linked to different pathological conditions such as certain types of neurodegenerative and heart diseases, hearing loss and cancer. In this chapter we summarize the complexity of PMCA regulation and function under normal and pathological conditions with particular attention to recent developments of the field.
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http://dx.doi.org/10.1007/978-3-030-12457-1_5DOI Listing
October 2019

Expression of calcium pumps is differentially regulated by histone deacetylase inhibitors and estrogen receptor alpha in breast cancer cells.

BMC Cancer 2018 Oct 23;18(1):1029. Epub 2018 Oct 23.

2nd Department of Pathology, Semmelweis University, Budapest, Hungary.

Background: Remodeling of Ca signaling is an important step in cancer progression, and altered expression of members of the Ca signaling toolkit including the plasma membrane Ca ATPases (PMCA proteins encoded by ATP2B genes) is common in tumors.

Methods: In this study PMCAs were examined in breast cancer datasets and in a variety of breast cancer cell lines representing different subtypes. We investigated how estrogen receptor alpha (ER-α) and histone deacetylase (HDAC) inhibitors regulate the expression of these pumps.

Results: Three distinct datasets displayed significantly lower ATP2B4 mRNA expression in invasive breast cancer tissue samples compared to normal breast tissue, whereas the expression of ATP2B1 and ATP2B2 was not altered. Studying the protein expression profiles of Ca pumps in a variety of breast cancer cell lines revealed low PMCA4b expression in the ER-α positive cells, and its marked upregulation upon HDAC inhibitor treatments. PMCA4b expression was also positively regulated by the ER-α pathway in MCF-7 cells that led to enhanced Ca extrusion capacity in response to 17β-estradiol (E2) treatment. E2-induced PMCA4b expression was further augmented by HDAC inhibitors. Surprisingly, E2 did not affect the expression of PMCA4b in other ER-α positive cells ZR-75-1, T-47D and BT-474. These findings were in good accordance with ChIP-seq data analysis that revealed an ER-α binding site in the ATP2B4 gene in MCF-7 cells but not in other ER-α positive tumor cells. In the triple negative cells PMCA4b expression was relatively high, and the effect of HDAC inhibitor treatment was less pronounced as compared to that of the ER-α positive cells. Although, the expression of PMCA4b was relatively high in the triple negative cells, a fraction of the protein was found in intracellular compartments that could interfere with the cellular function of the protein.

Conclusions: Our results suggest that the expression of Ca pumps is highly regulated in breast cancer cells in a subtype specific manner. Our results suggest that hormonal imbalances, epigenetic modifications and impaired protein trafficking could interfere with the expression and cellular function of PMCA4b in the course of breast cancer progression.
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http://dx.doi.org/10.1186/s12885-018-4945-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199715PMC
October 2018

Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation.

J Exp Clin Cancer Res 2017 06 26;36(1):87. Epub 2017 Jun 26.

Institut National de la Santé et de la Recherche Médicale, U978, Bobigny, France.

Background: Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis.

Methods: In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated.

Results: We show that E2A-PBX1 leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process.

Conclusion: These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.
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http://dx.doi.org/10.1186/s13046-017-0556-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5485704PMC
June 2017

Histone Deacetylase Inhibitor Treatment Increases the Expression of the Plasma Membrane Ca Pump PMCA4b and Inhibits the Migration of Melanoma Cells Independent of ERK.

Front Oncol 2017 24;7:95. Epub 2017 May 24.

2nd Institute of Pathology, Semmelweis University, Budapest, Hungary.

Several new therapeutic options emerged recently to treat metastatic melanoma; however, the high frequency of intrinsic and acquired resistance among patients shows a need for new therapeutic options. Previously, we identified the plasma membrane Ca ATPase 4b (PMCA4b) as a metastasis suppressor in BRAF-mutant melanomas and found that mutant BRAF inhibition increased the expression of the pump, which then inhibited the migratory and metastatic capability of the cells. Earlier it was also demonstrated that histone deacetylase inhibitors (HDACis) upregulated PMCA4b expression in gastric, colon, and breast cancer cells. In this study, we treated one BRAF wild-type and two BRAF-mutant melanoma cell lines with the HDACis, SAHA and valproic acid, either alone, or in combination with the BRAF inhibitor, vemurafenib. We found that HDACi treatment strongly increased the expression of PMCA4b in all cell lines irrespective of their BRAF mutational status, and this effect was independent of ERK activity. Furthermore, HDAC inhibition also enhanced the abundance of the housekeeping isoform PMCA1. Combination of HDACis with vemurafenib, however, did not have any additive effects on either PMCA isoform. We demonstrated that the HDACi-induced increase in PMCA abundance was coupled to an enhanced [Ca] clearance rate and also strongly inhibited both the random and directional movements of A375 cells. The primary role of PMCA4b in these characteristic changes was demonstrated by treatment with the PMCA4-specific inhibitor, caloxin 1c2, which was able to restore the slower Ca clearance rate and higher motility of the cells. While HDAC treatment inhibited cell motility, it decreased only modestly the ratio of proliferative cells and cell viability. Our results show that in melanoma cells the expression of both PMCA4b and PMCA1 is under epigenetic control and the elevation of PMCA4b expression either by HDACi treatment or by the decreased activation of the BRAF-MEK-ERK pathway can inhibit the migratory capacity of the highly motile A375 cells.
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http://dx.doi.org/10.3389/fonc.2017.00095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442207PMC
May 2017

Decreased calcium pump expression in human erythrocytes is connected to a minor haplotype in the ATP2B4 gene.

Cell Calcium 2017 07 3;65:73-79. Epub 2017 Feb 3.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar Tudosok krt. 2, Budapest, 1117, Hungary; MTA-SE Molecular Biophysics Research Group, Department of Biophysics and Radiation Biology, Semmelweis University, Tuzolto u. 37-43., Budapest, 1094, Hungary. Electronic address:

Plasma membrane Ca-ATPases are key calcium exporter proteins in most tissues, and PMCA4b is the main calcium transporter in the human red blood cells (RBCs). In order to assess the expression level of PMCA4b, we have developed a flow cytometry and specific antibody binding method to quantitatively detect this protein in the erythrocyte membrane. Interestingly, we found several healthy volunteers showing significantly reduced expression of RBC-PMCA4b. Western blot analysis of isolated RBC membranes confirmed this observation, and indicated that there are no compensatory alterations in other PMCA isoforms. In addition, reduced PMCA4b levels correlated with a lower calcium extrusion capacity in these erythrocytes. When exploring the potential genetic background of the reduced PMCA4b levels, we found no missense mutations in the ATP2B4 coding regions, while a formerly unrecognized minor haplotype in the predicted second promoter region closely correlated with lower erythrocyte PMCA4b protein levels. In recent GWA studies, SNPs in this ATP2B4 haplotype have been linked to reduced mean corpuscular hemoglobin concentrations (MCHC), and to protection against malaria infection. Our data suggest that an altered regulation of gene expression is responsible for the reduced RBC-PMCA4b levels that is probably linked to the development of human disease-related phenotypes.
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http://dx.doi.org/10.1016/j.ceca.2017.02.001DOI Listing
July 2017

The plasma membrane Ca pump PMCA4b inhibits the migratory and metastatic activity of BRAF mutant melanoma cells.

Int J Cancer 2017 06 17;140(12):2758-2770. Epub 2016 Nov 17.

2nd Department of Pathology, Semmelweis University, Budapest, Hungary.

Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca signaling is a well-known regulator of tumor progression, the crosstalk between Ca signaling and the Ras-BRAF-MEK-ERK pathway is much less explored. Here we show that in BRAF mutant melanoma cells the abundance of the plasma membrane Ca ATPase isoform 4b (PMCA4b, ATP2B4) is low at baseline but markedly elevated by treatment with the mutant BRAF specific inhibitor vemurafenib. In line with these findings gene expression microarray data also shows decreased PMCA4b expression in cutaneous melanoma when compared to benign nevi. The MEK inhibitor selumetinib-similarly to that of the BRAF-specific inhibitor-also increases PMCA4b levels in both BRAF and NRAS mutant melanoma cells suggesting that the MAPK pathway is involved in the regulation of PMCA4b expression. The increased abundance of PMCA4b in the plasma membrane enhances [Ca ] clearance from cells after Ca entry. Moreover we show that both vemurafenib treatment and PMCA4b overexpression induce marked inhibition of migration of BRAF mutant melanoma cells. Importantly, reduced migration of PMCA4b expressing BRAF mutant cells is associated with a marked decrease in their metastatic potential in vivo. Taken together, our data reveal an important crosstalk between Ca signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor.
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http://dx.doi.org/10.1002/ijc.30503DOI Listing
June 2017

Multifaceted plasma membrane Ca(2+) pumps: From structure to intracellular Ca(2+) handling and cancer.

Biochim Biophys Acta 2016 Jun 17;1863(6 Pt B):1351-63. Epub 2015 Dec 17.

2(nd) Department of Pathology, Semmelweis University, Budapest, Hungary; Molecular Oncology Research Group of the Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary. Electronic address:

Plasma membrane Ca(2+) ATPases (PMCAs) are intimately involved in the control of intracellular Ca(2+) concentration. They reduce Ca(2+) in the cytosol not only by direct ejection, but also by controlling the formation of inositol-1,4,5-trisphosphate and decreasing Ca(2+) release from the endoplasmic reticulum Ca(2+) pool. In mammals four genes (PMCA1-4) are expressed, and alternative RNA splicing generates more than twenty variants. The variants differ in their regulatory characteristics. They localize into highly specialized membrane compartments and respond to the incoming Ca(2+) with distinct temporal resolution. The expression pattern of variants depends on cell type; a change in this pattern can result in perturbed Ca(2+) homeostasis and thus altered cell function. Indeed, PMCAs undergo remarkable changes in their expression pattern during tumorigenesis that might significantly contribute to the unbalanced Ca(2+) homeostasis of cancer cells. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.
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http://dx.doi.org/10.1016/j.bbamcr.2015.12.011DOI Listing
June 2016

Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling.

Sci Rep 2015 Aug 3;5:12645. Epub 2015 Aug 3.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.

In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na(+)/Ca(2+) exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies.
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http://dx.doi.org/10.1038/srep12645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522653PMC
August 2015

Selective upregulation of the expression of plasma membrane calcium ATPase isoforms upon differentiation and 1,25(OH)2D3-vitamin treatment of colon cancer cells.

Biochem Biophys Res Commun 2015 Aug 23;464(1):189-94. Epub 2015 Jun 23.

Membrane Biology Research Group, Hungarian Academy of Sciences, Budapest, Hungary; Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary. Electronic address:

We have previously presented co-expression of the plasma membrane calcium ATPase isoforms 4b (PMCA4b) and 1b (PMCA1b) in colon carcinoma cells, and selective upregulation of PMCA4b during differentiation initiated by short chain fatty acids or post-confluent growth. Here we show that the induction of PMCA4b expression is a characteristic feature of the post-confluency-induced differentiation of both enterocyte-type and goblet cell-type colon cancer cells. Vitamin D3 (1,25(OH)2D3) is a well-known regulator of intestinal Ca(2+) absorption and of basic cell functions such as growth and differentiation in various cell types. As PMCA proteins are involved both in intestinal Ca(2+) absorption and adenocarcinoma cell differentiation, we investigated the effect of 1,25(OH)2D3 on PMCA expression in enterocyte-like colon carcinoma cells, and monitored its effect on the expression of various differentiation markers. 1,25(OH)2D3 stimulated PMCA1b, but not PMCA4b expression without modulating the expression of the majority of the differentiation markers examined. Caco-2 cells differentiated in post-confluent cultures present normal enterocyte-like intestinal epithelial phenotype. To better understand the role of PMCA proteins in vectorial Ca(2+) transport by enterocytes, we also studied their subcellular localization in mature polarized Caco-2 cells. Both PMCA isoforms were located to the basolateral membrane, and the PMCA-specific immunofluorescent signal was significantly higher in vitamin D3-treated cells, underlining the 1,25(OH)2D3-induced upregulation of PMCA (presumably 1b isoform) expression in differentiated Caco-2 cells. We suggest that while PMCA1b has a housekeeping function in colon cancer cells, PMCA4b participates in the reorganization of the Ca(2+) signalling machinery during cell differentiation. The subcellular localization of PMCA1b and its selective 1,25(OH)2D3-dependent upregulation indicate that this isoform may have a specific role in 1,25(OH)2D3-stimulated intestinal Ca(2+) absorption.
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http://dx.doi.org/10.1016/j.bbrc.2015.06.113DOI Listing
August 2015

Visualization of Calcium Dynamics in Kidney Proximal Tubules.

J Am Soc Nephrol 2015 Nov 18;26(11):2731-40. Epub 2015 Mar 18.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary; Department of Biophysics and Radiation Biology, MTA-SE Molecular Biophysics Research Group, and

Intrarenal changes in cytoplasmic calcium levels have a key role in determining pathologic and pharmacologic responses in major kidney diseases. However, cell-specific delivery of calcium-sensitive probes in vivo remains problematic. We generated a transgenic rat stably expressing the green fluorescent protein-calmodulin-based genetically encoded calcium indicator (GCaMP2) predominantly in the kidney proximal tubules. The transposon-based method used allowed the generation of homozygous transgenic rats containing one copy of the transgene per allele with a defined insertion pattern, without genetic or phenotypic alterations. We applied in vitro confocal and in vivo two-photon microscopy to examine basal calcium levels and ligand- and drug-induced alterations in these levels in proximal tubular epithelial cells. Notably, renal ischemia induced a transient increase in cellular calcium, and reperfusion resulted in a secondary calcium load, which was significantly decreased by systemic administration of specific blockers of the angiotensin receptor and the Na-Ca exchanger. The parallel examination of in vivo cellular calcium dynamics and renal circulation by fluorescent probes opens new possibilities for physiologic and pharmacologic investigations.
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http://dx.doi.org/10.1681/ASN.2014070705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625667PMC
November 2015

Plasma membrane Ca²⁺-ATPases can shape the pattern of Ca²⁺ transients induced by store-operated Ca²⁺ entry.

Sci Signal 2015 Feb 17;8(364):ra19. Epub 2015 Feb 17.

Hungarian National Blood Transfusion Service, Budapest H-1113, Hungary. 2nd Department of Pathology, Semmelweis University, Budapest H-1091, Hungary. Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest H-1117, Hungary.

Calcium (Ca(2+)) is a critical cofactor and signaling mediator in cells, and the concentration of cytosolic Ca(2+) is regulated by multiple proteins, including the plasma membrane Ca(2+)-ATPases (adenosine triphosphatases) (PMCAs), which use ATP to transport Ca(2+) out of cells. PMCA isoforms exhibit different kinetic and regulatory properties; thus, the presence and relative abundance of individual isoforms may help shape Ca(2+) transients and cellular responses. We studied the effects of three PMCA isoforms (PMCA4a, PMCA4b, and PMCA2b) on Ca(2+) transients elicited by conditions that trigger store-operated Ca(2+) entry (SOCE) and that blocked Ca(2+) uptake into the endoplasmic reticulum in HeLa cells, human embryonic kidney (HEK) 293 cells, or primary endothelial cell isolated from human umbilical cord veins (HUVECs). The slowly activating PMCA4b isoform produced long-lasting Ca(2+) oscillations in response to SOCE. The fast-activating isoforms PMCA2b and PMCA4a produced different effects. PMCA2b resulted in rapid and highly PMCA abundance-sensitive clearance of SOCE-mediated Ca(2+) transients, whereas PMCA4a reduced cytosolic Ca(2+), resulting in the establishment of a higher than baseline cytosolic Ca(2+) concentration. Mathematical modeling showed that slow activation was critical to the sustained oscillation induced by the "slow" PMCA4b pump. The modeling and experimental results indicated that the distinct properties of PMCA isoforms differentially regulate the pattern of SOCE-mediated Ca(2+) transients, which would thus affect the activation of downstream signaling pathways.
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http://dx.doi.org/10.1126/scisignal.2005672DOI Listing
February 2015

Histone deacetylase inhibitor- and PMA-induced upregulation of PMCA4b enhances Ca2+ clearance from MCF-7 breast cancer cells.

Cell Calcium 2014 Feb 28;55(2):78-92. Epub 2013 Dec 28.

Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary; Department of Molecular Cell Biology, Hungarian National Blood Transfusion Service, Budapest, Hungary. Electronic address:

The expression of the plasma membrane Ca2+ ATPase (PMCA) isoforms is altered in several types of cancer cells suggesting that they are involved in cancer progression. In this study we induced differentiation of MCF-7 breast cancer cells by histone deacetylase inhibitors (HDACis) such as short chain fatty acids (SCFAs) or suberoylanilide hydroxamic acid (SAHA), and by phorbol 12-myristate 13-acetate (PMA) and found strong upregulation of PMCA4b protein expression in response to these treatments. Furthermore, combination of HDACis with PMA augmented cell differentiation and further enhanced PMCA4b expression both at mRNA and protein levels. Immunocytochemical analysis revealed that the upregulated protein was located mostly in the plasma membrane. To examine the functional consequences of elevated PMCA4b expression, the characteristics of intracellular Ca2+ signals were investigated before and after differentiation inducing treatments, and also in cells overexpressing PMCA4b. The increased PMCA4b expression - either by treatment or overexpression - led to enhanced Ca2+ clearance from the stimulated cells. We found pronounced PMCA4 protein expression in normal breast tissue samples highlighting the importance of this pump for the maintenance of mammary epithelial Ca2+ homeostasis. These results suggest that modulation of Ca2+ signaling by enhanced PMCA4b expression may contribute to normal development of breast epithelium and may be lost in cancer.
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http://dx.doi.org/10.1016/j.ceca.2013.12.003DOI Listing
February 2014

Apart from its known function, the plasma membrane Ca²⁺ATPase can regulate Ca²⁺ signaling by controlling phosphatidylinositol 4,5-bisphosphate levels.

J Cell Sci 2014 Jan 6;127(Pt 1):72-84. Epub 2013 Nov 6.

Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1025 Budapest, Hungary.

Plasma membrane Ca(2+) ATPases (PMCAs, also known as ATP2B1-ATP2B4) are known targets of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], but if and how they control the PtdIns(4,5)P₂ pool has not been considered. We demonstrate here that PMCAs protect PtdIns(4,5)P₂ in the plasma membrane from hydrolysis by phospholipase C (PLC). Comparison of active and inactive PMCAs indicates that the protection operates by two mechanisms; one requiring active PMCAs, the other not. It appears that the mechanism requiring activity is the removal of the Ca(2+) required for sustained PLC activity, whereas the mechanism not requiring activity is PtdIns(4,5)P₂ binding. We show that in PMCA overexpressing cells, PtdIns(4,5)P₂ binding can lead to less inositol 1,4,5-triphosphate (InsP₃) and diminished Ca(2+) release from intracellular Ca(2+) pools. Inspection of a homology model of PMCA suggests that PMCAs have a conserved cluster of basic residues forming a 'blue collar' at the interface between the membrane core and the cytoplasmic domains. By molecular dynamics simulation, we found that the blue collar forms four binding pockets for the phosphorylated inositol head group of PtdIns(4,5)P₂; these pockets bind PtdIns(4,5)P₂ strongly and frequently. Our studies suggest that by having the ability to bind PtdIns(4,5)P₂, PMCAs can control the accessibility of PtdIns(4,5)P₂ for PLC and other PtdIns(4,5)P₂-mediated processes.
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http://dx.doi.org/10.1242/jcs.132548DOI Listing
January 2014

A C-terminal di-leucine motif controls plasma membrane expression of PMCA4b.

Biochim Biophys Acta 2013 Dec 2;1833(12):2561-2572. Epub 2013 Jul 2.

Hungarian National Blood Transfusion Service, Budapest, Hungary. Electronic address:

Recent evidences show that the localization of different plasma membrane Ca(2+) ATPases (PMCAs) is regulated in various complex, cell type-specific ways. Here we show that in low-density epithelial and endothelial cells PMCA4b localized mostly in intracellular compartments and its plasma membrane localization was enhanced upon increasing density of cells. In good correlation with the enhanced plasma membrane localization a significantly more efficient Ca(2+) clearance was observed in confluent versus non-confluent HeLa cell cultures expressing mCherry-PMCA4b. We analyzed the subcellular localization and function of various C-terminally truncated PMCA4b variants and found that a truncated mutant PMCA4b-ct24 was mostly intracellular while another mutant, PMCA4b-ct48, localized more to the plasma membrane, indicating that a protein sequence corresponding to amino acid residues 1158-1181 contained a signal responsible for the intracellular retention of PMCA4b in non-confluent cultures. Alteration of three leucines to alanines at positions 1167-1169 resulted in enhanced cell surface expression and an appropriate Ca(2+) transport activity of both wild type and truncated pumps, suggesting that the di-leucine-like motif (1167)LLL was crucial in targeting PMCA4b. Furthermore, upon loss of cell-cell contact by extracellular Ca(2+) removal, the wild-type pump was translocated to the early endosomal compartment. Targeting PMCA4b to early endosomes was diminished by the L(1167-69)A mutation, and the mutant pump accumulated in long tubular cytosolic structures. In summary, we report a di-leucine-like internalization signal at the C-tail of PMCA4b and suggest an internalization-mediated loss of function of the pump upon low degree of cell-cell contact.
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http://dx.doi.org/10.1016/j.bbamcr.2013.06.021DOI Listing
December 2013

B cell receptor-induced Ca2+ mobilization mediates F-actin rearrangements and is indispensable for adhesion and spreading of B lymphocytes.

J Leukoc Biol 2013 Apr 29;93(4):537-47. Epub 2013 Jan 29.

Eötvös Lóránd University, Pázmány Péter sétány 1/c, Budapest, Hungary.

B cells acquire membrane-bound cognate antigens from the surface of the APCs by forming an IS, similar to that seen in T cells. Recognition of membrane-bound antigens on the APCs initiates adhesion of B lymphocytes to the antigen-tethered surface, which is followed by the formation of radial lamellipodia-like structures, a process known as B cell spreading. The spreading response requires the rearrangement of the submembrane actin cytoskeleton and is regulated mainly via signals transmitted by the BCR. Here, we show that cytoplasmic calcium is a regulator of actin cytoskeleton dynamics in B lymphocytes. We find that BCR-induced calcium mobilization is indispensible for adhesion and spreading of B cells and that PLCγ and CRAC-mediated calcium mobilization are critical regulators of these processes. Measuring calcium and actin dynamics in live cells, we found that a generation of actin-based membrane protrusion is strongly linked to the dynamics of a cytoplasmic-free calcium level. Finally, we demonstrate that PLCγ and CRAC channels regulate the activity of actin-severing protein cofilin, linking BCR-induced calcium signaling to the actin dynamics.
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http://dx.doi.org/10.1189/jlb.0312169DOI Listing
April 2013

Characterization of calcium signals in human embryonic stem cells and in their differentiated offspring by a stably integrated calcium indicator protein.

Cell Signal 2013 Apr 7;25(4):752-9. Epub 2013 Jan 7.

Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.

Intracellular calcium signaling pathways play a major role in cellular responses such as proliferation, differentiation and apoptosis. Human embryonic stem cells (hESC) provide new possibilities to explore the development and differentiation of various cell types of the human body. Intracellular calcium responses to various ligands and the calcium signaling pathways, however, have not been thoroughly studied in embryonic stem cells and in their differentiated progenies. In our previous work we demonstrated that the use of the fluorescent calcium indicator Fluo-4 with confocal microscopy allows sensitive and reliable measurements of calcium modulation in human embryonic stem cells and stem-cell derived cardiomyocytes. Here we developed a human embryonic stem cell line stably expressing a genetically encoded Ca(2+) indicator (GCaMP2) using a transposon-based gene delivery system. We found that the differentiation properties were fully preserved in the GCaMP2-expressing hESC lines and Ca imaging could be performed without the need of toxic dye-loading of the cells. In undifferentiated hES cells the calcium signals induced by various ligands, ATP, LPA, trypsin or angiotensin II were comparable to those in Fluo-4 loaded cells. In accordance with previous findings, no calcium signal was evoked by thrombin, histamine or GABA. Cardiomyocyte colonies differentiated from hES-GCaMP2 cells could be recognized by spontaneous contractions and Ca(2+) oscillations. GCaMP2-expressing neural cells were identified based on their morphological and immuno-staining properties and Ca signals were characterized on those cells. Characteristics of both the spontaneous and ligand-induced Ca(2+) signals, as well as their pharmacological modification could be successfully examined in these model cells by fluorescence imaging.
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http://dx.doi.org/10.1016/j.cellsig.2012.12.024DOI Listing
April 2013

Modulation of endoplasmic reticulum calcium pump expression during lung cancer cell differentiation.

FEBS J 2013 Nov 11;280(21):5408-18. Epub 2012 Dec 11.

Institut National de la Santé et de la Recherche Médicale, UMR-S 940, Paris, France; Institut Universitaire d'Hématologie, Université Paris Diderot, PRES Sorbonne Paris-Cité, France.

Cellular calcium signaling plays important roles in several signal transduction pathways that control proliferation, differentiation and apoptosis. In epithelial cells calcium signaling is initiated mainly by calcium release from endoplasmic-reticulum-associated intracellular calcium pools. Because calcium is accumulated in the endoplasmic reticulum by sarco/endoplasmic reticulum calcium ATPases (SERCA), these enzymes play a critical role in the control of calcium-dependent cell activation, growth and survival. We investigated the modulation of SERCA expression and function in human lung adenocarcinoma cells. In addition to the ubiquitous SERCA2 enzyme, the SERCA3 isoform was also expressed at variable levels. SERCA3 expression was selectively enhanced during cell differentiation in lung cancer cells, and marked SERCA3 expression was found in fully differentiated normal bronchial epithelium. As studied by using a recombinant fluorescent calcium probe, induction of the expression of SERCA3, a lower calcium affinity pump, was associated with decreased intracellular calcium storage, whereas the amplitude of capacitative calcium influx remained unchanged. Our observations indicate that the calcium homeostasis of the endoplasmic reticulum in lung adenocarcinoma cells presents a functional defect due to decreased SERCA3 expression that is corrected during pharmacologically induced differentiation. The data presented in this work show, for the first time, that endoplasmic reticulum calcium storage is anomalous in lung cancer cells, and suggest that SERCA3 may serve as a useful new phenotypic marker for the study of lung epithelial differentiation.
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http://dx.doi.org/10.1111/febs.12064DOI Listing
November 2013

Plasma membrane calcium pump (PMCA) isoform 4 is targeted to the apical membrane by the w-splice insert from PMCA2.

Cell Calcium 2012 Feb 16;51(2):171-8. Epub 2012 Jan 16.

Department of Molecular Cell Biology, National Blood Center, Diószegi u. 64, Budapest, Hungary.

Local Ca(2+) signaling requires proper targeting of the Ca(2+) signaling toolkit to specific cellular locales. Different isoforms of the plasma membrane Ca(2+) pump (PMCA) are responsible for Ca(2+) extrusion at the apical and basolateral membrane of polarized epithelial cells, but the mechanisms and signals for differential targeting of the PMCAs are not well understood. Recent work demonstrated that the alternatively spliced w-insert in PMCA2 directs this pump to the apical membrane. We now show that inserting the w-insert into the corresponding location of the PMCA4 isoform confers apical targeting to this normally basolateral pump. Mutation of a di-leucine motif in the C-tail thought to be important for basolateral targeting did not enhance apical localization of the chimeric PMCA4(2w)/b. In contrast, replacing the C-terminal Val residue by Leu to optimize the PDZ ligand site for interaction with the scaffolding protein NHERF2 enhanced the apical localization of PMCA4(2w)/b, but not of PMCA4x/b. Functional studies showed that both apical PMCA4(2w)/b and basolateral PMCA4x/b handled ATP-induced Ca(2+) signals with similar kinetics, suggesting that isoform-specific functional characteristics are retained irrespective of membrane targeting. Our results demonstrate that the alternatively spliced w-insert provides autonomous apical targeting information in the PMCA without altering its functional characteristics.
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http://dx.doi.org/10.1016/j.ceca.2011.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279596PMC
February 2012

Regulation of apical membrane enrichment and retention of plasma membrane Ca ATPase splice variants by the PDZ-domain protein NHERF2.

Commun Integr Biol 2011 May 1;4(3):340-3. Epub 2011 May 1.

Department of Molecular Cell Biology; National Blood Center; Budapest, Hungary.

The localization of plasma membrane calcium ATPase (PMCA) isoforms in specified membrane compartments is crucial for their function in local Ca(2+) handling. PMCA2w/b is present in the apical membrane whereas alternative splice variants PMCA2x/b and 2z/b reside in the basolateral membrane in polarized epithelial cells. Here we found that the apical scaffolding protein NHERF2 greatly enhances the apical concentration of PMCA2w/b by tethering the pump to the underlying actin cytoskeleton. The interaction requires the C-terminal PDZ binding sequence in PMCA2b and results in increased membrane retention and decreased lateral mobility of the pump. In contrast, PMCA2x/b remains exclusively basolateral even when NHERF2 is overexpressed. Our results suggest that the alternatively spliced intracellular loop in PMCA2 imposes dominant membrane targeting information. NHERF2-mediated recruitment may be an effective means for polarized cells to regulate the abundance of PMCA2w/b in the apical membrane to meet an increased demand for local Ca(2+) extrusion.
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http://dx.doi.org/10.4161/cib.4.3.15040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187903PMC
May 2011

Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells.

Biochem Biophys Res Commun 2011 Jul 6;410(2):322-7. Epub 2011 Jun 6.

Department of Molecular Cell Biology, National Blood Center, Budapest, Hungary.

The "w" splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.
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http://dx.doi.org/10.1016/j.bbrc.2011.05.147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139432PMC
July 2011

Apical scaffolding protein NHERF2 modulates the localization of alternatively spliced plasma membrane Ca2+ pump 2B variants in polarized epithelial cells.

J Biol Chem 2010 Oct 27;285(41):31704-12. Epub 2010 Jul 27.

Department of Molecular Cell Biology, National Blood Center, H-1113 Budapest, Hungary.

The membrane localization of the plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na(+)/H(+) exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.
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http://dx.doi.org/10.1074/jbc.M110.164137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2951242PMC
October 2010

Apical localization of PMCA2w/b is lipid raft-dependent.

Biochem Biophys Res Commun 2009 Jun 18;384(1):32-6. Epub 2009 Apr 18.

Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA.

Alternative splicing of the first intracellular loop differentially targets plasma membrane calcium ATPase (PMCA) isoform 2 to the apical or basolateral membrane in MDCK cells. To determine if the targeting is affected by lipid interactions, we stably expressed PMCA2w/b and PMCA2z/b in MDCK cells, and analyzed the PMCA distribution by confocal fluorescence microscopy and membrane fractionation. PMCA2w/b showed clear apical and lateral distribution, whereas PMCA2z/b was mainly localized to the basolateral membrane. A significant fraction of PMCA2w/b partitioned into low-density membranes associated with lipid rafts. Depletion of membrane cholesterol by methyl-beta-cyclodextrin resulted in reduced lipid raft association and a striking loss of PMCA2w/b from the apical membrane, whereas the lateral localization of PMCA2z/b remained unchanged. Our data indicate that alternative splicing differentially affects the lipid interactions of PMCA2w/b and PMCA2z/b and that the apical localization of PMCA2w/b is lipid raft-dependent and sensitive to cholesterol depletion.
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http://dx.doi.org/10.1016/j.bbrc.2009.04.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731683PMC
June 2009

PSD-95 mediates membrane clustering of the human plasma membrane Ca2+ pump isoform 4b.

Biochim Biophys Acta 2009 Jun 27;1793(6):1023-32. Epub 2008 Nov 27.

National Blood Center, Department of Molecular Cell Biology, Diószegi u. 64, H-1113 Budapest, Hungary.

Besides the control of global calcium changes, specific plasma membrane calcium ATPase (PMCA) isoforms are involved in the regulation of local calcium signals. Although local calcium signaling requires the confinement of signaling molecules into microdomains, little is known about the specific organization of PMCA molecules within the plasma membrane. Here we show that co-expression with the postsynaptic density-95 (PSD-95) scaffolding protein increased the plasma membrane expression of PMCA4b and redistributed the pump into clusters. The clustering of PMCA4b was fully dependent on the presence of its PDZ-binding sequence. Using the fluorescence recovery after photobleaching (FRAP) technique, we show that the lateral membrane mobility of the clustered PMCA4b is significantly lower than that of the non-clustered molecules. Disruption of the actin-based cytoskeleton by cytochalasin D resulted in increased cluster size. Our results suggest that PSD-95 promotes the formation of high-density PMCA4b microdomains in the plasma membrane and that the membrane cytoskeleton plays an important role in the regulation of this process.
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http://dx.doi.org/10.1016/j.bbamcr.2008.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2693454PMC
June 2009

Cleavage of the plasma membrane Ca+ATPase during apoptosis.

Ann N Y Acad Sci 2007 Mar;1099:440-50

Membrane Research Group of the Hungarian Academy of Sciences, Budapest H-1051, Hungary.

Maintenance of Ca2+ homeostasis is essential for normal cellular function and survival. Recent evidences suggest that Ca2+ is also an important player of apoptosis. We demonstrated that the plasma membrane Ca2+ ATPase (PMCA) isoform 4b, a key element of cellular Ca2+ homeostasis, was cleaved by caspase-3 during the course of apoptosis. This cleavage of PMCA removed the entire regulatory region from the C terminus, leaving behind a 120-kDa catalytic fragment. Since loss of PMCA activity could lead to intracellular Ca2+ overload and consequently necrotic cell death, an important question is whether the apoptotic fragment of PMCA retains full activity or it is inactivated. To address this question, we constructed a C-terminally truncated mutant that corresponded to the caspase-3 fragment of PMCA4b and showed that it was fully and constitutively active. This mutant was targeted properly to the plasma membrane when it was expressed stably or transiently in several different cell lines. We followed truncation of PMCA during apoptosis induced by mitochondrial or receptor-mediated pathways and found that a similar fragment of 120 kDa was formed and remained intact for several hours after treatment. We have also demonstrated that the caspase-3 cleavage site is an important structural element of PMCA and found that the accessibility of the caspase-3 site depended strongly on the conformational state of the protein.
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http://dx.doi.org/10.1196/annals.1387.003DOI Listing
March 2007

Plasma membrane Ca2+ ATPases as dynamic regulators of cellular calcium handling.

Ann N Y Acad Sci 2007 Mar;1099:226-36

Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street S.W., Rochester, MN 55905, USA.

Plasma membrane Ca2+ ATPases (PMCAs) are essential components of the cellular toolkit to regulate and fine-tune cytosolic Ca2+ concentrations. Historically, the PMCAs have been assigned a housekeeping role in the maintenance of intracellular Ca2+ homeostasis. More recent work has revealed a perplexing multitude of PMCA isoforms and alternative splice variants, raising questions about their specific role in Ca2+ handling under conditions of varying Ca2+ loads. Studies on the kinetics of individual isoforms, combined with expression and localization studies suggest that PMCAs are optimized to function in Ca2+ regulation according to tissue- and cell-specific demands. Different PMCA isoforms help control slow, tonic Ca2+ signals in some cells and rapid, efficient Ca2+ extrusion in others. Localized Ca2+ handling requires targeting of the pumps to specialized cellular locales, such as the apical membrane of cochlear hair cells or the basolateral membrane of kidney epithelial cells. Recent studies suggest that alternatively spliced regions in the PMCAs are responsible for their unique targeting, membrane localization, and signaling cross-talk. The regulated deployment and retrieval of PMCAs from specific membranes provide a dynamic system for a cell to respond to changing needs of Ca2+ regulation.
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http://dx.doi.org/10.1196/annals.1387.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873821PMC
March 2007

Isoform-specific up-regulation of plasma membrane Ca2+ATPase expression during colon and gastric cancer cell differentiation.

Cell Calcium 2007 Dec 11;42(6):590-605. Epub 2007 Apr 11.

National Medical Centre, Institute of Haematology and Immunology, Budapest, Hungary.

In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca2+ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na+/K+-ATPase or that of the sarco/endoplasmic reticulum Ca2+ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca2+ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells. Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype.
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http://dx.doi.org/10.1016/j.ceca.2007.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2096732PMC
December 2007

Glutamate-induced protease-mediated loss of plasma membrane Ca2+ pump activity in rat hippocampal neurons.

J Neurochem 2006 Sep;98(5):1646-56

Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Ca2+ dysregulation is a hallmark of excitotoxicity, a process that underlies multiple neurodegenerative disorders. The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Here, we show that the rate of PMCA-mediated Ca2+ efflux from rat hippocampal neurons decreased following treatment with an excitotoxic concentration of glutamate. PMCA-mediated Ca2+ extrusion following a brief train of action potentials exhibited an exponential decay with a mean time constant (tau) of 8.8 +/- 0.2 s. Four hours following the start of a 30 min treatment with 200 microm glutamate, a second population of cells emerged with slowed recovery kinetics (tau = 16.5 +/- 0.3 s). Confocal imaging of cells expressing an enhanced green fluorescent protein (EGFP)-PMCA4b fusion protein revealed that glutamate treatment internalized EGFP and that cells with reduced plasma membrane fluorescence had impaired Ca2+ clearance. Treatment with inhibitors of the Ca2+-activated protease calpain protected PMCA function and prevented EGFP-PMCA internalization. PMCA internalization was triggered by activation of NMDA receptors and was less pronounced for a non-toxic concentration of glutamate relative to one that produces excitotoxicity. PMCA isoform 2 also internalized following exposure to glutamate, although the Na+/K+ ATPase did not. These data suggest that glutamate exposure initiated protease-mediated internalization of PMCAs with a corresponding loss of function that may contribute to the Ca2+ dysregulation that accompanies excitotoxicity.
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http://dx.doi.org/10.1111/j.1471-4159.2006.04063.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873837PMC
September 2006