Ágnes Enyedi - Academia.edu (original) (raw)

Papers by Ágnes Enyedi

Research paper thumbnail of The plasma membrane <scp>C</scp> a <sup>2+</sup> pump <scp>PMCA</scp> 4b inhibits the migratory and metastatic activity of <scp>BRAF</scp> mutant melanoma cells

International Journal of Cancer, Nov 17, 2016

Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth ... more Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca 2+ signaling is a well-known regulator of tumor progression, the crosstalk between Ca 2+ 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 2+ 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 selumetinibsimilarly to that of the BRAFspecific 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 2+ ] i clearance from cells after Ca 2+ 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 2+ signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor.

Research paper thumbnail of The lipid-binding peptide from the plasma membrane Ca2+ pump binds calmodulin, and the primary calmodulin-binding domain interacts with lipid

Journal of Biological Chemistry, 1992

Research paper thumbnail of Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles

Journal of Biological Chemistry, 1991

In mixed membrane vesicles prepared from human platelets, the presence of two distinct calcium pu... more In mixed membrane vesicles prepared from human platelets, the presence of two distinct calcium pump enzymes (molecular mass 100 and 97 kDa) was demonstrated by 32P autoradiography, immunoblotting, and thapsigargin inhibition. Both the 100-and 97-kDa membrane proteins showed calcium-dependent phosphoenzyme formation and reacted with a polyclonal anti-sarcoplasmic reticulum calcium pump antiserum, while only the 100-kDa protein reacted with the antiserum specific for the sarco-endoplasmic reticulumtype calcium transport ATPase 2b isoform. Thapsigargin, inhibiting active calcium transport in platelet membrane vesicles, predominantly blocked the phosphoenzyme formation of the 100-kDa isoform and of the tryptic calcium pump fragments of 55 and 35 kDa, while lanthanum specifically increased the phosphoenzyme formation of the 97-kDa enzyme and of the tryptic fragment of 80 kDa. These results indicate the presence of the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform and of a yet unidentified, 97-kDa calcium pump protein in human platelet membranes. Calcium transporting ATPases (Ca2+ pumps) translocate calcium ions through cellular membranes at the expense of ATP hydrolysis to the extracellular space or into intracellular calcium storage sites. Plasma membrane-type Ca2+ pumps are approximately 140-kDa calmodulin-binding proteins encoded by several genes which, by additional alternative splicing, give rise to several structurally related but distinct proteins (1-3). For the approximately 100-kDa sarco-endoplasmic reticulum-type calcium pumps (SERCA),' three genes have already been identified. The SERCA 1 gene codes for the skeletal muscle sarcoplasmic reticulum calcium-pump; the SERCA l a and Ib are alternatively spliced forms corresponding to the adult and neonatal pump isoforms, respectively. The SERCA *This work has been supported by Grants OTKA-968 and OKKFT-Tt 1.5.1.3. from the Hungarian Academy of Sciences, le

Research paper thumbnail of The Calmodulin Binding Domain of the Plasma Membrane Ca2+ Pump Interacts Both with Calmodulin and with Another Part of the Pump

Journal of Biological Chemistry, 1989

Synthetic peptides corresponding to the calmodulinbinding domain of the human erythrocyte Ca2+ pu... more Synthetic peptides corresponding to the calmodulinbinding domain of the human erythrocyte Ca2+ pump were prepared representing residues 2-29 (C28W), 2-2 1 (C20W), 2-16 (C15W), and 16-29 (C14) of the sequence (

Research paper thumbnail of Autoinhibitory domains of various Ca2+ transporters cross-react

Journal of Biological Chemistry, 1993

Synthetic peptides having the sequence of the calmodulin-binding autoinhibitory domain of the Na+... more Synthetic peptides having the sequence of the calmodulin-binding autoinhibitory domain of the Na+/ Caz+ exchanger (XIP) and of the plasma membrane Ca2+ pump (C28R2) inhibited the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca2+ pumps. XIP was nearly as effective in inhibiting both systems as C28R2, which had previously been considered to be the most powerful peptide inhibitor of PMCA. In contrast, calmodulin-binding peptides from non-Ca2+ transport proteins (calmodulin-dependent protein kinase I1 and myosin light chain kinase) showed only weak inhibition. The relative specificity and effectiveness of the peptides from the Ca2+ transport proteins, as well as the characteristics of their actions were similar in both SERCA and PMCA, suggesting a high degree of functional relatedness between these autoinhibitory regions. Secondary structure predictions show that the calmodulin-binding autoinhibitory domains of the Na+/Ca2+ exchanger and PMCA are predicted to form a structure which might be necessary for the observed cross-inhibition. The results also indicate that other domains of the Ca" transporters have common structural elements which interact with the autoinhibitory domains.

Research paper thumbnail of A highly active 120-kDa truncated mutant of the plasma membrane Ca2+ pump

Journal of Biological Chemistry, 1993

Research paper thumbnail of Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation

Journal of Experimental & Clinical Cancer Research, 2017

Background: Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymp... more 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.

Research paper thumbnail of Abstract 1883: Treatment with histone deacetylase (HDAC) and BRAF inhibitors upregulates the expression of the plasma membrane Ca2+ pump, PMCA4b and alters intracellular Ca2+ handling in BRAF mutant melanoma cells

Cancer Research, 2016

The aim of our study was to test the effects of histone deacetylase (HDAC) and BRAF inhibitors on... more The aim of our study was to test the effects of histone deacetylase (HDAC) and BRAF inhibitors on the expression and activity of the plasma membrane Ca2+ pump isoform 4b (PMCA4b) in BRAF mutant melanoma cell lines. Remodeling of Ca2+ homeostasis during malignancy is caused by the rearrangement of the Ca2+ signaling machinery, including Ca2+ pumps, Na+/Ca2+ exchangers and channels. Plasma membrane calcium ATPases (ATP2B - or PMCA) maintain the resting low intracellular calcium concentration by pumping out excess calcium from the cytosol. Changes in PMCA expression during malignant transformation have been described previously in colorectal and breast cancer cells, and most recently by our group in melanomas. We found that in BRAF mutant melanoma cells the PMCA 4b protein level was markedly elevated by BRAF inhibitor treatment. Overexpression of PMCA4b suppressed motility and metastatic potential. Previously, it was shown that HDAC inhibitors-induced differentiation up-regulated PMCA4...

Research paper thumbnail of Inhibition of the purified human red-cell Ca2+ pump by a monoclonal antibody

Biochemical Journal, 1989

1. A monoclonal antibody (1G4) was raised against the red-cell Ca2+ pump, and it reacted with the... more 1. A monoclonal antibody (1G4) was raised against the red-cell Ca2+ pump, and it reacted with the pump, as verified by Western blot analysis and by the e.l.i.s.a. method. 2. At 1 mM-ATP and 10 microM-Ca2+, 1G4 inhibited the activity of the purified Ca2+ pump by 40%. 3. Ca2+ pump inhibition by the antibody was non-competitive with regard to Ca2+, calmodulin and the high-affinity portion of the ATP curve. Thus its mechanism was quite different from that of the antibody previously reported [Verbist, Wuytack, Raemaekers, VanLeuven, Cassiman & Casteels (1986) Biochem. J. 240, 633-640], which partially caused inhibition by competition at the ATP site. 4. Antibody 1G4 reduced the steady-state level of phosphorylated intermediate and increased by 50% the calmodulin-activated p-nitrophenyl phosphatase activity of the pump. 5. The experimental results are consistent with the hypothesis that 1G4 inhibits the Ca2+ pump by decreasing the rate of the transition from the E2 form to the E1 form, ca...

Research paper thumbnail of Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium

Biochemical Journal, 1988

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium u... more A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 14...

Research paper thumbnail of Simultaneous presence of two distinct endoplasmic-reticulum-type calcium-pump isoforms in human cells. Characterization by radio-immunoblotting and inhibition by 2,5-di-(t-butyl)-1,4-benzohydroquinone

Biochemical Journal, 1992

Phosphorylation, immunoblotting, limited proteolysis and drug-sensitivity analysis were used to c... more Phosphorylation, immunoblotting, limited proteolysis and drug-sensitivity analysis were used to characterize the sarcoendoplasmic-reticulum Ca2+ ATPases in a variety of human cell types. In platelets, several megakaryoblastoid and lymphoblastoid cell lines two distinct autophosphorylated forms of these ATPases with molecular mass of 100 and 97 kDa could be observed, whereas in several other cell types the 97 kDa form was absent. On immunoblots the 97 kDa species was specifically recognized by an inhibitory monoclonal antibody raised against the Ca2+ pump of platelet internal membranes, yielded on trypsinolysis a major fragment of 80 kDa, exhibited a distinct electrophoretic migration pattern as compared with the skeletal-, cardiac- and smooth-muscle Ca2+ pumps, and its autophosphorylation was strongly inhibited by the Ca(2+)-mobilizing agent 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ). The 100 kDa species reacted with an antibody specific for the cardiac- and smooth-muscle Ca2+ pu...

Research paper thumbnail of Oxytocin regulates the plasma membrane Ca2+ transport in rat myometrium

Biochemical Journal, 1989

Development of myometrium in young female rats was stimulated by administration of diethylstilboe... more Development of myometrium in young female rats was stimulated by administration of diethylstilboestrol. Plasma membrane and sarcoplasmic reticulum from rat myometrium were separated by a new and rapid method using a Percoll gradient. Calcium uptake was inhibited in plasma membrane vesicles isolated from oxytocin-treated myometrium, while no consistent effect of oxytocin was found on the Ca2+ uptake in the sarcoplasmic reticulum. Oxytocin regulated the plasma membrane Ca2+ pump by decreasing its apparent affinity for Ca2+ without affecting its maximal velocity. The K1/2 for Ca2+ in the absence of calmodulin was 0.41 +/- 0.04 microM in normal membranes; this was increased to 0.93 +/- 0.12 microM in oxytocin-treated membranes. Calmodulin decreased the K1/2 for Ca2+ to 0.27 +/- 0.027 microM and oxytocin also increased this, to 0.46 +/- 0.061 microM. The effect of oxytocin on the plasma membrane Ca2+ pump was highly dependent on the hormonal status of the animals. When the diethylstilboe...

Research paper thumbnail of Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies

Biochemical Journal, 1996

The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against t... more The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51–75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156–1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719–738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, ...

Research paper thumbnail of More Residues Are Required for Full Calmodulin Affinity in Plasma-Membrane Calcium-Pump Isoform 4A Than in 4B

Research paper thumbnail of MEMBRANE TRANSPORT, STRUCTURE, FUNCTION, AND BIOGENESIS-Plasma membrane Ca2+ ATPase isoform 4b is cleaved and activated by caspase-3 during the early phase of apoptosis

Research paper thumbnail of MEMBRANE TRANSPORT, STRUCTURE, FUNCTION, AND BIOGENESIS-Intramolecular Interactions of the Regulatory Region with the Catalytic Core in the Plasma Membrane Calcium Pump

Research paper thumbnail of The caspase-3 cleavage product of PMCA4b is activated and is targeted to the basolateral membrane of polarized MDCKII cells

Research paper thumbnail of A plazmamembrán Ca2+ ATPáz 4b izoforma apoptotikus fragmentjét reprezentáló mutáns sejten belüli lokalizációja, stabilitása, hatásai a sejtek Ca2+ háztartására és szerepe az apoptózis folyamatban= Intracellular localization and stability of a mutant representing the apoptotic fragment of the plas...

Research paper thumbnail of Plasma Membrane Calcium Pump Isoform 4a Has a Longer Calmodulin-Binding Domain Than 4b

Journal of Biological Chemistry, 1996

Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes th... more Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes the expression of two variants, hPMCA4a and hPMCA4b, which have different downstream regulatory regions. Of the two, hPMCA4a has a lower affinity for calmodulin and a lower effective affinity for Ca 2؉ (Enyedi, A.,

Research paper thumbnail of Tryptophan 1093 Is Largely Responsible for the Slow Off Rate of Calmodulin from Plasma Membrane Ca2+ Pump 4b

Journal of Biological Chemistry, 2002

Tryptophan 1093 resides in the 28-residue calmodulinbinding/autoinhibitory domain of the plasma m... more Tryptophan 1093 resides in the 28-residue calmodulinbinding/autoinhibitory domain of the plasma membrane Ca 2؉ pump (PMCA). Previous studies with the isolated calmodulin-binding/autoinhibitory peptide from PMCA have shown that mutations of the tryptophan residue decrease the affinity of the peptide for calmodulin and its affinity as an inhibitor of proteolytically activated pump. In this study, the PMCA mutation in which tryptophan 1093 is converted to alanine (W1093A) was constructed in the full-length PMCA isoform 4b. The mutant pump was expressed in COS cells, and its steady state and pre-steady state kinetic properties were examined. The W1093A pump exhibited an increased basal activity in the absence of calmodulin, so the activation was ϳ2fold (it is 10-fold in the wild type). The W1093A mutation also lowered the steady state affinity for calmodulin from K 0.5 of 9 nM for wild type to 144 nM (assayed at 700 nM free Ca 2؉). Pre-steady state measurements of the rate of activation by Ca 2؉-calmodulin revealed that the W1093A mutant responded 2.5-fold faster to calmodulin. In contrast to these relatively modest effects, the halftime of inactivation of the mutant was reduced by more than 2 orders of magnitude from 41 min to 7 s. We conclude that tryptophan 1093 does not play a substantial role in Ca 2؉-calmodulin recognition; rather it functions primarily to slow the inactivation of the calmodulinactivated pump.

Research paper thumbnail of The plasma membrane <scp>C</scp> a <sup>2+</sup> pump <scp>PMCA</scp> 4b inhibits the migratory and metastatic activity of <scp>BRAF</scp> mutant melanoma cells

International Journal of Cancer, Nov 17, 2016

Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth ... more Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca 2+ signaling is a well-known regulator of tumor progression, the crosstalk between Ca 2+ 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 2+ 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 selumetinibsimilarly to that of the BRAFspecific 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 2+ ] i clearance from cells after Ca 2+ 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 2+ signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor.

Research paper thumbnail of The lipid-binding peptide from the plasma membrane Ca2+ pump binds calmodulin, and the primary calmodulin-binding domain interacts with lipid

Journal of Biological Chemistry, 1992

Research paper thumbnail of Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles

Journal of Biological Chemistry, 1991

In mixed membrane vesicles prepared from human platelets, the presence of two distinct calcium pu... more In mixed membrane vesicles prepared from human platelets, the presence of two distinct calcium pump enzymes (molecular mass 100 and 97 kDa) was demonstrated by 32P autoradiography, immunoblotting, and thapsigargin inhibition. Both the 100-and 97-kDa membrane proteins showed calcium-dependent phosphoenzyme formation and reacted with a polyclonal anti-sarcoplasmic reticulum calcium pump antiserum, while only the 100-kDa protein reacted with the antiserum specific for the sarco-endoplasmic reticulumtype calcium transport ATPase 2b isoform. Thapsigargin, inhibiting active calcium transport in platelet membrane vesicles, predominantly blocked the phosphoenzyme formation of the 100-kDa isoform and of the tryptic calcium pump fragments of 55 and 35 kDa, while lanthanum specifically increased the phosphoenzyme formation of the 97-kDa enzyme and of the tryptic fragment of 80 kDa. These results indicate the presence of the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform and of a yet unidentified, 97-kDa calcium pump protein in human platelet membranes. Calcium transporting ATPases (Ca2+ pumps) translocate calcium ions through cellular membranes at the expense of ATP hydrolysis to the extracellular space or into intracellular calcium storage sites. Plasma membrane-type Ca2+ pumps are approximately 140-kDa calmodulin-binding proteins encoded by several genes which, by additional alternative splicing, give rise to several structurally related but distinct proteins (1-3). For the approximately 100-kDa sarco-endoplasmic reticulum-type calcium pumps (SERCA),' three genes have already been identified. The SERCA 1 gene codes for the skeletal muscle sarcoplasmic reticulum calcium-pump; the SERCA l a and Ib are alternatively spliced forms corresponding to the adult and neonatal pump isoforms, respectively. The SERCA *This work has been supported by Grants OTKA-968 and OKKFT-Tt 1.5.1.3. from the Hungarian Academy of Sciences, le

Research paper thumbnail of The Calmodulin Binding Domain of the Plasma Membrane Ca2+ Pump Interacts Both with Calmodulin and with Another Part of the Pump

Journal of Biological Chemistry, 1989

Synthetic peptides corresponding to the calmodulinbinding domain of the human erythrocyte Ca2+ pu... more Synthetic peptides corresponding to the calmodulinbinding domain of the human erythrocyte Ca2+ pump were prepared representing residues 2-29 (C28W), 2-2 1 (C20W), 2-16 (C15W), and 16-29 (C14) of the sequence (

Research paper thumbnail of Autoinhibitory domains of various Ca2+ transporters cross-react

Journal of Biological Chemistry, 1993

Synthetic peptides having the sequence of the calmodulin-binding autoinhibitory domain of the Na+... more Synthetic peptides having the sequence of the calmodulin-binding autoinhibitory domain of the Na+/ Caz+ exchanger (XIP) and of the plasma membrane Ca2+ pump (C28R2) inhibited the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca2+ pumps. XIP was nearly as effective in inhibiting both systems as C28R2, which had previously been considered to be the most powerful peptide inhibitor of PMCA. In contrast, calmodulin-binding peptides from non-Ca2+ transport proteins (calmodulin-dependent protein kinase I1 and myosin light chain kinase) showed only weak inhibition. The relative specificity and effectiveness of the peptides from the Ca2+ transport proteins, as well as the characteristics of their actions were similar in both SERCA and PMCA, suggesting a high degree of functional relatedness between these autoinhibitory regions. Secondary structure predictions show that the calmodulin-binding autoinhibitory domains of the Na+/Ca2+ exchanger and PMCA are predicted to form a structure which might be necessary for the observed cross-inhibition. The results also indicate that other domains of the Ca" transporters have common structural elements which interact with the autoinhibitory domains.

Research paper thumbnail of A highly active 120-kDa truncated mutant of the plasma membrane Ca2+ pump

Journal of Biological Chemistry, 1993

Research paper thumbnail of Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation

Journal of Experimental & Clinical Cancer Research, 2017

Background: Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymp... more 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.

Research paper thumbnail of Abstract 1883: Treatment with histone deacetylase (HDAC) and BRAF inhibitors upregulates the expression of the plasma membrane Ca2+ pump, PMCA4b and alters intracellular Ca2+ handling in BRAF mutant melanoma cells

Cancer Research, 2016

The aim of our study was to test the effects of histone deacetylase (HDAC) and BRAF inhibitors on... more The aim of our study was to test the effects of histone deacetylase (HDAC) and BRAF inhibitors on the expression and activity of the plasma membrane Ca2+ pump isoform 4b (PMCA4b) in BRAF mutant melanoma cell lines. Remodeling of Ca2+ homeostasis during malignancy is caused by the rearrangement of the Ca2+ signaling machinery, including Ca2+ pumps, Na+/Ca2+ exchangers and channels. Plasma membrane calcium ATPases (ATP2B - or PMCA) maintain the resting low intracellular calcium concentration by pumping out excess calcium from the cytosol. Changes in PMCA expression during malignant transformation have been described previously in colorectal and breast cancer cells, and most recently by our group in melanomas. We found that in BRAF mutant melanoma cells the PMCA 4b protein level was markedly elevated by BRAF inhibitor treatment. Overexpression of PMCA4b suppressed motility and metastatic potential. Previously, it was shown that HDAC inhibitors-induced differentiation up-regulated PMCA4...

Research paper thumbnail of Inhibition of the purified human red-cell Ca2+ pump by a monoclonal antibody

Biochemical Journal, 1989

1. A monoclonal antibody (1G4) was raised against the red-cell Ca2+ pump, and it reacted with the... more 1. A monoclonal antibody (1G4) was raised against the red-cell Ca2+ pump, and it reacted with the pump, as verified by Western blot analysis and by the e.l.i.s.a. method. 2. At 1 mM-ATP and 10 microM-Ca2+, 1G4 inhibited the activity of the purified Ca2+ pump by 40%. 3. Ca2+ pump inhibition by the antibody was non-competitive with regard to Ca2+, calmodulin and the high-affinity portion of the ATP curve. Thus its mechanism was quite different from that of the antibody previously reported [Verbist, Wuytack, Raemaekers, VanLeuven, Cassiman & Casteels (1986) Biochem. J. 240, 633-640], which partially caused inhibition by competition at the ATP site. 4. Antibody 1G4 reduced the steady-state level of phosphorylated intermediate and increased by 50% the calmodulin-activated p-nitrophenyl phosphatase activity of the pump. 5. The experimental results are consistent with the hypothesis that 1G4 inhibits the Ca2+ pump by decreasing the rate of the transition from the E2 form to the E1 form, ca...

Research paper thumbnail of Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium

Biochemical Journal, 1988

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium u... more A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 14...

Research paper thumbnail of Simultaneous presence of two distinct endoplasmic-reticulum-type calcium-pump isoforms in human cells. Characterization by radio-immunoblotting and inhibition by 2,5-di-(t-butyl)-1,4-benzohydroquinone

Biochemical Journal, 1992

Phosphorylation, immunoblotting, limited proteolysis and drug-sensitivity analysis were used to c... more Phosphorylation, immunoblotting, limited proteolysis and drug-sensitivity analysis were used to characterize the sarcoendoplasmic-reticulum Ca2+ ATPases in a variety of human cell types. In platelets, several megakaryoblastoid and lymphoblastoid cell lines two distinct autophosphorylated forms of these ATPases with molecular mass of 100 and 97 kDa could be observed, whereas in several other cell types the 97 kDa form was absent. On immunoblots the 97 kDa species was specifically recognized by an inhibitory monoclonal antibody raised against the Ca2+ pump of platelet internal membranes, yielded on trypsinolysis a major fragment of 80 kDa, exhibited a distinct electrophoretic migration pattern as compared with the skeletal-, cardiac- and smooth-muscle Ca2+ pumps, and its autophosphorylation was strongly inhibited by the Ca(2+)-mobilizing agent 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ). The 100 kDa species reacted with an antibody specific for the cardiac- and smooth-muscle Ca2+ pu...

Research paper thumbnail of Oxytocin regulates the plasma membrane Ca2+ transport in rat myometrium

Biochemical Journal, 1989

Development of myometrium in young female rats was stimulated by administration of diethylstilboe... more Development of myometrium in young female rats was stimulated by administration of diethylstilboestrol. Plasma membrane and sarcoplasmic reticulum from rat myometrium were separated by a new and rapid method using a Percoll gradient. Calcium uptake was inhibited in plasma membrane vesicles isolated from oxytocin-treated myometrium, while no consistent effect of oxytocin was found on the Ca2+ uptake in the sarcoplasmic reticulum. Oxytocin regulated the plasma membrane Ca2+ pump by decreasing its apparent affinity for Ca2+ without affecting its maximal velocity. The K1/2 for Ca2+ in the absence of calmodulin was 0.41 +/- 0.04 microM in normal membranes; this was increased to 0.93 +/- 0.12 microM in oxytocin-treated membranes. Calmodulin decreased the K1/2 for Ca2+ to 0.27 +/- 0.027 microM and oxytocin also increased this, to 0.46 +/- 0.061 microM. The effect of oxytocin on the plasma membrane Ca2+ pump was highly dependent on the hormonal status of the animals. When the diethylstilboe...

Research paper thumbnail of Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies

Biochemical Journal, 1996

The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against t... more The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51–75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156–1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719–738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, ...

Research paper thumbnail of More Residues Are Required for Full Calmodulin Affinity in Plasma-Membrane Calcium-Pump Isoform 4A Than in 4B

Research paper thumbnail of MEMBRANE TRANSPORT, STRUCTURE, FUNCTION, AND BIOGENESIS-Plasma membrane Ca2+ ATPase isoform 4b is cleaved and activated by caspase-3 during the early phase of apoptosis

Research paper thumbnail of MEMBRANE TRANSPORT, STRUCTURE, FUNCTION, AND BIOGENESIS-Intramolecular Interactions of the Regulatory Region with the Catalytic Core in the Plasma Membrane Calcium Pump

Research paper thumbnail of The caspase-3 cleavage product of PMCA4b is activated and is targeted to the basolateral membrane of polarized MDCKII cells

Research paper thumbnail of A plazmamembrán Ca2+ ATPáz 4b izoforma apoptotikus fragmentjét reprezentáló mutáns sejten belüli lokalizációja, stabilitása, hatásai a sejtek Ca2+ háztartására és szerepe az apoptózis folyamatban= Intracellular localization and stability of a mutant representing the apoptotic fragment of the plas...

Research paper thumbnail of Plasma Membrane Calcium Pump Isoform 4a Has a Longer Calmodulin-Binding Domain Than 4b

Journal of Biological Chemistry, 1996

Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes th... more Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes the expression of two variants, hPMCA4a and hPMCA4b, which have different downstream regulatory regions. Of the two, hPMCA4a has a lower affinity for calmodulin and a lower effective affinity for Ca 2؉ (Enyedi, A.,

Research paper thumbnail of Tryptophan 1093 Is Largely Responsible for the Slow Off Rate of Calmodulin from Plasma Membrane Ca2+ Pump 4b

Journal of Biological Chemistry, 2002

Tryptophan 1093 resides in the 28-residue calmodulinbinding/autoinhibitory domain of the plasma m... more Tryptophan 1093 resides in the 28-residue calmodulinbinding/autoinhibitory domain of the plasma membrane Ca 2؉ pump (PMCA). Previous studies with the isolated calmodulin-binding/autoinhibitory peptide from PMCA have shown that mutations of the tryptophan residue decrease the affinity of the peptide for calmodulin and its affinity as an inhibitor of proteolytically activated pump. In this study, the PMCA mutation in which tryptophan 1093 is converted to alanine (W1093A) was constructed in the full-length PMCA isoform 4b. The mutant pump was expressed in COS cells, and its steady state and pre-steady state kinetic properties were examined. The W1093A pump exhibited an increased basal activity in the absence of calmodulin, so the activation was ϳ2fold (it is 10-fold in the wild type). The W1093A mutation also lowered the steady state affinity for calmodulin from K 0.5 of 9 nM for wild type to 144 nM (assayed at 700 nM free Ca 2؉). Pre-steady state measurements of the rate of activation by Ca 2؉-calmodulin revealed that the W1093A mutant responded 2.5-fold faster to calmodulin. In contrast to these relatively modest effects, the halftime of inactivation of the mutant was reduced by more than 2 orders of magnitude from 41 min to 7 s. We conclude that tryptophan 1093 does not play a substantial role in Ca 2؉-calmodulin recognition; rather it functions primarily to slow the inactivation of the calmodulinactivated pump.