Cyclic AMP-dependent protein kinase and Ca2+-calmodulin stimulate the formation of polyphosphoinositjdes in a sarcoplasmic reticulum preparation of rabbit heart

AbstractA rabbit heart membrane fraction enriched in sarcoplasmic reticulum was incubated in a reaction mixture containing [γ-32P]ATP. The catalytic subunit of cyclic AMP-dependent protein kinase enhanced the 32P-labelling of both phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. Ca2+-calmodulin also increased the 32P-incorporation into both polyphosphoinositides. Upon SDS gel-electrophoretic analysis of the membrane proteins, phospholamban was found to be concurrently phosphorylated by the exogenous catalytic subunit as well as by an endogenous Ca2+-calmodulm-dependent protein kinase

A szarko/endoplazmatikus retikulum Ca2+ ATPáz 3 szerkezet-elemzése és a nem-izom típusú sejtek Ca2+ -transzport fehérje készletének változásai sejtdifferenciáció során = Structural study of the sarco/endoplasmic reticulum Ca2+ ATPase 3 proteins and the modulation of the machinery of Ca2+-transport proteins during the differentiation of non-muscle cells

A Ca2+ függő sejtélettani folyamatok szabályozásában fontos szerepet betöltő szarko/endoplazmás retikulum (SERCA) és plazmamembrán (PMCA) Ca2+ATPázokat vizsgáltuk. Endogén, valamint vad típusú és mutáns rekombináns SERCA3 fehérjék tripszines proteolízisét elemezve kimutattuk két, kinetikailag eltérő fragmentációs profil párhuzamos megjelenését. Irányított mutagenezissel azonosítottunk néhány tripszines hasítási helyet. Eredményeink arra utalnak, hogy natív membránkörnyezetben a fragmentációs profilok a SERCA3 fehérje eltérő konformációs állapotaihoz rendelhetőek. Korábbi munkáinkhoz kapcsolódva elemeztük a gyomor-/béltumorsejtek differenciációja, és a SERCA és PMCA fehérjék expressziója, funkciója közötti összefüggéseket. Számos differenciációs modellben igazoltuk, hogy az éretlen sejtekben alacsony szinten kifejeződő SERCA3 és PMCA4b izoformák expressziója erősen indukálódik a tumorsejtek differenciációja során. A SERCA fehérjék funkcionális gátlása elősegítette egyes béltumorsejtek differenciációját. A béltumorok kialakulásában fontos APC/b-katenin/TCF4 jelátviteli út gátlása indukálta a SERCA3 expressziót. Különböző stádiumú béltumorokból nyert szöveti metszetek immunhisztokémiai vizsgálatai szerint a SERCA3 expresszió defektusa már a tumorok kialakulásának korai szakaszában jelentkezik. Eredményeink alapján felvetjük annak lehetőségét, hogy a gyomor-/béltraktusban egyes Ca2+ transzporterek, mint markerfehérjék, segíthetik malignus elváltozások kialakulásának és fenotípusának meghatározását, és diagnosztikai fejlesztések potenciális célpontjai lehetnek. | We studied the sarco/endoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca2+ATPases. The proper functions of these proteins are essential in the regulation of Ca2+-dependent cellular processes. Limited tryptic digestion of endogenously expressed or wild-type and mutant recombinant SERCA3 proteins resulted in two distinct fragmentation profiles. Using site-directed mutagenesis approach some of the tryptic sites were determined. Our data indicated that the SERCA3 fragmentation profiles were related to different conformational states of the enzyme. To further explore our previous work, we investigated the expression and function of various SERCA and PMCA isoforms during the differentiation of gastric/colon cancer cells. Using a wide range of model cells and differentiation protocols, strong induction of SERCA3 and PMCA4b expression were detected in differentiating cancer cells. Inhibition of SERCA function induced the maturation of colon cancer cells. Inhibition of the APC/b-catenin/TCF4 signaling pathway, essential during colon carcinogenesis, resulted in up-regulated SERCA3 expression. Immunohystochemical analysis of various tissue sections from colonic lesions, adenomas and adenocarcinomas showed that loss of SERCA3 expression is an early event during colon carcinogenesis. Our data suggest that some Ca2+ transport proteins could serve as new biomarkers for the analysis of the formation and phenotype of gastric/colon tumors, and should help in novel diagnostic development

Reaktív oxigén származékok szerepe a fibrózis kialakulásában = Reactive oxygen species in the development of organ fibrosis

Kutatásaink legfontosabb eredményei a következők: 1. Kimutattuk, hogy a peroxidazin fehérje expressziója fokozódik a miofibroblasztok differenciálódása folyamán és a fehérje szekretálódik a sejtek közötti térbe. Azt is kimutattuk, hogy a vese fibrotikus átalakulása során a peroxidazin felszaporodik a tubulus hámsejtek közötti térben. A peroxidazin sejtek közötti térbe történő szekréciója fontos, eddig ismeretlen eleme lehet a szöveti fibrózisnak. 2. Kimutattuk, hogy az emlős peroxidázok családjába tartozó laktoperoxidáz enzim hatékonyan katalizálja tirozin aminosavak összekapcsolását. Az emlős peroxidázok ditirozin-képző aktivitásának szerepe lehat a sejtek közötti állomány módosításában. 3. A NADPH oxidáz enzimcsalád Duox1 tagjáról kimutattuk, hogy szerepet játszhat a húgyhólyag hámsejtjeinek jelátviteli folyamataiban. 4. Elsőként mutattuk ki élő sejtekben, hogy az endoplazmás retikulum lumenében magas a H2O2 szintje, ami elsősorban az Ero-1L enzim aktivitásának köszönhető és független a Nox enzimek aktivitásától. 5. Genetikai modellekkel alátámasztva kimutattuk, hogy a fibroblaszt-miofibroblaszt átalakulás közben megfigyelhető H2O2 termelés a Nox4-p22phox enzimkomplex aktivitásának köszönhető. | The most important results of the research project are the followings: 1. We demonstrated the increased expression and secretion of peroxidasin during myofibroblastic differentiation. We showed that during the course of kidney fibrosis, peroxidasin accumulates in the peritubular space. The secretion of peroxidasin represents a previously unknown mechanism in tissue fibrosis. 2. We showed that lactoperoxidase, a member of the mammalian peroxidase family, efficiently catalyzes the formation of dityrosine residues. Dityrosine formation by mammalian peroxidases may play a role in the modification of the extracellular matrix. 3. We showed that the NADPH oxidase Duox1 has a role in the signaling mechanisms of urothelial cells. 4. We were the first to show in live cells that lumen of the mammalian endoplasmic reticulum is highly oxidative. The high level of H2O2 in the lumen is mainly due to Ero-1L activity and seems to be independent of Nox enzymes. 5. Using genetic models we showed that H2O2 production during myofibroblastic differentiation is due to the activity of the Nox4-p22phox enzyme complex

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 plasma membrane Ca2+ ATPase 4b and its role in cellular Ca2+ homeostasis and apoptosis

A jelen kutatási periódus alatt egy fontos Ca2+ transzport fehérje, a plazma membrán Ca2+ ATPáz (PMCA4b) struktúra/funkció változásait tanulmányoztuk az apoptózis valamint a nekrózis folyamatai alatt. Eredményeink azt bizonyítják, hogy a PMCA4b fehérjét - függetlenül az apoptózist kiváltó októl - a kaszpáz-3 proteáz hasítja és egy 120 kDa molekulatömegű fragment képződik. A fehérje közben elveszíti C-terminális regulátor régióját, és -szuper aktívvá - válik. A "szuper aktív" PMCA fragmentnek megfelelő mutáns a plazmamembránban lokalizálódik és képes a citoszólikus Ca2+ szint szabályozására. Ezzel szemben oxidatív stresszt kiváltó szerek hatására a PMCA jelentős mértékű internalizációja és a citoszólikus Ca2+ szint hosszan tartó megemelkedése figyelhető meg. A C-terminális régió meghatározónak bizonyult a PMCA4b plazmamembránban történő eloszlásának szempontjából is. Kimutattuk, hogy a PSD-95 állványfehérje a PMCA C-terminálisán található PDZ-kötő motívumon keresztül elősegíti a PMCA kijutását a plazmamembránba, és itt a képződő fehérje-komplex szigetszerű csoportokba rendeződik. Vizsgálataink elősegíthetik a Ca2+ homeosztázis felborulására visszavezethető egyes malignus elváltozások illetve degeneratív betegségek közötti összefüggések jobb megismerését. | During the present research period we studied structural and functional changes of an essential Ca2+ extrusion protein, the plasma membrane Ca2+ pump (PMCA), during apoptosis and necrosis. We followed truncation of PMCA4b 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 constructed a C-terminally truncated mutant that corresponded to this 120 kDa fragment and showed that it was fully and constitutively active, and targeted properly to the plasma membrane. In contrast, arsenate or excitotoxic concentration of glutamate induced PMCA internalization and consequently, resulted in an impaired Ca2+ clearance from the cytoplasm. We also showed that interaction 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 C-terminus. We suggest that loss of function internalization of PMCAs and/or disruption of specific Ca2+ signaling microdomains may contribute to the Ca2+ dysregulation that accompanies a number of degenerative diseases

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

Calcium (Ca2+) is a critical cofactor and signaling mediator in cells, and the concentration of cytosolic Ca2+ is regulated by multiple proteins, including the plasma membrane Ca2+-ATPases (adenosine triphosphatases) (PMCAs), which use ATP to transport Ca2+ out of cells. PMCA isoforms exhibit different kinetic and regulatory properties; thus, the presence and relative abundance of individual isoforms may help shape Ca2+ transients and cellular responses. We studied the effects of three PMCA isoforms (PMCA4a, PMCA4b, and PMCA2b) on Ca2+ transients elicited by conditions that trigger store-operated Ca2+ entry (SOCE) and that blocked Ca2+ 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 Ca2+ 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 Ca2+ transients, whereas PMCA4a reduced cytosolic Ca2+, resulting in the establishment of a higher than baseline cytosolic Ca2+ 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 Ca2+ transients, which would thus affect the activation of downstream signaling pathways

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

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

The plasma membrane Ca2+ pump PMCA4b inhibits the migratory and metastatic activity of BRAF mutant melanoma cells

Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca2+ signaling is a well-known regulator of tumor progression, the crosstalk between Ca2+ 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 Ca2+ 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 [Ca2+ ]i clearance from cells after Ca2+ 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 Ca2+ signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor

Multifaceted plasma membrane Ca2+ pumps: From structure to intracellular Ca2+ handling and cancer

Plasma membrane Ca2+ ATPases (PMCAs) are intimately involved in the control of intracellular Ca2+ concentration. They reduce Ca2+ in the cytosol not only by direct ejection, but also by controlling the formation of inositol-1,4,5-trisphosphate and decreasing Ca2+ release from the endoplasmic reticulum Ca2+ 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 Ca2+ with distinct temporal resolution. The expression pattern of variants depends on cell type; a change in this pattern can result in perturbed Ca2+ homeostasis and thus altered cell function. Indeed, PMCAs undergo remarkable changes in their expression pattern during tumorigenesis that might significantly contribute to the unbalanced Ca2+ homeostasis of cancer cells

Interaction between p22(phox) and Nox4 in the endoplasmic reticulum suggests a unique mechanism of NADPH oxidase complex formation.

The p22(phox) protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22(phox) in the TGF-beta1-stimulated H2O2 production of primary human and murine fibroblasts. TGF-beta1 induced H2O2 release of the examined cells, and the response was dependent on the expression of both Nox4 and p22(phox). Interestingly, the p22(phox) protein was present in the absence of any detectable Nox/Duox expression, and the p22(phox) level was unaffected by TGF-beta1. On the other hand, Nox4 expression was dependent on the presence of p22(phox), establishing an asymmetrical relationship between the two proteins. Nox4 and p22(phox) proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-beta1. We used a chemically induced protein dimerization method to study the orientation of p22(phox) and Nox4 in the endoplasmic reticulum membrane. This technique is based on the rapamycin-mediated heterodimerization of the mammalian FRB domain with the FK506 binding protein. The results of these experiments suggest that the enzyme complex produces H2O2 into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle