RESUMO
Calmodulin (CaM) is a Ca2+ binding protein modulating multiple targets, several of which are associated with cardiac pathophysiology. Recently, CaM mutations were linked to heart arrhythmia. CaM is crucial for cell growth and viability, yet the effect of the arrhythmogenic CaM mutations on cell viability, as well as heart rhythm, remains unknown, and only a few targets with relevance for heart physiology have been analyzed for their response to mutant CaM. We show that the arrhythmia-associated CaM mutants support growth and viability of DT40 cells in the absence of WT CaM except for the long QT syndrome mutant CaM D129G. Of the six CaM mutants tested (N53I, F89L, D95V, N97S, D129G, and F141L), three showed a decreased activation of Ca2+/CaM-dependent kinase II, most prominently the D129G CaM mutation, which was incapable of stimulating Thr286 autophosphorylation. Furthermore, the CaM D129G mutation led to bradycardia in zebrafish and an arrhythmic phenotype in a subset of the analyzed zebrafish.
Assuntos
Arritmias Cardíacas/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/genética , Proliferação de Células/genética , Mutação/genética , Taquicardia Ventricular/patologia , Animais , Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Células Cultivadas , Humanos , Síndrome do QT Longo/etiologia , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Conformação Proteica , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca(2+)/CaM complexes, which interact with and activate target proteins. In the present study the role of Ca(2+)/CaM in the regulation of the ligand-dependent activation of the epidermal growth factor receptor (EGFR) has been examined in living cells. We show that addition of different cell permeable CaM antagonists to cultured cells or loading cells with a Ca(2+) chelator inhibited ligand-dependent EGFR auto(trans)phosphorylation. This occurred also in the presence of inhibitors of protein kinase C, CaM-dependent protein kinase II and calcineurin, which are known Ca(2+)- and/or Ca(2+)/CaM-dependent EGFR regulators, pointing to a direct effect of Ca(2+)/CaM on the receptor. Furthermore, we demonstrate that down-regulation of CaM in conditional CaM knock out cells stably transfected with the human EGFR decreased its ligand-dependent phosphorylation. Substitution of six basic amino acid residues within the CaM-binding domain (CaM-BD) of the EGFR by alanine resulted in a decreased phosphorylation of the receptor and of its downstream substrate phospholipase Cγ1. These results support the hypothesis that Ca(2+)/CaM regulates the EGFR activity by directly interacting with the CaM-BD of the receptor located at its cytosolic juxtamembrane region.
Assuntos
Calmodulina/metabolismo , Receptores ErbB/metabolismo , Animais , Calcineurina/genética , Calcineurina/metabolismo , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/genética , Linhagem Celular , Receptores ErbB/agonistas , Receptores ErbB/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação/fisiologiaRESUMO
Calmodulin (CaM) was shown to be essential for survival of lower eukaryotes by gene deletion experiments. So far, no CaM gene deletion was reported in higher eukaryotes. In vertebrates, CaM is expressed from several genes, which encode an identical protein, making it difficult to generate a model system to study the effect of CaM gene deletion. Here, we present a novel genetic system based on the chicken DT40 cell line, in which the two functional CaM genes were deleted and one allele replaced with a CaM transgene that can be artificially regulated. We show that CaM is essential for survival of vertebrate cells as they die in the absence of CaM expression. Reversal of CaM repression or ectopic expression of HA-tagged CaM rescued the cells. Cells exclusively expressing HA-CaM with impaired individual calcium binding domains as well as HA-CaM lacking the ability to be phosphorylated at residues Tyr(99)/Tyr(138) or trimethylated at Lys(115) survived and grew well. CaM mutated at both Ca(2+) binding sites 3 and 4 as well as at both sites 1 and 2, but to a lesser degree, showed decreased ability to support cell growth. Cells expressing CaM with all calcium binding sites impaired died with kinetics similar to that of cells expressing no CaM. This system offers a unique opportunity to analyze CaM structure-function relationships in vivo without the use of pharmacological inhibitors and to analyze the function of wild type and mutated CaM in modulating the activity of different target systems without interference of endogenous CaM.
Assuntos
Cálcio/metabolismo , Calmodulina/fisiologia , Lisina/metabolismo , Tirosina/metabolismo , Animais , Calmodulina/genética , Calmodulina/metabolismo , Linhagem Celular , Galinhas , Deleção de Genes , Metilação , Fosforilação , Ligação ProteicaRESUMO
The Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) is well known for transmitting Ca2+-signals, which leads to a multitude of physiological responses. Its functionality is believed to involve CaMKII holoenzyme dynamics where trans-autophosphorylation of the crucial phosphorylation site, T286 occurs. Phosphorylation of this site does not occur when stimulated exclusively with the arrhythmia associated D130G mutant form of CaM in vitro. Here, we present evidence that the loss-of-CaMKII function correlates with premature phosphorylation of its inhibitory phosphosite T306 in CaMKIIα and T307 in CaMKIIδ as this site was up to 20-fold more phosphorylated in the presence of D130G CaM compared to wildtype CaM. Indeed, changing this phosphosite to a non-phosphorylatable alanine reversed the inhibitory effect of D130G both in vitro and in live cell experiments. In addition, several phosphosites with so far undescribed functions directing the Ca2+-sensitivity of the CaMKII sensor were also affected by the presence of the D130G mutation implicating a role of several additional autophosphosites (besides T286 and T306/T307) so far not known to regulate CaMKII Ca2+ sensitivity. Furthermore, we show that introducing a D130G mutation in the CALM2 gene of the P19CL6 pluripotent mouse embryonic carcinoma cell line using CRISPR/Cas9 decreased the spontaneous beat frequency compared to wildtype cells when differentiated into cardiomyocytes supporting an alteration of cardiomyocyte physiology caused by this point mutation. In conclusion, our observations shed for the first time light on how the D130G CaM mutation interferes with the function of CaMKII and how it affects the beating frequency of cardiomyocyte-like cells.
Assuntos
Arritmias Cardíacas/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/genética , Mutação de Sentido Incorreto , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Linhagem Celular Tumoral , Camundongos , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , FosforilaçãoRESUMO
ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2 downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate that ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose , Proteínas de Ligação ao Cálcio/fisiologia , Neoplasias/enzimologia , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Caspases/metabolismo , Divisão Celular/genética , Regulação para Baixo , Fase G2/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Neoplasias/patologiaRESUMO
The calcium binding protein ALG-2 is upregulated in several types of cancerous tissues and cancer cell death may be a consequence of ALG-2 downregulation. Novel research suggests that ALG-2 is involved in membrane repair mechanisms, in line with several published studies linking ALG-2 to processes of membrane remodeling and transport, which may contribute to the fitness of cells or protect them from damage. To investigate the involvement of ALG-2 in cell recovery after membrane damage we disrupted the PDCD6 gene encoding the ALG-2 protein in DT-40 cells and exposed them to electroporation. ALG-2 knock-out cells were more sensitive to electroporation as compared to wild type cells. This phenotype could be reversed by reestablishing ALG-2 expression confirming that ALG-2 plays an important role in cell recovery after plasma membrane damage. We found that overexpression of wild type ALG-2 but not a mutated form unable to bind Ca2+ partially protected HeLa cells from digitonin-induced cell death. Further, we were able to inhibit the cell protective function of ALG-2 after digitonin treatment by adding a peptide with the ALG-2 binding sequence of ALIX, which has been proposed to serve as the ALG-2 downstream target in a number of processes including cell membrane repair. Our results suggest that ALG-2 may serve as a novel therapeutic target in combination with membrane damaging interventions.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Membrana Celular/metabolismo , Digitonina/toxicidade , Eletroporação , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Cátions Bivalentes/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Técnicas de Inativação de Genes , Células HeLa , Humanos , MutaçãoRESUMO
ALG-2 (apoptosis linked gene 2 product) is a calcium binding protein for which no clear cellular function has been established. In this study we identified Scotin as a novel ALG-2 target protein containing 6 PXY and 4 PYP repeats, earlier identified in the ALG-2 binding regions of AIP1/ALIX and TSG101, respectively. An in vitro synthesized C-terminal fragment of Scotin bound specifically to immobilized recombinant ALG-2 and tagged ALG-2 and Scotin were shown by immunoprecipitation to interact in MCF7 and U2OS cell lines. Furthermore ALG-2 bound to endogenous Scotin in extracts from mouse NIH3T3 cells. Overexpression of ALG-2 led to accumulation of Scotin in MCF7 and H1299 cells. In vitro and in vivo binding of ALG-2 to Scotin was demonstrated to be strictly calcium dependent indicating a role of this interaction in calcium signaling pathways.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Guanilato Quinases , Humanos , Camundongos , Células NIH 3T3 , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/químicaRESUMO
The penta-EF hand (PEF) family of calcium binding proteins includes grancalcin, peflin, sorcin, calpain large and small subunits as well as ALG-2. Systematic testing of the heterodimerization abilities of the PEF proteins using the yeast two-hybrid and glutathione S-transferase pull-down assays revealed the new finding that grancalcin interacts strongly with sorcin. In addition, sorcin and grancalcin can be co-immunoprecipitated from lysates of human umbilical vein endothelial cells. Our results indicate that heterodimerization, in addition to differential interactions with target proteins, might be a way to regulate and fine tune processes mediated by calcium binding proteins of the penta-EF hand type.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Motivos EF Hand , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Dimerização , Endotélio Vascular/metabolismo , Glutationa Transferase/metabolismo , Humanos , Técnicas In Vitro , Testes de Precipitina , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Veias Umbilicais/citologiaRESUMO
Coated vesicles mediate the traffic of secretory and membrane cargo proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The coat protein complex (COPII) involved in vesicle budding is constituted by a GTPase, Sar1, the inner coat components of Sec23/Sec24 and the components of the outer coat Sec13/Sec31A. The Ca(2+)-binding protein ALG-2 was recently identified as a Sec31A binding partner and a possible link to Ca(2+) regulation of COPII vesicle budding. Here we show that ALG-2/Ca(2+) is capable of attenuating vesicle budding in vitro through interaction with an ALG-2 binding domain in the proline rich region of Sec31A. Binding of ALG-2 to Sec31A and inhibition of COPII vesicle budding is furthermore dependent on an intact Ca(2+)-binding site at EF-hand 1 of ALG-2. ALG-2 increased recruitment of COPII proteins Sec23/24 and Sec13/31A to artificial liposomes and was capable of mediating binding of Sec13/31A to Sec23. These results introduce a regulatory role for ALG-2/Ca(2+) in COPII tethering and vesicle budding.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Ligação Proteica , Estabilidade Proteica , Transporte ProteicoRESUMO
The apoptosis linked gene-2 (ALG-2), discovered as a proapoptotic calcium binding protein, has recently been found upregulated in lung cancer tissue indicating that this protein may play a role in the pathology of cancer cells and/or may be a tumor marker. Using immunohistochemistry on tissue microarrays we analysed the expression of ALG-2 in 7371 tumor tissue samples of various origin as well as in 749 normal tissue samples. Most notably, ALG-2 was upregulated in mesenchymal tumors. No correlation was found between ALG-2 staining intensity and survival of patients with lung, breast or colon cancer. siRNA mediated ALG-2 downregulation led to a significant reduction in viability of HeLa cells indicating that ALG-2 may contribute to tumor development and expansion.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/patologia , Neoplasias da Mama , Sobrevivência Celular/genética , Neoplasias do Colo , Regulação para Baixo/efeitos dos fármacos , Células HeLa , Humanos , Neoplasias Pulmonares , Mesoderma/patologia , Neoplasias/genética , RNA Interferente Pequeno/farmacologia , Análise Serial de TecidosRESUMO
A variety of stimuli can trigger intracellular calcium oscillations. Relatively little is known about the molecular mechanisms decoding these events. We show that ALG-2, a Ca2+-binding protein originally isolated as a protein associated with apoptosis, is directly linked to Ca2+ signalling. We discovered that the subcellular distribution of a tagged version of ALG-2 could be directed by physiological external stimuli (including ATP, EGF, prostaglandin, histamine), which provoke intracellular Ca2+ oscillations. Cellular stimulation led to a redistribution of ALG-2 from the cytosol to a punctate localization in an oscillatory fashion unitemporally with Ca2+ oscillations, whereas a Ca2+-binding deficient mutant of ALG-2 did not redistribute. Using tagged ALG-2 as bait we identified its novel target protein Sec31A and based on the partial colocalization of endogenous ALG-2 and Sec31A we propose that ALG-2 temporarily binds to the COPII vesicles providing a link between Ca2+ signalling and ER to Golgi trafficking.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Trifosfato de Adenosina/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Histamina/farmacologia , Humanos , Imuno-Histoquímica , Imunoprecipitação , Microscopia Confocal , Mutação , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transfecção , Proteínas de Transporte VesicularRESUMO
ALG-2 was isolated in a screen for proteins involved in programmed cell death and is the first Ca(2+)-binding protein found to be directly involved in apoptosis. We have generated polyclonal antibodies that are suitable for detecting ALG-2 using different immunological methods. Three commercial antibodies against ALG-2 did neither detect mouse recombinant ALG-2 nor endogenous ALG-2 in Jurkat cell lysates, whereas our own affinity-purified antibody recognized recombinant as well as endogenous ALG-2. The specificity of the antibody was shown by preabsorbtion experiments and on ALG-2-deficient cells using Western blot analysis and immunohistochemistry. Western blot analysis of 15 different adult mouse tissues demonstrated that ALG-2 is ubiquitously expressed. We found that ALG-2 was more than threefold overexpressed in rat liver hepatoma compared to normal rat liver using Western blot analysis, a result confirmed by immunohistochemical analysis. Staining of four different lung cancer tissue microarrays including specimens of 263 patients showed that ALG-2 is mainly localized to epithelial cells and significantly up-regulated in small-cell lung cancers and in non-small-cell lung cancers. Our results lead to the conclusion that ALG-2 beside its known proapoptotic functions may be a player in survival pathways.