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The appearance of modular proteins is a widespread phenomenon during the evolution of proteins. The combinatorial arrangement of different functional and/or structural domains within a single polypeptide chain yields a wide variety of activities and regulatory properties to the modular proteins. In this review, we will discuss proteins, that in addition to their catalytic, transport, structure, localization or adaptor functions, also have segments resembling the helix-loop-helix EF-hand motifs found in Ca2+-binding proteins, such as calmodulin (CaM). These segments are denoted CaM-like domains (CaM-LDs) and play a regulatory role, making these CaM-like proteins sensitive to Ca2+ transients within the cell, and hence are able to transduce the Ca2+ signal leading to specific cellular responses. Importantly, this arrangement allows to this group of proteins direct regulation independent of other Ca2+-sensitive sensor/transducer proteins, such as CaM. In addition, this review also covers CaM-binding proteins, in which their CaM-binding site (CBS), in the absence of CaM, is proposed to interact with other segments of the same protein denoted CaM-like binding site (CLBS). CLBS are important regulatory motifs, acting either by keeping these CaM-binding proteins inactive in the absence of CaM, enhancing the stability of protein complexes and/or facilitating their dimerization via CBS/CLBS interaction. The existence of proteins containing CaM-LDs or CLBSs substantially adds to the enormous versatility and complexity of Ca2+/CaM signaling.

[ES] El receptor del factor de crecimiento epidérmico (EGFR) es un receptor transmembrana con actividad tirosina quinasa. Este trabajo se centra en comprender el funcionamiento de este receptor, más concretamente en cómo se produce su dimerización y activación. El EGFR tiene una secuencia citosólica yuxtamembranal muy básica denominada dominio de unión a calmodulina (CaMBD) y otra secuencia acídica, localizada distalmente del dominio catalítico, que se denomina dominio parecido a calmodulina (CaMLD). Debido a estudios previos se postuló que la dimerización del receptor ocurre de la siguiente forma: tras su unión con el ligando factor de crecimiento epidérmico (EGF), el receptor se dimeriza y al mismo tiempo el complejo Ca2+/calmodulina (Ca2+/CaM) se une al CaMBD, induciendo la separación de este dominio de la membrana celular, a la que está unida electrostáticamente, favoreciendo la activación del receptor. El CaMBD de uno de los monómeros interacciona electrostáticamente con el CaMLD del monómero opuesto, liberando Ca2+/CaM del primero, y estabilizando el dímero. Para intentar validar este modelo, se han realizado experimentos de coinmunoprecipitación y cromatografía de gradiente de masas con la proteína recombinante glutatión S-transferasa (GST) fusionada a la secuencias del CaMBD y del CaMLD, para comprobar si existe dicha interacción. Los resultados de los experimentos, sin embargo, fueron inconcluyentes debido a que la proteína GST sola interacciona consigo misma formando dímeros y posiblemente oligómeros y con otras proteínas., [EN] The epidermal growth factor receptor (EGFR) is a transmembrane receptor with intrinsic tyrosine-kinase activity. This project tries to understand some aspects of the EGFR functionality. More specifically, the mechanism of dimerization and activation of the EGFR. This receptor has a highly basic cytosolic juxtamembrane sequence denoted the calmodulin-binding domain (CaMBD), and another acidic sequence denoted the calmodulin-like domain (CaMLD), located distally of the catalytic domain. Based on previous studies, it was proposed a hypothetical model in which the dimerization and activation of the EGFR happen in the following manner: the epidermal growth factor (EGF) binds to the EGFR inducing its dimerization. At the same time, Ca2+/calmodulin (Ca2+/CaM) binds to the CaMBD inducing the separation of this domain from the inner leaflet of the plasma membrane, where is bound electrostatically. Thereafter, the CaMBD of one monomer interacts electrostatically with the CaMLD of the apposed monomer, releasing the Ca2+/CaM complex, and stabilizing the dimer. Two types of experiments were performed in this project to validate this model. These experiments were coimmunoprecipitation and gel filtration chromatography with the recombinant protein glutathione-S-transferase (GST) fused to the CaMBD and CaMLD sequences. In this manner, the project tries to confirm whether both sequences are able to interact which each other. However, the results of the experiments were inconclusive because the protein GST alone was able of self-association forming dimers, and perhaps oligomers.

[ES] La metástasis causa el 90% de las muertes asociadas al cáncer. Este proceso depende de alteraciones en la célula tumoral como son una motilidad incrementada y desarrollo de capacidad invasiva. Las células tumorales A431 sobreexpresan el receptor del factor de crecimiento epidérmico (EGFR), receptor que se localiza en la membrana plasmática y participa en el envío de señales internas que activan mecanismos que estimulan la división celular y su motilidad. Dicha señalización es regulada por la calmodulina (CaM), proteína que a su vez regula la funcionalidad de la proteína adaptadora Grb7 de manera dependiente de Ca2+ y que también se asocia al EGFR y participa en procesos de proliferación y migración celular uniéndose a ella en su dominio 243RKLWKRFFCFLRRS256. Este estudio pretende determinar si células tumorales tratadas con péptidos sintéticos constituidos por la secuencia del sitio de unión de la CaM de Grb7 ven afectada su capacidad proliferativa y migratoria bajo la premisa de que estos péptidos pudieran secuestrar e inactivar la CaM e impedir por tanto que regule dichos procesos. Para ello, se realizaron ensayos de proliferación celular y cierre de herida artificial con células A431 y se compararon los resultados de estos experimentos en presencia de los péptidos, inhibidores de CaM de alta y baja afinidad y controles sin ningún tratamiento. Se observó un efecto inhibidor sobre la proliferación y migración celular por parte de los péptidos sintéticos aunque no de la forma que se esperaba, siendo este efecto además diferente que el provocado por los inhibidores conocidos de CaM. No obstante, los resultados si ofrecieron suficiente evidencia para considerar los péptidos aptos para continuar investigando en la misma dirección ya que todavía es necesaria la realización de distintos experimentos para acercarse a los objetivos planteados., [EN] Metastases causes the 90% of cancer-associated deaths. This process depends on tumor cells alterations including increased motility and invasiveness. A431 tumor cells over-express the epidermal growth-factor receptor (EGFR), that is located in the plasma membrane and plays a role in intracellular signaling which actives cell proliferation and migration. That signaling is regulated by calmodulin (CaM), a protein that also regulates the functionality of the adaptor protein Grb7 (growth factor receptor-bound protein 7) in a Ca2+ dependent manner to its 243RKLWKRFFCFLRRS256 domain. Grb7 also participates in cell proliferation and migration processes. This study expects to determinate whether tumor cells treated with synthetic peptides based in the CaM-binding domain of Grb7 sequence shows any effect in their proliferative and migratory capacity, under the premise of these peptides could sequestrate and inactivate CaM and therefore prevents the regulation of these processes. Different cell proliferation and wound healing assays with A431 cells were performed, and the results in the presence of the synthetic peptides, high and low affinity CaM inhibitors and non-treated controls were compared. Inhibitory effects of the synthetic peptides on cell proliferation and migration was observed, despite the fact that they were not as expected, when compared with the effects caused by the CaM inhibitors. Nevertheless, the results offered enough evidence to consider the peptides adequate to continue working in the same direction considering that it is still necessary to perform additional experiments to get closer to the objetives marked in this project.

Calmodulin (CaM) is a versatile Ca2+-sensor/transducer protein that modulates hundreds of enzymes, channels, transport systems, transcription factors, adaptors and other structural proteins, controlling in this manner multiple cellular functions. In addition to its capacity to regulate target proteins in a Ca2+-dependent and Ca2+-independent manner, the posttranslational phosphorylation of CaM by diverse Ser/Thr- and Tyr-protein kinases has been recognized as an important additional manner to regulate this protein by fine-tuning its functionality. In this review, we shall cover developments done in recent years in which phospho-CaM has been implicated in signalling pathways that are relevant for the onset and progression of diverse pathophysiological processes. These include diverse systems playing a major role in carcinogenesis and tumour development, prion-induced encephalopathies and brain hypoxia, melatonin-regulated neuroendocrine disorders, hypertension, and heavy metal-induced cell toxicity.

Calmodulin (CaM) is a universal regulator for a huge number of proteins in all eukaryotic cells. Best known is its function as a calcium-dependent modulator of the activity of enzymes, such as protein kinases and phosphatases, as well as other signaling proteins including membrane receptors, channels and structural proteins. However, less well known is the fact that CaM can also function as a Ca2+-dependent adaptor protein, either by bridging between different domains of the same protein or by linking two identical or different target proteins together. These activities are possible due to the fact that CaM contains two independently-folded Ca2+ binding lobes that are able to interact differentially and to some degree separately with targets proteins. In addition, CaM can interact with and regulates several proteins that function exclusively as adaptors. This review provides an overview over our present knowledge concerning the structural and functional aspects of the role of CaM as an adaptor protein and as a regulator of known adaptor/scaffold proteins.

Calcium-mediated signaling and the functionality of Src-family tyrosine kinases (SFKs) are two interconnected processes. Activation of these kinases, which are coupled to a series of receptors, mediates Ca2+ mobilization by regulating Ca2+ channels, and the generated Ca2+ signal in turn exerts control on the kinase activity via calmodulin. In this review, we shall cover the regulation of selected processes where crosstalk between the functionality of SFKs and the Ca2+ signal occurs during the lifespan of the cell, when subjected to different extracellular or intracellular stimuli. These events result in the modulation of many physiological processes, which are essential to maintain organismal homeostasis. We discuss the importance of these mechanisms, and the implicated signaling pathways on essential cellular processes, comprising proliferation, differentiation, cell adhesion, migration, cytoskeletal remodeling, oocyte fertilization, apoptosis and autophagy. Thereafter, we discuss the role that Ca2+ and SFKs exert in the control of selected physiological functions, including hormones/neurotransmitters release, striated and smooth muscle contraction, glomerular filtration, stress response, and the cellular response to viral and bacterial infections.

[Introduction]: Grb7 (growth factor receptor bound 7) is a mammalian adaptor protein that transduces signals from tyrosine kinase receptors. This protein is overexpressed, together with ErbB2, in different human carcinomas. Grb7 is implicated in cell migration, contributing to the invasiveness and metastatic capacity of tumour cells. Also, Grb7 controls cell proliferation and tumour-associated angiogenesis. Our group demonstrated that calmodulin (CaM) binds to the proximal region of the pleckstrin homology (PH) domain of human Grb7 in a Ca2+-dependent manner, comprising the sequence 243RKLWKRFFCFLRRS256. A deletion mutant of Grb7, lacking its CaM-binding domain (CaM-BD), impairs the adhesion and migratory capacity of transfected cells. Moreover, the expression of this deletion mutant in rat C6 glioblastoma cells strongly inhibited its proliferation, the growth of brain tumours derived from stereotaxically implanted tumour cells, and tumour-associated angiogenesis. Therefore, the CaM-BD of Grb7 could be a potential target to inhibit tumour cells invasiveness and metastasis development. [Materials and Methods]: Human carcinoma epidermoid A431 cells were used in all the experiments. Custom-made synthesis of peptides (>99% purity) with the amino acid sequence RKLWKRFFCFLRRS, and a derivative with a myristoyl group at its N-terminus (Myr-RKLWKRFFCFLRRS) to facilitate cell internalization, were manufactured by Wuxi Nordisk Biotech Ltd. (China). Cell migration was measured by artificial wound healing assays and time-lapse microscopy, and cell proliferation was measured by Crystal Violet staining. The assays were done in the absence and presence of 10 nM EGF, and 20-50 µg/ml of the different peptides, or 10-50 µM W-7 and W-12 (CaM antagonists). [Results]: The addition of EGF first induced a 12-18 h lag phase in which the migration of A431 cells was arrested, and a significant retraction of the border of the artificial wound was observed. Thereafter, the migration rate of th

We identified the Grb7 family members, Grb10 and Grb14, as Ca2+-dependent CaM-binding proteins using Ca2+-dependent CaM-affinity chromatography as we previously did with Grb7. The potential CaM-binding sites were identified and experimentally tested using fluorescent-labeled peptides corresponding to these sites. The apparent affinity constant of these peptides for CaM, and the minimum number of calcium ions bound to CaM that are required for effective binding to these peptides were also determined. We prepared deletion mutants of the three adaptor proteins lacking the identified sites and determined that they lost or strongly diminished their CaM-binding capacity following the sequence Grb7 > > Grb14 > Grb10. More than one CaM-binding site and/or accessory CaM-binding sites appear to exist in Grb10 and Grb14, as compared to a single one present in Grb7.

In this review, we shall describe the rich crosstalk between non-receptor Src-family kinases (SFKs) and the Ca2+ transient generated in activated cells by a variety of extracellular and intracellular stimuli, resulting in diverse signaling events. The exchange of information between SFKs and Ca2+ is reciprocal, as it flows in both directions. These kinases are main actors in pathways leading to the generation of the Ca2+ signal, and reciprocally, the Ca2+ signal modulates SFKs activity and functions. We will cover how SFKs participate in the generation of the cytosolic Ca2+ rise upon activation of a series of receptors and the mechanism of clearance of this Ca2+ signal. The role of SFKs modulating Ca2+-translocating channels participating in these events will be amply discussed. Finally, the role of the Ca2+ sensor protein calmodulin on the activity of c-Src, and potentially on other SFKs, will be outlined as well. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

We identified the Grb7 family members, Grb10 and Grb14, as Ca2+ -dependent CaM-binding proteins using Ca2+ -dependent CaM-affinity chromatography as we previously did with Grb7. The potential CaM-binding sites were identified and experimentally tested using fluorescent-labeled peptides corresponding to these sites. The apparent affinity constant of these peptides for CaM, and the minimum number of calcium ions bound to CaM that are required for effective binding to these peptides were also determined. We prepared deletion mutants of the three adaptor proteins lacking the identified sites and determined that they lost or strongly diminished their CaM-binding capacity following the sequence Grb7 > > Grb14 > Grb10. More than one CaM-binding site and/or accessory CaM-binding sites appear to exist in Grb10 and Grb14, as compared to a single one present in Grb7.

The epidermal growth factor receptor (EGFR/HER1/ErbB1) is a tyrosine kinase that belongs to the ErbB family, and is implicated in multiple cellular functions including: cell proliferation, cell survival, differentiation and cell migration, and is mutated or overexpressed in multiple solid tumors. We have demonstrated that the EGFR, and its cousin ErbB2/HER2, binds calmodulin (CaM) at the cytosolic juxtamembrane region in a Ca2+-dependent manner, participating in the ligand-dependent activation of the receptor as an intracellular co-activator. Deletion or mutation of the CaM-binding domain (CaMBD) of the EGFR inactivated or strongly decreased the tyrosine kinase activity of the receptor. Inhibition of CaM with specific cell-permeable chemical antagonists or downregulation of CaM in conditionalknockout cells stably expressing the human EGFR also inhibited the ligand-dependent activation of the receptor. Furthermore, CaM can be phosphorylated by the EGFR at both Tyr99 and Tyr138 in the absence of Ca2+. We will present results showing that phospho-Tyr-CaM also acts as an activator of the EGFR in the presence but not in the absence of receptor ligand, increasing therefore the tyrosine kinase activity of the receptor toward exogenous substrates. A cell-permeable peptide corresponding to a modified sequence of the CaM-BD of the EGFR also inhibited the ligand-dependent activation of the receptor and the proliferative capacity of cells overexpressing this receptor. A model describing how Ca2+/CaM and phospho-Tyr-CaM exert its activator action, and how targeting the CaM-BD of the EGFR, and ErbB2, could be a valid therapeutic strategy against tumors with overexpressed or dysregulated hyperactive receptors will be discussed.

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2 +-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca2+, but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca2+. This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca2+, absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized antiphospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (645R-R-R-H-I-V-R-K-R- T-L-R-R-L-L-Q660) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.

O-GlcNAcylation is the reversible addition of single O-linked ß-D-N-acetylglucosamine (GlcNAc) moieties to serine or threonine residues of proteins. This post-translational modification has been shown to occur in many nuclear and cytosolic proteins. An extensive crosstalk between O-GlcNAcylation and phosphorylation has been described. There are two enzymes implicated in this process: ß-N-acetyl-glucosaminyltransferase (OGT) adding GlcNAc moieties, and ß-N-acetylglucosaminidase (OGA) removing these moieties. It has been demonstrated using high throughput proteomic analysis that the epidermal growth factor receptor (EGFR) type III of Drosophila melanogaster undergoes O-GlcNAcylation. We present experimental evidences suggesting that the EGFR from human carcinoma epidermoid A431 cells is subjected to O-GlcNAcylation. We detected a positive O-GlcNAcylation signal in immunoprecipitated EGFR using immunoblot and two distinct specific anti-O-GlcNAc antibodies. Conversely, the presence of EGFR was detected by immunoblot among the O-GlcNAcylated proteins immunoprecipitated with an anti-O-GlcNAc antibody. These signals were enhanced when Thiamet G, a highly specific OGA inhibitor, was present. Most significantly, we detected a positive O-GlcNAcylation signal in immunoprecipitated and N-deglycosylated EGFR using peptide-N-glycosidase F (PNGase F), and from tunicamycin-treated cells when were metabolically labeled with azido-GlcNAc, biotinylated and probed with streptavidin-labeled peroxidase. Finally, we performed O-GlcNAcylation assay in vitro using immunoprecipitated EGFR and OGT in the presence of the substrate UDP-GlcNAc, which resulted in the enhancement of the EGFR O-GlcNAcylation signal as detected by immunoblot. We conclude that the EGFR from A431 tumor cells is subjected to O-GlcNAcylation and this may regulate the functionality of the receptor.

Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro.

[ES]: La calmodulina es una proteína receptora de calcio que interviene en multitud de procesos celulares regulando una gran diversidad de proteínas, entre ellas diversas tirosina quinasas. Aunque el aumento de calcio citosólico permite la activación de la calmodulina; también es capaz de regular distintas funciones celulares en forma de apocalmoculina (calmodulina no unida a calcio) y tras sufrir modificaciones postraduccionales como la fosforilación. Se ha demostrado in vivo que, en presencia de ligando, el complejo Ca2+/calmodulina se une al dominio de unión de la calmodulina del receptor del factor de crecimiento epidérmico (EGFR), que se encuentra en la región citosólica y uxtamembranal, activando la tirosina quinasa del receptor. En cambio, se ha observado in vitro que la calmodulina inhibe esta actividad tirosina quinasa cuando el receptor se activa por su ligando. Además, es sabido que diferentes especies de fosfo-calmodulina realizan distintas funciones celulares. En este trabajo, hemos querido profundizar acerca de la regulación que ejerce la calmodulina sobre las proteína tirosina quinasas. Hemos estudiado, en células vivas, el papel regulador de la misma sobre receptores ErbB y sobre la tirosina quinasa no receptora Src. Asimismo, se ha realizado un ensayo in vitro para determinar el papel de la calmodulina y de un mutante fosfo-mimético de la misma sobre la fosforilación del EGFR., [EN]: Calmodulin is a calcium-binding protein which is involved in the regulation of many cellular processes and a wide variety of proteins, including several protein tyrosine kinases. The increase of free cytosolic calcium allows calmodulin activation; but its function is not only due to the presence of calcium because this protein is also capable to regulate various cell functions as apocalmoculin (calcium-free calmodulin) and after undergoing post-translational modifications (such as phosphorylation). It has been shown in vivo that the Ca2+/ calmodulin complex binds to the calmodulin-binding domain of the EGFR, which is located in its citosolic juxtamembrane region in the region of the EGFR. This region activates the tyrosine kinase activity of the EGFR. In contrast, it has been demostrated in vitro that calmodulin inhibits the ligand-dependent activity of the EGFR. Furthermore, it is known that different phospho-calmodulin species perform different cellular functions. Therefore, in this work, for understanding more about the regulation that calmodulin exerts on protein tyrosine kinases, we have studied in living cells the role of calmodulin on ErbB receptors and the non-receptor tyrosine kinase Src. Likewise, it has been performed a study in vitro to determine the role of wild type calmodulin and a phospho-mimetic calmodulin mutant on EGFR phosphorylation.

Sex hormones regulate vascular function through nitric oxide (NO), thromboxane A2 (TXA2), and prostaglandin E2 (PGE2) among other factors. Since NO, TXA2 and PGE2 are known to regulate the epidermal growth factor receptor (EGFR) and downstream signaling pathways, the purpose of this study was to investigate whether the loss of ovarian function alters the production of these effectors regulating the activity of the mitogen-activated protein kinase/extracellular-regulated kinases 1 and 2 (MAPK-ERK1/2), an EGFR downstream pathway. For this purpose, mesenteric arteries from control and ovariectomized Sprague-Dawley rats (6-months old) were used to analyze: i) the release of NO, TXA2 and PGE2; ii) the vascular reactivity to acetylcholine (Ach), the NO donor sodium nitroprusside, the TXA2-mimetic compound U-46619, and exogenous PGE2; and iii) the activation status of MAPK(ERK1/2). Our results show that ovariectomy: i) does not change NO release while decreases its vasodilator action; ii) increases the release and vasoconstrictor action of TXA2 and PGE2; and iii) increases the basal activity of the MAPK(ERK1/2) pathway. We conclude that activation of the EGFR/MAPK axis by these effectors could contribute to increase the proliferation rate of vascular smooth muscle cells, leading to increase peripheral resistance and hypertension.

Calmodulin (CaM) is a Ca2+-receptor protein that regulates more than one hundred target proteins with and without enzymatic activity in Ca2+- dependent and independent manners and modulates a myriad of cellular processes, some of which are vital for tumorigenesis (i.e. cell proliferation, apoptosis and autophagy). c-Src is a non-receptor tyrosine kinase that participates in signaling pathways that transduces events initiated by different types of cellular receptors and adhesion molecules, and controls many cellular functions including: cell migration, proliferation, differentiation, apoptosis and stress-response, among others. It has been previously demonstrated that CaM directly interacts with c-Src in a partial Ca2+-dependent manner and that trifl uoperazine, a CaM antagonist, inhibits the phosphorylation of c-Src, suggesting that CaM plays an important role in survival signals in pancreatic tumor cells. We demonstrate in this work that recombinant c-Src directly binds CaM in Ca2+-dependent and independent manners, suggesting that this kinase likely has more than one CaM-binding domain. A couple of IQ-like motifs identified in silico in c-Src could be responsible for the binding of CaM to this kinase in the absence of Ca2+. We also show that CaM enhances the autophosphorylation and tyrosine kinase activity of c-Src in the absence and presence of Ca2+, most strongly in the former condition. We are testing whether the CaM antagonist W-7 inhibits or not the hydrogen peroxideinduced phosphorylation of c-Src in human breast adenocarcinoma SKBR-3 cells, and whether or not CaM-dependent protein kinase II and/or the CaM-regulated protein phosphatase calcineurin, play a role in this process.

Calmodulin (CaM) is a ubiquitous Ca2+ receptor protein mediating a large number of signaling processes in all eukaryotic cells. CaM plays a central role in regulating a myriad of cellular functions via interaction with multiple target proteins. This review focuses on the action of CaM and CaM-dependent signaling systems in the control of vertebrate cell proliferation, programmed cell death and autophagy. The significance of CaM and interconnected CaM-regulated systems for the physiology of cancer cells including tumor stem cells, and processes required for tumor progression such as growth, tumor-associated angiogenesis and metastasis are highlighted. Furthermore, the potential targeting of CaM-dependent signaling processes for therapeutic use is discussed. © 2013 Elsevier B.V.

This study analyzes whether the release of nitric oxide (NO) and thromboxane A2 (TXA2) depends on the time lapsed since gonadal function is lost, and their correlation with the proliferation of vascular smooth muscle cells (VSMC) mediated by the epidermal growth factor receptor (EGFR). For this purpose, aortic and mesenteric artery segments from control and 6-weeks or 5-months orchidectomized rats were used to measure NO and TXA2 release. The results showed that the basal and acetylcholine (ACh)-induced NO release were decreased 6 weeks post-orchidectomy both in aorta and mesenteric artery, but were recovered 5 months thereafter up to levels similar to those found in arteries from control rats. The basal and AChinduced TXA2 release increased in aorta and mesenteric artery 6 weeks post-orchidectomy, and was maintained at high levels 5 months thereafter. Since we previously observed that orchidectomy, which decreased testosterone level, enlarged the muscular layer of mesenteric arteries, the effect of testosterone on VSMC proliferation was analyzed. The results showed that treatment of cultured VSMC with testosterone downregulated mitogenic signaling pathways initiated by the liganddependent activation of the EGFR. In contrast, the EGFR pathways were constitutively active in mesenteric arteries of longterm orchidectomized rats. Thus, the exposure of mesenteric arteries from control rats to epidermal growth factor (EGF) induced the activation of EGFR signaling pathways. However, the addition of EGF to arteries from orchidectomized rats failed to induce a further activation of these pathways. In conclusion, this study shows that the release of NO depends on the time lapsed since the gonadal function is lost, while the release of TXA2 is already increased after short periods post-orchidectomy. The alterations in these signaling molecules could contribute to the constitutive activation of the EGFR and its downstream signaling pathways after long period post-orchidectomy e

Our success in producing an active epidermal growth factor receptor (EGFR) tyrosine kinase in Escherichia coli encouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST) that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest.