O-GlcNAcylation of the human epidermal growth factor receptor

The reversible O-linked attachment of single β-D-N-acetylglucosamine (GlcNAc) moieties to serine/threonine residues in target proteins is a frequently occurring post-translational modification affecting the functionality of many cellular systems. In this report we present experimental evidence suggesting that the epidermal growth factor receptor (EGFR) is subjected to O-GlcNAcylation in human carcinoma epidermoid A431 cells and human lung carcinoma A549 cells. However, no signal was detected in human cervix adenocarcinoma HeLa cells or in mouse EGFR-T17 fibroblasts ectopically expressing the human EGFR. We detected a positive O-GlcNAcylation signal in the immunoprecipitated EGFR by Western blotting using two distinct specific anti-O-GlcNAc antibodies even after N-deglycosylation of the receptor using peptide-N-glycosidase F (PNGase F). Conversely, the presence of EGFR was detected by Western blotting using an anti-EGFR antibody in the immunocomplex of O-GlcNAcylated proteins immunoprecipitated with an anti-O-GlcNAc antibody. These signals were enhanced when the O-linked β-N-acetylglucosaminidase (OGA) inhibitor Thiamet G was added to prevent the deglycosylation of the GlcNAc moiety(ies). Moreover, we also detected a positive signal in the immunoprecipitated and N-deglycosylated EGFR using PNGase F, and tunicamycin when the cells were metabolically labeled with azido-GlcNAc (GlcNAz), biotinylated and probed with a streptavidin-labeled peroxidase. Finally, EGFR and O-linked β-N-acetylglucosamine transferase (OGT) co-immunoprecipitate, and incubation of the immunoprecipitated EGFR with the immunoprecipitated OGT in the presence of uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) resulted in a significant enhancement of the EGFR O-GlcNAcylation signal as detected by Western blotting using an anti-O-GlcNAc antibody. We conclude that the human EGFR is subjected to O-GlcNAcylation in the A431 and A549 tumor cell lines.This work was funded by grants to AV from the European Commission (contract no. PITN-GA-2011-289033), the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2011-23494 and SAF2014-52048-R), and the Consejería de Educación de la Comunidad de Madrid (S2011/BMD-2349). SRS received funding from the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program FP7/2007-2013 under REA grant agreement PITN-GA-2011-289033.Peer Reviewe

O-GlcNAcylation of the human epidermal growth factor receptor

The reversible O-linked attachment of single β-D-N-acetylglucosamine (GlcNAc) moieties to serine/threonine residues in target proteins is a frequently occurring post-translational modification affecting the functionality of many cellular systems. In this report we present experimental evidence suggesting that the epidermal growth factor receptor (EGFR) is subjected to O-GlcNAcylation in human carcinoma epidermoid A431 cells and human lung carcinoma A549 cells. However, no signal was detected in human cervix adenocarcinoma HeLa cells or in mouse EGFR-T17 fibroblasts ectopically expressing the human EGFR. We detected a positive O-GlcNAcylation signal in the immunoprecipitated EGFR by Western blotting using two distinct specific anti-O-GlcNAc antibodies even after N-deglycosylation of the receptor using peptide-N-glycosidase F (PNGase F). Conversely, the presence of EGFR was detected by Western blotting using an anti-EGFR antibody in the immunocomplex of O-GlcNAcylated proteins immunoprecipitated with an anti-O-GlcNAc antibody. These signals were enhanced when the O-linked β-N-acetylglucosaminidase (OGA) inhibitor Thiamet G was added to prevent the deglycosylation of the GlcNAc moiety(ies). Moreover, we also detected a positive signal in the immunoprecipitated and N-deglycosylated EGFR using PNGase F, and tunicamycin when the cells were metabolically labeled with azido-GlcNAc (GlcNAz), biotinylated and probed with a streptavidin-labeled peroxidase. Finally, EGFR and O-linked β-N-acetylglucosamine transferase (OGT) co-immunoprecipitate, and incubation of the immunoprecipitated EGFR with the immunoprecipitated OGT in the presence of uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) resulted in a significant enhancement of the EGFR O-GlcNAcylation signal as detected by Western blotting using an anti-O-GlcNAc antibody. We conclude that the human EGFR is subjected to O-GlcNAcylation in the A431 and A549 tumor cell lines.This work was funded by grants to AV from the European Commission (contract no. PITN-GA-2011-289033), the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2011-23494 and SAF2014-52048-R), and the Consejería de Educación de la Comunidad de Madrid (S2011/BMD-2349). SRS received funding from the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program FP7/2007-2013 under REA grant agreement PITN-GA-2011-289033.Peer Reviewe

Regulatory role of calmodulin on systems relevant in tumor cells signaling

Memoria de Tesis Doctoral presentada por Silviya Raykova Stateva licenciada y Máster en Biología Celular, para obtar al grado de Doctor por la Universidad Autónoma de Madrid, y realizada en el Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-IIBM).-- Esta obra está bajo una licencia Creative Commons Reconocimiento-No Comercial-Sin Obra Derivada 4.0 Internacional.[EN]: Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on different CaM-binding proteins as compared to non-phosphorylated CaM. In this work 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. We demonstrated some biological 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), as well as c-Src and the epidermal growth factor receptor (EGFR). We demonstrated that CaM directly interacts with c-Src in both Ca2+-dependent and Ca2+-independent manners in vitro, and that in living cells the CaM antagonist W-7 inhibits this kinase induced by the upstream activation of 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. Most relevant and for the first time, we demonstrated that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action than Ca2+/CaM on Src auto-phosphorylation. We also present experimental evidences suggesting that the EGFR from A431 and A549 cells is subjected to O-GlcNAcylation, and that CaM may be involved in the regulation of this process through binding to the O-GlcNAc transferase (OGT). 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 a highly specific O-linked β-N-acetylglucosaminidase (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 GlcNAz. We also performed O-GlcNAcylation assay in vitro using immunoprecipitated EGFR and OGT, which resulted in the enhancement of the EGFR O-GlcNAcylation signal. We concluded that the EGFR from A431 and A549 tumor cells is subjected to O-GlcNAcylation. Furthermore, we present preliminary data using in silico studies, combined with binding assays such as Ca2+-dependent CaM-affinity chromatography and co-immunoprecipitation experiments, showing that OGT is a putative CaM-binding protein.[ES]: La calmodulina (CaM) fosforilada en diferentes residuos de serina/treonina y tirosina ejerce efectos reguladores diferenciales en una diversidad de proteínas de unión a CaM en comparación con CaM no fosforilada. En esta Tesis se describe la preparación y caracterización de una serie de mutantes fosfo-(Y)-miméticos de la CaM en el que uno o los dos residuos de tirosina presentes en la CaM (Y99 y Y138) fueron sustituidos por ácido aspártico o ácido glutámico. Hemos demostrado algunas propiedades biológicas de estos mutantes de la CaM, tales como su fosforilación diferencial por la tirosina quinasa c-Src y su acción, en comparación con CaM tipo salvaje, sobre la actividad de varios enzimas CaM-dependientes: la fosfodiesterasa de nucleótidos cíclicos 1 (PDE1), la óxido nítrico sintasa endotelial (eNOS), c-Src y el receptor del factor de crecimiento epidérmico (EGFR). Hemos demostrado que la CaM interactúa directamente con Src de forma dependiente e independiente de Ca2+ in vitro, y que en células vivas el antagonista de CaM W-7 inhibe esta quinasa inducida por la activación aguas arriba del EGFR, en células A431 de carcinoma epidermoide human, y por estrés oxidativo inducido por peróxido de hidrógeno, tanto en células A431 como en células de adenocarcinoma de mama humano SK-BR-3. De mayor relevencia y por primera vez, hemos demostrado que la CaM libre de Ca2+ (apo-CaM) ejerce una acción activadora mucho mayor que el complejo Ca2+/CaM sobre la auto-fosforilación de Src. También presentamos evidencias experimentales que sugieren que el EGFR de células A431 yA549 está sometido a O-GlcNAcylatión, y que la CaM puede estar implicada en la regulación de este proceso mediante su unión a la O-GlcNAc transferasa (OGT). Hemos detectado una señal positiva de O-GlcNAcylación en el EGFR inmunoprecipitado usando inmunoblot y dos anticuerpos específicos anti-O-GlcNAc distintos. Adicionalmente, la presencia de EGFR se detectó por inmunoblot entre las proteínas O-GlcNAcyladas e inmnoprecipitadas con un anticuerpo anti-O-GlcNAc. Estas señales se incrementaron cuando un inhibidor altamente específico de la O-ligado β-N-acetilglucosaminidasa (OGA) estuvo presente. Más significativamente, se detectó una señal de O-GlcNAcylación positiva en el EGFR inmunoprecipitado y N-deglicosilado usando péptido-N-glicosidasa F (PNGasa F), y a partir de células tratadas con tunicamicina cuando se marcaron metabólicamente con azido-GlcNAc. También se realizaron ensayos de O-GlcNAcylación in vitro utilizando EGFR y OGT inmunoprecipitados, lo que resultó en el incremento de la señal de O-GlcNAcylación en el EGFR. Llegamos a la conclusión de que el EGFR de las células tumorales A431 y A549 están sometidos a O-GlcNAcylación. Además, se presentan datos preliminares de estudios in silico, así como ensayos experimentales de unión, tales como cromatografía de afinidad de CaM dependiente de Ca2+ y co-inmunoprecupitación, demostrando que la enzima OGT es putativamente una proteína de unión a CaM.This work was done with the support of an European Commission Marie Curie DYNANO ITN fellowship.Peer Reviewe

Detection of O-linked N-acetylglucosamine post-translational modification of the epidermal growth factor receptor in A431 tumor cells

Póster presentado al 2nd Symposium on Biomedical Research: "Advances and perspectives in cancer", celebrado en Madrid el 17 de abril de 2015.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.This project was funded by grants from the EU (PITN-GA-2011-289033), MINECO (SAF2011-23494) and CAM (S2011/BMD-2349).Peer Reviewe

Regulation of different cellular systems by calmodulin and phospho-(Tyr)-calmodulin

Trabajo presentado al 4th DYNANO Network, celebrado en Namur (Bélgica) del 5 al 9 de abril de 2014.Peer Reviewe

O-GlcNacylation of the EGFR and systems regulated by calmodulin and phospho-(Tyr)-calmodulin

Trabajo presentado al 4th DYNANO Network, celebrado en Namur (Bélgica) del 5 al 9 de abril de 2014.Trabajo presentado al 4th DYNANO Network, celebrado en Namur (Bélgica) del 5 al 9 de abril de 2014.Peer Reviewe

Targeting the calmodulin-regulated ErbB/Grb7 signaling axis in cancer therapy

Signal transduction pathways essential for the survival and viability of the cell and that frequently present aberrant expression or function in tumors are attractive targets for pharmacological intervention in human cancers. In this short review we will describe the regulation exerted by the calcium-receptor protein calmodulin (CaM) on signaling routes involving the family of ErbB receptors - highlighting the epidermal growth factor receptor (EGFR/ErbB1) and ErbB2 - and the adaptor protein Grb7, a downstream signaling component of these receptors. The signaling mechanism of the ErbB/Grb7 axis and the regulation exerted by CaM on this pathway will be described. We will present a brief overview of the current efforts to inhibit the hyperactivity of ErbB receptors and Grb7 in tumors. The currently available information on targeting the CaM-binding site of these signaling proteins will be analyzed, and the pros and cons of directly targeting CaM versus the CaM-binding domain of the ErbB receptors and Grb7 as potential anti-cancer therapy will be discussed.The work in the authors laboratory was funded in part by grants (to AV) from the Secretaría de Estado de Investigación Desarrollo e Innovación - SEIDEI (SAF2011-23494), the Consejería de Educación de la Comunidad de Madrid (S2010/BMD-2349), the Agencia Española de Cooperación Internacional para el Desarrollo - AECID (AP/040803/11), and the European Commission (contract PITN-GA-2011-289033). IG-P, SRS and KJ were respectively supported by a fellowship from the Ministerio de Educación Cultura y Deporte, a Madame Curie contract from the European Commission, and a grant from the AECID.Peer reviewe

Ca2+/calmodulin and apo-calmodulin both bind to and enhance the tyrosine kinase activity of c-Src

This is an open access article distributed under the terms of the Creative Commons Attribution License.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.This work was funded by grants to AV from the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2011-23494 & SAF2014-52048-R), the Consejería de Educación de la Comunidad de Madrid (S2011/BMD-2349), the CSIC program iCOOP+ 2014 (COOPA20053), and the European Commission (contract PITN-GA-2011-289033). SRS received funding from the People Program (Marie Curie Actions) of the European Union's Seventh Framework Program FP7/2007-2013 under REA grant agreement n° PITN-GA-2011-289033. VS and GB were supported by fellowship and grants from the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela (03-00-6057-2005 & PG-03-8728-2013) and Fondo Nacional de Ciencia, Tecnología e Innovación (P-2011000884)Peer Reviewe

Characterization of phospho-(tyrosine)-mimetic calmodulin mutants

This is an open access article distributed under the terms of the Creative Commons Attribution License.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.This work was funded by grants to AV from the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2011-23494) and the Consejería de Educación de la Comunidad de Madrid (S2011/BMD-2349), and grant to AV and DS from the European Commission (contract PITN-GA-2011-289033 DYNANO). Additional funding to DS and MM was provided by the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias. SRS was funded by a Marie Curie contract from the European Commission and VS and GB were supported by fellowship and grants (03-00-6057-2005 and PG-03-8728-2013) from the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela.Peer Reviewe

Testing the function of calmodulin (CaM) in epidermal growth factor signaling using conditional CaM-knockout cells

Resumen del póster presentado al Keystone Symposium on PI 3-Kinase and Interplay with Other Signaling Pathways celebrado en Keystone-Colorado (US) del 24 de febrero al 1 de marzo de 2013.Calmodulin (CaM) is a major regulator of Ca2+-dependent cellular processes including phosphatidyl inositol-3 kinase related signaling. In contrast to lower eukaryotes, phylogenetically higher organisms have two or three functional genes encoding distinct transcripts for the expression of a single CaM protein sequence identical in all vertebrates. This has been a challenge for achieving conditional CaM-knockout mutants cell lines from vertebrates. Recently, we have described CaM-knockout chicken B lymphoma DT40 cell lines expressing a rat CaM transgene, which is repressible by tetracycline. These cell lines have been shown to be useful to study the functionality of CaM mutants lacking the ability of binding Ca2+ in one or multiple Ca2+-binding sites on the cell viability and proliferative capacity. Using this CaM knock out system we have demonstrated that CaM activates the epidermal growth factor receptor (EGFR) tyrosine kinase by direct binding to the receptor. Although the EGFR expressed in these cells is under the control of an exogenous promoter unrelated to the endogenous one, the expression of the transcript as well as the EGFR protein were found to be regulated by CaM in a biphasic manner. This points to the possibility of using these conditional CaM-knockout cells to study putative CaM-regulated transcriptional and translational events. We have previously demonstrated that CaM is phosphorylated at tyrosine residues by the EGFR. Thus, we are also pursuing studies in these cells on the action of transfected non-phosphorylatable and phosphomimetic CaM mutants on the functionality of the EGFR. Our novel genetic system offers the unique opportunity to study the function of CaM in connection with the multitude of CaM targets in signaling process without the use of pharmacological inhibitors, which are frequently unspecific.Peer Reviewe