title: The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor.
abstract: The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca(2+), 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 Ca(2+). This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca(2+), 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 anti-phospho-(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 ((645)R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q(660)) 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.
authors.affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain.
authors.forename: Silviya R
authors.initials: SR
authors.lastname: Stateva
authors.affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, 1010 Caracas, Venezuela.
authors.forename: Valentina
authors.initials: V
authors.lastname: Salas
authors.affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain Centro Nacional de Investigaciones Cardiovasculares, E-28029 Madrid, Spain.
authors.forename: Alberto
authors.initials: A
authors.lastname: Benguría
authors.affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain Centro Nacional de Investigaciones Cardiovasculares, E-28029 Madrid, Spain.
authors.forename: Itziar
authors.initials: I
authors.lastname: Cossío
authors.affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain.
authors.forename: Estefanía
authors.initials: E
authors.lastname: Anguita
authors.affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain Departamento de Fisiología, Genética y Microbiología, Facultad de Ciencias, Universidad de Alicante, E-03080 Alicante, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Martín-Nieto
authors.affiliation: Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, 1010 Caracas, Venezuela Instituto de Estudios Avanzados (IDEA), 1080 Caracas, Venezuela.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Benaim
authors.affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas & Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain antonio.villalobo@iib.uam.es.
authors.forename: Antonio
authors.initials: A
authors.lastname: Villalobo
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008297:Male; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D034421:Sus scrofa; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D008024:Ligands; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014443:Tyrosine; C512478:EGFR protein, human; C483803:Egfr protein, rat; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src)
keywords: calcium; calmodulin; epidermal growth factor receptor; phospho-(Tyr)-calmodulin; tyrosine kinase