The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

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 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 (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.This work was funded by the Secretaría de Estado de Investigación, Desarrollo e Innovación [grant number SAF2014-52048-R (to A.V.)]; the Consejería de Educación de la Comunidad de Madrid [grant number S2011/BMD-2349 (to A.V.)]; the CSIC program i-COOP+ 2014 [grant number COOPA20053 (to A.V.)] and the European Commission [grant number PITNGA-2011-289033 (to A.V.)]; the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program [grant number PITN-GA-2011-289033 (to S.R.S.)]; the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela [grant number CDCH-UCV 03-00-6057-2005 (to V.S.)]; and [grant number PG-03-8728-2013 (to G.B.)]; and the Fondo Nacional de Ciencia, Tecnología e Innovación [grant number P-2011000884 (to G.B.)