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