Regulatory role of calmodulin on systems relevant in tumor cells signaling

Resumen

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 ß-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.