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

Silviya R Stateva,José Martín-Nieto,Alberto Benguría,Itziar Cossío,Valentina Salas,Gustavo Benaim,Antonio Villalobo,Estefanía Anguita

doi:10.1042/bj20150851

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 invitro 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.

Highlights

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that belongs to the avian erythroblastosis oncogene B homolog (ErbB) receptor tyrosine kinase family and is implicated in the control of important cellular functions including cell proliferation, cell survival and apoptosis, differentiation and cell migration [1,2,3]
We show that tyrosine-phosphorylated CaM elicits a strong increase in the ligand-dependent activity of the EGFR in vitro, suggesting that phospho-(Tyr)-CaM may contribute to the ligand-dependent activation of this receptor in living cells, thereby enhancing EGFR-mediated signalling
To ascertain that the ligand-dependent extra-activation observed on the EGFR tyrosine kinase was due to the presence of P-(Tyr)-CaM, additional experiments were performed in the presence of CaM, but in which its phosphorylation during the first step of the assay was prevented by the presence of Ca2+, as this cation acts as an inhibitor of CaM phosphorylation as previously demonstrated [17,18,33,34]

Summary

Introduction

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that belongs to the avian erythroblastosis oncogene B homolog (ErbB) receptor tyrosine kinase family and is implicated in the control of important cellular functions including cell proliferation, cell survival and apoptosis, differentiation and cell migration [1,2,3]. The phosphorylation of 100 μg/ml PGT by the EGFR preparation (50 μl) was performed at 37 ◦C for appropriate periods of time in 100 μl of a medium containing 15 mM Hepes– NaOH (pH 7.4), 6 mM MgCl2, 0.5 mM EGTA, 0.5 % (w/w) Triton X-100, 2.5 % (w/v) glycerol and 30 nM histone, in the absence and presence of purified 32P-(Tyr)-CaM (6 nM) free of non-phosphorylated CaM [histone/P-(Tyr)CaM molar ratio = 5], 1 μM EGF (when added) and 10 μM (2 μCi) [γ -32P]ATP.

Results

Conclusion