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 antiphospho-(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 (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.

The multifunctional role of phospho-calmodulin in pathophysiological processes

“…As CaM is also a good substrate for the EGFR [17], it was expected that phospho-Tyr-CaM could also interact with the receptor. This indeed was the case [18,19], and the site of interaction of phospho-Tyr-CaM with the receptor was stablished to be the same that the one for non-phosphorylated CaM located at the cytosolic juxtamembrane region encompassing the sequence 645 RRRHIVRKRTLRRLLQ 660 [19]. The NMR-derived structure of a peptide corresponding to the transmembrane (TM) and the cytosolic juxtamembrane (JM cyt ) segment, where the CaM-BD is located, reconstituted in lipid bicelles shows that they form dimers and that the CaM-BD presents two helical segments divided by a flexible linker [20,21] (see Figure 2).…”

“…evidenceBiological effect (Ref. )Functional and pathological implicationsp-Y-CaMDGNG Y 99 ISAAAffect EF-hand IIIEGFR in vitro Phospho-Y-CaM enhances ligand-dependent EGFR activation [19]. CancerGQVN Y 138 EEFVAffect EF-hand IVc-Src In vivo The pleiotrophin/PTPRZ1 pathway enhances CaM phosphorylation in SCLC [37].…”

“…This flexible linker is important to understand the expected bending of the JM cyt to facilitate electrostatic interaction with the phosphoinositide-rich negatively charged inner leaflet of the plasma membrane to attain auto-inhibition of the EGFR in the absence of ligand [22]. In vitro studies show that phospho-Tyr-CaM enhances the ligand-dependent activation of the EGFR, and this effect was observed as well using the phospho-mimetic CaM(Y99D/Y138D) mutant [19]. This suggests that phospho-Tyr-CaM could be an intracellular co-activator of the EGFR more efficient than non-phosphorylated CaM in detaching the CaM-binding domain of the inner leaflet of the plasma membrane to which is electrostatically bound [22], most provably due to the extra negative charges of phosphate.…”

Calmodulin and PI3K Signaling in KRAS Cancers

“…Alternatively, the phosphorylated CaM can bind at the same site as the non-phosphorylated site. It has already been observed that phosphorylated and non-phosphorylated CaM share the same binding site ( 645 RRRHIVRKRTLRRLLQ 660 ) at the cytosolic juxtamembrane region of the EGFR [72]. …”

CALM1 Promotes ESCC Progression and Dampens Chemosensitivity to EGFR Inhibitor

“…Ca 2+ -CALM binding to the CALM binding domain (CALM-BD) of cytosolic juxtamembrane region of the receptor plays an important trigger role in ligand-dependent activation EGFR in living cells [42][43][44]. A further study was shown that nonphosphorylated CaM only interacts with the EGFR when is not phosphorylated at Tyr 1173 (tyrosine 1173 ) [45].Herein, We found here for the rst time that treatment with knock-out of CALM1 and EGFR inhibitors have signi cant effects against tumors in vivo and in vitro in ESCC. Recent studies have pointed to the potential for combinations of EGFR inhibitors with TKIs to overcome a certain degree of resistance for EGFR mutations [46].…”