Phosphorylation of Calmodulin by Permeabilized Fibroblasts Overexpressing the Human Epidermal Growth Factor Receptor

Abstract

Detergent-permeabilized EGFR-T17fibroblasts, which overexpress the human epidermal growth factor (EGF) receptor, phosphorylate both poly-L-(glutamic acid, tyrosine) and exogenous calmodulin in an EGF-stimulated manner. Phosphorylation of calmodulin requires the presence of cationic polypeptides, such as poly-L-(lysine) or histones, which exert a biphasic effect toward calmodulin phosphorylation. Optimum cationic polypeptide/calmodulin molar ratios of 0.3 and 7 were determined for poly-L-(lysine) and histones, respectively. Maximum levels of calmodulin phosphorylation were attained in the absence of free calcium, and a strong inhibition of this process was observed at very low concentrations (Ki= 0.2 μΜ) of this cation. The incorporation of phosphate into calmodulin occurred predominantly on tyrosine residue(s) and was stimulated 34-fold by EGF.

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

“…Figure 1(A) shows the phosphorylation of CaM in the absence and presence of EGF, performed in the first step of the assay in the absence of Ca 2 + (presence of EGTA) as indicated and the phosphorylation of PGT, subsequently assayed in the second step within the same reaction mixture either in the absence of Ca 2 + (EGTA) or in its presence (Ca 2 + ). As we have previously reported, the addition of Ca 2 + just before starting the phosphorylation of PGT in the second step should prevent further phosphorylation of CaM by the EGFR [17,18,33,34]. It can be observed that the phosphorylation of PGT, detected as a smear along the electrophoretic tracks, was higher when P-(Tyr)-CaM became accumulated in the first part of the sequential assay, as compared to control experiments performed in the absence of CaM.…”

“…Histone acts as a cationic cofactor for the phosphorylation of CaM and given that, the amount of P-(Tyr)-CaM formed in the assay depends on the CaM-histone ratio used [33,34]. Upon changing this ratio, we observed that the activation of the EGFR in the presence of EGF was directly proportional to the amount of accumulated P-(Tyr)-CaM (result not shown).…”

“…(i) In the absence of ligand the EGFR is auto-inhibited; (ii) upon EGF stimulation, the low cytosolic concentration of free Ca 2 + initially prevailing under such conditions [57] could favour an ultra-fast phosphorylation of apo-CaM by the EGFR [17,18,33,34], particularly if CaM were to be already tethered to the receptor; (iii) the generated P-(Tyr)-CaM could help to partially activate the EGFR, initiating the generation of the Ca 2 + signal; (iv) as the cytosolic concentration of free Ca 2 + rises [56], additional phosphorylation of CaM should come to a halt [17,18,33,34]; and (v) the increase in the cytosolic concentration of free Ca 2 + would induce the formation of the Ca 2 + -CaM and/or Ca 2 + /P-(Tyr)-CaM complexes allowing the complete detachment of the CaM-BD of the EGFR from the inner leaflet of the plasma membrane according to the model proposed by McLaughlin et al [25], thereby maintaining the receptor in a fully active state.…”

“…The Ca 2 + -induced electrophoretic mobility shift of accumulated P-(Tyr)-CaM at 21 kDa was attained by including EGTA in the electrophoresis sample buffer [43], which allowed 32 P-(Tyr)-CaM to be resolved from the 32 P-histone bands. The auto(trans)-phosphorylated receptor (P-EGFR) was also visible as a 32 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 Ca 2 + , as this cation acts as an inhibitor of CaM phosphorylation as previously demonstrated [17,18,33,34]. As can be observed in Figure 1(D), in the presence of Ca 2 + P-(Tyr)-CaM was not accumulated and the subsequent extra activation of the EGFR in the presence of EGF was absent.…”

“…We have previously demonstrated that the EGFR phosphorylates CaM in the absence of Ca 2 + and presence of different cationic polypeptides with a stoichiometry close to 1 (mol/mol), preferentially at Tyr 99 as compared with Tyr 138 [17,18,[33][34][35][36][37]]. Hence, we tested whether phospho-(Tyr)-CaM exerts an effect aimed at modulating the ligand-dependent activation of EGFR.…”