Phosphorylation of Calmodulin by the Epidermal‐growth‐factor‐receptor Tyrosine Kinase

Abstract

An epidermal-growth-factor(EGF)-receptor preparation isolated by calmodulin-affinity chromatography from rat liver plasma membranes is able to phosphorylate calmodulin. Calmodulin phosphorylation was enhanced 3-8-fold by EGF, was dependent on the presence of a polycation or basic protein and was inhibited by micromolar concentrations of Ca2+. Phosphate incorporation into calmodulin occurs predominantly on tyrosine residues. Partial proteolysis of phosphocalmodulin by thrombin identifies Tyr99, located in the third calcium-binding domain of calmodulin, as the phosphorylated residue. Stoichiometric measurements show a 3ZP/calmodulin molar ratio of approximately 1 when optimal phosphorylation conditions are used.Calmodulin is an intracellular calcium receptor that mediates multiple essential functions in eukaryotic cells (Means and Dedman, 1980; Klee et al., 1980;Klee and Vanaman, 1982;Manalan and Klee, 1984;Veigl et al., 1984;Strynadka and James, 1989;Bachs et al., 1992), including the regulation of cell proliferation (Veigl et al., 1984). In this context, it has been demonstrated that this ubiquitous regulator can control multiple nuclear processes (Bachs et al., 1992). Nevertheless, the role of calmodulin in cell proliferation does not appear to be exclusively exerted at the level of the nucleus. Recently we have demonstrated that the epidermal growth factor (EGF) receptor can be isolated by calmodulin-affinity chromatography, and that calmodulin inhibits its tyrosine lunase activity (San JosC et al., 1992). Calmodulin, therefore, could potentially play a regulatory role in the EGF-mediated mitogenic-signal pathway.Calmodulin has been shown to be a substrate for several different protein kinases (Plancke and Lazarides, 1983 ;Hiiring et al., 1985;Fukami et al., 1985;Graves et al., 1986;Lin et al., 1986;Nakajo et al., 1986Nakajo et al., , 1988Meggio et al., 1987Meggio et al., , 1992Kubo and Strott, 1988;Sacks and McDonald, 1988;Laurino et al., 1988; Sacks et al., 1989aSacks et al., , 1992aSan JosC et al., 1992;Benguria et al., 1993;Saville and Houslay, 1994). Since this phosphorylation process occurs in intact

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).…”

“…The non-receptor tyrosine kinase Src [48], the insulin receptor [51] and the EGFR [33], are kinases involved in the phosphorylation of CaM at both Tyr 99 and Tyr 138 . The dynamic nature of this process and the distinct phosphorylated forms of CaM at a given time could be difficult to track in living cells due to the short half-life of P-(Tyr)-CaM.…”

“…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.…”