Phosphorylation of Thr654 but not Thr669 within the juxtamembrane domain of the EGF receptor inhibits calmodulin binding

Calcium-calmodulin (CaM) binding to the epidermal growth factor receptor (EGFR) has been shown to both inhibit and stimulate receptor activity. CaM binds to the intracellular juxtamembrane (JM) domain (Met(645)-Phe(688)) of EGFR. Protein kinase C (PKC) mediated phosphorylation of Thr(654) occurs within this domain. CaM binding to the JM domain inhibits PKC phosphorylation and conversely PKC mediated phosphorylation of Thr(654) or Glu substitution of Thr(654) inhibits CaM binding. A second threonine residue (Thr(669)) within the JM domain is phosphorylated by the mitogen-activated protein kinase (MAPK). Previous results have shown that CaM interferes with EGFR-induced MAPK activation. If and how phosphorylation of Thr(669) affects CaM-EGFR interaction is however not known. In the present study we have used surface plasmon resonance (BIAcore) to study the influence of Thr(669) phosphorylation on real time interactions between the intracellular juxtamembrane (JM) domain of EGFR and CaM. The EGFR-JM was expressed as GST fusion proteins in Escherichia coli and phosphorylation was mimicked by generating Glu substitutions of either Thr(654) or Thr(669). Purified proteins were coupled to immobilized anti-GST antibodies at the sensor surface and increasing concentration of CaM was applied. When mutating Thr(654) to Glu(654) no specific CaM binding could be detected. However, neither single substitutions of Thr669 (Gly(669) or Glu(669)) nor double mutants Gly(654)/Gly(669) or Gly(654)/Glu(669) influenced the binding of CaM to the EGFR-JM. This clearly shows that PKC may regulate EGF-mediated CaM signalling through phosphorylation of Thr(654) whereas phosphorylation of Thr(669) seems to play a CaM independent regulatory role. The role of both residues in the EGFR-calmodulin interaction was also studied in silico. Our modelling work supports a scenario where Thr(654) from the JM domain interacts with Glu(120) in the calmodulin molecule. Phosphorylation of Thr(654) or Glu(654) substitution creates a repulsive electrostatic force that would diminish CaM binding to the JM domain. These results are in line with the Biacore experiments showing a weak binding of the CaM to the JM domain with Thr(654) mutated to Glu. Furthermore, these results provide a hypothesis to how CaM binding to EGFR might both positively and negatively interfere with EGFR-activity. (c) 2006 Elsevier Inc. All rights reserved.