Abstract
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 Thr
669 (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.