title: Phosphorylation of Thr654 but not Thr669 within the juxtamembrane domain of the EGF receptor inhibits calmodulin binding.
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 (Met645-Phe688) of EGFR. Protein kinase C (PKC) mediated phosphorylation of Thr654 occurs within this domain. CaM binding to the JM domain inhibits PKC phosphorylation and conversely PKC mediated phosphorylation of Thr654 or Glu substitution of Thr654 inhibits CaM binding. A second threonine residue (Thr669) 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 Thr669 affects CaM-EGFR interaction is however not known. In the present study we have used surface plasmon resonance (BIAcore) to study the influence of Thr669 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 Thr654 or Thr669. Purified proteins were coupled to immobilized anti-GST antibodies at the sensor surface and increasing concentration of CaM was applied. When mutating Thr654 to Glu654 no specific CaM binding could be detected. However, neither single substitutions of Thr669 (Gly669 or Glu669) nor double mutants Gly654/Gly669 or Gly654/Glu669 influenced the binding of CaM to the EGFR-JM. This clearly shows that PKC may regulate EGF-mediated CaM signalling through phosphorylation of Thr654 whereas phosphorylation of Thr669 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 Thr654 from the JM domain interacts with Glu120 in the calmodulin molecule. Phosphorylation of Thr654 or Glu654 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 Thr654 mutated to Glu. Furthermore, these results provide a hypothesis to how CaM binding to EGFR might both positively and negatively interfere with EGFR-activity.
authors.affiliation: Department of Pharmacology, Faculty of Health Sciences, University of Linköping, SE-58185 Linköping, Sweden.
authors.forename: Sami
authors.initials: S
authors.lastname: Aifa
authors.forename: Fakher
authors.initials: F
authors.lastname: Frikha
authors.forename: Nabil
authors.initials: N
authors.lastname: Miled
authors.forename: Knut
authors.initials: K
authors.lastname: Johansen
authors.forename: Ingemar
authors.initials: I
authors.lastname: Lundström
authors.forename: Samuel P S
authors.initials: SP
authors.lastname: Svensson
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance / Q000379:methods; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D008565:Membrane Proteins; D013912:Threonine; D066246:ErbB Receptors; D011493:Protein Kinase C; D002118:Calcium