Aifa Sami, Frikha Fakher, Miled Nabil, Johansen Knut, Lundström Ingemar, Svensson Samuel P S
Department of Pharmacology, Faculty of Health Sciences, University of Linköping, SE-58185 Linköping, Sweden.
Biochem Biophys Res Commun. 2006 Aug 25;347(2):381-7. doi: 10.1016/j.bbrc.2006.05.200. Epub 2006 Jun 9.
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.
钙调蛋白（CaM）与表皮生长因子受体（EGFR）的结合已被证明既能抑制也能刺激受体活性。CaM与EGFR的细胞内近膜（JM）结构域（Met645 - Phe688）结合。蛋白激酶C（PKC）介导的Thr654磷酸化发生在该结构域内。CaM与JM结构域的结合会抑制PKC磷酸化，反之，PKC介导的Thr654磷酸化或Thr654被Glu取代会抑制CaM结合。JM结构域内的第二个苏氨酸残基（Thr669）由丝裂原活化蛋白激酶（MAPK）磷酸化。先前的结果表明CaM会干扰EGFR诱导的MAPK激活。然而，Thr669的磷酸化是否以及如何影响CaM - EGFR相互作用尚不清楚。在本研究中，我们使用表面等离子体共振（BIAcore）来研究Thr669磷酸化对EGFR细胞内近膜（JM）结构域与CaM实时相互作用的影响。EGFR - JM以GST融合蛋白的形式在大肠杆菌中表达，通过将Thr654或Thr669替换为Glu来模拟磷酸化。纯化的蛋白在传感器表面与固定的抗GST抗体偶联，并施加浓度递增的CaM。当将Thr654突变为Glu654时，未检测到特异性的CaM结合。然而，Thr669的单取代（Gly669或Glu669）以及双突变体Gly654 / Gly669或Gly654 / Glu669均未影响CaM与EGFR - JM的结合。这清楚地表明PKC可能通过Thr654的磷酸化来调节EGF介导的CaM信号传导，而Thr669的磷酸化似乎发挥着不依赖CaM的调节作用。还通过计算机模拟研究了这两个残基在EGFR - 钙调蛋白相互作用中的作用。我们的建模工作支持一种情况，即JM结构域中的Thr654与钙调蛋白分子中的Glu120相互作用。Thr654的磷酸化或Glu654取代会产生排斥性静电力，这会减少CaM与JM结构域的结合。这些结果与Biacore实验一致，该实验表明当Thr654突变为Glu时，CaM与JM结构域的结合较弱。此外，这些结果为CaM与EGFR的结合如何正向和负向干扰EGFR活性提供了一个假设。
