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Calmodulin regulates intracellular trafficking of epidermal growth factor receptor and the MAPK signaling pathway
Francesc Tebar1, Priam Villalonga, Tatiana Sorkina
1Departament de Biologia Cel.lular, Facultat de Medicina, Institut d'Investigacions August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain 08036.
Abstract:
The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family involved in signal transduction and the regulation of cellular proliferation and differentiation. It is also a calmodulin-binding protein. To examine the role of calmodulin in the regulation of EGFR, the effect of calmodulin antagonist, W-13, on the intracellular trafficking of EGFR and the MAPK signaling pathway was analyzed. W-13 did not alter the internalization of EGFR but inhibited its recycling and degradation, thus causing the accumulation of EGF and EGFR in enlarged early endosomal structures. In addition, we demonstrated that W-13 stimulated the tyrosine phosphorylation of EGFR and consequent recruitment of Shc adaptor protein with EGFR, presumably through inhibition of the calmodulin-dependent protein kinase II (CaM kinase II). W-13-mediated EGFR phosphorylation was blocked by metalloprotease inhibitor, BB94, indicating a possible involvement of shedding in this process. However, MAPK activity was decreased by W-13; dissection of this signaling pathway showed that W-13 specifically interferes with Raf-1 activity. These data are consistent with the regulation of EGFR by calmodulin at several steps of the receptor signaling and trafficking pathways.
Insights
Calmodulin regulates epidermal growth factor receptor (EGFR) signaling and trafficking. An antagonist, W-13, inhibits EGFR recycling and degradation, impacting MAPK signaling by interfering with Raf-1 activity.
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor crucial for cellular processes.
- EGFR is implicated in signal transduction, proliferation, and differentiation.
- EGFR is also identified as a calmodulin-binding protein.
Purpose of the Study:
- To investigate the role of calmodulin in regulating EGFR.
- To analyze the effects of the calmodulin antagonist W-13 on EGFR intracellular trafficking and the MAPK signaling pathway.
Main Methods:
- Treatment of cells with W-13, a calmodulin antagonist.
- Analysis of EGFR internalization, recycling, and degradation.
- Assessment of EGFR tyrosine phosphorylation and Shc protein recruitment.
- Evaluation of MAPK signaling pathway activity, specifically Raf-1.
- Use of metalloprotease inhibitor BB94 to investigate shedding.
Main Results:
- W-13 inhibited EGFR recycling and degradation, leading to EGFR accumulation in endosomes.
- W-13 stimulated EGFR tyrosine phosphorylation and Shc recruitment, potentially via CaM kinase II inhibition.
- EGFR phosphorylation was blocked by BB94, suggesting shedding involvement.
- W-13 decreased MAPK activity by interfering with Raf-1.
Conclusions:
- Calmodulin plays a regulatory role in multiple steps of EGFR signaling and trafficking.
- W-13 affects EGFR trafficking and signaling, highlighting calmodulin's importance.
- The findings provide insights into the complex regulation of EGFR by calmodulin.