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Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living
A M Honegger1, A Schmidt, A Ullrich
1Rorer Biotechnology, Inc., King of Prussia, Pennsylvania 19406.
Abstract:
In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries.
Insights
Epidermal Growth Factor Receptor (EGFR) cross-phosphorylation occurs in living cells. A kinase-negative EGFR mutant suppresses EGFR signaling, highlighting its role in mitogenic response.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Epidermal Growth Factor (EGF) binding activates the intrinsic protein tyrosine kinase of the EGF Receptor (EGFR).
- EGFR activation leads to tyrosine phosphorylation of cellular substrates and autophosphorylation of the EGFR itself.
- Understanding EGFR autophosphorylation mechanisms in living cells is crucial for deciphering signal transduction pathways.
Purpose of the Study:
- To investigate the mechanism of EGFR autophosphorylation in living cells.
- To demonstrate EGFR cross-phosphorylation between receptor molecules in real-time.
- To assess the impact of a kinase-negative EGFR mutant on cellular response to EGF.
Main Methods:
- Established transfected cell lines coexpressing a kinase-negative EGFR mutant (K721A) and an active EGFR mutant.
- Stimulated cells with EGF and analyzed tyrosine phosphorylation of the kinase-negative mutant.
- Investigated the trafficking pathways of the kinase-negative mutant and CD63 after internalization.
- Compared the mitogenic response to EGF in cells expressing active EGFR alone versus coexpressing active and kinase-negative EGFR.
Main Results:
- Demonstrated EGFR cross-phosphorylation in living cells, where the active EGFR phosphorylated the kinase-negative mutant (K721A).
- Observed separate trafficking pathways for the kinase-negative mutant and CD63 post-internalization, limiting their association.
- Showed that coexpression of the kinase-negative mutant suppressed the mitogenic response to EGF compared to cells with active EGFR alone.
- Identified the kinase-negative mutant as a dominant-negative mutation interfering with EGF-induced signal transduction.
Conclusions:
- EGFR cross-phosphorylation is a key event occurring in living cells.
- Separate endocytic itineraries of EGFR mutants influence their interactions and signaling outcomes.
- The kinase-negative EGFR mutant acts as a dominant-negative inhibitor, suppressing EGF-mediated mitogenesis by interfering with signal transduction pathways prior to receptor separation.