Epidermal growth factor receptor signaling intensity determines intracellular protein interactions,ubiquitination, and internalization |
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Authors: | Schmidt Mirko H H Furnari Frank B Cavenee Webster K Bögler Oliver |
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Institution: | William and Karen Davidson Laboratory of Brain Tumor Biology, Hermelin Brain Tumor Center, Department of Neurosurgery, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202, USA. |
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Abstract: | Ligand activation of the epidermal growth factor receptor (EGFR) causes the binding of Cbls, which leads to EGFR polyubiquitination and internalization through endophilin complexes that contain the adaptor protein SH3-domain encoding, expressed in tumorigenic astrocytes/Cbl-interacting protein of 85 kDa/regulator of ubiquitous kinase (SETA/CIN85/Ruk). In cells grown at high density, high levels of SETA interfered in the recruitment of Casitas B-lineage (Cbl) proteins to the EGFR and reduced its polyubiquitination, suggesting that SETA has a regulatory function in the formation of the EGFR-Cbl-endophilin complex and in EGFR down-regulation. In a situation where there is EGFR signaling but no internalization or down-regulation, as is the case with the EGFR with exons 2-7 deleted (DeltaEGFR) oncogene, these proteins were absent altogether. By using mAb 806, which recognizes an EGFR-activation state and preferentially immunoprecipitates DeltaEGFR, we show that DeltaEGFR did not interact with Cbls, SETA, or endophilin A1, providing a mechanistic explanation for its lack of internalization. As would be expected by the absence of Cbl proteins in the DeltaEGFR complex, the mutant receptor was also not polyubiquitinated. The intracellular C terminus and tyrosine autophosphorylation pattern of DeltaEGFR are similar to wild-type EGFR, but it signals at a lower intensity as determined by levels of EGFR phosphotyrosine. To test the implication that the lack of interaction with the Cbl-SETA-endophilin complex is because of differences in signal intensity, EGFR-expressing cells were treated with tyrphostin AG1478 EGFR inhibitor. Attenuation of wild-type EGFR signal to levels similar to that found in DeltaEGFR resulted in the dissociation of SETA and Cbl proteins and a concomitant attenuation of receptor internalization. |
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