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Interaction between two ubiquitin-protein isopeptide ligases of different classes, CBLC and AIP4/ITCH
Jean-Rémy Courbard1, Frédéric Fiore, José Adélaïde
1Département d'Oncologie Moléculaire, U119 INSERM, 27 Boulevard Lei Roure, 13009 Marseille and Ipsogen SA, Institut Paoli-Calmettes, 3009 Marseille, France.
Abstract:
In metazoans, CBL proteins are RING finger type ubiquitin-protein isopeptide (E3) ligases involved in the down-regulation of epidermal growth factor tyrosine kinase receptors (EGFR). Among the three CBL proteins described in humans, CBLC (CBL3) remains poorly studied. By screening in parallel a human and a Caenorhabditis elegans library using the two-hybrid procedure in yeast, we found a novel interaction between Hsa-CBLC and Hsa-AIP4 or its C. elegans counterpart Cel-WWP1. Hsa-AIP4 and Cel-WWP1 are also ubiquitin E3 ligases. They contain a HECT (homologous to E6-AP C terminus) catalytic domain and four WW domains known to bind proline-rich regions. We confirmed the interaction between Hsa-CBLC and Hsa-AIP4 by a combination of glutathione S-transferase pull-down, co-immunoprecipitation, and colocalization experiments. We show that these two E3 ligases are involved in EGFR signaling because both become phosphorylated on tyrosine following epidermal growth factor stimulation. In addition, we observed that CBLC increases the ubiquitination of EGFR, and that coexpressing the WW domains of AIP4 exerts a dominant negative effect on EGFR ubiquitination. Finally, coexpressing CBLC and AIP4 induces a down-regulation of EGFR signaling. In conclusion, our data demonstrate that two E3 ligases of different classes can interact and cooperate to down-regulate EGFR signaling.
Insights
Two ubiquitin E3 ligases, CBLC and AIP4, interact to down-regulate epidermal growth factor receptor (EGFR) signaling. This study reveals a novel cooperation between different E3 ligase classes in EGFR pathway regulation.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- CBL proteins are E3 ubiquitin ligases crucial for down-regulating receptor tyrosine kinases.
- CBLC (CBL3) is a less-studied human CBL protein involved in signaling pathways.
- Epidermal growth factor receptor (EGFR) signaling is vital in metazoan development and disease.
Purpose of the Study:
- To investigate the function and interactions of the human CBLC protein.
- To identify novel binding partners of CBLC using a yeast two-hybrid screen.
- To elucidate the role of CBLC and its interacting partners in EGFR signaling regulation.
Main Methods:
- Yeast two-hybrid screening of human and C. elegans libraries.
- Glutathione S-transferase (GST) pull-down assays.
- Co-immunoprecipitation and colocalization experiments.
- Analysis of EGFR ubiquitination and signaling pathway activity.
Main Results:
- A novel interaction was identified between human CBLC and human AIP4 (HECT-type E3 ligase).
- Both CBLC and AIP4 are phosphorylated upon epidermal growth factor stimulation, indicating involvement in EGFR signaling.
- CBLC enhances EGFR ubiquitination, and AIP4's WW domains inhibit this process.
- Co-expression of CBLC and AIP4 leads to EGFR signaling down-regulation.
Conclusions:
- CBLC and AIP4, distinct E3 ligase classes, physically interact.
- These E3 ligases cooperate to regulate EGFR signaling.
- The findings reveal a novel mechanism for EGFR pathway control involving the coordinated action of multiple E3 ligases.