Interaction between two ubiquitin-protein isopeptide ligases of different classes, CBLC and AIP4/ITCH.
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.
Courbard
Jean-Rémy
JR
Département d'Oncologie Moléculaire, U119 INSERM, 27 Boulevard Lei Roure, 13009 Marseille and Ipsogen SA, Institut Paoli-Calmettes, 3009 Marseille, France.
Fiore
Frédéric
F
Adélaïde
José
J
Borg
Jean-Paul
JP
Birnbaum
Daniel
D
Ollendorff
Vincent
V
D000818:Animals; D019556:COS Cells; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D016335:Zinc Fingers
D029742:Caenorhabditis elegans Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D025801:Ubiquitin; C432966:ITCH protein, human; C471637:Itch protein, mouse; C471642:WWP1 protein, human; C413402:wwp-1 protein, C elegans; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D008025:Ligases; C119570:CBLC protein, human