Ubiquitin ligase activity of c-Cbl guides the epidermal growth factor receptor into clathrin-coated pits by two distinct modes of Eps15 recruitment.
We have demonstrated previously that c-Cbl requires the presence of a functional ubiquitin interacting motif (UIM) in Eps15 to mediate epidermal growth factor receptor (EGFR) endocytosis. Both the ubiquitin ligase activity of c-Cbl and the UIM of Eps15 were necessary for plasma membrane recruitment of Eps15 and entry of ligand-bound EGFR into coated pits and vesicles containing Eps15. This is consistent with a scenario in which ubiquitin moieties appended to activated EGFR complexes act as docking sites for Eps15 and thereby recruit receptors into clathrin coated pits. Here, we have investigated which additional structural features of c-Cbl are required for this process. We find that c-Cbl can guide ligand-bound EGFR into the Eps15 internalization route by two distinct mechanisms. These are either dependent on the phosphotyrosine binding domain of c-Cbl that directly binds to the EGFR or on the region C-terminal of the Ring finger, which allows for indirect binding to an alternative site on the receptor. No strict requirement exists for either ubiquitin modified EGFR or the Cbl binding ubiquitination substrate CIN85 as docking site for the UIM of Eps15. Only in the phosphotyrosine binding-dependent pathway, the EGFR is ubiquitinated and may serve as a site of recruitment for Eps15. Only in this pathway, Eps15 is tyrosine-phosphorylated, but this appears unrelated to its capacity to participate in EGFR internalization.
Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Annemieke A
AA
de Melker
Gerda
G
van der Horst
Jannie
J
Borst
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism*; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008957:Models, Genetic; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; C486206:EPS15 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D025801:Ubiquitin; D014443:Tyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; C493849:Cbl protein, mouse