c-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route.
Cbl family members have an evolutionarily conserved role in attenuating receptor tyrosine kinase function. Their negative regulatory capacity depends on a Ring finger domain that interacts with ubiquitin conjugating enzymes. Cbl molecules constitute a novel type of E3 or ubiquitin ligase family that is recruited to phosphotyrosine motifs. Ubiquitination of the receptor system is coupled to its downregulation, but it is unclear at which point in the endocytic pathway Cbl molecules come into play. Using low temperature and a dynamin mutant, we find that c-Cbl associates with and ubiquitinates the activated epidermal growth factor (EGF) receptor at the plasma membrane in the absence of endocytosis. With the aid of confocal microscopy and immunogold electron microscopy, we could demonstrate that c-Cbl associates with the EGF receptor at the plasma membrane prior to receptor recruitment into clathrin-coated pits and remains associated throughout the clathrin-mediated endocytic pathway. c-Cbl and the EGF receptor also colocalize in internal vesicles of multivesicular endosomes. Our data are consistent with a role for c-Cbl in clathrin-mediated endocytosis of tyrosine kinase receptors, as well as their intracellular sorting.
de Melker
A A
AA
Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
van der Horst
G
G
Calafat
J
J
Jansen
H
H
Borst
J
J
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D006224:Cricetinae; D034281:Dynamins; D004705:Endocytosis; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D009154:Mutation; D011485:Protein Binding; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D011518:Proto-Oncogene Proteins; D025801:Ubiquitin; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D020558:GTP Phosphohydrolases; D034281:Dynamins