The evolutionarily conserved N-terminal region of Cbl is sufficient to enhance down-regulation of the epidermal growth factor receptor.
The mammalian proto-oncoprotein Cbl and its homologues in Caenorhabditis elegans and Drosophila are evolutionarily conserved negative regulators of the epidermal growth factor receptor (EGF-R). Overexpression of wild-type Cbl enhances down-regulation of activated EGF-R from the cell surface. We report that the Cbl tyrosine kinase-binding (TKB) domain is essential for this activity. Whereas wild-type Cbl enhanced ligand-dependent EGF-R ubiquitination, down-regulation from the cell surface, accumulation in intracellular vesicles, and degradation, a Cbl TKB domain-inactivated mutant (G306E) did not. Furthermore, the transforming truncation mutant Cbl-N (residues 1-357), comprising only the Cbl TKB domain, functioned as a dominant negative protein. It colocalized with EGF-R in intracellular vesicular structures, yet it suppressed down-regulation of EGF-R from the surface of cells expressing endogenous wild-type Cbl. Therefore, Cbl-mediated down-regulation of EGF-R requires the integrity of both the N-terminal TKB domain and additional C-terminal sequences. A Cbl truncation mutant comprising amino acids 1-440 functioned like wild-type Cbl in down-regulation assays. This mutant includes the evolutionarily conserved TKB and RING finger domains but lacks the less conserved C-terminal sequences. We conclude that the evolutionarily conserved N terminus of Cbl is sufficient to effect enhancement of EGF-R ubiquitination and down-regulation from the cell surface.
Lill
N L
NL
Lymphocyte Biology Section, Division of Rheumatology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Douillard
P
P
Awwad
R A
RA
Ota
S
S
Lupher
M L
ML
Miyake
S
S
Meissner-Lula
N
N
Hsu
V W
VW
Band
H
H
D000595:Amino Acid Sequence; D001692:Biological Transport; D017124:Conserved Sequence; D015536:Down-Regulation; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
D006388:Hemagglutinins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases