View Program - asbmb

33
Cbl Amino Acids at a Putative Dimer Interface Regulate
EGF Receptor Trafficking
Nancy Lill 1 , Rajeev Sachdeva 1 , Solomon Abay 1 , Ilter Sever 1 , Gina Visser Smit 2 , Trenton Place 2 ,
John Koland 2 .
The Ohio State University, Pathology and OSUCCC, Columbus, OH 43210 1 , University of Iowa,
Pharmacology, Iowa City, IA 52242 2
Abstract:
The ubiquitin ligase c-Cbl controls epidermal growth factor receptor (EGF-R) signaling by
enhancing receptor ubiquitination, downregulation, and degradation. Cbl residues 1-434 are
the minimal evolutionarily conserved sequences sufficient to regulate EGF-R. They contain a
variant SH2 domain, linker region, RING finger (RF), and a subset of the RF tail amino acids
420-436. Data from a solved Cbl/UbcH7 crystal structure suggest that Cbl dimerizes through its
RF tail. Our objective was to perform structure/function studies to identify amino acids at the
putative Cbl dimer interface that regulate EGF-R fate. Constructs encoding the Cbl RF tail and
opposing interface alanine substitution mutants were developed by oligonucleotide-directed
mutagenesis. Wild type and mutant proteins were expressed in HEK 293 and COS-7 cells,
which were then assayed for EGF-R downregulation, trafficking, and degradation. Trafficking
was followed by fluorescence localization of GFP-tagged Cbl proteins, EGF and markers of
endocytic compartments in fixed and live cell imaging studies. The integrity of RF tail residues
V431 and F434 is essential for Cbl-dependent EGF-R degradation in lysosomes. Full-length Cbl
V431A failed to effect the ubiquitin-dependent degradation of hSprouty2 and blocked EGF-R
internalization. Full-length Cbl F434A compromised EGF-R degradation by stabilizing
phosphorylated Hrs and retarding sorting endosome fusion. Using these mutants with different
epitope tags, we determined that Cbl forms dimers and that dimer levels are altered by RF tail
amino acid substitutions. The mutation of residues on the opposite side of the putative Cbl
dimer interface revealed that only some interface residues are critical for EGF-R regulation. Our
results suggest that Cbl dimers can regulate EGFR fate at multiple trafficking checkpoints.