Cbl-b interacts with ubiquitinated proteins; differential functions of the UBA domains of c-Cbl and Cbl-b.
Cbl proteins are ubiquitin protein ligases, which ubiquitinate activated tyrosine kinases and target them for degradation. Both c-Cbl and Cbl-b have an ubiquitin associated (UBA) domain at their C-terminal end. We observed that high molecular weight ubiquitinated proteins constitutively coimmunoprecipitated with transfected and endogenous Cbl-b, but not c-Cbl. The binding site for these ubiquitinated proteins was mapped to the UBA domain of Cbl-b (UBAb). GST-fusion proteins containing the UBAb interacted with ubiquitinated proteins and polyubiquitin chains in vitro, whereas those containing the UBA domain of c-Cbl (UBAc) did not. The UBAb had a much greater affinity for polyubiquitin chains than for monoubiquitin. Analysis of the UBAb and UBAc demonstrate that the affinity for ubiquitin is determined by multiple amino-acid differences between the two domains. Overexpression of the UBAb, but not overexpression of the UBAc, inhibited a variety of ubiquitin-mediated processes such as degradation of ubiquitinated proteins (i.e. EGFR, Mdm-2, and Siah-1). This in vivo result is consistent with the differences in ubiquitin binding observed in vitro between the UBAb and UBAc. This difference in ubiquitin-binding may reflect distinct regulatory functions of c-Cbl and Cbl-b.
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, Bethesda, MD 20892, USA.
Gareth C
GC
Davies
Seth A
SA
Ettenberg
Ashley O
AO
Coats
Mark
M
Mussante
Sarangan
S
Ravichandran
Jack
J
Collins
Marion M
MM
Nau
Stan
S
Lipkowitz
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; C492211:CBLB protein, human; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases