The 120-kDa proto-oncogenic protein c-Cbl is a multidomain adaptor protein that is phosphorylated in response to the stimulation of a broad range of cell surface receptors and participates in the assembly of signaling complexes that are formed as a result of the activation of various signal transduction pathways. Several structural features of c-Cbl, including the phosphotyrosine-binding domain, proline-rich domain, and motifs containing phosphotyrosine and phosphoserine residues, mediate the association of c-Cbl with other components of these complexes. In addition to those domains that have been demonstrated to play a role in the binding of c-Cbl to other signaling molecules, c-Cbl also contains a RING finger motif and a putative leucine zipper. In this study, we demonstrate that the previously identified putative leucine zipper mediates the formation of Cbl homodimers. Using the yeast two-hybrid system, we show that deletion of the leucine zipper domain is sufficient to abolish Cbl homodimerization, while Cbl mutants carrying extensive N-terminal truncations retain the ability to dimerize with the full-length Cbl. The requirement of the leucine zipper for the homodimerization of Cbl was confirmed by in vitro binding assays, using deletion variants of the C-terminal half of Cbl with and without the leucine zipper domain, and in cells using Myc and green fluorescent protein (GFP) N-terminal-tagged Cbl variants. In cells, the deletion of the leucine zipper caused a decrease in both the tyrosine phosphorylation of Cbl and its association with the epidermal growth factor receptor following stimulation with epidermal growth factor, thus demonstrating a role for the leucine zipper in c-Cbl's signaling functions. Thus, the leucine zipper domain enables c-Cbl to homodimerize, and homodimerization influences Cbl's signaling function, modulating the activity of Cbl itself and/or affecting Cbl's associations with other signaling proteins in the cell.
Pubmed ID: 10521482 RIS Download
Mesh terms: Cell Line | Dimerization | Humans | Leucine Zippers | Phosphorylation | Phosphoserine | Phosphotyrosine | Proto-Oncogene Proteins | Proto-Oncogene Proteins c-cbl | Receptor, Epidermal Growth Factor | Recombinant Fusion Proteins | Recombinant Proteins | Saccharomyces cerevisiae | Transfection | Ubiquitin-Protein Ligases
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