Leucine zipper-mediated homodimerization of the adaptor protein c-Cbl. A role in c-Cbl's tyrosine phosphorylation and its association with epidermal growth factor receptor.
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.
Bartkiewicz
M
M
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Houghton
A
A
Baron
R
R
D002460:Cell Line; D019281:Dimerization; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D016350:Leucine Zippers; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D019000:Phosphotyrosine / Q000032:analysis; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection; D044767:Ubiquitin-Protein Ligases
D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010768:Phosphoserine; D019000:Phosphotyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; C067419:CBL protein, human