Cbl-b interacts with ubiquitinated proteins; differential functions of the UBA domains of c-Cbl and Cbl-b

Proc Amer Assoc Cancer Res, Volume 45, 2004 3935 Cbl proteins are ubiquitin protein ligases (E3s) which ubiquitinate activated tyrosine kinases, such as the EGFR, and target them for degradation. Thus, they act as negative regulators of tyrosine kinase receptor signaling. The three mammalian Cbl proteins, c-Cbl, Cbl-b, and Cbl-3, have several highly conserved domains including an N-terminal tyrosine kinase binding domain and a RING finger domain which are essential for their E3 activity. c-Cbl and Cbl-b, but not Cbl-3, contain an extensive C-terminal region containing proline rich and ubiquitin-associated (UBA) domains. We observed that high molecular weight ubiquitinated proteins constitutively coimmunoprecipitated with 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. The significance of the difference in ubiquitin binding between the UBAb and UBAc was confirmed in vivo . Overexpression of the UBAb, but not overexpression of the UBAc, inhibited a variety of ubiquitin-mediated processes such as degradation of ubiquitinated proteins (EGFR, Mdm-2, and Siah-1). The difference in ubiquitin-binding may reflect distinct regulatory functions of c-Cbl and Cbl-b.