April 2006
Volume 5
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April 2006
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Differential Functions of the Ubiquitin-associated Domains of Cbl and Cbl-b Proteins: “Cblings,” But Not Twins
There are three mammalian Cbl proteins: Cbl, Cbl-b, and Cbl-c (also known as Cbl-3). The Cbl proteins are characterized by several highly conserved domains. They contain an N-terminal tyrosine kinase binding (TKB) domain that mediates interactions with tyrosine phosphorylated proteins, a C3HC4 RING finger (which is the catalytic domain for ubiquitin ligase [E3] activity), and proline-rich regions (which mediate interactions with SH3-containing proteins). Cbl and Cbl-b share additional areas of homology in the C-terminal region, including more extensive proline-rich regions and a ubiquitin-associated (UBA) domain. This study characterized biochemical differences in ubiquitin binding of the Cbl and Cbl-b proteins in order to better understand the unique function of each. While studying EGFR downregulation by Cbl-b, we observed that high molecular weight ubiquitinated proteins constitutively coimmunoprecipitated with transfected and endogenous Cbl-b, but not Cbl. The binding site for these ubiquitinated proteins was mapped to the UBA domain of Cbl-b (UBAb). A glutathione S-transferase (GST) fusion protein containing the UBAb interacted with ubiquitinated proteins from cell lysates and purified polyubiquitin chains in vitro. The UBAb had a much greater affinity for polyubiquitin chains than for monoubiquitin. The UBA domain of Cbl-b is necessary and sufficient for the interaction of Cbl-b with ubiquitin chains and ubiquitinated proteins. Interestingly, the homologous UBA domain of Cbl (UBAc) did not bind to ubiquitin or ubiquitin chains and did not mediate association with high molecular weight ubiquitinated proteins in cells. UBA domains are short domains consisting of three alpha helices that are found in a number of proteins associated with ubiquitin-mediated processes. UBA domains from a number of proteins bind to ubiquitin. Proteins containing UBA domains or structurally related ubiquitin-binding domains (i.e., CUE and UIM domains) have been shown to bind to ubiquitinated membrane proteins via these domains and to mediate ubiquitin-driven endocytosis. Because the Cbl proteins mediate ubiquitination and endocytosis of activated receptor tyrosine kinases, the interaction of Cbl-b with ubiquitinated proteins via its UBA domain was very intriguing. It is also somewhat surprising that the homologous UBA region of Cbl did not bind ubiquitin. This work is the first description of such a dramatic difference in the ubiquitin binding ability of two closely related UBA domains. Receptor endocytosis in both yeast and mammalian cells is mediated, in part, by ubiquitination. In yeast, proteins containing UBA or other ubiquitin-binding domains have been shown to mediate the internalization of monoubiquitinated proteins and their trafficking to the vacuole. Thus, we investigated whether UBAb plays a role in EGFR downregulation. We did not find a significant difference in the downregulation of EGFR by wild-type Cbl-b or Cbl-b with the UBA deleted. Thus, UBAb does not seem necessary for EGFR trafficking. In contrast to the deletion of the UBA domain of Cbl-b, overexpression of UBAb, but not of UBAc, inhibited degradation of ubiquitinated EGFR as well as other proteins (i.e., Mdm-2 and Siah-1). This is most likely attributable to non-specific ubiquitin binding of isolated UBAb. This in vivo result is consistent with the differences in ubiquitin binding observed in vitro between UBAb and UBAc. The difference in ubiquitin-binding of the UBA domains of Cbl-b and Cbl likely reflects distinct regulatory functions of the proteins and warrants further investigation. One possible function of the ubiquitin binding of UBAb would be to target Cbl-b to a specific protein or subcellular localization. Another possible function would be the regulation of ubiquitin-mediated protein degradation. One mechanism by which the UBA domains can regulate ubiquitin-mediated processes is by inhibition of chain elongation of nascent ubiquitin chains. Interestingly, Cbl proteins have been shown to monoubiquitinate activated EGFR at multiple sites and target it for lysosomal degradation. Other published data suggest that Cbl proteins can polyubiquitinate some substrates and target them for proteasomal degradation. Thus, it is possible that the UBA domain of Cbl-b can regulate the length of the ubiquitin chains added to substrates. Another mechanism proposed for the inhibition of proteasomal degradation by UBA domains is masking of the ubiquitin chains, thus preventing the ubiquitinated protein from binding to the proteasome. In our work, we have found that overexpression of UBAb (but not of UBAc) inhibits ubiquitin-mediated protein degradation of a variety of proteins. This is likely due to the masking of the ubiquitin molecules attached to these proteins by the overexpressed UBAb, which in turn, prevents the proper recognition and degradation of the ubiquitinated proteins by the proteasome or the lysosome. Although further work is necessary to understand the physiologic function of UBAb, the differences in ubiquitin binding between the UBA domains of Cbl and Cbl-b provide clear evidence that these highly homologous proteins have distinct roles in epithelial cells. |
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bl
proteins are a highly conserved family of proteins found in metazoans. Upon
activation of a variety of tyrosine kinases, the Cbl proteins are tyrosine phosphorylated,
and they associate with other proteins via SH2- and SH3-mediated interactions.
These diverse interactions regulate signaling through numerous pathways. Our
laboratory has focused on the downregulation of epidermal growth factor receptor
(EGFR) as a model in which to study Cbl protein function in epithelial cells.
Mechanistic data from many labs, including our own, have demonstrated that Cbl
proteins mediate ubiquitination of the activated EGFR and enhance its endocytosis
and degradation. Thus, they are negative regulators of EGFR signaling. Parallel
studies on other receptor and non-receptor tyrosine kinases have demonstrated
that Cbl proteins similarly regulate a wide range of signaling pathways. Together,
these data indicate that the Cbl proteins are important regulators of intracellular
signaling and, consequently, of cell function and development.