Characterization of the Ubiquitin Ligase Cbl-c

Cbl proteins are ubiquitin ligases (E3s) which a play significant role in regulating tyrosine kinase (TK) signaling. There are three mammalian family members: c-Cbl, Cbl-b and Cbl-c.  All have a highly conserved N-terminal tyrosine kinase binding domain (TKB), a catalytic RING-finger domain (RF) and a C-terminal proline rich domain (PR) which mediates interactions with SH3 containing proteins.  While the activity and cellular roles of c-Cbl and Cbl-b have been studied widely, little is known about Cbl-c.  The purpose of this research has been to understand better Cbl-c through biochemical analysis of its E3 activity as well as through cellular studies of its interacting partners and function.  This study was accomplished in three parts:  I) studying the regulation of the E3 activity of Cbl-c; II) identifying a Cbl-c interacting protein and characterizing the interaction; and III) studying the ability of the Cbl proteins, including Cbl-c, to downregulate the Epidermal Growth Factor Receptor variant III.

I)  Published reports have demonstrated that the N-terminus of c-Cbl and Cbl-b have an inhibitory effect on their respective E3 activity.  An in vitro auto-ubiquitination assay was used to measure the E3 activity of Cbl-c wild type (WT) proteins compared to various deletion mutations.  Like c-Cbl and Cbl-b the N-terminus of Cbl-c inhibits the E3 activity of Cbl-c.  Deletion mapping shows that a 170 amino acid region of the TKB is responsible for this inhibition.  The phosphorylation of Cbl-c by Src leads to an increase in activity.  Mutational analysis demonstrated that Src phosphorylated tyrosine 341 (Y341) of Cbl-c and that this phosphorylation is both necessary and sufficient for activation of Cbl-c E3 activity.  However, there are additional sites of tyrosine phosphorylation.   An activating mutation that mimics phosphorylation (Y341E) was used to determine the mechanism by which the N-terminus inhibits and by which phosphorylation activates the protein.  The Cbl-c Wild Type (WT) protein binds to the ubiquitin-conjugating enzyme (E2) with a higher affinity than Y341E.  Since the process of poly-ubiquitination is a cyclical process that requires the release and rebinding of an E2, the slower release of the E2 from the unactivated form of Cbl-c leads to a lower enzymatic rate.  In conclusion, the N-terminal TKB increases the affinity of the RF of Cbl-c to the E2, and this affinity can be decreased by phosphorylation of Y341 on Cbl-c by Src thus leading to an increase in E3 activity.

II) To begin to study the physiologic role of Cbl-c, we sought to identify Cbl-c interacting proteins.  A yeast-two hybrid screen identified a new Cbl-c interaction protein, Hydrogen peroxide Inducible Construct 5 (Hic-5).  Coimmunoprecipitation experiments confirm this interaction and demonstrate that the interaction is mediated by the RF of Cbl-c and the LIM2 domain of Hic-5.  The use of various point mutations showed that disruption of the second zinc coordinating complex of the RF, but not the first, abrogates the interaction.  Likewise, disruption of the first zinc coordinating complex of the LIM2 domain of Hic-5, but not the second, abrogates the interaction.  Preliminary results suggest that Hic-5 can block Cbl-c mediated degradation of the EGFR but that it does not inhibit Cbl-c mediated ubiquitination of the receptor.   In conclusion, Cbl-c and Hic-5 interact through a novel RF/LIM mediated interaction and this interaction provides for a more directed study of the physiologic role of Cbl-c.

III) The most common EGFR mutant in glioblastomas (EGFRvIII) results from the deletion of exons 2–7.  This mutant receptor cannot bind EGF but is spontaneously active instead.  Since the EGFRvIII is transforming, we investigated whether it could be downregulated by the Cbl proteins.  The overexpression of all three Cbl proteins resulted in the ubiquitination and degradation of the EGFRvIII.  As with the EGFR WT, the TK binding domain and the RF of Cbl-b are sufficient for the downregulation of the EGFRvIII.  Also, we found that Cbl-b is recruited to the EGFRvIII and inhibits the transformation of NIH 3T3 cells by the EGFRvIII.  Mutation of the Cbl binding site (Y1045F) in the EGFRvIII inhibits its ubiquitination and downregulation by Cbl-b and enhances its ability to transform.  Furthermore, the EGFR TK inhibitor, AG 1478, prevents the downregulation of the EGFRvIII by the Cbl proteins and antagonizes the ability of an immunotoxin directed against the EGFRvIII to kill cells expressing this receptor.  In conclusion, the EGFRvIII does not transform by escaping regulation by Cbl proteins and this activation-induced downregulation of the EGFRvIII has an important role in mediating the toxicity of anti-EGFRvIII immunotoxins.