The RING Finger of c-Cbl Mediates Desensitization of the Epidermal Growth Factor Receptor

Abstract

Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both downregulation and ubiquitination of EGFR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitro reconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of cCbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.We have reported recently that c-Cbl can accelerate the rate of EGFR 1 degradation by increasing conjugation of polyubiquitin to an endocytosed EGFR (1). Consistent with sorting to degradation, genetic studies in Caenorhabditis elegans attributed a negative regulatory role to Sli-1, the c-Cbl ortholog of worms (2). This function is reminiscent of the action of c-Cbl downstream to the PGDFR (3), and it depends on a catalytically intact kinase domain of EGFR. The present study addressed the structural motif of c-Cbl involved in ubiquitination of EGFR. Although the 120-kDa c-Cbl protein possesses no identifiable catalytic activity, its NH 2 -terminal half carries a novel phosphotyrosine-binding domain (PYB) (4), that presumably mediates binding to the activated receptor (5). The carboxyl terminus of c-Cbl comprises several proline-rich tracts that allow constitutive binding of Src homology 3 (SH3)-containing proteins, such as Grb2, Nck, and the Cbl-associated protein, CAP (reviewed in Ref. 6). In addition, the COOH terminus is involved in inducible interactions with SH2-containing proteins, such as the regulatory subunit of the phosphoinositide 3-kinase, and the guanine nucleotide exchange protein Vav. An evolutionarily conserved RING-type zinc finger domain separates the adaptor domains of c-Cbl. RING fingers (RFs) are zinc-binding domains that differ from other zinc finger motifs in terms of structure and the zinc ligation scheme (reviewed in Ref. 7). Noteworthy is the fact that the two other family members of c-Cbl carry an intact RF (8, 9), but oncogenic Cbl variants are defective in the RF (10, 11). Our present study concentrated on the role the RF motif plays in Cbl-mediated degradation of EGFR. EXPERIMENTAL PROCEDURESMaterials, Buffers, and Antibodies-EGF was purchased from Sigma, and radioactive materials were from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom). Rabbit anti-c-Cbl (C-15) antibodies, ...

Differential roles of ERK and Akt pathways in regulation of EGFR-mediated signaling and motility in prostate cancer cells

“…The ERK pathway affects EGFR trafficking by threonine phosphorylation of EGFR Consequent to EGF stimulation, activated EGFR is rapidly ubiquitinated by c-Cbl, an SH2 domain-containing ubiquitin ligase that itself becomes phosphorylated and binds to EGFR, which promotes postinternalization of EGFR and sorting to endosomes and lysosomes for degradation (Levkowitz et al, 1998;Joazeiro et al, 1999;Waterman et al, 1999). In DU145 cells, EGF stimulation led to EGFR ubiquitination (Figure 3a, upper panel, lane 2), which was enhanced by PD98059 (lane 4 vs 2), but not by LY294002 (lane 6 vs 2).…”

EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins

“…The overexpression of Cbl proteins enhances EGF-induced ubiquitination and downregulation of the WT EGFR (Ettenberg et al, 1999b(Ettenberg et al, , 2001Levkowitz et al, 1999;Waterman et al, 1999b;Yokouchi et al, 1999). Therefore, we investigated whether the Cbl proteins also regulate the constitutively active mutant EGFRvIII in a cell line Chinese hamster ovary (CHO) that does not express the WT EGFR.…”

The IAP family: endogenous caspase inhibitors with multiple biological activities

“…These results suggest that the RING domain may directly participate the transfer of ubiquitin from E2 to substrate. Furthermore, ubiquitination of EGFR and p53 by Cbl and Mdm2, respectively, modulates their intracellular levels [45][46][47], indicating that these ubiquitination reactions are specific and physiologically important. This is strongly supported by the finding that the E3 activity of IAPs is important in regulating their own level and apoptosis-inhibiting activity [37].…”