Cbl-b-dependent Coordinated Degradation of the Epidermal Growth Factor Receptor Signaling Complex

Abstract

The Cbl proteins are a family of proteins found in metazoans from nematodes to vertebrates. These proteins have several highly conserved domains including an N-terminal tyrosine kinase binding (TKB) 1 domain and a RING finger (1-9). The three mammalian Cbl proteins,2,[6][7][8], are tyrosine-phosphorylated upon activation of a wide variety of growth factor receptors, and they associate with many signaling proteins via SH2 and SH3 interactions (reviewed in Ref. 10 and 11). These diverse interactions modulate signaling through many pathways (10,11). Recent work has shown that c-Cbl-and Cblb-deficient mice have hyperplastic tissues, consistent with a negative regulatory role in cellular proliferation for Cbl proteins (12-15). Together, these data indicate that the Cbl proteins are important regulators of intracellular signaling and consequently of cell function and development.Cbl proteins are negative regulators of epidermal growth factor receptor (EGFR) signaling. This was first shown by genetic studies in Caenorhabditis elegans, which demonstrated that Sli-1 (the C. elegans Cbl homologue) is a negative regulator of the Let-23 receptor tyrosine kinase (the EGFR homologue) in vulva development (3, 16). The Drosophila Cbl protein (D-Cbl) has been shown to associate with the EGFR, and overexpression of D-Cbl in the eye of Drosophila embryos inhibits EGFR-dependent photoreceptor cell development (4, 5). Several studies have shown that mammalian Cbl proteins become phosphorylated and recruited to the EGFR upon stimulation (11, 17) and that they inhibit EGFR function (7, 18 -20).The mechanism underlying the negative regulation of activated tyrosine kinases by Cbl proteins has recently been described. Cbl proteins function as ubiquitin protein ligases, which mediate the ubiquitination of activated tyrosine kinases including the EGFR and target them for degradation (20 -31). Ubiquitination of proteins occurs via the sequential activation and conjugation of ubiquitin to target proteins by the ubiquitinactivating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) (32). The E3 confers specificity to the ubiquitination process. An increasing number of RING finger proteins has been demonstrated to function as E3 proteins or as part of E3 complexes, and in each of them the RING finger is essential to this activity (33-43). The highly conserved TKB and RING finger domains of Cbl proteins are essential and sufficient for their E3 activity, and together these domains target the ubiquitination of activated tyrosine kinases such as the EGFR (20 -31).Here, we show that EGF activation induces a coordinated degradation of the EGFR, Cbl proteins, and other proteins of the EGFR signaling complex. These results suggest that Cbl proteins regulate degradation of multiple proteins in the active EGFR-signaling complex. EXPERIMENTAL PROCEDURESExpression Constructs-The expression plasmid for HA epitopetagged Cbl-b, c-Cbl, and the control vector (pCEFL) have been previously described (18). HA epitope-tagged C...

The Shb signalling scaffold binds to and regulates constitutive signals from the Epstein–Barr virus LMP2A membrane protein

“…Previous work has shown that Cbl is recruited to the autophosphorylated Y323 of Syk (Lupher et al, 1998) and that Cbl is targeted by the Nedd4/AIP4 family of E3 ubiquitin ligases (Magnifico et al, 2003). The regulation of CblC by AIP4 is an important part of the mechanism for downregulation of the epidermal growth factor receptor signaling complex (Ettenberg et al, 2001;Courbard et al, 2002). The interactions of Cbl with Syk and AIP4 may explain why we detect Cbl in immunoprecipitates of LMP2A (data not shown).…”

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.…”

“…In contrast, the deletion of the TKB domain containing the aminoterminus of Cbl-b (C2/3 Cbl-b) prevented the downregulation of the EGFRvIII by Cbl-b (Figure 3b, lane 5). Finally, a RING finger mutant of Cbl-b (C373A) that has been shown to lack E3 activity (Ettenberg et al, 2001) was unable to downregulate the EGFRvIII (Figure 3b, lane 6). Quantification of the downregulation of the EGFRvIII by the various constructs of Cbl-b revealed that N1/2 and WT Cbl-b downregulate the EGFRvIII to a similar extent, that the overexpression of C2/3 Cbl-b did not affect EGFRvIII levels, and that the RING finger mutant of Cbl-b tended to increase the amount of the EGFRvIII protein ( Figure 3c).…”

“…Quantification of the downregulation of the EGFRvIII by the various constructs of Cbl-b revealed that N1/2 and WT Cbl-b downregulate the EGFRvIII to a similar extent, that the overexpression of C2/3 Cbl-b did not affect EGFRvIII levels, and that the RING finger mutant of Cbl-b tended to increase the amount of the EGFRvIII protein ( Figure 3c). Therefore, like the WT EGFR (Ettenberg et al, 2001), the TKB and RING finger domains of Cbl-b are sufficient for the downregulation of the EGFRvIII. Also, the E3 activity of Cbl-b is necessary for the downregulation of the EGFRvIII by Cbl-b.…”

“…They are recruited to the activated EGFR either by the direct binding of their TKB domain to the phosphotyrosine residue at position 1045 in the EGFR (Levkowitz et al, 1999) or by an indirect mechanism mediated by Grb2 -the SH3 domains of Grb2 bind the proline-rich region of the Cbl proteins and the SH2 domain of Grb2 binds the phosphorylated EGFR (Waterman et al, 2002;Jiang et al, 2003). The RING finger domain of the Cbl proteins allows them to function as ubiquitin ligases (E3s) and so to target the EGFR signaling complex for internalization and subsequent degradation in the lysosome (Ettenberg et al, 1999b(Ettenberg et al, , 2001Levkowitz et al, 1999;Waterman et al, 1999a;Yokouchi et al, 1999;Duan et al, 2003). Thus, the Cbl proteins mediate the downregulation of the EGFR following stimulation with EGF.…”