Abstract
Proper control of Epidermal Growth Factor Receptor (EGFR) signaling is critical for normal development and regulated cell behaviors. Abnormal EGFR signaling is associated with tumorigenic process of various cancers. Complicated feedback networks control EGFR signaling through ligand production, and internalization-mediated destruction of ligand-receptor complexes. Previously, we found that two isoforms of D-Cbl, D-CblS and D-CblL, regulate EGFR signaling through distinct mechanisms. While D-CblL plays a crucial role in dose-dependent down-regulation of EGFR signaling, D-CblS acts in normal restriction of EGFR signaling and does not display dosage effect. Here, we determined the underlying molecular mechanism, and found that Drk facilitates the dose-dependent regulation of EGFR signaling through binding to the proline-rich motif of D-CblL, PR. Furthermore, the RING finger domain of D-CblL is essential for promoting endocytosis of the ligand-receptor complex. Interestingly, a fusion protein of the two essential domains of D-CblL, RING- PR, is sufficient to down-regulate EGFR signal in a dose-dependent manner by promoting internalization of the ligand, Gurken. Besides, RING-SH2Drk, a fusion protein of the RING finger domain of D-Cbl and the SH2 domain of Drk, also effectively down-regulates EGFR signaling in Drosophila follicle cells, and suppresses the effects of constitutively activated EGFR. The RING-SH2Drk suppresses EGFR signaling by promoting the endosomal trafficking of ligand-receptor complexes, suggesting that Drk plays a negative role in EGFR signaling by enhancing receptor endocytosis through cooperating with the RING domain of D-Cbl. Interfering the recruitment of signal transducer, Drk, to the receptor by the RING-SH2Drk might further reduces EGFR signaling. The fusion proteins we developed may provide alternative strategies for therapy of cancers caused by hyper-activation of EGFR signaling.
Highlights
Ubiquitination occurs via sequential activation and conjugation of ubiquitin to target proteins by ubiquitin activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3) [1]
We applied our results to generate a chimeric protein, containing the RING finger domain of Casitas Blineage lymphoma (Cbl) fused to the SH2 domain of Downstream of receptor kinase (Drk), which can very effectively down-regulate Epidermal Growth Factor Receptor (EGFR) signaling
This study investigated the role of these molecules in D-CblL mediated regulation, and found that elimination of Drk interaction resulted in significant reduction of the effect of D-CblL over-expression (Table 1)
Summary
Ubiquitination occurs via sequential activation and conjugation of ubiquitin to target proteins by ubiquitin activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3) [1]. Aside from protein degradation, ubiquitination represents a crucial signal for the endocytosis of signaling molecules such as EGFR. A critical E3 ubiqutin ligase mediating the ubiquitiationdependent receptor endocytosis is the proto-oncogene Casitas Blineage lymphoma (Cbl), which was first identified as the cellular homolog of v-cbl, which induces pre-B-cell lymphomas and myeloid tumors [8,9]. In Drosophila, D-Cbl negatively regulates EGFR signaling in dorsoventral patterning during oogenesis [11], in eye development [12,13,14], and in border cell migration [15]. The RING finger domain in Cbl is highly conserved during evolution, for critical amino acids related to E3 ligase activity [16,20]. In Drosophila, two major isoforms, D-CblL and D-CblS are generated from the single D-Cbl gene [22]; the shorter isoform D-CblS lacks the proline-rich and UBA domains
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