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D-Cbl binding to Drk leads to dose-dependent down-regulation of EGFR signaling and increases receptor-ligand endocytosis.

Wang PY, Pai LM - PLoS ONE (2011)

Bottom Line: 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.Interfering the recruitment of signal transducer, Drk, to the receptor by the RING-SH2(Drk) might further reduces EGFR signaling.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biomedical Science, Chang Gung University, Tao-Yuan, Taiwan.

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-SH2(Drk), 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-SH2(Drk) 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-SH2(Drk) 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.

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The amount of Drk in the EGFR complex.The EGFR complex were immunoprecipitated by mouse monoclonal anti-EGFR anti-bodies from ovariant lysates extracted from wild-type females or females expressing D-CblL, or RING-SH2Drk in their follicle cells. The immunoprecipitate was separated by SDS-PAGE, and analyzed by rabbit polyclonal anti-EGFR (upper panel) or anti-Drk antibodies (lower panel). The EGFR signal appeared near 170kD (arrowhead in upper panel), and the Drk signal was near 24kD (arrowhead in lower panel).
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pone-0017097-g006: The amount of Drk in the EGFR complex.The EGFR complex were immunoprecipitated by mouse monoclonal anti-EGFR anti-bodies from ovariant lysates extracted from wild-type females or females expressing D-CblL, or RING-SH2Drk in their follicle cells. The immunoprecipitate was separated by SDS-PAGE, and analyzed by rabbit polyclonal anti-EGFR (upper panel) or anti-Drk antibodies (lower panel). The EGFR signal appeared near 170kD (arrowhead in upper panel), and the Drk signal was near 24kD (arrowhead in lower panel).

Mentions: Because the SH2 domain of RING-SH2Drk was derived from Drk, we assumed that the docking site for RING-SH2Drk on EGFR is the same as that for Drk. Therefore, this chimeric protein might compete with endogenous Drk for binding to EGFR. This possibility was tested by immunoprecipitation using anti-EGFR antibodies to determine the amount of Drk in the receptor complex. 40% of Drk in the EGFR complex was reduced in egg chambers expressing RING-SH2Drk or full length D-CblL, compared to the wild-type egg chambers (Figure 6). This result indicates that RING-SH2Drk interferes with the interaction between endogenous Drk and EGFR, which may lead to reduced signal transduction. Taken together, the chimeric protein RING-SH2Drk may down-regulate EGFR signaling through promoting the endosomal trafficking of the EGFR complex and reducing the recruitment of Drk/Sos in signal transduction.


D-Cbl binding to Drk leads to dose-dependent down-regulation of EGFR signaling and increases receptor-ligand endocytosis.

Wang PY, Pai LM - PLoS ONE (2011)

The amount of Drk in the EGFR complex.The EGFR complex were immunoprecipitated by mouse monoclonal anti-EGFR anti-bodies from ovariant lysates extracted from wild-type females or females expressing D-CblL, or RING-SH2Drk in their follicle cells. The immunoprecipitate was separated by SDS-PAGE, and analyzed by rabbit polyclonal anti-EGFR (upper panel) or anti-Drk antibodies (lower panel). The EGFR signal appeared near 170kD (arrowhead in upper panel), and the Drk signal was near 24kD (arrowhead in lower panel).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3038869&req=5

pone-0017097-g006: The amount of Drk in the EGFR complex.The EGFR complex were immunoprecipitated by mouse monoclonal anti-EGFR anti-bodies from ovariant lysates extracted from wild-type females or females expressing D-CblL, or RING-SH2Drk in their follicle cells. The immunoprecipitate was separated by SDS-PAGE, and analyzed by rabbit polyclonal anti-EGFR (upper panel) or anti-Drk antibodies (lower panel). The EGFR signal appeared near 170kD (arrowhead in upper panel), and the Drk signal was near 24kD (arrowhead in lower panel).
Mentions: Because the SH2 domain of RING-SH2Drk was derived from Drk, we assumed that the docking site for RING-SH2Drk on EGFR is the same as that for Drk. Therefore, this chimeric protein might compete with endogenous Drk for binding to EGFR. This possibility was tested by immunoprecipitation using anti-EGFR antibodies to determine the amount of Drk in the receptor complex. 40% of Drk in the EGFR complex was reduced in egg chambers expressing RING-SH2Drk or full length D-CblL, compared to the wild-type egg chambers (Figure 6). This result indicates that RING-SH2Drk interferes with the interaction between endogenous Drk and EGFR, which may lead to reduced signal transduction. Taken together, the chimeric protein RING-SH2Drk may down-regulate EGFR signaling through promoting the endosomal trafficking of the EGFR complex and reducing the recruitment of Drk/Sos in signal transduction.

Bottom Line: 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.Interfering the recruitment of signal transducer, Drk, to the receptor by the RING-SH2(Drk) might further reduces EGFR signaling.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biomedical Science, Chang Gung University, Tao-Yuan, Taiwan.

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-SH2(Drk), 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-SH2(Drk) 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-SH2(Drk) 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.

Show MeSH
Related in: MedlinePlus