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CIN85 participates in Cbl-b-mediated down-regulation of receptor tyrosine kinases
Iwona Szymkiewicz1, Katarzyna Kowanetz, Philippe Soubeyran
1Ludwig Institute for Cancer Research, Box 595, Husargatan 3, Uppsala, S-75124, Sweden.
Abstract:
The Cbl family of ubiquitin ligases in mammals contains three members, Cbl, Cbl-b, and Cbl-3, that are involved in down-regulation of receptor tyrosine kinases (RTKs) by mediating receptor ubiquitination and degradation. More recently, a novel pathway has been identified whereby Cbl promotes internalization of EGF receptor via a CIN85/endophilin pathway that is functionally separable from the ubiquitin ligase activity of Cbl (1). Here we show that Cbl-b, but not Cbl-3, utilize the same mechanism to down-regulate multiple RTKs. CIN85 was shown to bind to the minimal binding domain identified in the carboxyl terminus of Cbl-b. Ligand-induced phosphorylation of Cbl-b further increased their interactions and led to a rapid and sustained recruitment of CIN85 in the complex with EGF or PDGF receptors. Inhibition of binding between CIN85 and Cbl-b was sufficient to impair Cbl-b-mediated internalization of EGF receptors, while being dispensable for Cbl-b-directed polyubiquitination of EGF receptors. Moreover, CIN85 and Cbl/Cbl-b were constitutively associated with activated PDGF, EGF, or c-Kit receptors in several tumor cell lines. Our data reveal a common pathway utilized by Cbl and Cbl-b that may have an important and redundant function in negative regulation of ligand-activated as well as oncogenically activated RTKs in vivo.
Insights
The Cbl-b protein regulates receptor tyrosine kinases (RTKs) through a pathway involving CIN85, distinct from its ubiquitin ligase activity. This mechanism is crucial for down-regulating RTKs in both normal and cancer cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The Cbl family of ubiquitin ligases (Cbl, Cbl-b, Cbl-3) regulates receptor tyrosine kinases (RTKs) via ubiquitination and degradation.
- A novel Cbl-mediated pathway involving CIN85/endophilin facilitates EGF receptor internalization, separate from its ligase function.
Purpose of the Study:
- To investigate if Cbl-b utilizes the CIN85/endophilin pathway for RTK down-regulation.
- To elucidate the role of CIN85 in Cbl-b-mediated receptor internalization and RTK regulation.
Main Methods:
- Investigated Cbl-b interactions with CIN85 using binding assays.
- Analyzed the effect of CIN85-Cbl-b binding inhibition on EGF receptor internalization.
- Examined the association of CIN85, Cbl/Cbl-b with activated RTKs in tumor cell lines.
Main Results:
- Cbl-b, but not Cbl-3, uses the CIN85/endophilin pathway to down-regulate multiple RTKs.
- CIN85 binds to Cbl-b's carboxyl terminus; phosphorylation enhances this interaction.
- Inhibiting CIN85-Cbl-b binding impairs Cbl-b-mediated EGF receptor internalization but not ubiquitination.
- CIN85 and Cbl/Cbl-b associate with activated PDGF, EGF, and c-Kit receptors in tumor cells.
Conclusions:
- Cbl-b employs a common pathway with Cbl for RTK internalization via CIN85.
- This pathway plays a significant and potentially redundant role in negatively regulating activated RTKs in vivo.