CD2AP and Cbl-3/Cbl-c Constitute a Critical Checkpoint in the Regulation of Ret Signal Transduction

Abstract

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are critical for nervous system development and maintenance. GFLs promote survival and growth via activation of the receptor tyrosine kinase (RTK) Ret. In sympathetic neurons, the duration of Ret signaling is governed by how rapidly Ret is degraded after its activation. In an effort to elucidate mechanisms that control the half-life of Ret, we have identified two novel Ret interactors, CD2-associated protein (CD2AP) and Cbl-3. CD2AP, an adaptor molecule involved in the internalization of ubiquitinated RTKs, is associated with Ret under basal, unstimulated conditions in neurons. After Ret activation by GDNF, CD2AP dissociates. Similarly, the E3-ligase Cbl-3 interacts with unphosphorylated Ret and dissociates from Ret after Ret activation. In contrast to their dissociation from autophosphorylated Ret, an interaction between CD2AP and Cbl-3 is induced by GDNF stimulation of sympathetic neurons, suggesting that CD2AP and Cbl-3 dissociate from Ret as a complex. In neurons, the overexpression of CD2AP enhances the degradation of Ret and inhibits GDNF-dependent survival, and gene silencing of CD2AP blocks Ret degradation and promotes GDNF-mediated survival. Surprisingly, Cbl-3 overexpression dramatically stabilizes activated Ret and enhances neuronal survival, even though Cbl-family E3 ligases normally function to trigger RTK downregulation. In combination with CD2AP, however, Cbl-3 promotes Ret degradation rapidly and almost completely blocks survival promotion by GDNF, suggesting that Cbl-3 acts as a switch that is triggered by CD2AP and oscillates between inhibition and promotion of Ret degradation. Consistent with the hypothesis, Cbl-3 silencing in neurons only inhibited Ret degradation and enhanced neuronal survival in combination with CD2AP silencing. CD2AP and Cbl-3, therefore, constitute a checkpoint that controls the extent of Ret downregulation and, thereby, the sensitivity of neurons to GFLs.

CD2-associated Protein (CD2AP) Enhances Casitas B Lineage Lymphoma-3/c (Cbl-3/c)-mediated Ret Isoform-specific Ubiquitination and Degradation via Its Amino-terminal Src Homology 3 Domains

“…The cells were then detergent-extracted, and Ret51 was immunoprecipitated. Similar to sympathetic neurons (8), knockdown of Cbl-3/c inhibited the degradation of Ret triggered by GDNF stimulation (Fig. 1A).…”

“…Gene silencing in sympathetic neurons and podocytes was accomplished using siRNA, as we have done previously (8,23). Briefly, siRNA against Cbl-3/c, or a scrambled control, were transfected into the cells using i-Fect according to the manufacturer's instructions (Neuromics).…”

“…The Cbl family of E3 ligases appears to be critical for the ubiquitination of Ret51, with both c-Cbl and Cbl-3/c capable of binding and ubiquitinating Ret51, thereby promoting its degradation (6,8). In primary sympathetic neurons, Ret51 degradation occurs primarily via Cbl-3/c, although Cbl-b also can associate with Ret51 (5,8). Cbl-3/c differs significantly from the other two mammalian Cbls in that it lacks a large portion of the carboxyl-terminal region present in c-Cbl and Cbl-b.…”

“…Nevertheless, Cbl-3/c has an active catalytic ring finger domain and can ubiquitinate proteins, thus targeting them for internalization and degradation (10,11). In sympathetic neurons, Cbl-3/c associates with the adaptor protein CD2AP, which appears to promote Ret51 degradation via Cbl-3/c (8). CD2AP, along with its paralog CIN85, regulate the association of Cbl proteins with receptors, thereby linking these receptor complexes to the internalization machinery (13)(14)(15)(16)(17).…”

Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3)

“…CD2AP plays an integral role in actin dynamics, endocytosis of cell surface receptors, and their degradation (e.g. binding to F-actin and promoting actin bundling (6); promoting internalization of EGF receptor (7) and VEGF receptor (8); and down-regulation of the T-cell receptor (9), the HER2 (ErbB2) receptor in a subset of breast cancers (10), and the RET (rearranged during transfection) receptor (11,12)). Conversely, CD2AP positively regulates the plasmacytoid dendritic cell receptor BDCA2; its degradation occurs only with a lack of CD2AP activity (13).…”