Differential recruitment of E3 ubiquitin ligase complexes regulates RET isoform internalization

Abstract

The RET receptor tyrosine kinase is implicated in normal development and cancer. RET is expressed as two isoforms, RET9 and RET51, with unique C-terminal tail sequences that recruit distinct protein complexes to mediate signals. Upon activation, RET isoforms are internalized with distinct kinetics, suggesting differences in regulation. Here, we demonstrate that RET9 and RET51 differ in their abilities to recruit E3 ubiquitin ligases to their unique C-termini. RET51, but not RET9, interacts with, and is ubiquitylated by CBL, which is recruited through interactions with the GRB2 adaptor protein. RET51 internalization was not affected by CBL knockout but was delayed in GRB2-depleted cells. In contrast, RET9 ubiquitylation requires phosphorylation-dependent changes in accessibility of key RET9 C-terminal binding motifs that facilitate interactions with multiple adaptor proteins, including GRB10 and SHANK2, to recruit the NEDD4 ubiquitin ligase. We showed that NEDD4-mediated ubiquitylation is required for RET9 localization to clathrin-coated pits and subsequent internalization. Our data establish differences in the mechanisms of RET9 and RET51 ubiquitylation and internalization that may influence the strength and duration of RET isoform signals and cellular outputs.This article has an associated First Person interview with the first authors of the paper.

A specific Ret receptor isoform is required for pioneer axon outgrowth and growth cone dynamics

“…Live imaging of RET51/Jip3 retrograde co-transport as well as the disruption of RET51 retrograde axonal transport and pRet localization followed by loss of Jip3, suggests Ret51 retrograde transport may be required for this signaling role. In other cell types, Ret51 is much more rapidly internalized from the plasma membrane and differentially ubiquitylated compared to Ret9 18,40 . In mouse sympathetic neuron culture, Ret51 is more rapidly degraded in axon terminals compared to Ret9 following GDNF treatment 16 .…”

Exploiting Ret Isoforms in Managing Medullary and Papillary Thyroid Cancer

“…RET isoforms recruit distinct signaling complexes, leading to different patterns of target gene expression [14,15]. Although both RET9 and RET51 promote downstream signaling through multiple pathways, including PI3K/Akt/mTOR, and ERK/MAPK, distinct protein interactions with RET isoform unique C-terminal sequences affect the timing, duration and subcellular localization of these events [4,14,16,17]. Most notably, RET51 interacts with adaptor proteins containing SH2 domains, such as GRB2, through a unique phosphorylated tyrosine residue (pY1096) in its C-terminal tail [16,18].…”

“…Although both RET9 and RET51 promote downstream signaling through multiple pathways, including PI3K/Akt/mTOR, and ERK/MAPK, distinct protein interactions with RET isoform unique C-terminal sequences affect the timing, duration and subcellular localization of these events [4,14,16,17]. Most notably, RET51 interacts with adaptor proteins containing SH2 domains, such as GRB2, through a unique phosphorylated tyrosine residue (pY1096) in its C-terminal tail [16,18]. In contrast, RET9 has distinct binding capabilities at pY1062, and also contains a unique C-terminal binding motif (FTRF 1072 ), which allows it to interact with PDZ domain proteins such as SHANK family members [4,16] (Figure 1).…”

“…Most notably, RET51 interacts with adaptor proteins containing SH2 domains, such as GRB2, through a unique phosphorylated tyrosine residue (pY1096) in its C-terminal tail [16,18]. In contrast, RET9 has distinct binding capabilities at pY1062, and also contains a unique C-terminal binding motif (FTRF 1072 ), which allows it to interact with PDZ domain proteins such as SHANK family members [4,16] (Figure 1). RET9 and RET51 have been found to differ in their membrane localization, and in their internalization into the cell and intracellular trafficking upon activation [16,17,19].…”

“…In contrast, RET9 has distinct binding capabilities at pY1062, and also contains a unique C-terminal binding motif (FTRF 1072 ), which allows it to interact with PDZ domain proteins such as SHANK family members [4,16] (Figure 1). RET9 and RET51 have been found to differ in their membrane localization, and in their internalization into the cell and intracellular trafficking upon activation [16,17,19]. RET51 matures more efficiently than RET9, and is present in relatively greater quantities at the cell membrane, while RET9 matures slowly and accumulates as immature protein [17,20].…”