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 ubiquitinated 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 ubiquitination requires phosphodependent 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 ubiquitination is required for RET9 localization to clathrin coated pits and subsequent internalization. Our data establish differences in the mechanisms of RET9 and RET51 ubiquitination and internalization that may influence the strength and duration of RET isoform signals and cellular outputs.