Division of Cancer Biology and Genetics, Cancer Research Institute, and Department of Pathology and Molecular Medicine, Queen's University, Kingston ON Canada, K7L 3N6.
Department of Chemistry and Biology, Ryerson University, Toronto, ON, Canada, M5B 2K3.
J Cell Sci. 2017 Oct 1;130(19):3282-3296. doi: 10.1242/jcs.203885. Epub 2017 Aug 9.
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.
RET 受体酪氨酸激酶参与正常发育和癌症。RET 有两种异构体，即 RET9 和 RET51，它们具有独特的 C 端尾序列，可以招募不同的蛋白复合物来介导信号。激活后，RET 异构体以不同的动力学内化，表明其调节存在差异。在这里，我们证明了 RET9 和 RET51 在招募其独特 C 端的 E3 泛素连接酶的能力上存在差异。RET51 而不是 RET9 与 CBL 相互作用并被其泛素化，CBL 通过与 GRB2 衔接蛋白的相互作用被募集。CBL 敲除不影响 RET51 的内化，但会延迟 GRB2 耗尽细胞中的内化。相比之下，RET9 的泛素化需要 RET9 C 端关键结合基序的磷酸化依赖性变化，以促进与多个衔接蛋白（包括 GRB10 和 SHANK2）的相互作用，从而募集 NEDD4 泛素连接酶。我们表明，NEDD4 介导的泛素化对于 RET9 定位到网格蛋白包被的陷窝和随后的内化是必需的。我们的数据建立了 RET9 和 RET51 泛素化和内化的机制差异，这可能影响 RET 异构体信号的强度和持续时间以及细胞输出。本文附有该论文第一作者的第一人称采访。
