RET(MEN 2B) is active in the endoplasmic reticulum before reaching the cell surface

Abstract

MEN 2B (multiple endocrine neoplasia type 2B) is an autosomal dominant cancer syndrome caused by an oncogenic form of the receptor tyrosine kinase REarranged during transfection (RET). The MEN 2B syndrome is associated with an abnormal autophosphorylation of the mutated receptor even without ligand-stimulation. Here, we characterize the activation of a RET MEN 2B variant carrying the point mutation Met918Thr, and show that the 150 kDa precursor of RET MEN 2B becomes phosphorylated already during synthesis in the endoplasmic reticulum (ER). At least three different tyrosine residues (Tyr905, Tyr1062, Tyr1096) of the RET MEN 2B precursor are phosphorylated before the oncogenic receptor reaches the cell surface. We also demonstrate that the precursor of RET MEN 2B interacts with both growth factor receptorbound protein and Src homology 2 domain-containing already in the ER, and that this interaction is dependent on the kinase activity of RET. With the aid of two RET mutants (RET MEN 2B/S32L and RET MEN 2B/F393L ), which accumulate in the ER, we show that the oncogenic precursor of the receptor has the capacity to activate AKT, extracellular signal-regulated kinase and signal transducer and activator of transcription 3 from the ER. Taken together, our data demonstrate that the oncogenic precursor of RET MEN 2B is phosphorylated, interacts with adapter proteins and induces downstream signalling from the ER.Oncogene ( Keywords: RET; MEN 2B; precursor; downstream signalling; endoplasmic reticulum RE arranged during transfection (RET) is a transmembrane receptor tyrosine kinase, which in the presence of a glycosyl phosphatidylinositol-anchored co-receptor GDNF family receptor (GFRa) can be activated by glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs). There are four different GFLs (GDNF, neurturin, artemin and persephin), which bind to the coreceptors GFRa1-4, respectively. As RET is the signal transducer of four different ligand/co-receptor complexes, it has important physiological functions in different tissues. In spite of its broad expression pattern and vitally important functions during development (Airaksinen and Saarma, 2002), the oncogenic mutant forms of RET cause quite limited clinical symptoms -activating mutations in RET are only responsible for hereditary endocrine cancer syndromes (Kodama et al., 2005). This paradox has not yet been explained, and is likely due to some still uncharacterized activation or inactivation mechanisms of RET.Based on the clinical symptoms, the multiple endocrine neoplasia type 2 (MEN 2) is divided into three groups: MEN 2A, MEN 2B and familiar medullary thyroid carcinoma (FMTC). In MEN 2A, the disease has been linked to point mutations, which are mostly located to cysteine residues in the extracellular cysteine-rich domain of RET. These point mutations introduce abnormal intermolecular cysteine bridges, and cause an autoactivation of RET MEN 2A by dimerization. In the case of MEN 2B, the point mutations are located in the intracellular domain of RET where...

Familial associations in medullary thyroid carcinoma with Hirschsprung disease: the role of the RET-C620 “Janus” genetic variation

“…It is known that the endoplasmic reticulum links to adaptor proteins, to influence RET function in different directions. For example, in MEN2B, a RET 150-kDa precursor has been shown to be phosphorylated during its synthesis in the endoplasmic reticulum from which it induces downstream signaling and activation [28]. The endoplasmic reticulum has also been shown to decrease cell surface RET expression in HSCR [29].…”

A direct role for Met endocytosis in tumorigenesis

“…Thus, it has been assumed that D1246N and M1268T mutants are oncogenic simply because they are highly activated 27,40 . Recent studies however have reported signalling of mutated activated RTKs, in an immature form, from intracellular compartments of the secretory pathway [41][42][43][44] , leading to tumorigenesis 41,45 . We suggest that Met mutants D1246N and M1268T are oncogenic not only because they are activated but also because they signal on endosomes.…”

Medullary Thyroid Cancer: Molecular Biology and Novel Molecular Therapies

“…As a result, the mutated RET no longer requires dimerization to become active [37]. In a recent study, it was shown that codon 918 RET mutant (MEN 2B) is already active in the endoplasmic reticulum where it interacts with adaptor proteins [38]. Interestingly, several types of RET mutation can be further activated by GDNF as well [39].…”