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RET-Familial Medullary Thyroid Carcinoma Mutants Y791F and S891A Activate a Src/JAK/STAT3 Pathway, Independent of Glial Cell Line–Derived Neurotrophic Factor

Departments of ¹ Medical Genetics, ² Pharmaceutical Biology, ³ Pathology, and ⁴ Developmental Genetics, University of Groningen, Groningen, the Netherlands; ⁵ Department of Surgery, Ernst-Grube-Strasse, Martin-Luther-University, Halle-Wittenberg, Germany

Requests for reprints: Robert M.W. Hofstra, Department of Medical Genetics. University of Groningen, Antonius Deusinglaan 4, 9713 AW Groningen, the Netherlands. Phone: 31-50-363-2925; Fax: 31-50-363-2947; E-mail: r.m.w.hofstra{at}medgen.umcg.nl.

The RET proto-oncogene encodes a receptor tyrosine kinase whose dysfunction plays a crucial role in the development of several neural crest disorders. Distinct activating RET mutations cause multiple endocrine neoplasia type 2A (MEN2A), type 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). Despite clear correlations between the mutations found in these cancer syndromes and their phenotypes, the molecular mechanisms connecting the mutated receptor to the different disease phenotypes are far from completely understood. Luciferase reporter assays in combination with immunoprecipitations, and Western and immunohistochemistry analyses were done in order to characterize the signaling properties of two FMTC-associated RET mutations, Y791F and S891A, respectively, both affecting the tyrosine kinase domain of the receptor. We show that these RET-FMTC mutants are monomeric receptors which are autophosphorylated and activated independently of glial cell line–derived neurotrophic factor. Moreover, we show that the dysfunctional signaling properties of these mutants, when compared with wild-type RET, involve constitutive activation of signal transducers and activators of transcription 3 (STAT3). Furthermore, we show that STAT3 activation is mediated by a signaling pathway involving Src, JAK1, and JAK2, differing from STAT3 activation promoted by RET^C634R which was previously found to be independent of Src and JAKs. Three-dimensional modeling of the RET catalytic domain suggested that the structural changes promoted by the respective amino acids substitutions lead to a more accessible substrate and ATP-binding monomeric conformation. Finally, immunohistochemical analysis of FMTC tumor samples support the in vitro data, because nuclear localized, Y705-phosphorylated STAT3, as well as a high degree of RET expression at the plasma membrane was observed.

Key Words: RET • GDNF • MEN2 • FMTC • STAT3 • JAK1 • JAK2 • Src

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