Chapter 2 RET - FMTC mutants Y 791 F and S 891 A activate a Src / JAK / STAT 3 pathway , independent of GDNF Cancer Research , 2005 ; 65 : 1729 - 37

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 immunoprecipitation, western and immunohistochemistry analyses were performed in order to characterize the signalling 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 GDNF. Moreover, we demonstrate that the dysfunctional signalling properties of these mutants, when compared to wild type RET, involve constitutive activation of STAT3. Furthermore, we demonstrate that STAT3 activation is mediated by a signalling pathway involving SRC, JAK1 and JAK2, differing from STAT3 activation promoted by RETC634R which previously was 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, as nuclear localized, Y705-phosphorylated STAT3 as well as a high degree of RET expression at the plasma membrane was observed.