1. RET-familial medullary thyroid carcinoma mutants Y791F and S891A activate a Src/JAK/STAT3 pathway, independent of glial cell line-derived neurotrophic factor
- Author
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Ivan Plaza Menacho, Tineke van der Sluis, Charles H.C.M. Buys, Roelof Koster, Almer M. van der Sloot, Grzegorz M. Burzynski, Bart J. L. Eggen, Wim J. Quax, Harry Hollema, Jan Osinga, Oliver Gimm, Robert M.W. Hofstra, Nanotechnology and Biophysics in Medicine (NANOBIOMED), Biopharmaceuticals, Discovery, Design and Delivery (BDDD), and Restoring Organ Function by Means of Regenerative Medicine (REGENERATE)
- Subjects
Cancer Research ,Protein Conformation ,PROTEIN ,Multiple Endocrine Neoplasia Type 2a ,RECEPTOR TYROSINE KINASE ,Proto-Oncogene Mas ,Receptor tyrosine kinase ,Immunoenzyme Techniques ,Adenosine Triphosphate ,DOMAIN ,Neurotrophic factors ,Chlorocebus aethiops ,Glial cell line-derived neurotrophic factor ,CRYSTAL-STRUCTURE ,Phosphorylation ,Receptor ,Luciferases ,Cells, Cultured ,Oncogene Proteins ,biology ,INSULIN-RECEPTOR ,Protein-Tyrosine Kinases ,DNA-Binding Proteins ,Oncology ,Carcinoma, Medullary ,Signal transduction ,Tyrosine kinase ,Proto-oncogene tyrosine-protein kinase Src ,Protein Binding ,Signal Transduction ,STAT3 Transcription Factor ,Blotting, Western ,Proto-Oncogene Proteins pp60(c-src) ,AUTOPHOSPHORYLATION SITES ,Proto-Oncogene Proteins ,Animals ,Humans ,Immunoprecipitation ,Glial Cell Line-Derived Neurotrophic Factor ,Nerve Growth Factors ,Thyroid Neoplasms ,Cell Nucleus ,MUTATIONS ,Cell Membrane ,Proto-Oncogene Proteins c-ret ,Receptor Protein-Tyrosine Kinases ,Janus Kinase 1 ,PROTOONCOGENE ,Janus Kinase 2 ,Enzyme Activation ,Insulin receptor ,Amino Acid Substitution ,HIRSCHSPRUNGS-DISEASE ,Mutation ,biology.protein ,Cancer research ,Trans-Activators ,MICE LACKING GDNF - Abstract
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 RETC634R 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.
- Published
- 2005