RET-familial medullary thyroid carcinoma mutants Y791F and S891A activate a Src/JAK/STAT3 pathway, independent of glial cell line-derived neurotrophic factor.
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.
Plaza Menacho
Ivan
I
Department of Medical Genetics, University of Groningen, Groningen, the Netherlands.
Koster
Roelof
R
van der Sloot
Almer M
AM
Quax
Wim J
WJ
Osinga
Jan
J
van der Sluis
Tineke
T
Hollema
Harry
H
Burzynski
Grzegorz M
GM
Gimm
Oliver
O
Buys
Charles H C M
CH
Eggen
Bart J L
BJ
Hofstra
Robert M W
RM
D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D018276:Carcinoma, Medullary / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D051100:Glial Cell Line-Derived Neurotrophic Factor; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D053613:Janus Kinase 1; D053614:Janus Kinase 2; D008156:Luciferases / Q000378:metabolism; D018813:Multiple Endocrine Neoplasia Type 2a / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009414:Nerve Growth Factors / Q000378:metabolism*; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret; D016392:Proto-Oncogene Proteins pp60(c-src); D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D013964:Thyroid Neoplasms / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism
D004268:DNA-Binding Proteins; C495039:GDNF protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; C000715470:MAS1 protein, human; D009414:Nerve Growth Factors; D015513:Oncogene Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D000255:Adenosine Triphosphate; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D020794:Receptor Protein-Tyrosine Kinases; C507904:JAK1 protein, human; C507924:JAK2 protein, human; D053613:Janus Kinase 1; D053614:Janus Kinase 2; D016392:Proto-Oncogene Proteins pp60(c-src)