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RET activation by germline MEN2A and MEN2B mutations
M G Borrello1, D P Smith, B Pasini
1Division of Experimental Oncology A, Istituto Nazionale Tumori, Milan, Italy.
Abstract:
The RET proto-oncogene encodes a receptor tyrosine kinase (TK). It has been shown that distinct germline mutations in the RET proto-oncogene are associated with the dominantly inherited cancer syndromes multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma (FMTC) as well as Hirschsprung disease (HSCR), a congenital disorder characterised by absent enteric innervation. In this study, we have transfected NIH3T3 and PC12 phaeochromocytoma cells with MEN2A (Cys634-> Arg) and MEN2B (Met918-> Thr) RET constructs. Both caused transformation of the NIH3T3 cells and differentiation of PC12 cells. The Ret (MEN2A) and Ret (MEN2B) proteins were constitutively phosphorylated on tyrosine, and their in vitro kinase activity was significantly higher than that of the wild type protein. The MTC cell line TT carries a CYs634-> Trp MEN2A mutation, and we have shown by immunoelectronmicroscopy that Ret is clustered on the cell surface in a manner reminiscent of ligand-induced aggregation of cell surface receptors. RET is activated, as RET/PTC oncogene, by somatic rearrangements which link the TK domain to a constitutive dimerization interface in papillary thyroid carcinomas. We have compared the biological and biochemical activity of the TK domains of the wild type and MEN 2B Ret in the context of the RET/PTC. The results show that the MEN 2B mutation significantly increases the TK domain enzymatic activity suggesting that dimerization may be still necessary for MEN 2B Ret to express its full activity.
Insights
Germline mutations in the RET proto-oncogene drive cancers like MEN2A/2B and Hirschsprung disease. This study shows specific RET mutations enhance tyrosine kinase activity and cell transformation, impacting cancer development.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- The RET proto-oncogene, encoding a receptor tyrosine kinase, is implicated in cancers and congenital disorders.
- Germline mutations in RET are linked to multiple endocrine neoplasia (MEN 2A/2B), familial medullary thyroid carcinoma (FMTC), and Hirschsprung disease (HSCR).
Purpose of the Study:
- To investigate the functional impact of specific RET proto-oncogene mutations associated with MEN 2A and MEN 2B.
- To compare the biochemical and biological activity of wild-type RET with MEN 2A and MEN 2B mutant forms.
Main Methods:
- Transfection of NIH3T3 and PC12 cells with MEN2A (Cys634->Arg) and MEN2B (Met918->Thr) RET constructs.
- Assessment of cell transformation and differentiation.
- Analysis of constitutive tyrosine phosphorylation and in vitro kinase activity.
- Immunoelectronmicroscopy to study RET clustering on cell surfaces.
- Comparison of wild-type and MEN 2B RET tyrosine kinase (TK) domain activity within RET/PTC oncogenes.
Main Results:
- MEN2A and MEN2B RET constructs induced NIH3T3 cell transformation and PC12 cell differentiation.
- Both mutant RET proteins exhibited constitutive tyrosine phosphorylation and significantly elevated in vitro kinase activity compared to wild-type.
- RET (MEN2A) with a Cys634->Trp mutation showed cell surface clustering.
- The MEN 2B mutation markedly increased the enzymatic activity of the RET TK domain in the context of RET/PTC.
Conclusions:
- Specific RET mutations, such as those in MEN 2A and MEN 2B, lead to constitutive activation and enhanced kinase function.
- RET activation through dimerization, particularly in RET/PTC oncogenes, is crucial for its full activity, even with pre-existing mutations like MEN 2B.
- These findings elucidate mechanisms underlying RET-driven cancers and congenital disorders.