Signalling Pathways of M918t Ret Mutant in Multiple Endocrine Neoplasia Type 2b

This dissertation, "Signalling Pathways of M918T RET Mutant in Multiple Endocrine Neoplasia Type 2B" by 陳展豪, Chin-ho, Chan, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Signalling pathways of M918T RET mutant in Multiple Endocrine Neoplasia type 2B Submitted by Chan Chin Ho For the degree of Master of Philosophy at The University of Hong Kong in August 2005 Multiple endocrine neoplasia type 2B (MEN2B) is an inherited cancer syndrome characterized by the presence of medullary thyroid carcinoma and phaeochromocytoma. A single amino acid substitution (M918T) in the RET receptor tyrosine kinase was found in more than 95% of the patients. This mutation is believed to lead to constitutive activation and altered substrate specificity of the RET receptor, which resulted in cellular transformation. There are two predominant isoforms of RET, designated as RET51 and RET9, as a result of alternative splicing. To screen for the possible components of M918T mutant RET (RET/MEN2B) signaling pathways, the profiles of the phosphorylated proteins of RET51/MEN2B and RET9/MEN2B transfected cells were compared with that of either wild type RET or empty vector transfected cells. In brief, serine-, threonine- and tyrosine-phosphorylated proteins of the transfected NIH3T3 cells were isolated by affinity chromatography and resolved by SDS-PAGE. Then the protein bands differentially displayed in the lane of phosphorylated proteins from cells expressing iRET/MEN2B were identified by mass spectrometry. Four protein bands were identified to be upregulated in RET51/MEN2B cells: Tubulin beta-2 chain, UDP- glucose dehydrogenase (UGDH), alpha enolase, and placenta and embryonic expression gene; while peroxiredoxin 1 was upregulated in RET9/MEN2B cells. UGDH is a key enzyme responsible for extracellular matrix production and its activity is upregulated by phosphorylation. UGDH is important in Wg signaling in Drosophila, the homologue of mammalian Wnt. Therefore the regulation of β-catenin, the main component of Wnt signaling pathway, was studied in RET51/MEN2B expressing cells. The results suggested that the β-catenin signaling pathway was activated by RET51/MEN2B. First, immunocytochemistry showed that RET51/MEN2B may promote the translocation of plasma membrane bound β-catenin into the cytoplasm; Second, western analysis showed that RET51/MEN2B may stabilize cytosolic β-catenin by inhibiting the association between β-catenin and GSK3β, a protein which normally targets β-catenin to the degradation pathway. This outweighed the opposite destabilizing effect exerted by both RET/MEN2B and GDNF stimulated RET51/WT. In both cell types, GSK3β is dephosphorylated at Ser9 which would lead to association between β-catenin and GSK3β. Third, this was supported by the data that there was an elevation of nuclear β-catenin in RET51/MEN2B cells, which may result from stabilisation of cytosolic β-catenin followed by transfer of β-catenin into the nucleus. Finally, preliminary results suggested that blocking of UGDH activity by its specific inhibitor, piperine (100μM), negatively regulates the β-catenin signaling in RET51/MEN2B and empty vector transfected clones. Taken together, the findings suggested that UGDH might act as an upstream regulator of the RET51/MEN2B mediated β-catenin signaling pathway. ii DOI: 10.5353/th_b3460324 Subjects: Oncogenes Protein-tyrosine kinase Cellular signal transduction Endocrine glands - Cancer - Genetic aspects