Alfano, Luigi (2009) The receptor tyrosine kinase RET is a Hsp90 client protein and is degraded upon exposure to 17-AAG. [Tesi di dottorato] (Unpublished)

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Item Type: Tesi di dottorato
Language: English
Title: The receptor tyrosine kinase RET is a Hsp90 client protein and is degraded upon exposure to 17-AAG.
Creators:
CreatorsEmail
Alfano, Luigialfanoluigi@hotmail.com
Date: 30 November 2009
Institution: Università degli Studi di Napoli Federico II
Department: Biologia e patologia cellullare e molecolare "L. Califano"
Doctoral School: Medicina molecolare
PHD name: Oncologia ed endocrinologia molecolare
PHD cycle: 22
PHD Coordinator:
nameemail
Vecchio, Giancarlovecchio@unina.it
Tutor:
nameemail
Santoro, Massimomasantor@unina.it
Carlomagno, Francescapersfra@tin.it
Date: 30 November 2009
Uncontrolled Keywords: RET; Hsp90; 17-AAG.
MIUR S.S.D.: Area 06 - Scienze mediche > MED/04 - Patologia generale
Date Deposited: 27 May 2010 07:36
Last Modified: 30 Apr 2014 19:39
URI: http://www.fedoa.unina.it/id/eprint/3936

Abstract

Molecular alterations of the receptor tyrosin kinase RET are involved in the pathogenesis of thyroid cancer. Germline point mutations in the extracellular and intracellular domains of the receptor are responsible of a group of inherited cancer diseases defined as Multiple Endocrine Neplasia type 2 syndromes (MEN2), whose major feature is represented by Medullary Thyroid Carcinoma (MTC). Somatic point mutations of RET are also found in around 40% of sporadic MTC. Hsp90 is part of the molecular chaperone machinery involved in mediating correct folding and stabilization of client proteins. Hsp90 chaperone fuctions in concert with the action of a multitude of other chaperone and co-chaperone proteins like Hsp70, Aha1 and CDC37; its activity depends on ATP and can be hindred by Geldanamycin-like compounds. Several kinases implicated in the process of neoplastic transformation are clients of Hsp90. In this thesis, we demonstrated that RET is a Hsp90 client protein. In RAT1 murine fibroblasts, stably transfected with RET wt or RET C634R oncogenic mutant, the Geldanamycin-derived drug 17- AAG was able to induce a proteasome dependent degradation of the receptor. Treatment with 17-AAG caused dissociation of Hsp90-RET complex and stabilized the interaction between RET and Hsp70 leading to recruitment of the Hsp70 interacting E3-ligase CHIP. Polyubiquitination by E3-ligases is commonly a destruction signal that mediates recognition of the targetted proteins by proteasome. Overexpression of CHIP wt, but not of two different CHIP defective mutants, CHIP-TPR and CHIP-ΔU, induced RET polyubiquitination and degradation. Interestingly, 17-AAG obstructed RET oncogenic signalling, decreasing RET C634R mediated activation of Ras-MAPK pathway and blocking transactivation of AP1-responsive and Mycgene promoters. We could not observe any changes in sensitivity to 17-AAG-induced degradation among several RET MTC-associated mutants carrying mutations in the intracellular domain of the receptor, the region where Hsp90 chaperone has been shown to bind to receptor tyrosine kinases. In human MTC cells carrying oncogenic RET mutants, Hsp90 inhibition by 17-AAG induced receptor degradation and signalling hindrance as shown by reduced phosphorylation of Shc and MAPK proteins. In such cells, 17-AAG caused a robust growth arrest, measured by decreased incorporation of BrdU, but failed to induce apoptosis. In conclusion we demonstrated that RET is a Hsp90 client protein and the chaperone is required for folding and stabilization of the receptor.

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