The role of Cbl-mediated ubiquitination in the regulation of the Met receptor tyrosine kinase /

The Met receptor tyrosine kinase (RTK) and its ligand, the hepatocyte growth factor/scatter factor (HGF/SF), which regulate epithelial cell remodelling, dispersal and invasion, are deregulated in many human cancers. In the last twenty years, mechanisms of RTK activation and signalling have been studied extensively. In contrast, very little is known about the mechanisms of down-regulation of RTKs. At the time I began my thesis work, the family of Cbl ubiquitin-protein ligases emerged as negative regulators for RTKs. In this thesis, I demonstrate that c-Cbl is an important negative regulator of the Met receptor. Upon HGF stimulation, c-Cbl promotes ubiquitination of the Met RTK. This requires the association of the c-Cbl tyrosine kinase binding (TKB) domain with a DpYR binding motif, including Y1003, present in the juxtamembrane of the Met receptor. A Met Y1003F receptor, which lacks the c-Cbl TKB binding site, is not ubiquitinated, transforms fibroblast and epithelial cells in vitro and is tumorigenic in vivo. I demonstrate that ubiquitination of the Met receptor is not required for its internalization from the plasma membrane, but is essential for its lysosomal degradation. In the presence of HGF, the Met Y1003F receptor is poorly degraded and remains phosphorylated, leading to sustained activation of the Ras-MAPK pathway. Moreover, fusion of a ubiquitin moiety to the carboxy-terminus of Met Y1003F is sufficient to decrease Met receptor stability, prevent sustained MEK1/2 activation and reduce cell transformation.To examine the tumorigenicity of the Met Y1003F receptor in animals, I generated a murine model expressing Met Y1003F under the MMTV promoter, and observed that Y1003F substitution increases Met receptor tumorigenicity in mammary glands. This demonstrates for the first time that ubiquitination of a RTK is required for its biological functions in vivo. Furthermore, this highlights synergy between an activating mutation (M1250T) and a loss of down-regulation mutation (Y1003F) in the tumorigenicity of the Met receptor. Based on these results and on the observation that several RTKs escape the lysosomal degradative pathway in human tumours, I propose that loss of RTK down-regulation is a common mechanism for oncogenic activation of RTKs in human tumours