Met/Hepatocyte Growth Factor Receptor Ubiquitination Suppresses Transformation and Is Required for Hrs Phosphorylation

Abstract

The Met receptor tyrosine kinase (RTK) regulates epithelial remodeling, dispersal, and invasion and is deregulated in many human cancers. It is now accepted that impaired down-regulation, as well as sustained activation, of RTKs could contribute to their deregulation. Down-regulation of the Met receptor involves ligand-induced internalization, ubiquitination by Cbl ubiquitin ligases, and lysosomal degradation. Here we report that a ubiquitination-deficient Met receptor mutant (Y1003F) is tumorigenic in vivo. The Met Y1003F mutant is internalized, and undergoes endosomal trafficking with kinetics similar to the wild-type Met receptor, yet is inefficiently targeted for degradation. This results in sustained activation of Met Y1003F and downstream signals involving the Ras-mitogen-activated protein kinase pathway, cell transformation, and tumorigenesis. Although Met Y1003F undergoes endosomal trafficking and localizes with the cargo-sorting protein Hrs, it is unable to induce phosphorylation of Hrs. Fusion of monoubiquitin to Met Y1003F is sufficient to decrease Met receptor stability and prevent sustained MEK1/2 activation. In addition, this rescues Hrs tyrosine phosphorylation and decreases transformation in a focus-forming assay. These results demonstrate that Cbl-dependent ubiquitination is dispensable for Met internalization but is critical to target the Met receptor to components of the lysosomal sorting machinery and to suppress its inherent transforming activity.

From Tpr-Met to Met, tumorigenesis and tubes

“…The recruitment of the Cbl family of ubiquitin-protein ligases is required for ligand-induced degradation of many RTKs, among them the epidermal growth factor receptor (EGFR), the platelet-derived growth factor receptor, the colony-stimulating factor-1 receptor (CSF-1R) (reviewed by Thien and Langdon, 2001) and the Met receptor (Peschard et al, 2001Abella et al, 2005) (Figure 3). Growing evidence indicates that ubiquitination of RTKs is critical for their lysosomal degradation, through their ubiquitin-dependent targeting to intralumenal vesicles of MVBs and lysosomal degradation (Urbe et al, 2000;Raiborg et al, 2002;Duan et al, 2003;Jiang et al, 2003;Yamasaki et al, 2003).…”

“…Stimulation of the Met receptor with HGF/SF induces tyrosine phosphorylation of the receptor, and stimulation with high levels of HGF/SF leads to detectable Met receptor ubiquitination and enhanced degradation (Jeffers et al, 1997b;Kamei et al, 1999;Shen et al, 2000;Abella et al, 2005). The juxtamembrane region of Met contains an additional docking site, Y1003, which acts as a negative regulator of Met biological activity , and that is absent in the Tpr-Met oncogene (Figure 1).…”

“…Met receptor in normal and tumor biology P Peschard and M Park stability of Met and sustained signaling of downstream pathways (Peschard et al, 2001;Abella et al, 2005). Although a consensus for c-Cbl TKB domain binding has been established (D/NxpYxxD/EF), this motif is not present in Met, and instead, a DpYR motif, including Y1003, is required for the direct recruitment of the c-Cbl TKB domain and for ubiquitination of the Met receptor .…”

Analysis of Receptor Tyrosine Kinase Internalization Using Flow Cytometry

“…Receptor internalization from the cell surface represents a key first step in the inactivation process, by targeting the internalized receptor for lysosomal degradation. Corruption of these mechanisms contributes to prolonged cMet signaling (5,6,13) suggesting that defects in cMet endocytic trafficking could be a contributing factor to increased cMet levels and signaling in some human metastatic cancers.…”

“…Acid stripping cells by washing with low pH solutions readily dissociates most ligands from their receptors (4,13,16). However the optimal acid stripping conditions for removing cell surface associated ligand without damaging the cells must be empirically determined for each cell line and for each receptor/ligand combination.…”