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.

ACKNOWLEDGMENTS

We thank Marisa Ponzo and Naila Chughtai for help with transient transfection assays and Dongmei Zuo and Laurent Sansregret for help with confocal imaging and flow cytometry, respectively. We are grateful to members of the Park laboratory for critical comments on the manuscript and Stephane Laporte for his helpful discussion. We thank Harald Stenmark for providing us with Hrs antibodies, Linda Hicke for the ubiquitin (7KtoR) plasmid, George Vande Woude for HGF, and Robert Lodge for GFP-Rab7.

J.A. is a recipient of an Alexander McFee studentship and P. P. is a recipient of a Terry Fox research studentship from National Cancer Institute of Canada. Sylvie Urbé is a Wellcome Trust Career Development Fellow. M.P. is recipient of a Canadian Institute of Health Research (CIHR) senior scientist award. This research was supported by an operating grant to M.P. from the CIHR.