The receptor tyrosine kinase Ax1 has been shown to be activated by its ligand Gash and by oxidative stress in the form of hydrogen peroxide. However, the regulatory mechanisms controlling the levels of Ax1 upon Gash binding or oxidative stress have not been elucidated. This report demonstrates that Gash-induced downregulation of Ax1 is blocked by inhibitors of endocytosis and lysosomal degradation, but not by inhibitors of proteosomal activity. Furthermore, it is shown that binding of Ax1 to Gash induces the phosphorylation and ubiquitination of Ax1 and the interaction of Ax1 with the ubiquitin ligase c-Cb1. Importantly, hydrogen peroxide induces Ax1 tyrosine phosphorylation but not its ubiquitination, determining the inhibition of Ax1 downregulation. These results suggest that as shown for other receptor tyrosine kinases, ubiquitination of Ax1 is needed to ensure its proper degradation in the lysosome, and that oxidative stress may inhibit Ax1 ubiquitination and downregulation. (c) 2005 Elsevier Inc. All rights reserved.