Negative Regulation of c-Kit Is Dependent on Direct Binding of Cbl to Tyrosines 568 and 936.

C-Kit belongs to the family of type III receptor tyrosine kinases and is mainly expressed in hematopoietic precursor cells. Binding of stem cell factor (Kit ligand) leads to receptor dimerization, activation of intrinsic tyrosine kinase activity and cross-autophosphorylation of receptor tyrosine residues which provide specific docking sites for molecules that transduce the signal further downstream. Signaling through c-Kit results in several different cellular responses, most of which are directly involved in growth control and survival. Negative regulation of the receptor activity is therefore of much importance concerning pathogenic processes such as tumorigenesis in which activated c-Kit often plays a role. Here we present data showing that the ubiquitin E3 ligase Cbl must be activated by the Src family kinases and recruited to c-Kit in order to trigger receptor degradation. We demonstrate that the autophosphorylation sites Y568 and Y936 in c-Kit, previously reported to be binding sites for APS, Src or Grb2, respectively, are involved in Cbl recruitment and activation, possibly in a direct manner. I571 and L939 of c-Kit hereby determine the specificity for binding the N-terminal tyrosine kinase binding domain of Cbl. We show that activation of Cbl by Src family kinase members on one hand and direct or indirect recruitment of active Cbl to the pY568/I571 and pY936/L939 motifs of c-Kit on the other, are separable events and are both necessary for Cbl-mediated c-Kit degradation. Consistently, both the Y568/570F double mutant lacking Src kinase activity and the I571A/L939A double mutant lacking Cbl-specific interaction sites fail to ubiquitinate and degrade c-Kit upon ligand stimulation in stably transfected PAE and Ba/F3 cells. Thus, we could reveal novel aspects of the dynamic process of docking and activating signaling molecules at receptor phosphotyrosines that negatively regulate c-Kit-mediated responses. Moreover, we demonstrate that Cbl mediates monoubiquitination of c-Kit and that the receptor subsequently is targeted for lysosomal degradation. Taken together, our findings reveal novel insights on how c-Kit -mediated signal transduction is negatively regulated by Cbl.