KITY568F/Y570F and Cbl RF domains are essential for SCF-induced degredation of KIT and Cbl proteins. The 293 cells were transfected with different combinations of cDNA constructs or empty vectors as indicated. Cells were starved and stimulated with or without SCF for 10 minutes (A) or 5 minutes (B-C). (A) Y568/Y570, the KIT juxtamembrane SH2 domain docking site, is required for tyrosine phosphorylation of Cbl-b and the degradation of both KIT and Cbl-b induced by SCF stimulation. Cell lysates were immunoprecipitated with anti-HA antibody followed by blotting with α-pTyr to show tyrosine phosphorylation of Cbl-b protein stimulated with SCF. Membrane was stripped and reprobed with α-HA to show Cbl-b levels, or lysates were subjected directly to probe with anti-HA antibody to detect Cbl-b and then stripped and reprobed with anti-KIT antibody to detect the expression of different KIT proteins (bottom 2 panels). (B) Y568/570 in KIT and Cbl-b TKB domain are essential for SCF/KIT-induced tyrosine phosphorylation and degradation of Cbl-b and KIT. Cell lysates were subjected directly to probe with α-pTyr to detect tyrosine phosphorylation of wild-type and mutant Cbl-b or KIT proteins (top), with anti-KIT antibody to detect the expression of different KIT proteins (middle), or with α-HA antibody to detect the levels of wild-type and mutant Cbl-b (bottom). (C) RF domain in c-Cbl is essential for the degradation of KIT and itself induced by SCF, and Y568/Y570 in KIT is essential for the tyrosine phosphorylation of c-Cbl and the degradation of both KIT and c-Cbl. Cell lysates were subjected to probe as in panel B except that α-HA antibody was used to detect the levels of wild-type and mutant c-Cbl.
