C-KIT phosphorylation mutants Y721F, Y568F and Y568F/Y570F are unable to activate Mitf.

<p>(A) HEK293T cells were transfected with wt or mutant forms of c-KIT and Mitf. C-KIT with mutated binding site for the PI3 kinase regulatory subunit p85 (Y721F), did not result in a Mitf band shift. C-KIT mutants Y568F and Y568F/Y570F, lacking the ability to activated Src, did not activate Mitf upon SCF stimulation. However, the c-KIT Y703F/Y936F mutant was able to mediate SCF-induced Mitf activation. (B) Mouse melanocytes Melan-A that were treated as above showed the same Mitf activation pattern. To statistically verify the results, Mitf mobility shift densitometric quantitation was performed on (A) HEK293T cells and (B) Melan-A cells. Each bar represents the mean ± SEM for at least three independent experiments. *Denotes significant difference from positive control <i>p = </i>0.00025. SFC treatment is indicated by gray bars. (C) Immunoprecipitation and subsequent immunoblotting of Gab2 protein reveal that neither HEK293T cells nor Melan- A melanocytes maintain an endogenous expression. In contrast, Gab2 protein is detected in the positive control BaF3 cell line.