Figure 1.
C-KIT activation induces a mobility shift of Mitf.
(A) HEK293T cells transfected with c-KIT and Mitf and (B) Melan-A cells transfected with only Mitf displays mobility shift, where the upper band becomes more intense than the lower band, after 5–15 min of SCF (100 ng/ml) stimulation.
Figure 2.
Mitf upper band is abolished by S73A, S409A and S73/409A substitution mutations.
Transfection with wt Mitf in HEK293T cells shows a mobility shift after 15 minutes of SCF stimulation. In contrast, the S73A, S409A and S73/409A mutations of Mitf completely eliminate the heavy molecular weight band of Mitf, independent of hSCF treatment.
Figure 3.
Human SCF (hSCF) does not activate mouse c-KIT.
(A) Endogenous mouse c- kit immunoprecipitated from Melan-A cells stimulated with mouse SCF (mSCF) was autophosphorylated. (B) However, the addition of hSCF to the Melan-A cells did not activate endogenous mouse c-KIT. Immunoblotting with 4G10 antibody was used to detect general tyrosine phosphorylation to assess the c-KIT phosphorylation/ activation.
Figure 4.
C-KIT phosphorylation mutants Y721F, Y568F and Y568F/Y570F are unable to activate Mitf.
(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 p = 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.
Figure 5.
Inhibitors against Src kinase, Mek, PI3 kinase, Akt and p38 antagonize c-KIT mediated Mitf activation.
Thirty minutes prior to SCF stimulation (A) HEK293T cells and (B) Melan-A cells were treated with Src family kinase inhibitor (SU6656), Mek inhibitor (U0126), PI3 kinase inhibitor (LY294002) or Akt inhibitor (Akt IV), respectively, all of which prevented c-KIT from activating Mitf. Densitometry analysis was done to statistically present Mitf mobility shift in (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 p = 0.00025. SFC treatment is indicated by gray bars.
Figure 6.
Effects of C-KIT and Mitf on cell proliferation.
HEK293T cells transfected with either c-KIT or Mitf did not influence the level of cell proliferation regardless of SCF stimulation. However, when wt c-KIT or c-KIT Y703F/Y936F and Mitf were introduced, SCF treatment for 48 hours resulted in an increase in cell numbers. While the c-KIT mutants Y568F and Y721F were able to mediate a proliferative response to SCF stimulation, the level of increase was significantly (p<0.01) lower compared to wt c-KIT or c-KIT Y703F/Y936F. Each bar represents the average of nine independent experiments. Error bars signify 95% confidence interval.
Figure 7.
Proposed model for c-KIT mediated Mitf activation in HEK293T cells and mouse melanocytes, Melan- A.
Phosphorylation of c-KIT Y568/Y570 recruits and Src kinase, triggering the activation of Ras/Raf/Mek/Erk and p38 kinase pathways which finally leads to Mitf activation. In contrast, phosphorylation of c-KIT Y721 results direct binding of p85 and subsequent Mitf activation through Akt. P85 can also indirectly interact with c-KIT through the binding of Gab 2. Since Gab 2 is absent in both HEK283T and Melan- A cells, the Y721 pathways is favored, rendering the Y703/Y936 dispensible for Mitf activation.