C-KIT Signaling Depends on Microphthalmia-Associated Transcription Factor for Effects on Cell Proliferation

The development of melanocytes is regulated by the tyrosine kinase receptor c-KIT and the basic-helix-loop-helix-leucine zipper transcription factor Mitf. These essential melanocyte survival regulators are also well known oncogenic factors in malignant melanoma. Despite their importance, not much is known about the regulatory mechanisms and signaling pathways involved. In this study, we therefore sought to identify the signaling pathways and mechanisms involved in c-KIT mediated regulation of Mitf. We report that c-KIT stimulation leads to the activation of Mitf specifically through the c-KIT phosphorylation sites Y721 (PI3 kinase binding site), Y568 and Y570 (Src binding site). Our study not only confirms the involvement of Ras-Erk signaling pathway in the activation of Mitf, but also establishes that Src kinase binding to Y568 and Y570 of c-KIT is required. Using specific inhibitors we observe and verify that c-KIT induced activation of Mitf is dependent on PI3-, Akt-, Src-, p38- or Mek kinases. Moreover, the proliferative effect of c-KIT is dependent on Mitf in HEK293T cells. In contrast, c-KIT Y568F and Y721F mutants are less effective in driving cell proliferation, compared to wild type c-KIT. Our results reveal novel mechanisms by which c-KIT signaling regulates Mitf, with implications for understanding both melanocyte development and melanoma

The interconnected KIT receptor and Microphthalmia-associated transcription factor axis in melanoma

Melanoma is an aggressive disease that arises from deregulated signaling events in melanocytes. The KIT receptor tyrosine kinase and the Microphthalmia-associated transcription factor (MITF) are two essential components that are required for the normal physiology of melanocyte development and function. Consequently, aberrant activities of these factors are involved in the development of melanoma. To better understand the molecular basis of disease development we studied the mechanisms that gatekeep and finetune the activity of both KIT and MITF. In this thesis we have identified novel KIT ligand-dependent signaling pathways that, through MITF, affect cell proliferation. Notably, we found that the specific KIT tyrosine phosphorylation sites Y721 (PI3 kinase binding site), Y568 and Y570 (SRC binding sites) affect phosphorylation status of the MITF protein. Using inhibitors against SRC, PI3K, AKT, MEK ERK and p38 during KIT activation, we identified that these signaling pathways are essential components in communicating signals between KIT and MITF. The activity of KIT is regulated by the tetrapeptide insert in the extracellular juxtamembrane domain of the receptor. By generating receptor mutants with different insert lengths, we showed that the length of this insert is critical for the fine tuning of receptor activation. Using mass spectrometry and phospho-site specific antibodies against MITF, we found and characterized several serine and tyrosine phosphorylation sites in the protein. More importantly, in stark contrast to previous results, we discovered that the S73 and S409 phosphorylation sites of MITF are neither dependent on KIT nor MAPK-ERK signaling. Furthermore, we show that the tyrosine phosphorylation sites of MITF are required for oncogenic KIT signaling and affect nuclear localization of MITF. The structural properties of MITF and the KIT receptor were also clarified. By solving the crystal structure of MITF we explain the restricted heterodimerization of MITF towards TFEB, TFEC and TFE3, the binding of M-box DNA sequence and the ability to produce interallelic complementation. In short, the detailing of KIT and MITF regulation has increased our understanding of key signaling processes that are important to understand the development of melanoma

Differential activity of c-KIT splice forms is controlled by extracellular peptide insert length.

Understanding receptor activation is important for disease intervention. Activation of the receptor tyrosine kinase c-KIT is involved in numerous diseases including melanoma, mastocytosis, multiple myeloma and gastrointestinal stromal tumors. To better understand the regulation of activation, we studied the two c-KIT isoforms, c-KIT(-) and c-KIT(+) which differ by a tetrapeptide insert GNNK, located in the extracellular juxtamembrane domain of the c-KIT(+) isoform. This region is important for regulating receptor activation. Here we show that the consecutive elimination of one amino acid at a time from the GNNK tetrapeptide insert gradually increases receptor tyrosine phosphorylation, ubiquitination, internalization and downstream MAP kinase-ERK activation. Successively decreasing the insert length progressively improves cell survival during drug treatment. Our results indicate that the length of the tetrapeptide fine-tunes receptor activity, thus providing deeper insight into c-KIT activation

Suppressor Of cytokine signaling 6 (SOCS6) negatively regulates Flt3 signal transduction through direct binding to phosphorylated Tyr 591 and Tyr 919 of Flt3.

The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). The suppressors of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can regulate receptor tyrosine kinases signal transduction. In this study we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation to Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phospho-peptide fishing indicates that SOCS6 binds directly to phospho-tyrosine 591 and 919 of Flt3. By using stable transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3 as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker activation of Erk1/2 but not Akt in transfected Ba/F3 and UT-7 cells, and in OCI-AML-5 cells. The absence of SOCS6 promotes Ba/F3 and UT-7 cell proliferation induced by oncogenic internal-tandem-duplications (ITDs) of Flt3. Taken together, these results suggest that SOCS6 negatively regulates Flt3 activation and downstream Erk signaling pathway and cell proliferation

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.</p

Mitf upper band is abolished by S73A, S409A and S73/409A substitution mutations.

<p>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.</p

Proposed model for c-KIT mediated Mitf activation in HEK293T cells and mouse melanocytes, Melan- A.

<p>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.</p

Inhibitors against Src kinase, Mek, PI3 kinase, Akt and p38 antagonize c-KIT mediated Mitf activation.

<p>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 <i>p</i> = 0.00025. SFC treatment is indicated by gray bars.</p