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

Sammanfattning: 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.