Abstract
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.
Highlights
Cell signaling plays an important role in the fine tuning of cellular function and behavior
Previous mobility shift studies were mostly performed with endogenous c-KIT and Microphthalmia associated transcription factor (Mitf) in melanocytes and melanoma cells
Our results suggest that the c-KIT phosphorylation sites Y586 and Y570 are involved in the signaling to Mitf and that Src might play a role in this event
Summary
Cell signaling plays an important role in the fine tuning of cellular function and behavior. Signaling cascades generated by the cell surface tyrosine kinase receptor c-KIT through the binding of its ligand stem cell factor (SCF) are involved in the regulation of many cell types including melanocytes [1], mast cells [2,3], germ cells [4] and interstitial cells of Cajal [5]. Phosphorylation of tyrosine residues enables c-KIT to recruit and bind to downstream signaling proteins for subsequent activation of signal transduction pathways. The transduction signal relayed from c-KIT to Src kinases triggers the activation of the Ras-Erk pathway, involving the pro-survival and anti-apoptotic Ras-Raf-Mek-Erk cascade [14,15]. Even though the importance of c-KIT in melanogenesis is recognized and a loss-of-function mutation of the receptor, or its downstream targets, can lead to developmental pigmentary diseases like piebaldism and Waardenburgs syndrome, the signaling cascades of c-KIT in melanocytes are not fully elucidated [20,21]
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