Oncogenic KIT mutations in different exons lead to specific changes in melanocyte phospho-proteome

• Different KIT mutations lead to activation of different downstream pathways.
• Single point mutations in KIT determine changes in the cell's phospho-proteome that are specific for each mutations site.
• Mutations in KIT559 lead to over activation of kinases members of the casein kinase 1 (CK1) family.
• Mutations in KIT816 lead to over activation of kinases members of the p21-activated kinases (PAKs) family.

Mutations in the proto-oncogene c-KIT (KIT) are found in several cancers, and the site of these mutations differs markedly between cancer types. We used site directed mutagenesis to induce KIT559, KIT642 and KIT816 mutations in primary human melanocytes (PHM) and we investigated the impact of each mutation on KIT function. We studied canonical KIT-signaling pathways by immunoblotting, and we used stable isotope labeling by amino acids in cell culture (SILAC) and kinase prediction models to identify kinases differently activated in respective mutants. We validated our results with the analysis of phosphorylation levels of selected substrates for each kinase. We concluded that CK1 ε and δ are more active in cell clones harboring KIT559 and KIT642 mutations, whereas PAK4 is more active in clones with KIT816 mutation. Our findings might help to develop further therapeutic options for tumors with specific KIT mutations in different domains.Biological significanceDifferent types of cancers harbor mutations in the oncogene KIT. The use of small molecules inhibitors directly targeting KIT had a limited success in the treatment of patients with KIT mutant cancers. Our study describes specific phospho-proteome changes due to different KIT mutations, and provides targets of further therapeutic options.

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