Líffræðifélag Íslands - biologia.is
Líffræðiráðstefnan 2021
Erindi/veggspjald / Talk/poster E72
Höfundar / Authors: Eyvindur Árni Sigurðarson (1), Valerie Fock (1,2) and Eiríkur Steingrímsson (1)
Starfsvettvangur / Affiliations: (1) Department of Biochemistry and Molecular Biology, Faculty of Medicine, BioMedical Center, University of Iceland. (2) Children’s Cancer Research Institute, Vienna, Austria.
Kynnir / Presenter: Eyvindur Árni Sigurðarson
MITF is a master regulator of melanocyte development where it regulates differentiation, proliferation, and invasion. It also plays an important role in melanoma. Phosphorylation has been shown to affect the activity, stability, and localization of MITF. Interestingly, pharmaceuticals that affect the signalling pathways that regulate MITF phosphorylation are used to treat melanoma but how they affect MITF is not known at present. There is an urgent need for better understanding MITF phosphorylation and its resulting effects. We have used mass spectroscopy to map phosphorylation sites in MITF and their response to Braf inhibitors and have identified multiple novel sites in MITF, in addition to the previously identified sites. Sites that were consistently phosphorylated were mutated from serine to alanine to block phosphorylation and wild type and mutant versions expressed in A375P cells using an inducible PiggyBac system. RNA-sequencing was then used to identify differentially expressed genes (DEG) in the mutants to identify the functional effects of the phosphorylation. Gene set enrichment analysis was done on the DEGs to unravel the effects of the mutations. The expression of the MITF protein and RNA was confirmed using western blotting and qPCR, respectively. The results suggest that some of the mutants may affect the ability of MITF to regulate specific sets of target genes and thus that specific phosphorylation events direct MITF to a subset of the genes that MITF regulates.