Main author: Eyvindur Árni Sigurðarson
Institution or Company: Háskóli Íslands
Co-Authors, Institution or Company:
Valerie Fock, Medical University of Vienna. Sara Sigurbjörnsdóttir, Háskóli Íslands. Florent Dingly, Institut Curie.. Damarys Loew, Institut Curie. Lionel Larue, Institut Curie. Eiríkur Steingrímsson, Háskóli Íslands.
Introduction: MITF is a transcription factor that is vital to both melanocyte and melanoma biology and plays a major role in differentiation, proliferation, and invasion. Although phosphorylation has been suggested to affect the activity, location, and stability of MITF, the results so far are incomplete. The current literature has focused primarily on two phosphorylation sites on the protein. As several emergent melanoma drugs target kinases that may phosphorylate MITF, there is an urgent need to understand the effects MITF phosphorylation.
Methods: To identify phosphorylation sites, tagged-MITF was transfected into cells and grown in large quantities in the presence or absence of kinase inhibitors, and the protein purified by immunoprecipitation. The purified MITF was run through liquid chromatography with tandem mass spectrometer to identify phosphorylation sites. Several sites were identified, both novel and previously known sites. Selected sites were mutated to alanine to block phosphorylation and stably expressed in A375P melanoma cells using the PiggyBac inducible transposon system. Western blots and qRT-PCR were used to equalise expression between the mutants. The effects of the mutants were then investigated using immunofluorescence to see the localization of MITF, RNA-sequencing to identify differentially expressed genes and growth curves were analysed using the Incucyte S3 live-cell imager.
Results: The mutants have different effects on localization, gene expression and growth with some mutants showing no effects in some categories.
Conclusion: These results suggest that some phosphorylation sites are redundant, and others have a more conserved role.
