401 Characterization of novel MAPK interactor with potential in therapeutic development

Abstract

MAPK signaling pathways are integral to cell proliferation and apoptosis (e.g. via ERK), so identifying potential therapeutic targets that can regulate MAPK activation remains an area of active research. We first investigated MAPK interactors through BioID. BirA-tagged MAPK-1 was expressed in three melanoma cell lines (A375, SKMEL28, COLO829) with oncogenic BRAF mutations, under two conditions: with or without drug inhibition of MAPK activation. The biotin MAPK-1 proximal proteins are biotin labeled and then captured by streptavidin pull-down and detected by LC-MS. These proximal interactor proteins, detected by LC-MS, were all previously annotated in MAPK1 pathways with one notable exception—TICRR (treslin). The treslin protein is part of initiating DNA replication, which suggests that elucidating its relationship with MAPK activity could be biologically significant. To identify which treslin residues are possibly modified by MAPK, we used a two-pronged approach and, in live cells, used in vitro kinase and phosphoproteomic assays. For the first, we incubated recombinant treslin with active MAPK for increasing periods of time and detected phosphorylated residues on treslin through mass spectometry. To complement this approach, we also looked at MAPK pathway inhibition and its result on TICRR phosphorylation—A375 cells were exposed to pharmacologic inhibition of ERK, MERK, and RAFi, and the exogenously expressed treslin was captured by a FLAG tag and its phosphorylated residues determined through mass spectrometry. We found several candidate treslin residues that are site of MAPK phosphorylation. Our findings show that treslin’s relationship to MAPK is relatively uncharacterized yet that understanding presents possible biological and ultimately clinical benefits.