Mediator Kinase Substrates in Human Myometrial Stem Cells Revealed by Using Chemical Inhibitors and Quantitative Phosphoproteomics

Abstract

Uterine fibroids are benign tumors that occur in the myometrium of the uterus. Although they are benign, they represent a significant health problem as they affect approximately 70% of women and cause pregnancy complications, infertility and menstrual disorders. These health issues are especially concerning as uterine fibroids affect women of reproductive age. To date, non-invasive, long-term treatment options for uterine fibroids are not available. Thus, a significant need remains to develop non-invasive long-term treatment options for uterine fibroids. Strikingly, 70% of uterine fibroids have mutations in a protein called MED12. MED12 is one of 3 proteins that constitute the Mediator kinase complex. Together, these proteins (MED12, MED13, CYCLIN C and CDK8/19) function as a complex to integrate and transduce signals to RNA polymerase II, and thereby regulate transcription in response to numerous signaling pathways. Interestingly, our lab has previously demonstrated that these MED12 mutations lead to a loss of Mediator kinase activity. Thus, we hypothesize that this loss of Mediator kinase activity promotes uterine fibroid formation by dysregulating cellular processes. The goal of this work was to identify substrates that are phosphorylated by Mediator kinase in uterine fibroid progenitor cells, myometrium side population (MyoSP) cells, in order to understand how loss of Mediator kinase activity might promote the development of uterine fibroids. In order to identify proteins that are phosphorylated by Mediator kinase, MyoSP cells were metabolically labeled with either heavy or light stable isotopes and treated with vehicle or a specific inhibitor of Mediator kinase. Mass spectrometry was used to identify and quantify phosphoproteins from each condition (inhibitor treated vs. vehicle treated). An Empirical Bayes analysis was used to identify 307 phosphoproteins that significantly decreased upon treatment with the Mediator kinase inhibitor. Of these proteins, 4 (STAT1, TP53BP1, FOXK1, and NELFB) were further validated as direct targets of Mediator kinase by in-vitro kinase assay. In conclusion, this work identifies direct substrates of Mediator kinase that can be investigated for their roles in promoting uterine fibroid formation and represent potential targets for novel therapies to treat uterine fibroids.