Identification and characterization of the Mediator kinase-dependent myometrial stem cell phosphoproteome

Abstract Objective To identify in myometrial stem/progenitor cells, the presumptive cell of origin for uterine fibroids, substrates of Mediator-associated CDK8/19 kinase, known to be disrupted by uterine fibroid driver mutations in Mediator subunit MED12. Design Experimental Study. Setting Academic Research Laboratory. Patients Women undergoing hysterectomy for uterine fibroids. Interventions Stable isotopic labeling of amino acids in myometrial stem/progenitor cell culture (SILAC) coupled with chemical inhibition of CDK8/19 and downstream quantitative phosphoproteomics and transcriptomic analyses. Main Outcome Measures High-confidence Mediator kinase substrates identified by SILAC-based quantitative phosphoproteomics were determined using an Empirical Bayes analysis and validated orthogonally by in vitro kinase assay featuring reconstituted Mediator kinase modules comprising wild-type or G44D mutant MED12 corresponding to the most frequent uterine fibroid driver mutation in MED12. Mediator kinase regulated transcripts identified by RNA-seq were linked to Mediator kinase substrates by computational analyses. Results 296 unique phosphosites in 166 proteins were significantly decreased ( > 2-fold; p Conclusion Altogether, these studies identify a new catalog of patho/biologically relevant Mediator kinase substrates implicated in the pathogenesis of MED12-mutation positive uterine fibroids, and further uncover a biochemical basis to link Mediator kinase activity with CUX1 and FOXK1 in the regulation of myometrial stem/progenitor cell fate.