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 cyclin dependent kinase 8/19 (CDK8/19), which is known to be disrupted by uterine fibroid driver mutations in Mediator complex subunit 12 (MED12).

Design: Experimental study.

Setting: Academic research laboratory.

Patient(s): Women undergoing hysterectomy for uterine fibroids.

Intervention(s): Stable isotopic labeling of amino acids in cell culture (SILAC) coupled with chemical inhibition of CDK8/19 and downstream quantitative phosphoproteomics and transcriptomic analyses in myometrial stem/progenitor cells.

Main outcome measure(s): 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 sequencing were linked to Mediator kinase substrates by computational analyses.

Result(s): A total of 296 unique phosphosites in 166 proteins were significantly decreased (≥ twofold) upon CDK8/19 inhibition, including 118 phosphosites in 71 nuclear proteins representing high-confidence Mediator kinase substrates linked to RNA polymerase II transcription, RNA processing and transport, chromatin modification, cytoskeletal architecture, and DNA replication and repair. Orthogonal validation confirmed a subset of these proteins, including Cut Like Homeobox 1 (CUX1) and Forkhead Box K1 (FOXK1), to be direct targets of MED12-dependent CDK8 phosphorylation in a manner abrogated by the most common uterine fibroid driver mutation (G44D) in MED12, implicating these substrates in disease pathogenesis. Transcriptome-wide profiling of Mediator kinase-inhibited myometrial stem/progenitor cells revealed alterations in cell cycle and myogenic gene expression programs to which Mediator kinase substrates could be linked directly. Among these, CUX1 is an established transcriptional regulator of the cell cycle whose corresponding gene on chromosome 7q is the locus for a recurrent breakpoint in uterine fibroids, linking MED12 and Mediator kinase with CUX1 for the first time in uterine fibroid pathogenesis. FOXK1, a transcriptional regulator of myogenic stem cell fate, was found to be coordinately enriched along with kinase, but not core, Mediator subunits in myometrial stem/progenitor cells compared with differentiated uterine smooth muscle cells.

Conclusion(s): These studies identify a new catalog of pathologically and 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.

Keywords: MED12 mutations; mediator kinase; myometrial stem/progenitor cells; phosphoproteomics; uterine fibroids.

FIGURE 1

Identification of high-confidence Mediator kinase substrates in myometrial (MM) side population (SP) cells. (A) Volcano plot of statistically significant phosphosite changes (empirical Bayes analysis) in MM SP cells after 45 minutes of treatment with CCT251545. Mediator kinase-dependent phosphosites considered ‘‘high-confidence’’ are those undergoing an inhibitor-dependent change in abundance of ≥ twofold with P<.05 (red dots). Putative direct Mediator kinase phosphosites (upper left quadrant) are those reduced in abundance upon Mediator kinase inhibition. Phosphopeptides in validated Mediator kinase substrates are indicated. NS = not significant. (B) Functional categorization of high-confidence Mediator kinase substrates.

FIGURE 2

Validation of select Mediator kinase substrates. (A) Purified three-subunit and four-subunit CDK8 kinase modules (3S CKM and 4S CKM, respectively) analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. For 3S CKM, CDK8 wild-type (WT) and kinase-dead (KD) (lanes 1 and 2), as well as MED12 (1–100) WT and G44D mutant derivatives (lanes 3 and 4), are indicated. For 4S CKM, MED12 WT and G44D mutant derivatives (lanes 5 and 6) are indicated. (B) In vitro kinase assays were performed using CCNC-CDK8 WT without (–) or with (+) MED12 (1–100) WT along with each of the indicated substrates (RNA Pol II [polymerase II] C-terminal domain [CTD] [lanes 1 and 2], STAT1 [lanes 3 and 4], TP53BP1 [lanes 5 and 6], Foxk1 [lanes 7 and 8], NELFB [lanes 9 and 10], CUX1 [lanes 11 and 12], or SRSF2 [lanes 13 and 14]). Kinase reactions were resolved by SDS-PAGE and subjected to Phosphoimager analysis to detect phosphorylated (³²P) substrates (top panels) or Coomassie blue staining to visualize input substrates (bottom panels). ³²P-CDK8 indicates autophosphorylation of CDK8, which is stimulated by MED12 (1–100). RNA Pol II CTD and SRSF2 are positive and negative controls for Mediator kinase activity, respectively. (C and D) In vitro kinase assays were performed using 3S CKM (C) or 4S CKM (D) and either WT or phosphomutant substrates bearing Ser to Ala substitution mutations at Mediator kinase-dependent phosphosites mapped by SILAC analysis. Where indicated, kinase reactions were performed using 3S CKM or 4S CKM reconstituted with CDK8 WT or CDK8 KD as well as MED12 WT or G44D mutant derivatives. Kinase reactions were resolved by SDS-PAGE and analyzed as described in (B).

FIGURE 3

Mediator kinase regulates myogenic and cell growth-related gene expression programs in myometrial side population (MM SP) cells. (A) Heat maps of 401 genes differentially expressed (FC≥1.5-fold; P<.05) in MM SP cells upon 3 hours of CCT251545 treatment, including both up-regulated (n = 123) and down-regulated (n=278) genes. Only a subset of genes are labeled. (B and C) Hallmark gene sets enriched in Mediator kinase-inhibited MM SP cells. GSEA =XX.

FIGURE 4

Mediator kinase-regulated genes are linked functionally with Mediator kinase substrates in myometrial side population (MM SP) cells. (A) Ciiider enrichment plots for 401 Mediator kinase-regulated genes, including up-regulated and down-regulated gene sets. Plots show the enrichment (proportional ratio of promoter regions bound by transcription factors [indicated by dots]) and average log proportion bound. Dot size and color reflect the log₂ P value (significance score). Significance scores greater than or less than zero indicate overrepresentation or underrepresentation of transcription factor-binding sites, respectively, in promoter regions of queried genes. Note that binding sites for Mediator kinase substrates FOXK1 and CUX1 (indicated) are overrepresented and underrepresented in up-regulated and down-regulated gene sets, respectively. (B) Ciiider enrichment plot for Hallmark myogenesis gene set. Note that binding sites for MEF2, a Mediator kinase-regulated gene that physically and functionally interacts with Mediator kinase substrates FOXK1 and TRIM28, are enriched in the myogenic gene set. (C) Whole cell lysates of paired SP and non-SP fractions from two different patient-derived myometria were processed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis using antibodies specific for Mediator kinase substrates (FOX1, NELFB), Mediator kinase module subunits (MED13, MED12, CDK8/CDK19, CCNC), Mediator core subunits (MED4, MED30), α-smooth muscle actin (ACTA2), and β-ACTIN (loading control). Paired SP and non-SP fractions were isolated from uteri bearing MED12 mutation-negative (patient 1) or MED12 mutation-positive (patient 2) uterine fibroids. Note that FOXK1, along with kinase, but not core, Mediator subunits is enriched in undifferentiated MM SP cells compared with differentiated non-SP (NSP) (ACTA2-expressing) cells. MED = Mediator.