The SCF-Fbw7 ubiquitin ligase degrades MED13 and MED13L and regulates CDK8 module association with Mediator

Abstract

The Mediator complex is an essential transcription regulator that bridges transcription factors with RNA polymerase II. This interaction is controlled by dynamic interactions between Mediator and the CDK8 module, but the mechanisms governing CDK8 module-Mediator association remain poorly understood. We show that Fbw7, a tumor suppressor and ubiquitin ligase, binds to CDK8-Mediator and targets MED13/13L for degradation. MED13/13L physically link the CDK8 module to Mediator, and Fbw7 loss increases CDK8 module-Mediator association. Our work reveals a novel mechanism regulating CDK8 module-Mediator association and suggests an expanded role for Fbw7 in transcriptional control and an unanticipated relationship with the CDK8 oncogene.

Figure 1.

MED13/13L bind Fbw7 as canonical substrates. (A) Mediator subunits that associated with wild-type (wt) Fbw7 but not Fbw7 RL. (Comp) CompPASS specificity; (TSC) average total spectral counts of all peptides in duplicate runs. Optimal Fbw7 degrons are indicated (bold). (B) Mediator binds Fbw7. 293A cells were cotransfected with Flag-Fbw7 or Fbw7 RL and HA-DN-Cul1 to inhibit SCF^Fbw7-mediated degradation. Endogenous Mediator subunits that coprecipitated with Fbw7 are shown. (WCE) Whole-cell extract; (IP) immunoprecipitation. (C) Schematic of the CDK8-bound Mediator complex. (Dark gray) CDK8 module; (light gray) Mediator core. (D,E) Stable interactions between MED13 or MED13L and Fbw7 require proteasomal inhibition. Cells were cotransfected with Flag-Fbw7 and/or myc-MED13/13L and treated with the proteasome inhibitor bortezomib. MED13 (D) or MED13L (E) only coprecipitated after bortezomib treatment (lane 6). (F) DN-Cul1 allows stable MED13-Fbw7 binding. 293 cells were transfected with Flag-Fbw7, HA-DN-Cul1, and myc-MED13 as indicated, and the amount of MED13 bound to immunoprecipitated Fbw7 is shown. (G,H) Mutating the MED13/13L CPDs (TA) or CPD-interacting residues in Fbw7 (RL) abrogates the Fbw7-MED13/13L interaction. Cells were transfected as indicated with wild-type or T326A CPD mutants of MED13/13L and/or wild-type Fbw7 or Fbw7 RL as well as DN-Cul1.

Figure 2.

MED13 and MED13L are Fbw7 substrates. (A) Fbw7 increases MED13/13L turnover. 293 cells were cotransfected with myc-MED13/13L or MED13/13L T326A and Flag-Fbw7, labeled with ³⁵S-Met, and chased with cold methionine as indicated. Turnover of labeled MED13/13L was assayed by phosphor-imaging of immunoprecipitated MED13, and the graphs show averages of two independent experiments. Entire gels are shown in Supplemental Figure 3. (B) Fbw7 specifically ubiquitylates MED13/13L in vitro. Myc-MED13/13L (wild-type [wt]) or myc-MED13/13L CPD mutants (TA) were immunoprecipitated and subjected to in vitro ubiquitylation assays containing recombinant wild-type Fbw7 (F7) or Fbw7 RL (RL) and other ubiquitylation components (E1, Cul1, E2, and ubiquitin). Controls were performed without Fbw7 (lanes 3,4) or ubiquitin (Ub) (lane 8). Membranes were reprobed to detect MED1 and MED14. (C) shRNA-mediated knockdown of Fbw7 increases endogenous MED13 and MED13L abundance. Cells were untreated or transduced with nonsilencing control shRNA (shNSC) or an Fbw7-specific shRNA (shFbw7). Two biological replicates are shown. (D) Quantitation of MED13/13L abundance in 293 cells expressing sh-Fbw7 or control shRNA (two biological replicates) (Fig 3A) or Fbw7-null DLD1 cells and controls (three biological replicates) (Fig. 3A). Bands were quantified with an ImageQuant Bioanalyzer and ImageQuant TL. (E) Knockdown of Fbw7 does not increase MED13 or MED13L transcription. MED13/13L mRNA was quantified by real-time PCR (normalized to GAPDH). (F) Knockdown of Fbw7 does not increase MED13 translation. Cells were pulse-labeled with ³⁵S-Met, and endogenous MED13 was immunoprecipitated.

Figure 3.

Fbw7 loss increases CDK8 module–Mediator association. (A) Fbw7 was silenced by shRNA in 293As as shown in Figure 2. The amount of endogenous CDK8 module–Mediator association was determined by immunoprecipitation with the Mediator core component MED1 and Western analyses of the indicated proteins. Four independent biological replicates were quantified with an ImageQuant Bioanalyzer and ImageQuant TL. Values were normalized to the amount of immunoprecipitated MED1 in each sample, and error bars show ±standard error of the mean (SEM). (B) Fbw7-dependent changes in CDK8 module–Mediator association were examined as in A, except CDK8 was immunoprecipitated. The normalized amounts of CDK8-bound core Mediator and CDK8 module subunits are shown (two biological replicates). (C,D) Lysates from wild-type and Fbw7-null DLD1 cells were analyzed as shown in A and B. The unnormalized quantified data used for this figure are shown in Supplemental Figure 5.

Figure 4.

Fbw7 ubiquitylates Mediator-bound Med13/13L. (A) Fbw7 ubiquitylates Mediator-bound MED13. Mediator was immunoprecipitated via MED1 and subjected to in vitro ubiquitylation. Cells were pretreated with bortezomib (lanes 3,4) or bortezomib and okadaic acid (lanes 5,6) prior to lysis. (B,C) Overexpression of MED13 or MED13 TA does not increase CDK8 module–Mediator association. Increasing amounts of MED13 or MED13 TA were transfected into 239As. Mediator was immunoprecipitated via MED1, and CDK8 module–Mediator association was assayed via Western analysis. Three experiments were quantified as in Figure 3C and normalized against immunoprecipitated MED1. Error bars show the standard error of the mean. (B) A representative Western blot is shown. (D) Overexpression of CDK8 does not increase the amount of CDK8 bound to Mediator. Increasing amounts of myc-tagged or untagged CDK8 were transfected into 293As. Mediator was immunoprecipitated through MED1, and the positions of myc-tagged CDK8 and endogenous CDK8 are shown. Controls include immunoprecipitation with nonspecific IgG (lane 12) or no cell lysate (lane 13). (E) Model: Fbw7 binds and ubiquitylates Mediator-bound MED13/13L, resulting in their degradation and the dissociation of the remaining CDK8 module components from the Mediator core (Med).