The human EKC/KEOPS complex is recruited to Cullin2 ubiquitin ligases by the human tumour antigen PRAME

Abstract

The human tumour antigen PRAME (preferentially expressed antigen in melanoma) is frequently overexpressed during oncogenesis, and high PRAME levels are associated with poor clinical outcome in a variety of cancers. However, the molecular pathways in which PRAME is implicated are not well understood. We recently characterized PRAME as a BC-box subunit of a Cullin2-based E3 ubiquitin ligase. In this study, we mined the PRAME interactome to a deeper level and identified specific interactions with OSGEP and LAGE3, which are human orthologues of the ancient EKC/KEOPS complex. By characterizing biochemically the human EKC complex and its interactions with PRAME, we show that PRAME recruits a Cul2 ubiquitin ligase to EKC. Moreover, EKC subunits associate with PRAME target sites on chromatin. Our data reveal a novel link between the oncoprotein PRAME and the conserved EKC complex and support a role for both complexes in the same pathways.

Figure 1. PRAME interacts with OSGEP and LAGE3.

(A) Coimmunoprecipitation assays verify the interaction of PRAME with the EKC subunits OSGEP and LAGE3. Constructs expressing the indicated proteins were transiently transfected in 293T cells. Anti-TAG immunoprecipitations were performed with rabbit HA antibody (Abcam). TAG-PRAME was detected with monoclonal anti-HA (Covance) and TTE-tagged proteins with monoclonal BB2 (Diagenode) which recognizes the TY1 tag. The lower panel shows a scheme of the tagged proteins used (tags and coding sequences are not on scale). (B-C) PRAME directly interacts with EKC complex through OSGEP and LAGE3. SF21 cells were co-infected with baculoviruses expressing the indicated proteins. Anti-FLAG immunoprecipitations were performed, and total cell lysates and eluates were analyzed by western blotting. Panels showing anti-HA immunoblots are from the same exposure of the same blot, as are the panels showing anti-cMyc immunoblots. Asterisks indicate light chains of the antibodies used in the immunoprecipitation.

Figure 2. OSGEP interacts with PRAME and Cul2 ubiquitin complex components.

OSGEP interacts with PRAME and Cul2-EloBC ligases. Immunoblot analysis of TAG-PRAME and TAG-OSGEP protein complexes purified from K562 cells to verify the mass spectrometry data. Mock purification was performed on wild type cells. 0.8% of input and 33% of IP were separated on NuPage 4–12% gels. Tagged proteins were detected with mouse HA antibody (Covance, top panel); endogenous PRAME and TAG-PRAME were detected in the second panel with affinity-purified PRAME antibody after staining for Cul2; the other proteins were detected as indicated. Asterisks indicate protein A that dissociated from the beads after elution.

Figure 3. Organization of human EKC complex.

SF21 cells were co-infected with baculoviruses expressing the indicated proteins. Anti-FLAG immunoprecipitations were performed, and total cell lysates and eluates were analyzed by western blotting. Interactions detected are summarized in the diagram on the right. Panels showing anti-HA immunoblots are from the same exposure of the same blot, as are the panels showing anti-cMyc immunoblots. Asterisk indicates light chains of the antibodies used in the immunoprecipitation.

Figure 4. OSGEP protein levels are modulated by protein-protein interactions.

(A) Multiple OSGEP moieties are present in the same complex. Constructs expressing the indicated proteins were transiently transfected in 293T cells. Total cell lysates and FLAG eluates were analyzed by western blotting. A plasmid expressing GFP was cotransfected to control for the transfection efficiency. (B) OSGEP levels are affected by protein-protein interactions. 293T cells were transfected with constructs expressing FLAG-OSGEP and the proteins indicated. The bar graph shows FLAG-OSGEP levels quantified by immunoblot with the Odyssey system (values are the average of two independent experiments). A plasmid expressing GFP was cotransfected to control for the transfection efficiency. (C) and (D) LAGE3-OSGEP interface mutants decrease OSGEP protein levels. Transient transfections in 293T cells with the mutant constructs indicated and immunoblot by Odyssey of total cell lysates. Graphs report intensities of FLAG-OSGEP quantified with Odyssey (A.U., arbitrary units).

Figure 5. PRAME recruits Cul2-EloBC ligases to EKC.

(A) PRAME does not require an intact BC-box to interact with EKC components. Coimmunoprecipitation assays as in Fig.1A with wild type and BC-box mutant M2 PRAME. (B) PRAME bridges Cullin2 ligases to EKC complex. Immunoblot analysis as in Fig. 2 of TAG-OSGEP or TAG-LAGE3 immunoprecipitates with or without knock down of endogenous PRAME. Asterisk indicates heavy chains of the antibodies used in the immunoprecipitation. (C) Models of the protein complexes architecture.

Figure 6. OSGEP and LAGE3 are present at PRAME-bound promoters on chromatin.

ChIP-qPCR experiments using BB2 antibody were performed on K562 cells lines stably expressing TTE-OSGEP or TTE-LAGE3. As control for specificity, ChIPs were performed on the parental cells (wt) and on cells transduced with the vector expressing the tag only (K562-TTE) using a panel of primer sets for PRAME biding sites. Values are expressed as mean recoveries ± standard deviation from three independent experiments.