The Elongin BC-box protein family includes the von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling proteins, which are substrate recognition subunits of structurally related classes of E3 ubiquitin ligases composed of Elongin C-Elongin B-Cullin 2-Rbx1 (Cul2 ubiquitin ligases) or of Elongin C-Elongin B-Cullin 5-Rbx2 (Cul5 ubiquitin ligases). The Elongin BC complex acts as an adaptor that links a substrate recognition subunit to heterodimers of either Cullin 2 (Cul2) and RING finger protein Rbx1 or Cullin 5 (Cul5) and Rbx2. It has been shown (Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R. C., Conaway, J. W., and Nakayama, K. I. (2004) Genes Dev. 18, 3055-3065) that interaction of BC-box proteins with their cognate Cul-Rbx module is determined by specific regions, called Cul2-or Cul5-boxes, located immediately downstream of their BC-boxes. Here, we investigate further the mechanisms governing assembly of BC-box proteins with their specific Cul-Rbx modules. Through purification and characterization of a larger collection of BC-box proteins that serve as substrate recognition subunits of Cul2 and Cul5 ubiquitin ligases and through structure-function studies, we define Cul2-and Cul5-boxes in greater detail. Although it previously appeared that there was little sequence similarity between Cul5-and Cul2-box motifs, analyses of newly identified BC-box proteins reveal that residues conserved in the Cul2-box represent a subset of those conserved in the Cul5-box. The sequence motif LPP, which is conserved in most Cul5-boxes and has been suggested to specify assembly of Cul5 ligases, is compatible with Cul2 interaction. Finally, the spacing between BC-and Cullin-boxes is much more flexible than has been appreciated and can vary from as few as 3 and as many as ϳ80 amino acids. Taken together, our findings shed new light on the mechanisms by which BC-box proteins direct recruitment of Cullin-Rbx modules during reconstitution of ubiquitin ligases.A growing body of evidence has implicated members of the families of Cullin 2 (Cul2)-and Cullin 5 (Cul5)-containing E3 ubiquitin ligases in regulation of diverse cellular processes including cell signaling, growth, and differentiation (reviewed in Ref.(1). Cul2 and Cul5 ubiquitin ligases each include the heterodimeric Elongin BC complex, composed of the 112-amino acid Elongin C protein, which is similar in sequence to the Skp1-Cullin1-F box ubiquitin ligase subunit Skp1, and the 118-amino acid Elongin B protein, which includes an N-terminal ubiquitin-like domain and a 34-amino acid C-terminal extension. In the context of Cul2 and Cul5 ubiquitin ligases, the Elongin BC complex functions as an adaptor that links a BCbox substrate recognition subunit to either a Cul2-Rbx1 or a Cul5-Rbx2 module that uses the RING finger domains of the related Rbx1 or Rbx2 proteins to recruit and activate an E2 ubiquitin-conjugating enzyme for ubiquitination of substrates.The founding member of the Cul2 family of E3 ubiquitin ligases is the von Hippel-Linda...

The human tumour antigen PRAME (preferentially expressed antigen of melanoma) is frequently overexpressed in tumours. High PRAME levels correlate with poor clinical outcome of several cancers, but the mechanisms by which PRAME could be involved in tumourigenesis remain largely elusive. We applied protein-complex purification strategies and identified PRAME as a substrate recognition subunit of a Cullin2-based E3 ubiquitin ligase. PRAME can be recruited to DNA in vitro, and genome-wide chromatin immunoprecipitation experiments revealed that PRAME is specifically enriched at transcriptionally active promoters that are also bound by NFY and at enhancers. Our results are consistent with a role for the PRAME ubiquitin ligase complex in NFY-mediated transcriptional regulation.

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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.