Characterization of Cullin-box Sequences That Direct Recruitment of Cul2-Rbx1 and Cul5-Rbx2 Modules to Elongin BC-based Ubiquitin Ligases

Abstract

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 CRL2LRR-1 ubiquitin ligase regulates cell cycle progression during C. elegans development

“…Multiple sequence alignment of LRR-1 with selected BC/Cul-2 boxcontaining proteins from C. elegans revealed that these two motifs are conserved in LRR-1. In particular, the key residues that mediate binding to ELC-1 and CUL-2 (Stebbins et al, 1999;Vasudevan et al, 2007;Mahrour et al, 2008) are invariant in LRR-1 (Fig. 1B), suggesting that it is a bona fide substrate-recruitment subunit of a CRL2 complex in C. elegans.…”

“…1A). The BC/Cul-2 box includes two short sequences, termed BC and the Cul-2 box, that are required for ELC-1 and CUL-2 binding, respectively (Vasudevan et al, 2007;Mahrour et al, 2008). Through proteomic screens in mammalian cells (HEK293T), we and others have identified LRR-1 as a potential substrate-recognition subunit of a CRL2 complex (Kamura et al, 2004;Olma et al, 2009).…”

An interaction network of the mammalian COP9 signalosome identifies Dda1 as a core subunit of multiple Cul4-based E3 ligases

“…2G; supplementary material Table S4). Therefore, among the 16 BC-box proteins that were recently identified in FLAG-ElonginB pulldowns (Mahrour et al, 2008), only Lrrc14, Lrr1 and Fem1 stably associated with the CSN. Similarly, only four BTB proteins (Btbd1, Btbd2, Klhldc5 and Klhl18) were systematically identified in the CSN immunoprecipitates (Fig.…”

Analysis of the Cullin Binding Sites of the E4orf6 Proteins of Human Adenovirus E3 Ubiquitin Ligases

“…Cellular proteins bind to Cul2-based ligase complexes through a VHL box and to Cul5 complexes through a SOCS box. Both are comprised of an Elongin B and C binding domain (BC box) followed by a Cul2 or Cul5 binding domain, with an average 10 to 25 amino acids separating the two (7,9). In some viral proteins, however, these domains can also be quite distant, as is the case for the LANA protein, where they are over 800 amino acids apart (26).…”

“…While the BC box domains have been identified in the early region 4 open reading frame 6 (E4 34K, or E4orf6) protein of human adenovirus type 5 (Ad5), the location of the Cullin domains has remained elusive (3,8). The Cullin binding domain within other viral proteins has been located in most of the examples listed in Table 1, only about half of which follow the cellular consensus sequence (LPxP for Cul5, and LxxxLxxxL for Cul2) (7,9). Thus, it seems equally likely that human adenoviruses might utilize either a cellular consensus domain or a novel sequence to bind Cullin 2 or 5.…”