title: 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 LPPhiP, 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 approximately 80 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.
authors.affiliation: Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.
authors.forename: Nawel
authors.initials: N
authors.lastname: Mahrour
authors.forename: William B
authors.initials: WB
authors.lastname: Redwine
authors.forename: Laurence
authors.initials: L
authors.lastname: Florens
authors.forename: Selene K
authors.initials: SK
authors.lastname: Swanson
authors.forename: Skylar
authors.initials: S
authors.lastname: Martin-Brown
authors.forename: William D
authors.initials: WD
authors.lastname: Bradford
authors.forename: Karen
authors.initials: K
authors.lastname: Staehling-Hampton
authors.forename: Michael P
authors.initials: MP
authors.lastname: Washburn
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Conaway
authors.forename: Joan W
authors.initials: JW
authors.lastname: Conaway
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D044842:Cullin Proteins / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D000075065:Elongin; D006801:Humans; D009097:Multienzyme Complexes / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics; D000099272:Replisomes
chemical_list: D002352:Carrier Proteins; D044842:Cullin Proteins; D004259:DNA-Directed DNA Polymerase; D000075065:Elongin; D009097:Multienzyme Complexes; D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D000099272:Replisomes; C105153:CUL2 protein, human; C496871:CUL5 protein, human; C000616396:ELOB protein, human; C000616395:ELOC protein, human; C479529:RBX1 protein, human; C118307:RNF7 protein, human; C497446:VHL protein, human