E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages.
An E3 ubiquitin ligase mediates the transfer of activated ubiquitin from an E2 ubiquitin-conjugating enzyme to its substrate lysine residues. Using a structure-based, yeast two-hybrid strategy, we discovered six previously unidentified interactions between the human heterodimeric RING E3 BRCA1-BARD1 and the human E2s UbcH6, Ube2e2, UbcM2, Ubc13, Ube2k and Ube2w. All six E2s bind directly to the BRCA1 RING motif and are active with BRCA1-BARD1 for autoubiquitination in vitro. Four of the E2s direct monoubiquitination of BRCA1. Ubc13-Mms2 and Ube2k direct the synthesis of Lys63- or Lys48-linked ubiquitin chains on BRCA1 and require an acceptor ubiquitin attached to BRCA1. Differences between the mono- and polyubiquitination activities of the BRCA1-interacting E2s correlate with their ability to bind ubiquitin noncovalently at a site distal to the active site. Thus, BRCA1 has the ability to direct the synthesis of specific polyubiquitin chain linkages, depending on the E2 bound to its RING.
Christensen
Devin E
DE
Department of Biochemistry, University of Washington, Box 357350, Seattle, Washington 98195-7350, USA.
Brzovic
Peter S
PS
Klevit
Rachel E
RE
D000595:Amino Acid Sequence; D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D025821:Polyubiquitin / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D054829:RING Finger Domains; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D025821:Polyubiquitin; D044763:Ubiquitin-Conjugating Enzymes; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases