Dynamic interactions of proteins in complex networks: identifying the complete set of interacting E2s for functional investigation of E3-dependent protein ubiquitination.
A ubiquitin ligase (E3) functions at the crossroad between ubiquitin activation and the attachment of ubiquitin to protein substrates. During this process, the E3 interacts with both a substrate and a ubiquitin-conjugating enzyme (E2). Although a major goal when investigating an E3 is to identify its substrates, recent evidence indicates that the E2 dictates the type of ubiquitin modification that will occur on the substrate. There are approximately 30 E2s identified in the human genome, many of which remain to be characterized. We found that the RING E3 BRCA1/BARD1 can interact with 10 different E2s. The ability of BRCA1 to interact with multiple E2s is likely to be a common feature among other RING and U-box E3s. We and others have also found that certain E2s show a preference for attaching either the first ubiquitin to a substrate lysine or ubiquitin to itself (chain building), suggesting that E2s may play a role in dictating product formation. Therefore, when investigating the functions of an E3 it is advisable to identify all E2s that interact with the E3 so that these can be used in E3-dependent substrate-ubiquitination assays. We describe a method used to identify all the E2s that interact with BRCA1. Defining the set of E2s that interact with other RING and U-box E3s will open the door for predictive models and lead to a better understand of substrate ubiquitination.
Christensen
Devin E
DE
Department of Biochemistry, University of Utah, Salt Lake City, UT 98195, USA.
Klevit
Rachel E
RE
D000595:Amino Acid Sequence; D019313:BRCA1 Protein / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D025941:Protein Interaction Mapping; D017386:Sequence Homology, Amino Acid; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination
D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D025521:Tumor Suppressor Proteins; D044763:Ubiquitin-Conjugating Enzymes; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases