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BMC Cell Biology

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Mass spectrometry, Proteins -- Research, Ubiquitin


Background: Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as leucine-rich repeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein.

Results: Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of Cul3-bound proteins that contain either the LRR or WD40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor.

To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex.

Conclusion: Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC/MS-MS analysis. Subsequently, our biochemical analysis revealed that some CLWs modify binding of BTB domain-containing proteins to the complex, causing degradation of the BTB domain-containing protein. As these CLWs were excluded from our list of substrates, we propose that CLWs serve as unique Cul3 binding proteins that provide an alternative regulatory mechanism for the complex.


Additional files are included. This is the publisher's final PDF. © 2014 Wimuttisuk et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.



Persistent Identifier

singer_1.xls (67 kB)
Additional file 1: Table S1: Potential Cul3-binding proteins that were identified by MudPIT

singer_2.xls (237 kB)
Additional file 2: Table S2: Potential Cul3-binding proteins that were identified by one peptide sequence from MudPIT analysis

singer_3.pdf (4134 kB)
Additional file 3: Table S3: Summary of conserved domains of potential Cul3 and actin-binding proteins. Figure S1. LRR3 binds Ctb62. HA-tagged LRR3 was transfected into HEK293 cells, either alone (third lane) or with MYC-tagged Ctb62 (first lane) or with MYC-tagged Ctb62 deleted for its BTB domain. Lower gel shows an immunoblot of levels of expression of LRR3 and the upper blot shows binding of LRR3 to Ctb62 by immunoprecipitating Ctb62 followed by an immunoblot for LRR3. Figure S2. Cul3 is ubiquitinated at the lysine 414 residue. HEK293 cells were transfected with vectors expressing wild-type Cul3, Cul3K414R mutant, and HA-tagged ubiquitin. Lysates were prepared, checked for protein expression (bottom), and immunoprecipitated with anti-HA antibody. The precipitates were separated by SDS-PAGE and analyzed by immunoblot for Cul3.

singer_4.xls (55 kB)
Additional file 4: Table S4: Summary of proteins that contain conserved domains of interest from MudPIT-identified Cul3-binding proteins.