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First published online March 18, 2009
doi: 10.1242/10.1242/jcs.043539

Journal of Cell Science 122, 1035-1044 (2009)
Published by The Company of Biologists 2009
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An interaction network of the mammalian COP9 signalosome identifies Dda1 as a core subunit of multiple Cul4-based E3 ligases

Michael Hans Olma1,*, Marcia Roy2,*,{ddagger}, Thierry Le Bihan3, Izabela Sumara1, Sarah Maerki1, Brett Larsen2, Manfredo Quadroni4, Matthias Peter1,§, Mike Tyers2,{ddagger},§ and Lionel Pintard2,5,§

1 Swiss Federal Institute of Technology Zürich (ETH), Institute of Biochemistry, 8093 Zürich, Switzerland
2 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Ontario Toronto, Canada M5G 1X5
3 Centre for Systems Biology at Edinburgh (CSBE), The University of Edinburgh, The Kings Buildings, Mayfield Road, Edinburgh EH9 3JU
4 Centre Intégratif de Génomique, plateforme de protéomique, Chemin des Boveresses 155, 1066 Epalinges, Switzerland
5 Institut Jacques Monod CNRS, Université Paris Diderot, 5 Rue Thomas Mann, 75205 Paris, Cedex 13, France


Figure 1
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  		Fig. 1. The protein interaction network of the mammalian CSN. (A) Protein extracts prepared from HEK 293T cells stably expressing the indicated FLAG-tagged CSN subunits were fractionated on a Superose 6 column (bed volume: 24 ml) and 1-ml fractions were collected and analyzed by SDS-PAGE with specific FLAG, Csn1 and Cul3 antibodies. (B) CSN-interacting CRL subunits are highlighted in purple (SCF), blue (CRL2 and CRL5), green (CRL3) and red (CRL4). Subunits highlighted in orange have been previously found associated with CRL4s. (C) CSN-cullin interactions observed by LC-MS-MS were confirmed by co-immunoprecipitation experiments combined with western blot identification.

 

Figure 2
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  		Fig. 2. The CSN stably interacts with a limited subset of CRLs. (A) Endogenous Cul3 immunocomplexes were affinity-purified with specific antibodies, and associated proteins were analyzed by LC-MS-MS (left panel). (B) Klhl9 and 13, and Btbd1 and 2 were immunoprecipitated from 293T cells expressing FLAG-Csn4, separated by SDS-PAGE and immunoblotted with specific antibodies against the indicated proteins. The arrows mark neddylated (Cul3Nedd8) and unneddylated Cul3. (C) Endogenous Cul4A immunocomplexes were separated by SDS-PAGE and analyzed by tandem mass spectrometry. (D) Cul4A immunocomplexes were separated by SDS-PAGE and blotted with specific antibodies directed against Cul4A, Ddb1, Ddb2, Cand1, Csn2, Csn3, Cul3, Vprbp, Wdr23 and Dda1. (E) HA-tagged DCAF proteins H326, Wdr23 and Ddb2 were expressed in HeLa cells and immunoprecipitated. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with specific antibodies directed against the HA peptide, Cul4A, Cul4B, Ddb1, Cul3, Csn2 and Csn3. (F) Summary of the LC-MS-MS analysis of FLAG-CSN, Cul3 and Cul4A immunoprecipitates. Green squares indicate presence in the immunoprecipitate and red indicate absence. Cul3 and CRL4 core subunits (Cul4A, Cul4B and Ddb1) as well as BTB and DCAF adaptors are presented. (G) Summary of the LC-MS-MS analysis of FLAG-Lrr1, -Lrrc14 immunoprecipitates and co-immunoprecipitation analysis of Btbd1 and 2, Klhl9 and 13 and HA-H326, -Ddb2 and -Wdr23 immunoprecipitates. Green squares indicate presence in the immunoprecipitate and red indicate absence.

 

Figure 3
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  		Fig. 3. Dda1 is an unstable core subunit of multiple CRL4s. (A) HEK 293T cells stably expressing FLAG-Dda1 were treated with MG132 or for control DMSO. After 3 hours, protein translation was inhibited with cycloheximide (CHX) and samples were collected at various times (in hours) after CHX addition. Protein extracts were analyzed by SDS-PAGE with specific FLAG and Csn1 antibodies. (B) HeLa cells incubated with oligonucleotides targeting Cul4A were transfected with a plasmid expressing FLAG-Dda1. Cells were fixed and immunostained with anti-Cul4A (upper right panel) and anti-FLAG antibodies (bottom left panel). DAPI was used to counterstain DNA (upper left panel), and merged images are presented in the bottom right panel. (C) Protein extracts prepared from cells incubated with control oligonucleotides (lanes 1 and 2), or oligonucleotides targeting Dda1 (lanes 3 and 4) or Cul4 paralogs (lanes 5 and 6) were separated by SDS-PAGE. Cul4A and B, Cdt1, and Dda1 levels were assessed by immunoblotting using specific antibodies. Tubulin was used as a loading control. (D) LC-MS-MS analysis of anti-FLAG-Dda1 immunoprecipitates. Note that FLAG-Dda1 co-purifies with all COP9 signalosome (subunits highlighted in yellow) and many CRL4 subunits (colored in red and orange). (E) Vprbp was immunoprecipitated with control antibodies or antibodies directed against the N- or C-terminal part of the protein. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with specific antibodies specific for Vprbp, Cul4A, Ddb1 and Dda1. (F) HA-tagged DCAF proteins H326, Wdr23 and Ddb2 were expressed in HeLa cells and immunoprecipitated. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with specific antibodies directed against the HA peptide, Ddb1 and Dda1. (G) Dda1 immunoprecipitates were treated with lambda phosphatase (lane 2) or an inactivated phosphatase (lane 3) and analyzed by western blotting using specific Dda1 antibodies.

 

Figure 4
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  		Fig. 4. Dda1 associates with the chromatin in a cell cycle-dependent manner. (A) Cells were arrested in S-phase using a double thymidine block and samples were collected at various times after release. Protein extracts were separated by SDS-PAGE and immunoblotted with antibodies directed against Dda1 and the indicated cell cycle markers (Cyclin E and PH3). Tubulin was used as a loading control. (B) The localization of Dda1 was analyzed by immunofluorescence in interphase cells pre-extracted (left panels) or not (right panels) with buffers that remove nucleoplasmatic proteins. DAPI staining was used to visualize the DNA. Merge images are shown in the right row. (C) Total lysates (T) were prepared from aphidicolin-arrested S-phase cells and separated into a nucleo-cytoplasmic (NC) and a chromatin-bound fraction (Chr), solubilized after digestion with micrococcal nuclease. Fractions were separated by SDS-PAGE and immunoblotted with antibodies directed against Dda1, Ddb1, Cul4A, tubulin and histone H3. (D,E) Dda1 and Cul4A localize to the nucleus but Dda1 is excluded from chromatin in mitosis. The localization of Dda1 (panel D) and Cul4A (panel E) was analyzed by immunofluorescence at different stages of mitosis. Antibodies against tubulin visualize the mitotic spindle, and the DNA was stained with DAPI (left row). Merge images are presented in the right row. Note that Cul4A remains associated with chromosomes throughout mitosis, whereas Dda1 staining is diffuse in metaphase and anaphase cells. (F) Cul4A immunoprecipitates from extracts prepared from cells arrested in S-phase or mitosis with aphidicolin or nocodazole, respectively, were analyzed by immunoblotting for the presence of Cul4A, Ddb1 and Dda1 as indicated.

 

Figure 5
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  		Fig. 5. Dda1 functionally interacts with a subset of CRL4s. (A) Cul4A- and B-, Dda1- and Wdr23-depleted cells accumulate {gamma}H2A.X foci. Cells were immunostained with anti-phospho H2A.X antibodies and DNA was counterstained with DAPI. (B,C) Quantification of the number of {gamma}H2A.X-positive cells by FACS analysis in control cells or cells depleted of Cul4A and B, Dda1 and Wdr23. Cells were fixed with ethanol, stained with an {gamma}H2A.X antibody and analyzed by FACS. The number of {gamma}H2A.X-positive cells (R6 area) was plotted. (D) Immunoblotting of Wdr23 in extracts prepared from control and Wdr23 siRNA-depleted HeLa cells. (E) Ddb1 is required for nuclear accumulation of Dda1. Endogenous Dda1 was localized by indirect immunofluorescence (left column) in control cells (upper panel), or cells treated with Ddb1 (middle panel) or Dda1 siRNA (bottom panel). DNA was counterstained with DAPI (middle column), and merged images are presented in the right panels. (F) Dda1 uses its N-terminal domain to bind Ddb1. Full-length and truncated versions of FLAG-Dda1 that remove the first 26 amino acids ({Delta}N26), or the last 17 and 29 amino acids, respectively ({Delta}C17 and {Delta}C29), were transiently expressed in HEK 293T and immunoprecipitated with M2-agarose. The immunoprecipitates were separated by SDS-PAGE and blotted with specific Ddb1 (upper panel) and FLAG antibodies (lower panel).

 

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© The Company of Biologists Ltd 2009