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First published online 9 September 2008
doi: 10.1242/jcs.030445

Journal of Cell Science 121, 3218-3223 (2008)
Published by The Company of Biologists 2008
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DEN1 deneddylates non-cullin proteins in vivo

Yaru Chan1,*, Jeongsook Yoon2,*, June-Tai Wu1,3, Hyung-Jun Kim2, Kuan-Ting Pan4, Jeongbin Yim2 and Cheng-Ting Chien1,{ddagger}

1 Institute of Molecular Biology, Academia Sinica, 128, Sec No. 2, Academia Road, Taipei 115, Taiwan
2 School of Biological Sciences, Seoul National University, 56-1 Shinlim-dong, Gwanak-gu, Seoul 151-742, Korea
3 Department of Medical Research, National Taiwan University Hospital, No. 7, Chung San South Road, Taipei, Taiwan
4 NRPGM Core Facilities for Proteomics, Institute of Biological Chemistry, Academia Sinica, 128, Sec No. 2, Academia Road, Taipei 115, Taiwan


Figure 1
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  		Fig. 1. Processing of pNedd8 by DEN1. (A) DEN1 processes His-pNedd8GG in a concentration-dependent manner (0.05-0.3 µg, lanes 2-5). The processed product is equivalent to the purified His-mNedd8 in mobility (lane 6, indicated by arrow). However, His-pNedd8AA (0.3 µg) was not processed by DEN1 (lane 7). (B) In a similar set of experiments, DEN1CA fails to process His-pNedd8GG. Asterisks in A,B indicate a non-specific signal present in the preparation of His-pNedd8GG.

 

Figure 2
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  		Fig. 2. Defective processing of Nedd8-CFP fusions in DEN1null mutants. Western blot using larval lysates expressing myc-mNedd8 (lane 2), myc-Nedd8GG-CFP (lanes 3, 4) or myc-Nedd8AA-CFP (lanes 5, 6) transgene by tubP-GAL4 in wild-type or DEN1null background probed with antibodies against Myc. The protein level of Myc-Nedd8GG-CFP at 40 kDa is more reduced in wild type compared with DEN1null (lanes 3, 4, arrow on left). However, Myc-Nedd8AA-CFP is expressed at identical levels in both wild type and DEN1null (lanes 5, 6). The product from the processing of Myc-Nedd8GG-CFP runs at 11 kDa with a mobility identical to Myc-mNedd8 (lane 2, arrowhead), but slightly slower than a non-specific proteolytic product in lanes 4-6 (asterisk). Lane 1 is wild-type control without transgene expression and the bottom panel shows {alpha}-Tub expression.

 

Figure 3
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  		Fig. 3. Neddylation of Cul1 and Cul3 in DEN1 mutants. (A,B) Deneddylation of Cul1 (A) and Cul3 (B) by DEN1. Neddylated and unneddylated Flag-Cul1 or Flag-Cul3 proteins were immunoprecipitated by the anti-Flag antibody from extracts of S2 cells transfected with pUAST-myc-mNedd8 and pUAST-flag-Cul1 or pUAST-flag-Cul13. The precipitates were reconstituted in reactions with purified DEN1 (0, 2.5, 5 µg in lanes 1, 2, 3, respectively) and DEN1CA (5 µg in lane 4). Western blots against Cul1 (A) or Cul3 (B) show that the protein levels of neddylated Cul1 and Cul3 are reduced in the presence of DEN1 but not DEN1CA. (C,D) Protein extracts prepared from wild-type and DEN1null larvae were analyzed by western blots for Cul1 (C) and Cul3 (D) expression. The levels of neddylated Cul1 and Cul3, denoted as Cul1Nedd8 and Cul3Nedd8, are reduced in DEN1null mutants. (E,F) UAS-myc-mNedd8 was expressed by tubP-GAL4 in wild-type and DEN1null, and larval lysates were immunoprecipitated by antibodies against Cul1 (E) or Cul3 (F). The immunocomplexes were analyzed using anti-Cul1 (left in E), anti-Cul3 (left in F) and anti-Myc antibodies (right panels) in western blots. Cul1Nedd8 levels are dramatically reduced, whereas the Cul3Nedd8 levels are only slightly reduced.

 

Figure 4
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  		Fig. 4. Accumulation of neddylated proteins in DEN1null mutants. (A-C) Transgenes UAS-myc-Nedd8GG-CFP (A), UAS-myc-Nedd8AA-CFP (B) and UAS-myc-mNedd8 (C) are expressed by the GAL4 driver ms1096 in wing pouches (outlined). Nedd8 expression, as shown by Myc staining (purple), was enhanced in GFP-negative DEN1null clones (arrows) for Myc-Nedd8GG-CFP (A) and Myc-mNedd8 (C), but not for Myc-Nedd8AA-CFP (B). Right panels show merged images. Mutant clones were located by the absence of the nuclear GFP signal (in left panels, see Materials and Methods for genotypes), which could be differentiated from the CFP signal (for the fusion proteins) by their emission spectra using Carl Zeiss LSM Meta 510 confocal microscope.

 

Figure 5
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  		Fig. 5. Ectopic neddylated proteins in DEN1null. (A) Expression of Myc-mNedd8-conjugated proteins was induced in DEN1null, as shown in western blot using the anti-Myc antibody for larval extracts prepared from tubP-GAL4;UAS-myc-mNedd8 in the wild type (lane 1) or DEN1null (lane 2) genetic background. (B) In western blot to detect endogenous neddylated proteins using anti-Nedd8 antibodies, ectopic neddylated proteins appear in DEN1null (lane 2) when compared with wild type (lane 1), and disappears in DEN1null;APP-BP1EX62 (lane 4), which displays a similar Nedd8 pattern to APP-BP1EX62 (lane 3). (C) Distinct neddylation patterns in DEN1null and CSN5null mutants in western blot using anti-Nedd8 antibodies for larval extracts prepared from wild type (lane 1), DEN1null (lane 2), CSN5null (lane 3) and DEN1null;CSN5null (lane 4).

 

Figure 6
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  		Fig. 6. Deneddylation activity of DEN1. Neddylated proteins in DEN1null larval lysates (lane 2) could be depleted by the co-incubated GST-DEN1 (lane 4), displaying a pattern largely identical to that detected in the wild-type lysates (lane 1). When the DEN1null lysates were treated with purified GST (lane 3) or GST-DEN1CA (lane 5), the neddylation pattern is similar to that without any treatment (lane 2).

 

Figure 7
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  		Fig. 7. Model for DEN1 activity in vivo. DEN1 functions in the processing the C terminus of Nedd8 precursor, generating mature Nedd8 with exposed di-Gly motif that is available for neddylation. The Nedd8 moiety on cullin proteins is deconjugated by the CSN. However, we propose that conjugated Nedd8 on other non-cullin proteins is deconjugated by DEN1.

 

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