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First published online 6 May 2008
doi: 10.1242/jcs.020552

Journal of Cell Science 121, 1825-1831 (2008)
Published by The Company of Biologists 2008
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UV-induced degradation of securin is mediated by SKP1-CUL1-βTrCP E3 ubiquitin ligase

M. Cristina Limón-Mortés1, Mar Mora-Santos1, Águeda Espina2, José A. Pintor-Toro2, Antonio López-Román1, María Tortolero1 and Francisco Romero1,*

1 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, Apdo 1095, 41080 Sevilla, Spain
2 Centro Andaluz de Biología y Medicina Regenerativa, CSIC, Sevilla, Spain


Figure 1
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  		Fig. 1. Reduction in the amount of securin after UV irradiation in the S and G2 phases of the cell cycle occurs in both cytoplasm and nucleus. HeLa cells were synchronized in S and G2 phases, and exposed to UV radiation where indicated. C, synchronized and nonirradiated cells. (A) Immunofluorescence experiments using an immunoaffinity-purified polyclonal anti-securin antibody, a monoclonal anti-CCNB1 (cyclin B) antibody and DAPI. In the merge, securin staining is shown in green, cyclin B in red, and DAPI in blue. Original magnification x403. (B) Detection of securin in subcellular fractions from synchronized cells. Fractions were separated by SDS-PAGE, transferred, and probed with the indicated antibodies. Anti-Raf and anti-hnRNP C were used to control for purity of the fractions. CE, cytosolic extracts; NE, nuclear extracts.

 

Figure 2
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  		Fig. 2. UV-induced degradation of securin is not mediated by APC/C. (A) HeLa cells were transiently transfected with pSUPER-Cdh1 and pSUPER-Cdc20 or pSUPER alone, and harvested 48 hours after transfection. Where indicated, cells were irradiated 1 hour 30 minutes before harvesting. Extracts were Western blotted for the detection of CDH1, CDC20 and securin (hSec). Note that the western blot for CDC20 shows three bands, the upper two (and most prominent) of which are crossreactive and not specific to CDC20. (B) HeLa cells were transiently transfected with siRNA oligonucleotides against CDC27/APC3 or nonspecific control oligonucleotides (mock) and, after 48 hours, were irradiated or left untreated, and collected 1 hour 30 minutes later. Western blots of extracts were analyzed for CDC27/APC3, securin and {alpha}-tubulin levels. HeLa, lysates from nontransfected HeLa cells.

 

Figure 3
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  		Fig. 3. SKP1-CUL1-βTrCP is the E3 ubiquitin ligase that mediates the UV-induced degradation of securin. (A) COS-7 cells transiently transfected with pCDNA3 or pCDNA3-Flag-CUL11-452 were irradiated (UV) or left untreated (C) 1 hour 30 minutes before harvesting. NP40 extracts were prepared and western blotted for CUL1, Flag-CUL11-452 and securin detection. (B) COS-7 cells were transiently transfected with the indicated plasmids and treated as above. Western blots were probed for the presence of CUL1, Flag-CUL11-452, securin-VSV and EGFP. The asterisk indicates an nonspecific band. (C) COS-7 cells were transiently transfected with pCDNA3 or pCDNA3-MT-Skp2{Delta}F, and irradiated (UV) or left untreated (C) 1 hour 30 minutes before harvesting. Extracts were western blotted for SKP2, MT-SKP2{Delta}F, and securin detection. (D) COS-7 cells were transfected with the indicated plasmids, treated as described, and blotted with anti-SKP2, anti-VSV, and anti-GFP. (E) COS-7 cells transiently transfected with pCS2-HA or pCS2-HA-βTrCP{Delta}F were left untreated (C), irradiated (UV), or treated with okadaic acid (OA) 1 hour 30 minutes before harvesting. NP40 extracts were prepared and western blotted for the detection of the indicated proteins. Cells treated with OA were used as a negative control of the experiment because phosphorylated forms of securin after OA treatment are degraded via the SCF complex (Gil-Bernabe et al., 2006Go), but βTrCP{Delta}F does not prevent this degradation. (F) Transfected COS-7 cells with the indicated plasmids were treated as described, and blotted for the presence of HA-βTrCP{Delta}F, securin-VSV, and EGFP.

 

Figure 4
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  		Fig. 4. Securin binds to βTrCP in vivo. (A) Anti-securin and preimmune (PI) sera were used in immunoprecipitations from NP40 extracts of COS-7 cells transiently transfected with pCS2-HA-βTrCP and treated with LLnL for 4 hours before harvesting. The immunoprecipitates were probed for the presence of HA-βTrCP and securin. Lysate, lysate from LLnL-treated COS-7 pCS2-HA-βTrCP cells. (B) COS-7 cells transiently transfected with pCS2-HA-βTrCP{Delta}F were left untreated (Control) or irradiated (UV) before harvesting. Extracts were used for coimmunoprecipitation experiments. Lysate, lysate from COS-7 pCS2-HA-βTrCP{Delta}F cells. Asterisk indicates IgG light chains.

 

Figure 5
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  		Fig. 5. Securin is ubiquitylated by SCFβTrCP. (A) COS-7 cells were transfected with the indicated plasmids, and treated with LLnL for 4 hours before harvesting. Extracts were prepared as indicated in the Materials and Methods. Proteins were separated by SDS-PAGE, electroblotted and probed with anti-HA and anti-GFP antibodies. (B) Transfected cells were left untreated (C) or irradiated (UV) 1 hour 30 minutes before harvesting.

 

Figure 6
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  		Fig. 6. Identification of a new securin destruction motif. (A) Alignment of βTrCP-binding motifs in securin proteins of various species. The arrow indicates Asp109 of securin (hSec1) that was mutated to Gly. (B) COS-7 cells were transfected with the indicated plasmids, and left untreated (C) or irradiated (UV) before harvesting. NP40 extracts were prepared and western blotted for the detection of the indicated proteins.

 

Figure 7
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  		Fig. 7. GSK-3β inhibitors reverse the effect of UV radiation on securin expression. HeLa cells were left untreated (C) or UV-irradiated (UV) in the presence or absence of LiCl (A) or TDZD-8 (B). Inhibitors were added 1 hour before irradiation. Where indicated, extracts were treated with {lambda}-PP. Equal amounts of extracts were resolved by SDS-PAGE. Immunoblotting was performed with anti-securin and anti-{alpha}-tubulin antibodies.

 

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