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Guido Tricot - One of the best experts on this subject based on the ideXlab platform.
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abstract 214 high expression of cks1b antagonizes mln4924 a nedd8 activating enzyme inhibitor that induces myeloma cell growth inhibition through decreased neddylation of SCF Complex
Cancer Research, 2012Co-Authors: Fenghuang Zhan, He Wang, Maurizio Zangari, Guido TricotAbstract:Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: MLN4924 binds to the NEDD8-activating enzyme (NAE), and promotes neddylation of SCF (Skp1-Cul1 / Cdc53-F-box protein) Complex resulting in inhibition of proteasomal degradation of several substrates. CKS1B acts as an adaptor of SCF Complexes involved in cell cycle progression and DNA damage repair in many cancers, including multiple myeloma (MM). In this study, we investigate the functional role of CKS1B expression on MLN4924 in myeloma and determine whether CKS1B can be used as a potential biomarker for sensitivity to MLN4924 treatment in MM. Methods: Three myeloma cell lines, KMS12, KMS28PE, and OCI-MY5 in which CKS1B was artificially over-expressed (CKS1B-OE) as well as their empty vector (EV) controls were treated with MLN4924 at different concentrations. The IC50 and growth inhibition of cells were analyzed via a fluorencent screeing assay. APC-labeled annexinV and PI were assessed by flow cytometry to detect apoptosis and cell cycle changes. The expression of Cullin-NEDD8 and a series of ubiquitined SCF substrates were analyzed by non-reducing western blot. Results: MLN4924 effectively inhibited cell growth and induced cell apoptosis in both CKS1B-OE and EV MM cells. However, MM cells transfected with EV were more sensitive than those transfected with CKS1B-OE as evidenced by the IC50 values (KMS28PE OE vs. EV 99: 65 nM; KMS12 OE vs. EV 125: 72 nM; and OCI-MY5 OE vs. EV 106:89 nM). The cell viabilities at 100 nM were 49.6% vs. 26.7%, 54.5% vs. 34.2%, and 53.6% vs. 36.8% in CKS1B-OE versus EV KMS28PE, KMS12, and OCI-MY5 cells (P<0.05). Western blots showed that over-expression of CKS1B significantly decreased expression of cullin1, nedd8, and the conjugated Complex of cullin-nedd8 in MLN4924 treated cells (100 nM, 24h) compared with EV in all tested three cell lines. We also found that high expression of CKS1B was negatively correlated with SCF substrates including p21, CDT-1, p27 and CDT-1. MLN4924 induced a dose-dependent accumulation of these substrates in all three MM cell lines. Conclusion: High expression of CKS1B induces SCF substrate ubiquitination and degradation in MM cells. The NAE inhibitor MLN4924 induces cell apoptosis through decreased neddylation of SCF Complex resulting in increase of p27, p21, CDT-1 and p130. CKS1B can be used as a marker of drug resistance to MLN4924, suggesting that a combination of MLN4924 with other drugs will be necessary to treat myeloma patients with high endogenous expression of CKS1B. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 214. doi:1538-7445.AM2012-214
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abstract 214 high expression of cks1b antagonizes mln4924 a nedd8 activating enzyme inhibitor that induces myeloma cell growth inhibition through decreased neddylation of SCF Complex
Cancer Research, 2012Co-Authors: Fenghuang Zhan, He Wang, Maurizio Zangari, Guido TricotAbstract:Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: MLN4924 binds to the NEDD8-activating enzyme (NAE), and promotes neddylation of SCF (Skp1-Cul1 / Cdc53-F-box protein) Complex resulting in inhibition of proteasomal degradation of several substrates. CKS1B acts as an adaptor of SCF Complexes involved in cell cycle progression and DNA damage repair in many cancers, including multiple myeloma (MM). In this study, we investigate the functional role of CKS1B expression on MLN4924 in myeloma and determine whether CKS1B can be used as a potential biomarker for sensitivity to MLN4924 treatment in MM. Methods: Three myeloma cell lines, KMS12, KMS28PE, and OCI-MY5 in which CKS1B was artificially over-expressed (CKS1B-OE) as well as their empty vector (EV) controls were treated with MLN4924 at different concentrations. The IC50 and growth inhibition of cells were analyzed via a fluorencent screeing assay. APC-labeled annexinV and PI were assessed by flow cytometry to detect apoptosis and cell cycle changes. The expression of Cullin-NEDD8 and a series of ubiquitined SCF substrates were analyzed by non-reducing western blot. Results: MLN4924 effectively inhibited cell growth and induced cell apoptosis in both CKS1B-OE and EV MM cells. However, MM cells transfected with EV were more sensitive than those transfected with CKS1B-OE as evidenced by the IC50 values (KMS28PE OE vs. EV 99: 65 nM; KMS12 OE vs. EV 125: 72 nM; and OCI-MY5 OE vs. EV 106:89 nM). The cell viabilities at 100 nM were 49.6% vs. 26.7%, 54.5% vs. 34.2%, and 53.6% vs. 36.8% in CKS1B-OE versus EV KMS28PE, KMS12, and OCI-MY5 cells (P<0.05). Western blots showed that over-expression of CKS1B significantly decreased expression of cullin1, nedd8, and the conjugated Complex of cullin-nedd8 in MLN4924 treated cells (100 nM, 24h) compared with EV in all tested three cell lines. We also found that high expression of CKS1B was negatively correlated with SCF substrates including p21, CDT-1, p27 and CDT-1. MLN4924 induced a dose-dependent accumulation of these substrates in all three MM cell lines. Conclusion: High expression of CKS1B induces SCF substrate ubiquitination and degradation in MM cells. The NAE inhibitor MLN4924 induces cell apoptosis through decreased neddylation of SCF Complex resulting in increase of p27, p21, CDT-1 and p130. CKS1B can be used as a marker of drug resistance to MLN4924, suggesting that a combination of MLN4924 with other drugs will be necessary to treat myeloma patients with high endogenous expression of CKS1B. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 214. doi:1538-7445.AM2012-214
Keiichi I Nakayama - One of the best experts on this subject based on the ideXlab platform.
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substrate binding promotes formation of the skp1 cul1 fbxl3 SCF fbxl3 protein Complex
Journal of Biological Chemistry, 2013Co-Authors: Kanae Yumimoto, Tetsuya Muneoka, Tomohiro Tsuboi, Keiichi I NakayamaAbstract:The Skp1–Cul1–F-box protein (SCF) Complex is one of the most well characterized types of ubiquitin ligase (E3), with the E3 activity of the Complex being regulated in part at the level of Complex formation. Fbxl3 is an F-box protein that is responsible for the ubiquitylation and consequent degradation of cryptochromes (Crys) and thus regulates oscillation of the circadian clock. Here we show that formation of the SCFFbxl3 Complex is regulated by substrate binding in vivo. Fbxl3 did not associate with Skp1 and Cul1 to a substantial extent in transfected mammalian cells. Unexpectedly, however, formation of the SCFFbxl3 Complex was markedly promoted by forced expression of its substrate Cry1 in these cells. A mutant form of Fbxl3 that does not bind to Cry1 was unable to form an SCF Complex, suggesting that interaction of Cry1 with Fbxl3 is essential for formation of SCFFbxl3. In contrast, recombinant Fbxl3 associated with recombinant Skp1 and Cul1 in vitro even in the absence of recombinant Cry1. Domain-swap analysis revealed that the COOH-terminal leucine-rich repeat domain of Fbxl3 attenuates the interaction of Skp1, suggesting that a yet unknown protein associated with the COOH-terminal domain of Fbxl3 and inhibited SCF Complex formation. Our results thus provide important insight into the regulation of both SCF ubiquitin ligase activity and circadian rhythmicity.
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substrate binding promotes formation of the skp1 cul1 fbxl3 SCFfbxl3 protein Complex
Journal of Biological Chemistry, 2013Co-Authors: Kanae Yumimoto, Tetsuya Muneoka, Tomohiro Tsuboi, Keiichi I NakayamaAbstract:The Skp1-Cul1-F-box protein (SCF) Complex is one of the most well characterized types of ubiquitin ligase (E3), with the E3 activity of the Complex being regulated in part at the level of Complex formation. Fbxl3 is an F-box protein that is responsible for the ubiquitylation and consequent degradation of cryptochromes (Crys) and thus regulates oscillation of the circadian clock. Here we show that formation of the SCF(Fbxl3) Complex is regulated by substrate binding in vivo. Fbxl3 did not associate with Skp1 and Cul1 to a substantial extent in transfected mammalian cells. Unexpectedly, however, formation of the SCF(Fbxl3) Complex was markedly promoted by forced expression of its substrate Cry1 in these cells. A mutant form of Fbxl3 that does not bind to Cry1 was unable to form an SCF Complex, suggesting that interaction of Cry1 with Fbxl3 is essential for formation of SCF(Fbxl3). In contrast, recombinant Fbxl3 associated with recombinant Skp1 and Cul1 in vitro even in the absence of recombinant Cry1. Domain-swap analysis revealed that the COOH-terminal leucine-rich repeat domain of Fbxl3 attenuates the interaction of Skp1, suggesting that a yet unknown protein associated with the COOH-terminal domain of Fbxl3 and inhibited SCF Complex formation. Our results thus provide important insight into the regulation of both SCF ubiquitin ligase activity and circadian rhythmicity.
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regulation of the cell cycle by SCF type ubiquitin ligases
Seminars in Cell & Developmental Biology, 2005Co-Authors: Keiichi I Nakayama, Keiko NakayamaAbstract:Regulation of the cell cycle is dependent on protein degradation by the ubiquitin-proteasome system. Two major ubiquitin ligases, the anaphase-promoting Complex or cyclosome (APC/C) and SCF Complex, are responsible for the periodic proteolysis of many regulators of the cell cycle. The receptor component of the SCF Complex is one of many F-box proteins, three of which--Skp2, Fbw7, and beta-TrCP--are well characterized and implicated in cell cycle regulation. We have generated mice deficient in Skp2, Fbw7, or beta-TrCP1 and have identified the roles of these proteins in both cell cycle regulation and mouse development. Clinical evidence also suggests that dysregulation of these F-box proteins contributes to human cancers.
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preferential interaction of tip120a with cul1 that is not modified by nedd8 and not associated with skp1
Biochemical and Biophysical Research Communications, 2003Co-Authors: Kiyotaka Oshikawa, Masayoshi Yada, Shigetsugu Hatakeyama, Takumi Kamura, Masaki Matsumoto, Keiichi I NakayamaAbstract:Abstract The SCF Complex, which consists of the invariable components Skp1, Cul1, and Rbx1 as well as a variable F-box protein, functions as an E3 ubiquitin ligase. The mechanism by which the activity of this Complex is regulated, however, has been unclear. The application of tandem affinity purification has now resulted in the identification of a novel Cul1-binding protein: TATA-binding protein-interacting protein 120A (TIP120A, also called CAND1). Immunoprecipitation, immunoblot, and immunofluorescence analyses with mammalian cells revealed that TIP120A physically associates with Cul1 in the nucleus and that this interaction is mediated by a central region of Cul1 distinct from its binding sites for Skp1 and Rbx1. Furthermore, TIP120A was shown to interact selectively with Cul1 that is not modified by NEDD8. The Cul1-TIP120A Complex does not include Skp1, raising the possibility that TIP120A competes with Skp1 for binding to Cul1. These observations thus suggest that TIP120A may function as a negative regulator of the SCF Complex by binding to nonneddylated Cul1 and thereby preventing assembly of this ubiquitin ligase.
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multiple skp1 related proteins in caenorhabditis elegans diverse patterns of interaction with cullins and f box proteins
Current Biology, 2002Co-Authors: Atsushi Yamanaka, Masayoshi Yada, Hiroyuki Imaki, Makoto Koga, Yasumi Ohshima, Keiichi I NakayamaAbstract:Background The ubiquitin-proteasome pathway of proteolysis controls the abundance of specific regulatory proteins. The SCF Complex is a type of ubiquitin-protein ligase (E3) that contributes to this pathway in many biological systems. In yeast and mammals, the SCF Complex consists of common components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Whereas only one functional Skp1 gene is present in the human genome, the genome of Caenorhabditis elegans has now been shown to contain at least 21 Skp1 -related ( skr ) genes. The biochemical properties, expression, and function of the C. elegans SKR proteins were examined. Results Of the 17 SKR proteins examined, eight (SKR-1, -2, -3, -4, -7, -8, -9, and -10) were shown to interact with C. elegans CUL1 by yeast two-hybrid analysis or a coimmunoprecipitation assay in mammalian cells. Furthermore, SKR proteins exhibited diverse binding specificities for C. elegans F-box proteins. The tissue specificity of expression of the CUL1-interacting SKR proteins was also varied. Suppression of skr-1 or skr-2 genes by double-stranded RNA interference resulted in embryonic death, whereas that of skr-7 , -8 , -9 , or -10 was associated with slow growth and morphological abnormalities. Conclusions The multiple C. elegans SKR proteins exhibit marked differences in their association with Cullins and F-box proteins, in tissue specificity of expression, and in phenotypes associated with functional suppression by RNAi. At least eight of the SKR proteins may, like F-box proteins, act as variable components of the SCF Complex in C. elegans .
Fenghuang Zhan - One of the best experts on this subject based on the ideXlab platform.
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abstract 214 high expression of cks1b antagonizes mln4924 a nedd8 activating enzyme inhibitor that induces myeloma cell growth inhibition through decreased neddylation of SCF Complex
Cancer Research, 2012Co-Authors: Fenghuang Zhan, He Wang, Maurizio Zangari, Guido TricotAbstract:Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: MLN4924 binds to the NEDD8-activating enzyme (NAE), and promotes neddylation of SCF (Skp1-Cul1 / Cdc53-F-box protein) Complex resulting in inhibition of proteasomal degradation of several substrates. CKS1B acts as an adaptor of SCF Complexes involved in cell cycle progression and DNA damage repair in many cancers, including multiple myeloma (MM). In this study, we investigate the functional role of CKS1B expression on MLN4924 in myeloma and determine whether CKS1B can be used as a potential biomarker for sensitivity to MLN4924 treatment in MM. Methods: Three myeloma cell lines, KMS12, KMS28PE, and OCI-MY5 in which CKS1B was artificially over-expressed (CKS1B-OE) as well as their empty vector (EV) controls were treated with MLN4924 at different concentrations. The IC50 and growth inhibition of cells were analyzed via a fluorencent screeing assay. APC-labeled annexinV and PI were assessed by flow cytometry to detect apoptosis and cell cycle changes. The expression of Cullin-NEDD8 and a series of ubiquitined SCF substrates were analyzed by non-reducing western blot. Results: MLN4924 effectively inhibited cell growth and induced cell apoptosis in both CKS1B-OE and EV MM cells. However, MM cells transfected with EV were more sensitive than those transfected with CKS1B-OE as evidenced by the IC50 values (KMS28PE OE vs. EV 99: 65 nM; KMS12 OE vs. EV 125: 72 nM; and OCI-MY5 OE vs. EV 106:89 nM). The cell viabilities at 100 nM were 49.6% vs. 26.7%, 54.5% vs. 34.2%, and 53.6% vs. 36.8% in CKS1B-OE versus EV KMS28PE, KMS12, and OCI-MY5 cells (P<0.05). Western blots showed that over-expression of CKS1B significantly decreased expression of cullin1, nedd8, and the conjugated Complex of cullin-nedd8 in MLN4924 treated cells (100 nM, 24h) compared with EV in all tested three cell lines. We also found that high expression of CKS1B was negatively correlated with SCF substrates including p21, CDT-1, p27 and CDT-1. MLN4924 induced a dose-dependent accumulation of these substrates in all three MM cell lines. Conclusion: High expression of CKS1B induces SCF substrate ubiquitination and degradation in MM cells. The NAE inhibitor MLN4924 induces cell apoptosis through decreased neddylation of SCF Complex resulting in increase of p27, p21, CDT-1 and p130. CKS1B can be used as a marker of drug resistance to MLN4924, suggesting that a combination of MLN4924 with other drugs will be necessary to treat myeloma patients with high endogenous expression of CKS1B. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 214. doi:1538-7445.AM2012-214
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abstract 214 high expression of cks1b antagonizes mln4924 a nedd8 activating enzyme inhibitor that induces myeloma cell growth inhibition through decreased neddylation of SCF Complex
Cancer Research, 2012Co-Authors: Fenghuang Zhan, He Wang, Maurizio Zangari, Guido TricotAbstract:Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: MLN4924 binds to the NEDD8-activating enzyme (NAE), and promotes neddylation of SCF (Skp1-Cul1 / Cdc53-F-box protein) Complex resulting in inhibition of proteasomal degradation of several substrates. CKS1B acts as an adaptor of SCF Complexes involved in cell cycle progression and DNA damage repair in many cancers, including multiple myeloma (MM). In this study, we investigate the functional role of CKS1B expression on MLN4924 in myeloma and determine whether CKS1B can be used as a potential biomarker for sensitivity to MLN4924 treatment in MM. Methods: Three myeloma cell lines, KMS12, KMS28PE, and OCI-MY5 in which CKS1B was artificially over-expressed (CKS1B-OE) as well as their empty vector (EV) controls were treated with MLN4924 at different concentrations. The IC50 and growth inhibition of cells were analyzed via a fluorencent screeing assay. APC-labeled annexinV and PI were assessed by flow cytometry to detect apoptosis and cell cycle changes. The expression of Cullin-NEDD8 and a series of ubiquitined SCF substrates were analyzed by non-reducing western blot. Results: MLN4924 effectively inhibited cell growth and induced cell apoptosis in both CKS1B-OE and EV MM cells. However, MM cells transfected with EV were more sensitive than those transfected with CKS1B-OE as evidenced by the IC50 values (KMS28PE OE vs. EV 99: 65 nM; KMS12 OE vs. EV 125: 72 nM; and OCI-MY5 OE vs. EV 106:89 nM). The cell viabilities at 100 nM were 49.6% vs. 26.7%, 54.5% vs. 34.2%, and 53.6% vs. 36.8% in CKS1B-OE versus EV KMS28PE, KMS12, and OCI-MY5 cells (P<0.05). Western blots showed that over-expression of CKS1B significantly decreased expression of cullin1, nedd8, and the conjugated Complex of cullin-nedd8 in MLN4924 treated cells (100 nM, 24h) compared with EV in all tested three cell lines. We also found that high expression of CKS1B was negatively correlated with SCF substrates including p21, CDT-1, p27 and CDT-1. MLN4924 induced a dose-dependent accumulation of these substrates in all three MM cell lines. Conclusion: High expression of CKS1B induces SCF substrate ubiquitination and degradation in MM cells. The NAE inhibitor MLN4924 induces cell apoptosis through decreased neddylation of SCF Complex resulting in increase of p27, p21, CDT-1 and p130. CKS1B can be used as a marker of drug resistance to MLN4924, suggesting that a combination of MLN4924 with other drugs will be necessary to treat myeloma patients with high endogenous expression of CKS1B. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 214. doi:1538-7445.AM2012-214
Gerd Sutter - One of the best experts on this subject based on the ideXlab platform.
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the highly conserved orthopoxvirus 68k ankyrin like protein is part of a cellular SCF ubiquitin ligase Complex
Virology, 2008Co-Authors: Karin M Sperling, Astrid Schwantes, Barbara S Schnierle, Gerd SutterAbstract:The 68k ankyrin-like protein (68k-ank) of unknown function is highly conserved among orthopoxviruses and contains ankyrin repeats and an F-box-like domain. We performed a yeast-two-hybrid screen with 68k-ank to find interacting proteins. From a human and a murine cDNA library, 99% of the interaction partners were S-phase kinase-associated protein 1a (Skp1a), a part of the SCF ubiquitin ligase Complex. 68k-ank co-immunoprecipitated with components of the endogenous, mammalian SCF ubiquitin ligase. This interaction was F-box domain dependent and could also be observed in infected cells, indicating that SCF Complex formation might be important for the viral life cycle.
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rapid communication the highly conserved orthopoxvirus 68k ankyrin like protein is part of a cellular SCF ubiquitin ligase Complex
2008Co-Authors: Karin M Sperling, Astrid Schwantes, Barbara S Schnierle, Gerd SutterAbstract:The 68k ankyrin-like protein (68k-ank) of unknown function is highly conserved among orthopoxviruses and contains ankyrin repeats and an F-box-like domain. We performed a yeast-two-hybrid screen with 68k-ank to find interacting proteins. From a human and a murine cDNA library, 99% of the interaction partners were S-phase kinase-associated protein 1a (Skp1a), a part of the SCF ubiquitin ligase Complex. 68k-ank coimmunoprecipitated with components of the endogenous, mammalian SCF ubiquitin ligase. This interaction was F-box domain dependent and could also be observed in infected cells, indicating that SCF Complex formation might be important for the viral life cycle.
Michele Pagano - One of the best experts on this subject based on the ideXlab platform.
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control of cell growth by the SCF and apc c ubiquitin ligases
Current Opinion in Cell Biology, 2009Co-Authors: Jeffrey R Skaar, Michele PaganoAbstract:The ubiquitin–proteasome system plays key roles in the control of cell growth. The cell cycle, in particular, is highly regulated by the functions of the SCF and APC/C ubiquitin ligases, and perturbation of their function can result in tumorigenesis. Although the SCF and APC/C Complexes are well established in growth control pathways, many aspects of their function remain unknown. Recent studies have shed light on the mechanism of SCF-mediated ubiquitination and new functions for the SCF Complex and APC/C. Our expanding understanding of the roles of the SCF and APC/C Complexes highlight the potential for targeted molecular therapies.
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involvement of the SCF Complex in the control of cdh1 degradation in s phase
Cell Cycle, 2005Co-Authors: Ramla Benmaamar, Michele PaganoAbstract:The anaphase promoting Complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that acts as a key regulator in the progression through mitosis (when mostly in Complex with Cdc20) and as a stabilizer of the G1 phase (when in Complex with Cdh1). Cdh1 is an activator of APC/C, and it has previously been reported that it is capable of mediating its own degradation during Go and G1. Herein, we show that the SCF Complex (Skp1/Cul1/F-box protein/Roc1) intervenes in the surveillance of Cdh1 cellular abundance in S-phase.
