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Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase.

Aghajan M, Jonai N, Flick K, Fu F, Luo M, Cai X, Ouni I, Pierce N, Tang X, Lomenick B, Damoiseaux R, Hao R, Del Moral PM, Verma R, Li Y, Li C, Houk KN, Jung ME, Zheng N, Huang L, Deshaies RJ, Kaiser P, Huang J - Nat. Biotechnol. (2010)

Bottom Line: We show here that SMER3 inhibits SCF(Met30) in vivo and in vitro, but not the closely related SCF(Cdc4).Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30.Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, and the Molecular Biology Institute, University of California, Los Angeles, California, USA.

ABSTRACT
The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)(Met30) ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCF(Met30) in vivo and in vitro, but not the closely related SCF(Cdc4). Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

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SMER3 targets SCFMet30a, Biochemical evidence for SCFMet30 inhibition by SMER3 but not rapamycin. Yeast cells were cultured in YPDA medium to mid-log 0.8×107 cells/ml, treated with indicated concentrations of SMER3 or rapamycin for 45 min, and total protein was extracted for Western blot analyses (Supplementary Information). Met4 ubiquitination in vivo can be directly assessed by immunoblotting because ubiquitinated forms of Met4 are not subjected to proteasomal degradation and can thus be detected due to a characteristic mobility shift on denaturing gels30. Asterisk (*) denotes a non-specific band immuno-reactive to the anti-Met4 antibody (generous gift from Mike Tyers). b, SMER3 resistance in met4Δ cells. Yeast cells were treated with either vehicle (DMSO) or 4 μM SMER3 and growth curve analysis was performed with an automated absorbance reader measuring O.D. at 595 nm every 30 min (Supplementary Information). Cell growth was measured in liquid because SMER3 activity is undetectable on agar. c, Genetic interaction between SCFMet30 and TOR. Temperature sensitive mutants as indicated were grown at 25°C to mid-log phase in YPDA medium and serial dilutions were spotted onto plates with or without 2.5 nM rapamycin. The plates were incubated at the permissive temperatures for the mutants: 28°C for cdc34-3, cdc53-1, cdc4-3 and met30-6 because these mutants exhibited fitness defects at 30°C even without rapamycin, or 30°C (standard growth temperature) for met30-9 and skp1-25 because these alleles are not temperature sensitive until at 37°C. d, SMER3 specifically inhibits SCFMet30 E3 ligase in vitro. Components of SCFMet30 were co-expressed in insect cells and the complex was purified based on a GST-tag fused to Skp1. Met4 expressed in insect cells was bound to SCFMet30 and the ligase-substrate complex eluted with glutathione. Purified ligase-substrate complexes were combined with purified SCFCdc4 and phosphorylated Sic1 and pre-incubated with DMSO or the indicated concentrations of SMER3 for 20 min at room temperature. The ubiquitination reaction was initiated by addition of E1, E2, ubiquitin, and ATP. The reaction was allowed to proceed for 25 min, with an aliquot of the reaction collected after the first 5 min to accommodate different reaction kinetics by the two SCFs. Reaction products were analyzed by immunoblotting. The asterisks indicate a protein cross-reacting with the anti-Met4 antibody. e, The amount of un-ubiquitinated substrate (Met4 and Sic1) was quantified on a Fuji LAS-4000 imaging system and inhibition was expressed as the ratio of un-ubiquitinated substrate in DMSO/SMER3.
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Figure 2: SMER3 targets SCFMet30a, Biochemical evidence for SCFMet30 inhibition by SMER3 but not rapamycin. Yeast cells were cultured in YPDA medium to mid-log 0.8×107 cells/ml, treated with indicated concentrations of SMER3 or rapamycin for 45 min, and total protein was extracted for Western blot analyses (Supplementary Information). Met4 ubiquitination in vivo can be directly assessed by immunoblotting because ubiquitinated forms of Met4 are not subjected to proteasomal degradation and can thus be detected due to a characteristic mobility shift on denaturing gels30. Asterisk (*) denotes a non-specific band immuno-reactive to the anti-Met4 antibody (generous gift from Mike Tyers). b, SMER3 resistance in met4Δ cells. Yeast cells were treated with either vehicle (DMSO) or 4 μM SMER3 and growth curve analysis was performed with an automated absorbance reader measuring O.D. at 595 nm every 30 min (Supplementary Information). Cell growth was measured in liquid because SMER3 activity is undetectable on agar. c, Genetic interaction between SCFMet30 and TOR. Temperature sensitive mutants as indicated were grown at 25°C to mid-log phase in YPDA medium and serial dilutions were spotted onto plates with or without 2.5 nM rapamycin. The plates were incubated at the permissive temperatures for the mutants: 28°C for cdc34-3, cdc53-1, cdc4-3 and met30-6 because these mutants exhibited fitness defects at 30°C even without rapamycin, or 30°C (standard growth temperature) for met30-9 and skp1-25 because these alleles are not temperature sensitive until at 37°C. d, SMER3 specifically inhibits SCFMet30 E3 ligase in vitro. Components of SCFMet30 were co-expressed in insect cells and the complex was purified based on a GST-tag fused to Skp1. Met4 expressed in insect cells was bound to SCFMet30 and the ligase-substrate complex eluted with glutathione. Purified ligase-substrate complexes were combined with purified SCFCdc4 and phosphorylated Sic1 and pre-incubated with DMSO or the indicated concentrations of SMER3 for 20 min at room temperature. The ubiquitination reaction was initiated by addition of E1, E2, ubiquitin, and ATP. The reaction was allowed to proceed for 25 min, with an aliquot of the reaction collected after the first 5 min to accommodate different reaction kinetics by the two SCFs. Reaction products were analyzed by immunoblotting. The asterisks indicate a protein cross-reacting with the anti-Met4 antibody. e, The amount of un-ubiquitinated substrate (Met4 and Sic1) was quantified on a Fuji LAS-4000 imaging system and inhibition was expressed as the ratio of un-ubiquitinated substrate in DMSO/SMER3.

Mentions: Induction of MET-gene expression in response to SMER3 exposure suggested that the cellular pathway controlling homeostasis of sulfur-containing compounds was a possible target for SMER3. The key regulator of this pathway is the ubiquitin ligase SCFMet30, which restrains the transcriptional activator Met4 in an inactive state in methionine-replete medium by attachment of a regulatory ubiquitin chain13. Inactivation of SCFMet30 prevents Met4 ubiquitination, permitting the formation of an active Met4-containing transcription complex that induces expression of the MET-genes and blocks cell proliferation. One hypothesis to explain the MET-gene activation and growth inhibition in SMER3-treated cells is that SMER3 inhibits SCFMet30. In agreement with this notion, Met4 ubiquitination was blocked in cells exposed to SMER3 (but not rapamycin) (Fig. 2a). Furthermore, genetic analyses have previously demonstrated that deubiquitinated Met4 mediates cell cycle arrest upon inactivation of SCFMet30 (ref 13), and deletion of MET4 rescues lethality of met30Δ (ref 14). Notably, met4Δ cells were also less susceptible to growth inhibition by SMER3 (but not rapamycin, exemplifying specificity) (Fig. 2b and Supplementary Fig. 2). These findings are consistent with SMER3 being an inhibitor of SCFMet30. However, the incomplete resistance of met4Δ to SMER3 (Fig. 2b) suggests that SMER3 likely has additional targets other than SCFMet30 and that cell growth inhibition by SMER3 is not solely due to SCFMet30 inhibition. This is not uncommon as even Gleevec, which was originally believed to be a highly specific inhibitor of BCR-Abl, is now appreciated to exert its biological effects through protein kinases in addition to its intended target15.


Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase.

Aghajan M, Jonai N, Flick K, Fu F, Luo M, Cai X, Ouni I, Pierce N, Tang X, Lomenick B, Damoiseaux R, Hao R, Del Moral PM, Verma R, Li Y, Li C, Houk KN, Jung ME, Zheng N, Huang L, Deshaies RJ, Kaiser P, Huang J - Nat. Biotechnol. (2010)

SMER3 targets SCFMet30a, Biochemical evidence for SCFMet30 inhibition by SMER3 but not rapamycin. Yeast cells were cultured in YPDA medium to mid-log 0.8×107 cells/ml, treated with indicated concentrations of SMER3 or rapamycin for 45 min, and total protein was extracted for Western blot analyses (Supplementary Information). Met4 ubiquitination in vivo can be directly assessed by immunoblotting because ubiquitinated forms of Met4 are not subjected to proteasomal degradation and can thus be detected due to a characteristic mobility shift on denaturing gels30. Asterisk (*) denotes a non-specific band immuno-reactive to the anti-Met4 antibody (generous gift from Mike Tyers). b, SMER3 resistance in met4Δ cells. Yeast cells were treated with either vehicle (DMSO) or 4 μM SMER3 and growth curve analysis was performed with an automated absorbance reader measuring O.D. at 595 nm every 30 min (Supplementary Information). Cell growth was measured in liquid because SMER3 activity is undetectable on agar. c, Genetic interaction between SCFMet30 and TOR. Temperature sensitive mutants as indicated were grown at 25°C to mid-log phase in YPDA medium and serial dilutions were spotted onto plates with or without 2.5 nM rapamycin. The plates were incubated at the permissive temperatures for the mutants: 28°C for cdc34-3, cdc53-1, cdc4-3 and met30-6 because these mutants exhibited fitness defects at 30°C even without rapamycin, or 30°C (standard growth temperature) for met30-9 and skp1-25 because these alleles are not temperature sensitive until at 37°C. d, SMER3 specifically inhibits SCFMet30 E3 ligase in vitro. Components of SCFMet30 were co-expressed in insect cells and the complex was purified based on a GST-tag fused to Skp1. Met4 expressed in insect cells was bound to SCFMet30 and the ligase-substrate complex eluted with glutathione. Purified ligase-substrate complexes were combined with purified SCFCdc4 and phosphorylated Sic1 and pre-incubated with DMSO or the indicated concentrations of SMER3 for 20 min at room temperature. The ubiquitination reaction was initiated by addition of E1, E2, ubiquitin, and ATP. The reaction was allowed to proceed for 25 min, with an aliquot of the reaction collected after the first 5 min to accommodate different reaction kinetics by the two SCFs. Reaction products were analyzed by immunoblotting. The asterisks indicate a protein cross-reacting with the anti-Met4 antibody. e, The amount of un-ubiquitinated substrate (Met4 and Sic1) was quantified on a Fuji LAS-4000 imaging system and inhibition was expressed as the ratio of un-ubiquitinated substrate in DMSO/SMER3.
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Figure 2: SMER3 targets SCFMet30a, Biochemical evidence for SCFMet30 inhibition by SMER3 but not rapamycin. Yeast cells were cultured in YPDA medium to mid-log 0.8×107 cells/ml, treated with indicated concentrations of SMER3 or rapamycin for 45 min, and total protein was extracted for Western blot analyses (Supplementary Information). Met4 ubiquitination in vivo can be directly assessed by immunoblotting because ubiquitinated forms of Met4 are not subjected to proteasomal degradation and can thus be detected due to a characteristic mobility shift on denaturing gels30. Asterisk (*) denotes a non-specific band immuno-reactive to the anti-Met4 antibody (generous gift from Mike Tyers). b, SMER3 resistance in met4Δ cells. Yeast cells were treated with either vehicle (DMSO) or 4 μM SMER3 and growth curve analysis was performed with an automated absorbance reader measuring O.D. at 595 nm every 30 min (Supplementary Information). Cell growth was measured in liquid because SMER3 activity is undetectable on agar. c, Genetic interaction between SCFMet30 and TOR. Temperature sensitive mutants as indicated were grown at 25°C to mid-log phase in YPDA medium and serial dilutions were spotted onto plates with or without 2.5 nM rapamycin. The plates were incubated at the permissive temperatures for the mutants: 28°C for cdc34-3, cdc53-1, cdc4-3 and met30-6 because these mutants exhibited fitness defects at 30°C even without rapamycin, or 30°C (standard growth temperature) for met30-9 and skp1-25 because these alleles are not temperature sensitive until at 37°C. d, SMER3 specifically inhibits SCFMet30 E3 ligase in vitro. Components of SCFMet30 were co-expressed in insect cells and the complex was purified based on a GST-tag fused to Skp1. Met4 expressed in insect cells was bound to SCFMet30 and the ligase-substrate complex eluted with glutathione. Purified ligase-substrate complexes were combined with purified SCFCdc4 and phosphorylated Sic1 and pre-incubated with DMSO or the indicated concentrations of SMER3 for 20 min at room temperature. The ubiquitination reaction was initiated by addition of E1, E2, ubiquitin, and ATP. The reaction was allowed to proceed for 25 min, with an aliquot of the reaction collected after the first 5 min to accommodate different reaction kinetics by the two SCFs. Reaction products were analyzed by immunoblotting. The asterisks indicate a protein cross-reacting with the anti-Met4 antibody. e, The amount of un-ubiquitinated substrate (Met4 and Sic1) was quantified on a Fuji LAS-4000 imaging system and inhibition was expressed as the ratio of un-ubiquitinated substrate in DMSO/SMER3.
Mentions: Induction of MET-gene expression in response to SMER3 exposure suggested that the cellular pathway controlling homeostasis of sulfur-containing compounds was a possible target for SMER3. The key regulator of this pathway is the ubiquitin ligase SCFMet30, which restrains the transcriptional activator Met4 in an inactive state in methionine-replete medium by attachment of a regulatory ubiquitin chain13. Inactivation of SCFMet30 prevents Met4 ubiquitination, permitting the formation of an active Met4-containing transcription complex that induces expression of the MET-genes and blocks cell proliferation. One hypothesis to explain the MET-gene activation and growth inhibition in SMER3-treated cells is that SMER3 inhibits SCFMet30. In agreement with this notion, Met4 ubiquitination was blocked in cells exposed to SMER3 (but not rapamycin) (Fig. 2a). Furthermore, genetic analyses have previously demonstrated that deubiquitinated Met4 mediates cell cycle arrest upon inactivation of SCFMet30 (ref 13), and deletion of MET4 rescues lethality of met30Δ (ref 14). Notably, met4Δ cells were also less susceptible to growth inhibition by SMER3 (but not rapamycin, exemplifying specificity) (Fig. 2b and Supplementary Fig. 2). These findings are consistent with SMER3 being an inhibitor of SCFMet30. However, the incomplete resistance of met4Δ to SMER3 (Fig. 2b) suggests that SMER3 likely has additional targets other than SCFMet30 and that cell growth inhibition by SMER3 is not solely due to SCFMet30 inhibition. This is not uncommon as even Gleevec, which was originally believed to be a highly specific inhibitor of BCR-Abl, is now appreciated to exert its biological effects through protein kinases in addition to its intended target15.

Bottom Line: We show here that SMER3 inhibits SCF(Met30) in vivo and in vitro, but not the closely related SCF(Cdc4).Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30.Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, and the Molecular Biology Institute, University of California, Los Angeles, California, USA.

ABSTRACT
The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)(Met30) ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCF(Met30) in vivo and in vitro, but not the closely related SCF(Cdc4). Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

Show MeSH
Related in: MedlinePlus