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CSN-mediated deneddylation differentially modulates Ci(155) proteolysis to promote Hedgehog signalling responses.

Wu JT, Lin WH, Chen WY, Huang YC, Tang CY, Ho MS, Pi H, Chien CT - Nat Commun (2011)

Bottom Line: Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCF(Slimb) is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells.The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation.We propose that the affinity of substrate-E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan.

ABSTRACT
The Hedgehog (Hh) morphogen directs distinct cell responses according to its distinct signalling levels. Hh signalling stabilizes transcription factor cubitus interruptus (Ci) by prohibiting SCF(Slimb)-dependent ubiquitylation and proteolysis of Ci. How graded Hh signalling confers differential SCF(Slimb)-mediated Ci proteolysis in responding cells remains unclear. Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCF(Slimb) is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells. As a consequence, expression of the low-threshold Hh target gene dpp is disrupted, highlighting the critical role of CSN deneddylation on low-to-intermediate Hh signalling response. The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation. We propose that the affinity of substrate-E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo.

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Effects of neddylation and deneddylation on F-box proteins Slimb and Ago.(a) CSN5 clones generated in Drosophila third-instar larval wing discs are revealed by the absence of GFP (green). The protein level of tubulin promoter-driven myc-slimb (red) decreases in GFP-negative CSN5 clones (arrow). (b) Nedd8AN015 clones marked by the absence of GFP (green) were generated in wing discs expressing Myc-Slimb. The level of Myc-Slimb (red) in Nedd8AN015 clones is higher (arrow) compared with wild type. (c) The level of Myc-SlimbΔfbx (red) expressed by ap-GAL4 is unaltered by CSN5 mutation (arrow) in the dorsal compartment of wing discs. (d) GFP-negative CSN5 clones are generated in wing discs (green), in which Flag-Ago (red) is expressed in wing pouches under the control of ms1096-GAL4. Flag-tag staining of Flag-Ago protein has a comparable signal level in wild-type and CSN5 cells (arrow). Scale bars, 50 μm.
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f1: Effects of neddylation and deneddylation on F-box proteins Slimb and Ago.(a) CSN5 clones generated in Drosophila third-instar larval wing discs are revealed by the absence of GFP (green). The protein level of tubulin promoter-driven myc-slimb (red) decreases in GFP-negative CSN5 clones (arrow). (b) Nedd8AN015 clones marked by the absence of GFP (green) were generated in wing discs expressing Myc-Slimb. The level of Myc-Slimb (red) in Nedd8AN015 clones is higher (arrow) compared with wild type. (c) The level of Myc-SlimbΔfbx (red) expressed by ap-GAL4 is unaltered by CSN5 mutation (arrow) in the dorsal compartment of wing discs. (d) GFP-negative CSN5 clones are generated in wing discs (green), in which Flag-Ago (red) is expressed in wing pouches under the control of ms1096-GAL4. Flag-tag staining of Flag-Ago protein has a comparable signal level in wild-type and CSN5 cells (arrow). Scale bars, 50 μm.

Mentions: Whereas neddylation of cullins promotes CRL activities, deneddylation of cullins also promotes substrate degradation, mainly through protecting cullins and substrate receptors from neddylation-induced degradation4243444546. To test whether protection of substrate receptors by deneddylation is a general rule, we first examined the protein stability of Slimb, a substrate receptor for the SCF complex that ubiquitylates several substrates including Ci155 (refs 13, 47). Mutant clones for the CSN catalytic subunit CSN5 (CSN5) were generated in third-instar wing discs that also carry the myc-slimb transgene under the control of the ubiquitous tubulin promoter. In CSN5 cells, the protein level of Myc-Slimb is diminished (arrow in Fig. 1a), suggesting that the CSN is required to stabilize Slimb in vivo. In contrast, neddylation promotes Myc-Slimb turnover, as Myc-Slimb accumulates to high levels in cells homozygous for Nedd8AN015 (arrow in Fig. 1b). To test whether the SCF-Slimb interaction is essential for neddylation-mediated Slimb turnover, we examined the protein stability of Myc-SlimbΔfbx that cannot be incorporated into the SCF complex because of the truncation of the F-box47. In contrast to the full-length Slimb, the protein levels of SlimbΔfbx in CSN5 mutant and neighbouring wild-type cells are indistinguishable (Fig. 1c). Taken together, we conclude that the substrate receptor Slimb, when incorporated into the SCF complex, is regulated by neddylation-induced degradation. However, neddylation-induced degradation of substrate receptors is unlikely a general rule, as the protein level of the Flag-conjugated F-box protein Archipelago (Flag-Ago) is unaltered in CSN5 cells (Fig. 1d), suggesting that neddylation and deneddylation regulate the protein stability of some but not all of the SCF substrate receptors in Drosophila.


CSN-mediated deneddylation differentially modulates Ci(155) proteolysis to promote Hedgehog signalling responses.

Wu JT, Lin WH, Chen WY, Huang YC, Tang CY, Ho MS, Pi H, Chien CT - Nat Commun (2011)

Effects of neddylation and deneddylation on F-box proteins Slimb and Ago.(a) CSN5 clones generated in Drosophila third-instar larval wing discs are revealed by the absence of GFP (green). The protein level of tubulin promoter-driven myc-slimb (red) decreases in GFP-negative CSN5 clones (arrow). (b) Nedd8AN015 clones marked by the absence of GFP (green) were generated in wing discs expressing Myc-Slimb. The level of Myc-Slimb (red) in Nedd8AN015 clones is higher (arrow) compared with wild type. (c) The level of Myc-SlimbΔfbx (red) expressed by ap-GAL4 is unaltered by CSN5 mutation (arrow) in the dorsal compartment of wing discs. (d) GFP-negative CSN5 clones are generated in wing discs (green), in which Flag-Ago (red) is expressed in wing pouches under the control of ms1096-GAL4. Flag-tag staining of Flag-Ago protein has a comparable signal level in wild-type and CSN5 cells (arrow). Scale bars, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105314&req=5

f1: Effects of neddylation and deneddylation on F-box proteins Slimb and Ago.(a) CSN5 clones generated in Drosophila third-instar larval wing discs are revealed by the absence of GFP (green). The protein level of tubulin promoter-driven myc-slimb (red) decreases in GFP-negative CSN5 clones (arrow). (b) Nedd8AN015 clones marked by the absence of GFP (green) were generated in wing discs expressing Myc-Slimb. The level of Myc-Slimb (red) in Nedd8AN015 clones is higher (arrow) compared with wild type. (c) The level of Myc-SlimbΔfbx (red) expressed by ap-GAL4 is unaltered by CSN5 mutation (arrow) in the dorsal compartment of wing discs. (d) GFP-negative CSN5 clones are generated in wing discs (green), in which Flag-Ago (red) is expressed in wing pouches under the control of ms1096-GAL4. Flag-tag staining of Flag-Ago protein has a comparable signal level in wild-type and CSN5 cells (arrow). Scale bars, 50 μm.
Mentions: Whereas neddylation of cullins promotes CRL activities, deneddylation of cullins also promotes substrate degradation, mainly through protecting cullins and substrate receptors from neddylation-induced degradation4243444546. To test whether protection of substrate receptors by deneddylation is a general rule, we first examined the protein stability of Slimb, a substrate receptor for the SCF complex that ubiquitylates several substrates including Ci155 (refs 13, 47). Mutant clones for the CSN catalytic subunit CSN5 (CSN5) were generated in third-instar wing discs that also carry the myc-slimb transgene under the control of the ubiquitous tubulin promoter. In CSN5 cells, the protein level of Myc-Slimb is diminished (arrow in Fig. 1a), suggesting that the CSN is required to stabilize Slimb in vivo. In contrast, neddylation promotes Myc-Slimb turnover, as Myc-Slimb accumulates to high levels in cells homozygous for Nedd8AN015 (arrow in Fig. 1b). To test whether the SCF-Slimb interaction is essential for neddylation-mediated Slimb turnover, we examined the protein stability of Myc-SlimbΔfbx that cannot be incorporated into the SCF complex because of the truncation of the F-box47. In contrast to the full-length Slimb, the protein levels of SlimbΔfbx in CSN5 mutant and neighbouring wild-type cells are indistinguishable (Fig. 1c). Taken together, we conclude that the substrate receptor Slimb, when incorporated into the SCF complex, is regulated by neddylation-induced degradation. However, neddylation-induced degradation of substrate receptors is unlikely a general rule, as the protein level of the Flag-conjugated F-box protein Archipelago (Flag-Ago) is unaltered in CSN5 cells (Fig. 1d), suggesting that neddylation and deneddylation regulate the protein stability of some but not all of the SCF substrate receptors in Drosophila.

Bottom Line: Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCF(Slimb) is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells.The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation.We propose that the affinity of substrate-E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan.

ABSTRACT
The Hedgehog (Hh) morphogen directs distinct cell responses according to its distinct signalling levels. Hh signalling stabilizes transcription factor cubitus interruptus (Ci) by prohibiting SCF(Slimb)-dependent ubiquitylation and proteolysis of Ci. How graded Hh signalling confers differential SCF(Slimb)-mediated Ci proteolysis in responding cells remains unclear. Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCF(Slimb) is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells. As a consequence, expression of the low-threshold Hh target gene dpp is disrupted, highlighting the critical role of CSN deneddylation on low-to-intermediate Hh signalling response. The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation. We propose that the affinity of substrate-E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo.

Show MeSH
Related in: MedlinePlus