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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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The CSN positively regulates Ci155 protein stability in wing discs.(a) CSN5 clones are revealed by the absence of GFP (green) in wing discs. Ci155 stained with the 2A1 antibody (red) is reduced in CSN5 clones located in the A compartment, with arrow indicating mutant cells in the low-to-intermediate Hh signalling region and arrowhead indicating cells in the low Hh signalling region. (b) GFP-negative CSN4 clones were generated in wing discs (green). Ci155 staining (red) is lower in the CSN4 cells in the A compartment (arrow) than in the wild-type cells. The Ci155 levels in CSN4 and wild-type cells located in A/P boundary are similar (arrowhead). (c) CSN4 clones revealed by the absence of GFP (green) were generated in wing discs that simultaneously express UAS-ci-myc under the control of ms1096-GAL4. Protein levels of Ci-Myc detected by the anti-Myc antibody (red) are reduced in CSN4 mutant clones (arrow). (d) The ci-lacZ expression (red) is not reduced in CSN4 mutant clones (arrow) generated in wing discs. (e) GFP-negative CSN5 clones (green) were generated in wing discs expressing wild-type CSN5 construct under the control of ms1096-GAL4. Expression of wild-type CSN5 rescues the Ci155 level (red) in CSN5 clones (arrow). (f) Expression of CSN5D148N, which loses deneddylation activity, fails to rescues the Ci155 level (red) in CSN5 clones (arrows). (g) slimbP1493, CSN5 clones revealed by the absence of GFP (green) in wing discs show Ci155 (red) accumulation in the A compartment (arrow) except when located adjacent to the A/P boundary (arrowhead). (h) The Ci-3P mutant protein (red), carrying mutations in three PKA phosphorylation sites, is ectopically expressed in CSN4 mosaic wing pouches under the control of ms1096-GAL4. There is no difference in Ci-3P expression in wild-type and GFP-negative CSN4 cells (arrow). Scale bars in all panels represent 50 μm.
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f2: The CSN positively regulates Ci155 protein stability in wing discs.(a) CSN5 clones are revealed by the absence of GFP (green) in wing discs. Ci155 stained with the 2A1 antibody (red) is reduced in CSN5 clones located in the A compartment, with arrow indicating mutant cells in the low-to-intermediate Hh signalling region and arrowhead indicating cells in the low Hh signalling region. (b) GFP-negative CSN4 clones were generated in wing discs (green). Ci155 staining (red) is lower in the CSN4 cells in the A compartment (arrow) than in the wild-type cells. The Ci155 levels in CSN4 and wild-type cells located in A/P boundary are similar (arrowhead). (c) CSN4 clones revealed by the absence of GFP (green) were generated in wing discs that simultaneously express UAS-ci-myc under the control of ms1096-GAL4. Protein levels of Ci-Myc detected by the anti-Myc antibody (red) are reduced in CSN4 mutant clones (arrow). (d) The ci-lacZ expression (red) is not reduced in CSN4 mutant clones (arrow) generated in wing discs. (e) GFP-negative CSN5 clones (green) were generated in wing discs expressing wild-type CSN5 construct under the control of ms1096-GAL4. Expression of wild-type CSN5 rescues the Ci155 level (red) in CSN5 clones (arrow). (f) Expression of CSN5D148N, which loses deneddylation activity, fails to rescues the Ci155 level (red) in CSN5 clones (arrows). (g) slimbP1493, CSN5 clones revealed by the absence of GFP (green) in wing discs show Ci155 (red) accumulation in the A compartment (arrow) except when located adjacent to the A/P boundary (arrowhead). (h) The Ci-3P mutant protein (red), carrying mutations in three PKA phosphorylation sites, is ectopically expressed in CSN4 mosaic wing pouches under the control of ms1096-GAL4. There is no difference in Ci-3P expression in wild-type and GFP-negative CSN4 cells (arrow). Scale bars in all panels represent 50 μm.

Mentions: We also tested how deneddylation regulates another SCFSlimb substrate Ci155 in wing discs1327. Unlike the increased level of Arm, the Ci155 protein level is instead downregulated in both CSN5 and CSN4 mutant cells (Fig. 2a,b), suggesting that neddylated SCFSlimb degrades more Ci155, despite the reduced levels of both Cul142 and the substrate receptor Slimb (Fig. 1a) in CSN mutants. Thus, Ci155 proteolysis is more sensitive to the level of neddylation, but less so to the bulk concentrations of SCFSlimb components. In comparison to mutant clones in the low Hh signalling region (arrowhead in Fig. 2a), the reduction in the Ci155 level is more prominent in low-to-intermediate Hh signalling regions (arrow in Fig. 2a) in which the Ci155 protein level starts to fall but has not yet reached the basal level. Deneddylation upregulates the Ci155 level by a post-transcriptional mechanism, as we found that the protein levels of ci-myc and HA-ci transgenes under the ms1096-GAL4 driver are reduced in CSN mutant cells (arrow in Fig. 2c and Supplementary Fig. S2). Furthermore, the expression of ci-lacZ that recapitulates ci transcription remains constant in CSN5 cells (Fig. 2d).


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)

The CSN positively regulates Ci155 protein stability in wing discs.(a) CSN5 clones are revealed by the absence of GFP (green) in wing discs. Ci155 stained with the 2A1 antibody (red) is reduced in CSN5 clones located in the A compartment, with arrow indicating mutant cells in the low-to-intermediate Hh signalling region and arrowhead indicating cells in the low Hh signalling region. (b) GFP-negative CSN4 clones were generated in wing discs (green). Ci155 staining (red) is lower in the CSN4 cells in the A compartment (arrow) than in the wild-type cells. The Ci155 levels in CSN4 and wild-type cells located in A/P boundary are similar (arrowhead). (c) CSN4 clones revealed by the absence of GFP (green) were generated in wing discs that simultaneously express UAS-ci-myc under the control of ms1096-GAL4. Protein levels of Ci-Myc detected by the anti-Myc antibody (red) are reduced in CSN4 mutant clones (arrow). (d) The ci-lacZ expression (red) is not reduced in CSN4 mutant clones (arrow) generated in wing discs. (e) GFP-negative CSN5 clones (green) were generated in wing discs expressing wild-type CSN5 construct under the control of ms1096-GAL4. Expression of wild-type CSN5 rescues the Ci155 level (red) in CSN5 clones (arrow). (f) Expression of CSN5D148N, which loses deneddylation activity, fails to rescues the Ci155 level (red) in CSN5 clones (arrows). (g) slimbP1493, CSN5 clones revealed by the absence of GFP (green) in wing discs show Ci155 (red) accumulation in the A compartment (arrow) except when located adjacent to the A/P boundary (arrowhead). (h) The Ci-3P mutant protein (red), carrying mutations in three PKA phosphorylation sites, is ectopically expressed in CSN4 mosaic wing pouches under the control of ms1096-GAL4. There is no difference in Ci-3P expression in wild-type and GFP-negative CSN4 cells (arrow). Scale bars in all panels represent 50 μm.
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f2: The CSN positively regulates Ci155 protein stability in wing discs.(a) CSN5 clones are revealed by the absence of GFP (green) in wing discs. Ci155 stained with the 2A1 antibody (red) is reduced in CSN5 clones located in the A compartment, with arrow indicating mutant cells in the low-to-intermediate Hh signalling region and arrowhead indicating cells in the low Hh signalling region. (b) GFP-negative CSN4 clones were generated in wing discs (green). Ci155 staining (red) is lower in the CSN4 cells in the A compartment (arrow) than in the wild-type cells. The Ci155 levels in CSN4 and wild-type cells located in A/P boundary are similar (arrowhead). (c) CSN4 clones revealed by the absence of GFP (green) were generated in wing discs that simultaneously express UAS-ci-myc under the control of ms1096-GAL4. Protein levels of Ci-Myc detected by the anti-Myc antibody (red) are reduced in CSN4 mutant clones (arrow). (d) The ci-lacZ expression (red) is not reduced in CSN4 mutant clones (arrow) generated in wing discs. (e) GFP-negative CSN5 clones (green) were generated in wing discs expressing wild-type CSN5 construct under the control of ms1096-GAL4. Expression of wild-type CSN5 rescues the Ci155 level (red) in CSN5 clones (arrow). (f) Expression of CSN5D148N, which loses deneddylation activity, fails to rescues the Ci155 level (red) in CSN5 clones (arrows). (g) slimbP1493, CSN5 clones revealed by the absence of GFP (green) in wing discs show Ci155 (red) accumulation in the A compartment (arrow) except when located adjacent to the A/P boundary (arrowhead). (h) The Ci-3P mutant protein (red), carrying mutations in three PKA phosphorylation sites, is ectopically expressed in CSN4 mosaic wing pouches under the control of ms1096-GAL4. There is no difference in Ci-3P expression in wild-type and GFP-negative CSN4 cells (arrow). Scale bars in all panels represent 50 μm.
Mentions: We also tested how deneddylation regulates another SCFSlimb substrate Ci155 in wing discs1327. Unlike the increased level of Arm, the Ci155 protein level is instead downregulated in both CSN5 and CSN4 mutant cells (Fig. 2a,b), suggesting that neddylated SCFSlimb degrades more Ci155, despite the reduced levels of both Cul142 and the substrate receptor Slimb (Fig. 1a) in CSN mutants. Thus, Ci155 proteolysis is more sensitive to the level of neddylation, but less so to the bulk concentrations of SCFSlimb components. In comparison to mutant clones in the low Hh signalling region (arrowhead in Fig. 2a), the reduction in the Ci155 level is more prominent in low-to-intermediate Hh signalling regions (arrow in Fig. 2a) in which the Ci155 protein level starts to fall but has not yet reached the basal level. Deneddylation upregulates the Ci155 level by a post-transcriptional mechanism, as we found that the protein levels of ci-myc and HA-ci transgenes under the ms1096-GAL4 driver are reduced in CSN mutant cells (arrow in Fig. 2c and Supplementary Fig. S2). Furthermore, the expression of ci-lacZ that recapitulates ci transcription remains constant in CSN5 cells (Fig. 2d).

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