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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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Related in: MedlinePlus

The CSN regulates Ci155 proteolysis predominantly in intermediate Hh signalling regions.(a) Mutant clones for CSN4 or Cul1EX were generated simultaneously in the same wing discs of the hsflp; FRT42D CSN4/FRT42D Cul1EX, nlsGFP genotype. Ci155 (red) levels stained by 2A1 antibody was measured in the following cells: CSN4 mutant clones marked by the absence of GFP, Cul1EX mutant clones marked by two copies of GFP and double-heterozygous CSN4/+, Cul1EX/+ cells marked by one copy of GFP (green). The lower panels in a show close-up images of a region near the A/P boundary (outlined by a dashed square in upper left panel), with some CSN4 clones highlighted by white dashed lines (lower left panel). (b) The A compartment of wing pouches showing expression of Ci155 (red) and dpp-lacZ (green) is divided equally into 12 regions along the A/P axis. Expression of dpp-lacZ is detected in region 11. Scale bars represent 50 μm (a, b). (c) The portions of Ci155 that undergo SCFSlimb-mediated proteolysis in regions 1–12 of wild-type (grey line) and CSN4 mutant cells (dashed line) were calculated. Averaged Ci155 pixel intensities for each region (n >= 6) are shown. Asterisks indicate a significant difference of Ci155 proteolysis between wild-type and CSN4 (P<0.05). Error bars present the standard deviation.
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f3: The CSN regulates Ci155 proteolysis predominantly in intermediate Hh signalling regions.(a) Mutant clones for CSN4 or Cul1EX were generated simultaneously in the same wing discs of the hsflp; FRT42D CSN4/FRT42D Cul1EX, nlsGFP genotype. Ci155 (red) levels stained by 2A1 antibody was measured in the following cells: CSN4 mutant clones marked by the absence of GFP, Cul1EX mutant clones marked by two copies of GFP and double-heterozygous CSN4/+, Cul1EX/+ cells marked by one copy of GFP (green). The lower panels in a show close-up images of a region near the A/P boundary (outlined by a dashed square in upper left panel), with some CSN4 clones highlighted by white dashed lines (lower left panel). (b) The A compartment of wing pouches showing expression of Ci155 (red) and dpp-lacZ (green) is divided equally into 12 regions along the A/P axis. Expression of dpp-lacZ is detected in region 11. Scale bars represent 50 μm (a, b). (c) The portions of Ci155 that undergo SCFSlimb-mediated proteolysis in regions 1–12 of wild-type (grey line) and CSN4 mutant cells (dashed line) were calculated. Averaged Ci155 pixel intensities for each region (n >= 6) are shown. Asterisks indicate a significant difference of Ci155 proteolysis between wild-type and CSN4 (P<0.05). Error bars present the standard deviation.

Mentions: To gain insights into how the CSN regulates SCFSlimb-mediated Ci155 proteolysis, the proportions of proteolysed and stable Ci155 along the A/P axis of wing discs were quantified. We generated random mosaic twin clones from double heterozygotes for the Cul1EX and CSN4 alleles, both located at chromosome 2R (Fig. 3a). One of the twin clones is homozygous for Cul1EX, marked with the double-brightness of green fluorescent protein (GFP) signal, and the other twin clone is homozygous for CSN4, marked by the complete absence of GFP signal. Cells marked by single brightness of GFP signal are double heterozygous for Cul1EX and CSN4, which are considered as wild-type control. The Ci155-expressing A compartment of wing pouches was divided into 12 equal regions, with the most anterior cells being in region 1 and cells receiving the highest level of Hh in region 12 (Fig. 3b). We measured the Ci155 protein levels in Cul1EX, CSN4 and wild-type control cells in regions 1–12. The percentage of Ci155 that has undergone SCFSlimb-dependent proteolysis in each region was calculated as the Ci155 level in Cul1EX cells (no proteolysis) subtracted by the Ci155 level in double-heterozygous cells (normal proteolysis in wild-type control), which is then normalized to the Ci155 level in Cul1EX cells: percentage of Ci155 proteolysis=([Ci155]Cul1EX−[Ci155]wild-type)/[Ci155]Cul1EX. We found that the extent of SCFSlimb-dependent Ci155 proteolysis is constantly high across regions 1–8, and gradually declines from region 8 to 12 (Fig. 3c). We also quantified the extents of SCFSlimb-dependent Ci155 proteolysis in CSN4 cells across regions 1–12 using the same methodology: percentage of Ci155 proteolysis=([Ci155]Cul1EX−[Ci155]CSN4)/[Ci155]Cul1EX. Interestingly, Ci155 proteolysis in CSN4 cells remained similar to wild-type cells in regions 1–8, and graded Ci155 proteolysis in CSN4 cells was also present from regions 8 to 12. Remarkably, Ci155 proteolysis is prominently enhanced in CSN4 cells in regions 9, 10 and 11 compared with wild-type cells in the same regions, with 30, 40 and 24% reductions in the Ci155 levels, respectively (Fig. 3c). In region 12 closest to the A/P boundary in which the protein level of Ci155 is regulated by the Cul3-organized CRL2751, the proportion of SCFSlimb-dependent Ci155 proteolysis is unaltered in CSN4 mutant cells (arrow in Fig. 3a and arrowhead in Fig. 2b). Mild enhancements of 1–6% of Ci155 proteolysis were noted in CSN4 cells in regions 1–8, although these differences were not statistically significant except for regions 6 and 7 (n≥6, P<0.05 by Student's t-test). On the basis of the proteolytic curves of Ci155 in wild-type and CSN4 cells, we propose that proteolysis-resistant Ci155 in wild-type cells is composed of 'stable' and 'conditionally stable' forms, the latter being stable in wild-type cells but proteolysed in CSN mutant cells by SCFSlimb, whereas the former is insensitive to the absence of CSN activity. The levels of conditionally stable Ci155 are highest in regions 9–11, in which cells are exposed to low-to-intermediate levels of Hh.


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 regulates Ci155 proteolysis predominantly in intermediate Hh signalling regions.(a) Mutant clones for CSN4 or Cul1EX were generated simultaneously in the same wing discs of the hsflp; FRT42D CSN4/FRT42D Cul1EX, nlsGFP genotype. Ci155 (red) levels stained by 2A1 antibody was measured in the following cells: CSN4 mutant clones marked by the absence of GFP, Cul1EX mutant clones marked by two copies of GFP and double-heterozygous CSN4/+, Cul1EX/+ cells marked by one copy of GFP (green). The lower panels in a show close-up images of a region near the A/P boundary (outlined by a dashed square in upper left panel), with some CSN4 clones highlighted by white dashed lines (lower left panel). (b) The A compartment of wing pouches showing expression of Ci155 (red) and dpp-lacZ (green) is divided equally into 12 regions along the A/P axis. Expression of dpp-lacZ is detected in region 11. Scale bars represent 50 μm (a, b). (c) The portions of Ci155 that undergo SCFSlimb-mediated proteolysis in regions 1–12 of wild-type (grey line) and CSN4 mutant cells (dashed line) were calculated. Averaged Ci155 pixel intensities for each region (n >= 6) are shown. Asterisks indicate a significant difference of Ci155 proteolysis between wild-type and CSN4 (P<0.05). Error bars present the standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: The CSN regulates Ci155 proteolysis predominantly in intermediate Hh signalling regions.(a) Mutant clones for CSN4 or Cul1EX were generated simultaneously in the same wing discs of the hsflp; FRT42D CSN4/FRT42D Cul1EX, nlsGFP genotype. Ci155 (red) levels stained by 2A1 antibody was measured in the following cells: CSN4 mutant clones marked by the absence of GFP, Cul1EX mutant clones marked by two copies of GFP and double-heterozygous CSN4/+, Cul1EX/+ cells marked by one copy of GFP (green). The lower panels in a show close-up images of a region near the A/P boundary (outlined by a dashed square in upper left panel), with some CSN4 clones highlighted by white dashed lines (lower left panel). (b) The A compartment of wing pouches showing expression of Ci155 (red) and dpp-lacZ (green) is divided equally into 12 regions along the A/P axis. Expression of dpp-lacZ is detected in region 11. Scale bars represent 50 μm (a, b). (c) The portions of Ci155 that undergo SCFSlimb-mediated proteolysis in regions 1–12 of wild-type (grey line) and CSN4 mutant cells (dashed line) were calculated. Averaged Ci155 pixel intensities for each region (n >= 6) are shown. Asterisks indicate a significant difference of Ci155 proteolysis between wild-type and CSN4 (P<0.05). Error bars present the standard deviation.
Mentions: To gain insights into how the CSN regulates SCFSlimb-mediated Ci155 proteolysis, the proportions of proteolysed and stable Ci155 along the A/P axis of wing discs were quantified. We generated random mosaic twin clones from double heterozygotes for the Cul1EX and CSN4 alleles, both located at chromosome 2R (Fig. 3a). One of the twin clones is homozygous for Cul1EX, marked with the double-brightness of green fluorescent protein (GFP) signal, and the other twin clone is homozygous for CSN4, marked by the complete absence of GFP signal. Cells marked by single brightness of GFP signal are double heterozygous for Cul1EX and CSN4, which are considered as wild-type control. The Ci155-expressing A compartment of wing pouches was divided into 12 equal regions, with the most anterior cells being in region 1 and cells receiving the highest level of Hh in region 12 (Fig. 3b). We measured the Ci155 protein levels in Cul1EX, CSN4 and wild-type control cells in regions 1–12. The percentage of Ci155 that has undergone SCFSlimb-dependent proteolysis in each region was calculated as the Ci155 level in Cul1EX cells (no proteolysis) subtracted by the Ci155 level in double-heterozygous cells (normal proteolysis in wild-type control), which is then normalized to the Ci155 level in Cul1EX cells: percentage of Ci155 proteolysis=([Ci155]Cul1EX−[Ci155]wild-type)/[Ci155]Cul1EX. We found that the extent of SCFSlimb-dependent Ci155 proteolysis is constantly high across regions 1–8, and gradually declines from region 8 to 12 (Fig. 3c). We also quantified the extents of SCFSlimb-dependent Ci155 proteolysis in CSN4 cells across regions 1–12 using the same methodology: percentage of Ci155 proteolysis=([Ci155]Cul1EX−[Ci155]CSN4)/[Ci155]Cul1EX. Interestingly, Ci155 proteolysis in CSN4 cells remained similar to wild-type cells in regions 1–8, and graded Ci155 proteolysis in CSN4 cells was also present from regions 8 to 12. Remarkably, Ci155 proteolysis is prominently enhanced in CSN4 cells in regions 9, 10 and 11 compared with wild-type cells in the same regions, with 30, 40 and 24% reductions in the Ci155 levels, respectively (Fig. 3c). In region 12 closest to the A/P boundary in which the protein level of Ci155 is regulated by the Cul3-organized CRL2751, the proportion of SCFSlimb-dependent Ci155 proteolysis is unaltered in CSN4 mutant cells (arrow in Fig. 3a and arrowhead in Fig. 2b). Mild enhancements of 1–6% of Ci155 proteolysis were noted in CSN4 cells in regions 1–8, although these differences were not statistically significant except for regions 6 and 7 (n≥6, P<0.05 by Student's t-test). On the basis of the proteolytic curves of Ci155 in wild-type and CSN4 cells, we propose that proteolysis-resistant Ci155 in wild-type cells is composed of 'stable' and 'conditionally stable' forms, the latter being stable in wild-type cells but proteolysed in CSN mutant cells by SCFSlimb, whereas the former is insensitive to the absence of CSN activity. The levels of conditionally stable Ci155 are highest in regions 9–11, in which cells are exposed to low-to-intermediate levels of Hh.

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