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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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Conditionally stable Ci155 is sensitive to phosphorylation levels.(a) CSN4 clones are generated in wing discs that express dominant-negative GSK3β (DN-GSK3β) by ms-1096-GAL4. DN-GSK3β increases Ci155 expression (red) in low Hh signalling regions (arrowhead). The Ci155 level is downregulated in CSN4 cells (arrow) in the presence of DN-GSK3β. (b) Inhibition of CKI activity by C765-GAL4-driven DN-DBT increases the accumulation of Ci155 (red) in wild-type cells. This accumulation of Ci155 is downregulated in CSN4 clones (arrow). (c) CSN4 clones marked by the absence of GFP (green) were generated in wing discs expressing UAS-ci-C1-3E-myc under the control of ms1096-GAL4. The Ci-C1-3E-Myc levels detected by anti-Myc antibody (red) were decreased in CSN4 clones (arrow). (d) CSN4 clones marked by the absence of GFP (green) were generated in wing discs expressing UAS-ci-G2-3E-myc. Ci-G2-3E-Myc (red) staining is decreased in CSN4 clones (arrow). (e) Ectopic expression of PKA-R*, the regulatory subunit of PKA, by C765-GAL4 increases Ci155 levels in the A compartment. The upregulated Ci155 levels (red) either in low or low-to-intermediate Hh regions (arrow and arrowhead, respectively) are not altered in CSN5 clones in wing discs. (f) CSN5 clones were generated in wing discs that express the catalytic subunit of PKA (PKA-mC*) by ms-1096-GAL4. Ci155 levels (red) near the A/P boundary in the high Hh signalling region is downregulated in CSN5 clones (arrows) compared with wild-type cells (arrowhead). Dashed lines indicate the A/P boundary. Scale bars in all panels represent 50 μm.
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f5: Conditionally stable Ci155 is sensitive to phosphorylation levels.(a) CSN4 clones are generated in wing discs that express dominant-negative GSK3β (DN-GSK3β) by ms-1096-GAL4. DN-GSK3β increases Ci155 expression (red) in low Hh signalling regions (arrowhead). The Ci155 level is downregulated in CSN4 cells (arrow) in the presence of DN-GSK3β. (b) Inhibition of CKI activity by C765-GAL4-driven DN-DBT increases the accumulation of Ci155 (red) in wild-type cells. This accumulation of Ci155 is downregulated in CSN4 clones (arrow). (c) CSN4 clones marked by the absence of GFP (green) were generated in wing discs expressing UAS-ci-C1-3E-myc under the control of ms1096-GAL4. The Ci-C1-3E-Myc levels detected by anti-Myc antibody (red) were decreased in CSN4 clones (arrow). (d) CSN4 clones marked by the absence of GFP (green) were generated in wing discs expressing UAS-ci-G2-3E-myc. Ci-G2-3E-Myc (red) staining is decreased in CSN4 clones (arrow). (e) Ectopic expression of PKA-R*, the regulatory subunit of PKA, by C765-GAL4 increases Ci155 levels in the A compartment. The upregulated Ci155 levels (red) either in low or low-to-intermediate Hh regions (arrow and arrowhead, respectively) are not altered in CSN5 clones in wing discs. (f) CSN5 clones were generated in wing discs that express the catalytic subunit of PKA (PKA-mC*) by ms-1096-GAL4. Ci155 levels (red) near the A/P boundary in the high Hh signalling region is downregulated in CSN5 clones (arrows) compared with wild-type cells (arrowhead). Dashed lines indicate the A/P boundary. Scale bars in all panels represent 50 μm.

Mentions: Our results suggest that low-to-intermediate Hh signalling (regions 9–11) renders the appearance of conditionally stable Ci155, whereas high (region 12) and low (regions 1–8) Hh signalling activities do not. It has also been suggested that graded Hh signalling activities counteract Ci155 proteolysis by inhibiting different levels of Ci155 phosphorylation55, leading to differential affinities to the F-box protein Slimb for SCFSlimb-mediated proteolysis26. We therefore tested whether the phosphorylation status of Ci155 alters the level of conditionally stable Ci155. Ci155 phosphorylation can be compromised by the ectopic expression of dominant-negative GSK3β (DN-GSK3β), resulting in the inhibition of Ci155 proteolysis in the low Hh signalling region24. Although proteolysed Ci155 in the low Hh region (regions 1–8) is 84±5% of total Ci155 in wild-type cells (Fig. 3c), it is reduced to 38±8% in the DN-GSK3β expression clones (averaged from seven clones in regions 1–8, arrowhead in Fig. 5a). Accumulation of Ci155 in regions 1–8, however, is suppressed by the CSN4 mutation (arrow in Fig. 5a), with 75±11% of Ci155 undergoing proteolysis in CSN4 mutant cells expressing DN-GSK3β (n=8). Thus, the accumulated Ci155 in regions 1–8 on DN-GSK3ββ wings is conditionally stable in nature, undergoing proteolysis in the absence of CSN activity. Likewise, inhibiting CKI kinase activity by expressing dominant-negative Doubletime (DN-DBT) causes Ci155 accumulation25, which is also suppressed by the CSN4 mutation, as shown by reduced Ci155 levels in CSN4 mutant cells expressing DN-DBT (arrow, Fig. 5b) compared with wild-type cells expressing DN-DBT. Thus, lowering the phosphorylation level by inhibiting either GSK3β or CKI produces conditionally stable Ci155 in the low Hh signalling region, mimicking the Ci155 behaviour in the region with low-to-intermediate Hh signalling.


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)

Conditionally stable Ci155 is sensitive to phosphorylation levels.(a) CSN4 clones are generated in wing discs that express dominant-negative GSK3β (DN-GSK3β) by ms-1096-GAL4. DN-GSK3β increases Ci155 expression (red) in low Hh signalling regions (arrowhead). The Ci155 level is downregulated in CSN4 cells (arrow) in the presence of DN-GSK3β. (b) Inhibition of CKI activity by C765-GAL4-driven DN-DBT increases the accumulation of Ci155 (red) in wild-type cells. This accumulation of Ci155 is downregulated in CSN4 clones (arrow). (c) CSN4 clones marked by the absence of GFP (green) were generated in wing discs expressing UAS-ci-C1-3E-myc under the control of ms1096-GAL4. The Ci-C1-3E-Myc levels detected by anti-Myc antibody (red) were decreased in CSN4 clones (arrow). (d) CSN4 clones marked by the absence of GFP (green) were generated in wing discs expressing UAS-ci-G2-3E-myc. Ci-G2-3E-Myc (red) staining is decreased in CSN4 clones (arrow). (e) Ectopic expression of PKA-R*, the regulatory subunit of PKA, by C765-GAL4 increases Ci155 levels in the A compartment. The upregulated Ci155 levels (red) either in low or low-to-intermediate Hh regions (arrow and arrowhead, respectively) are not altered in CSN5 clones in wing discs. (f) CSN5 clones were generated in wing discs that express the catalytic subunit of PKA (PKA-mC*) by ms-1096-GAL4. Ci155 levels (red) near the A/P boundary in the high Hh signalling region is downregulated in CSN5 clones (arrows) compared with wild-type cells (arrowhead). Dashed lines indicate the A/P boundary. Scale bars in all panels represent 50 μm.
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Related In: Results  -  Collection

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f5: Conditionally stable Ci155 is sensitive to phosphorylation levels.(a) CSN4 clones are generated in wing discs that express dominant-negative GSK3β (DN-GSK3β) by ms-1096-GAL4. DN-GSK3β increases Ci155 expression (red) in low Hh signalling regions (arrowhead). The Ci155 level is downregulated in CSN4 cells (arrow) in the presence of DN-GSK3β. (b) Inhibition of CKI activity by C765-GAL4-driven DN-DBT increases the accumulation of Ci155 (red) in wild-type cells. This accumulation of Ci155 is downregulated in CSN4 clones (arrow). (c) CSN4 clones marked by the absence of GFP (green) were generated in wing discs expressing UAS-ci-C1-3E-myc under the control of ms1096-GAL4. The Ci-C1-3E-Myc levels detected by anti-Myc antibody (red) were decreased in CSN4 clones (arrow). (d) CSN4 clones marked by the absence of GFP (green) were generated in wing discs expressing UAS-ci-G2-3E-myc. Ci-G2-3E-Myc (red) staining is decreased in CSN4 clones (arrow). (e) Ectopic expression of PKA-R*, the regulatory subunit of PKA, by C765-GAL4 increases Ci155 levels in the A compartment. The upregulated Ci155 levels (red) either in low or low-to-intermediate Hh regions (arrow and arrowhead, respectively) are not altered in CSN5 clones in wing discs. (f) CSN5 clones were generated in wing discs that express the catalytic subunit of PKA (PKA-mC*) by ms-1096-GAL4. Ci155 levels (red) near the A/P boundary in the high Hh signalling region is downregulated in CSN5 clones (arrows) compared with wild-type cells (arrowhead). Dashed lines indicate the A/P boundary. Scale bars in all panels represent 50 μm.
Mentions: Our results suggest that low-to-intermediate Hh signalling (regions 9–11) renders the appearance of conditionally stable Ci155, whereas high (region 12) and low (regions 1–8) Hh signalling activities do not. It has also been suggested that graded Hh signalling activities counteract Ci155 proteolysis by inhibiting different levels of Ci155 phosphorylation55, leading to differential affinities to the F-box protein Slimb for SCFSlimb-mediated proteolysis26. We therefore tested whether the phosphorylation status of Ci155 alters the level of conditionally stable Ci155. Ci155 phosphorylation can be compromised by the ectopic expression of dominant-negative GSK3β (DN-GSK3β), resulting in the inhibition of Ci155 proteolysis in the low Hh signalling region24. Although proteolysed Ci155 in the low Hh region (regions 1–8) is 84±5% of total Ci155 in wild-type cells (Fig. 3c), it is reduced to 38±8% in the DN-GSK3β expression clones (averaged from seven clones in regions 1–8, arrowhead in Fig. 5a). Accumulation of Ci155 in regions 1–8, however, is suppressed by the CSN4 mutation (arrow in Fig. 5a), with 75±11% of Ci155 undergoing proteolysis in CSN4 mutant cells expressing DN-GSK3β (n=8). Thus, the accumulated Ci155 in regions 1–8 on DN-GSK3ββ wings is conditionally stable in nature, undergoing proteolysis in the absence of CSN activity. Likewise, inhibiting CKI kinase activity by expressing dominant-negative Doubletime (DN-DBT) causes Ci155 accumulation25, which is also suppressed by the CSN4 mutation, as shown by reduced Ci155 levels in CSN4 mutant cells expressing DN-DBT (arrow, Fig. 5b) compared with wild-type cells expressing DN-DBT. Thus, lowering the phosphorylation level by inhibiting either GSK3β or CKI produces conditionally stable Ci155 in the low Hh signalling region, mimicking the Ci155 behaviour in the region with low-to-intermediate Hh signalling.

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