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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 differentially regulates Hh downstream gene expression.(a) A CSN4 clone (indicated by the dashed outline) is generated in dpp-lacZ wing discs. dpp-lacZ expression detected by anti-β-gal antibody (red) is markedly repressed in CSN4 cells (arrow). (b) CSN5 clones indicated by the absence of GFP are generated in wing discs. Ptc (red) protein level in CSN5 cells (arrow) detected by the monoclonal antibody Apa-1 is similar to that in wild-type cells. (c) ptc-lacZ staining (red) is comparable in wild-type and CSN5 cells that are marked by the absence of GFP (arrow). (d) En protein levels (red) are increased in the CSN5 cells (arrow) located in the A/P boundary as well as in the A and P compartments (arrowheads). Scale bars in all panels represent 50 μm.
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f4: The CSN differentially regulates Hh downstream gene expression.(a) A CSN4 clone (indicated by the dashed outline) is generated in dpp-lacZ wing discs. dpp-lacZ expression detected by anti-β-gal antibody (red) is markedly repressed in CSN4 cells (arrow). (b) CSN5 clones indicated by the absence of GFP are generated in wing discs. Ptc (red) protein level in CSN5 cells (arrow) detected by the monoclonal antibody Apa-1 is similar to that in wild-type cells. (c) ptc-lacZ staining (red) is comparable in wild-type and CSN5 cells that are marked by the absence of GFP (arrow). (d) En protein levels (red) are increased in the CSN5 cells (arrow) located in the A/P boundary as well as in the A and P compartments (arrowheads). Scale bars in all panels represent 50 μm.

Mentions: To examine whether the level of conditionally stable Ci155 is essential for distinct cell fates directed by graded Hh signalling, we assayed the activation of Hh-responsive, position-specific genes, such as ptc and dpp, whose expression represent high and low Hh signalling responses, respectively19. Interestingly, we found that the expression of dpp-lacZ is significantly repressed in CSN4 mutant cells (arrow in Fig. 4a), whereas the Ptc protein level and the expression of ptc-lacZ remain unaltered (arrows in Fig. 4b,c), indicating a critical role of the conditionally stable Ci155 for the Hh-mediated dpp expression. The expression of engrailed (en) is regulated by high Hh signalling activity in the A/P boundary52. We tested whether en expression in the A/P boundary is downregulated in CSN4 mutant because of Ci155 proteolysis. En protein level is instead enhanced not only at the A/P boundary but also in A and P compartments, in which en expression is independent from Hh signalling (Fig. 4d), suggesting that en expression is subjected to another layer of regulation by the CSN.


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 differentially regulates Hh downstream gene expression.(a) A CSN4 clone (indicated by the dashed outline) is generated in dpp-lacZ wing discs. dpp-lacZ expression detected by anti-β-gal antibody (red) is markedly repressed in CSN4 cells (arrow). (b) CSN5 clones indicated by the absence of GFP are generated in wing discs. Ptc (red) protein level in CSN5 cells (arrow) detected by the monoclonal antibody Apa-1 is similar to that in wild-type cells. (c) ptc-lacZ staining (red) is comparable in wild-type and CSN5 cells that are marked by the absence of GFP (arrow). (d) En protein levels (red) are increased in the CSN5 cells (arrow) located in the A/P boundary as well as in the A and P compartments (arrowheads). Scale bars in all panels represent 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105314&req=5

f4: The CSN differentially regulates Hh downstream gene expression.(a) A CSN4 clone (indicated by the dashed outline) is generated in dpp-lacZ wing discs. dpp-lacZ expression detected by anti-β-gal antibody (red) is markedly repressed in CSN4 cells (arrow). (b) CSN5 clones indicated by the absence of GFP are generated in wing discs. Ptc (red) protein level in CSN5 cells (arrow) detected by the monoclonal antibody Apa-1 is similar to that in wild-type cells. (c) ptc-lacZ staining (red) is comparable in wild-type and CSN5 cells that are marked by the absence of GFP (arrow). (d) En protein levels (red) are increased in the CSN5 cells (arrow) located in the A/P boundary as well as in the A and P compartments (arrowheads). Scale bars in all panels represent 50 μm.
Mentions: To examine whether the level of conditionally stable Ci155 is essential for distinct cell fates directed by graded Hh signalling, we assayed the activation of Hh-responsive, position-specific genes, such as ptc and dpp, whose expression represent high and low Hh signalling responses, respectively19. Interestingly, we found that the expression of dpp-lacZ is significantly repressed in CSN4 mutant cells (arrow in Fig. 4a), whereas the Ptc protein level and the expression of ptc-lacZ remain unaltered (arrows in Fig. 4b,c), indicating a critical role of the conditionally stable Ci155 for the Hh-mediated dpp expression. The expression of engrailed (en) is regulated by high Hh signalling activity in the A/P boundary52. We tested whether en expression in the A/P boundary is downregulated in CSN4 mutant because of Ci155 proteolysis. En protein level is instead enhanced not only at the A/P boundary but also in A and P compartments, in which en expression is independent from Hh signalling (Fig. 4d), suggesting that en expression is subjected to another layer of regulation by the CSN.

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