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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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CSN control of Ci155 levels upregulated by SlimbΔfbx or Uba1 depletion.(a) CSN5 clones are generated in wing discs expressing Myc-SlimbΔfbx in the dorsal compartment under the control of ap-GAL4. Ectopic expression of SlimbΔfbx increases the levels of Ci155 (red) in the dorsal compartment. Moreover, the upregulated Ci155 is maintained in CSN5 clones (arrow). (b) Under the control of ms1096-GAL4, the moderate level of ectopic Myc-SlimbΔfbx in the dorsal compartment causes similar accumulation of Ci155 (red) in both wild-type and CSN5 cells (arrowhead); the lower level of ectopic Myc-SlimbΔfbx in the ventral compartment also causes similar accumulation of Ci155 in both wild-type and CSN5 cells (arrow) (c) GFP-marked MARCM clones (green) expressing uba1-dsRNA by ms1096-GAL4 generated in wing discs show upregulation of Ci155 (red) levels compared with adjacent wild-type cells that are negative for GFP. (d) GFP-marked uba1-dsRNA MARCM clones that are also CSN5 show Ci155 levels (red) comparable with adjacent GFP-negative wild-type cells. Scale bars in all panels represent 50 μm.
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f6: CSN control of Ci155 levels upregulated by SlimbΔfbx or Uba1 depletion.(a) CSN5 clones are generated in wing discs expressing Myc-SlimbΔfbx in the dorsal compartment under the control of ap-GAL4. Ectopic expression of SlimbΔfbx increases the levels of Ci155 (red) in the dorsal compartment. Moreover, the upregulated Ci155 is maintained in CSN5 clones (arrow). (b) Under the control of ms1096-GAL4, the moderate level of ectopic Myc-SlimbΔfbx in the dorsal compartment causes similar accumulation of Ci155 (red) in both wild-type and CSN5 cells (arrowhead); the lower level of ectopic Myc-SlimbΔfbx in the ventral compartment also causes similar accumulation of Ci155 in both wild-type and CSN5 cells (arrow) (c) GFP-marked MARCM clones (green) expressing uba1-dsRNA by ms1096-GAL4 generated in wing discs show upregulation of Ci155 (red) levels compared with adjacent wild-type cells that are negative for GFP. (d) GFP-marked uba1-dsRNA MARCM clones that are also CSN5 show Ci155 levels (red) comparable with adjacent GFP-negative wild-type cells. Scale bars in all panels represent 50 μm.

Mentions: To further corroborate the SCF-dependent mechanism for CSN regulation, we tested whether Ci155 has to be incorporated into the SCF complex for the CSN to confer its conditional stability. To do this, the F-box-truncated SlimbΔfbx protein that can competitively bind Ci155 but cannot be incorporated into SCF was ectopically expressed by ap-GAL4 in wing discs. Higher levels of Ci155 detected in wing discs suggest that SlimbΔfbx-sequestered Ci155 is free from SCF-mediated proteolysis. Unlike the conditionally stable Ci155 upregulated by DN-GSK3β or DN-DBT expression, the upregulated Ci155 levels caused by SlimbΔfbx expression is insensitive to the absence of CSN activity in CSN5 cells (arrow, Fig. 6a). Thus, Ci155 that binds the substrate receptor Slimb, but fails to form SCFSlimb-Ci155 intermediates, is insufficient to produce conditionally stable Ci155.


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)

CSN control of Ci155 levels upregulated by SlimbΔfbx or Uba1 depletion.(a) CSN5 clones are generated in wing discs expressing Myc-SlimbΔfbx in the dorsal compartment under the control of ap-GAL4. Ectopic expression of SlimbΔfbx increases the levels of Ci155 (red) in the dorsal compartment. Moreover, the upregulated Ci155 is maintained in CSN5 clones (arrow). (b) Under the control of ms1096-GAL4, the moderate level of ectopic Myc-SlimbΔfbx in the dorsal compartment causes similar accumulation of Ci155 (red) in both wild-type and CSN5 cells (arrowhead); the lower level of ectopic Myc-SlimbΔfbx in the ventral compartment also causes similar accumulation of Ci155 in both wild-type and CSN5 cells (arrow) (c) GFP-marked MARCM clones (green) expressing uba1-dsRNA by ms1096-GAL4 generated in wing discs show upregulation of Ci155 (red) levels compared with adjacent wild-type cells that are negative for GFP. (d) GFP-marked uba1-dsRNA MARCM clones that are also CSN5 show Ci155 levels (red) comparable with adjacent GFP-negative wild-type cells. Scale bars in all panels represent 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: CSN control of Ci155 levels upregulated by SlimbΔfbx or Uba1 depletion.(a) CSN5 clones are generated in wing discs expressing Myc-SlimbΔfbx in the dorsal compartment under the control of ap-GAL4. Ectopic expression of SlimbΔfbx increases the levels of Ci155 (red) in the dorsal compartment. Moreover, the upregulated Ci155 is maintained in CSN5 clones (arrow). (b) Under the control of ms1096-GAL4, the moderate level of ectopic Myc-SlimbΔfbx in the dorsal compartment causes similar accumulation of Ci155 (red) in both wild-type and CSN5 cells (arrowhead); the lower level of ectopic Myc-SlimbΔfbx in the ventral compartment also causes similar accumulation of Ci155 in both wild-type and CSN5 cells (arrow) (c) GFP-marked MARCM clones (green) expressing uba1-dsRNA by ms1096-GAL4 generated in wing discs show upregulation of Ci155 (red) levels compared with adjacent wild-type cells that are negative for GFP. (d) GFP-marked uba1-dsRNA MARCM clones that are also CSN5 show Ci155 levels (red) comparable with adjacent GFP-negative wild-type cells. Scale bars in all panels represent 50 μm.
Mentions: To further corroborate the SCF-dependent mechanism for CSN regulation, we tested whether Ci155 has to be incorporated into the SCF complex for the CSN to confer its conditional stability. To do this, the F-box-truncated SlimbΔfbx protein that can competitively bind Ci155 but cannot be incorporated into SCF was ectopically expressed by ap-GAL4 in wing discs. Higher levels of Ci155 detected in wing discs suggest that SlimbΔfbx-sequestered Ci155 is free from SCF-mediated proteolysis. Unlike the conditionally stable Ci155 upregulated by DN-GSK3β or DN-DBT expression, the upregulated Ci155 levels caused by SlimbΔfbx expression is insensitive to the absence of CSN activity in CSN5 cells (arrow, Fig. 6a). Thus, Ci155 that binds the substrate receptor Slimb, but fails to form SCFSlimb-Ci155 intermediates, is insufficient to produce conditionally stable Ci155.

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