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PMID: 18625727 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

A unique protection signal in Cubitus interruptus prevents its complete proteasomal degradation.

Molecular and cellular biology ·Vol. 28 ·No. 18 ·2008-09-00 ·Pages 5555-68

Wang Y, Price MA

Abstract

The limited proteolysis of Cubitus interruptus (Ci), the transcription factor for the developmentally and medically important Hedgehog (Hh) signaling pathway, triggers a critical switch between transcriptional repressor and activator forms. Ci repressor is formed when the C terminus of full-length Ci is degraded by the ubiquitin-proteasome pathway, an unusual reaction since the proteasome typically completely degrades its substrates. We show that several regions of Ci are required for generation of the repressor form: the zinc finger DNA binding domain, a single lysine residue (K750) near the degradation end point, and a 163-amino-acid region at the C terminus. Unlike other proteins that are partially degraded by the proteasome, dimerization is not a key feature of Ci processing. Using a pulse-chase assay in cultured Drosophila cells, we distinguish between regions required for initiation of degradation and those required for the protection of the Ci N terminus from degradation. We present a model whereby the zinc finger region and K750 together form a unique protection signal that prevents the complete degradation of Ci by the proteasome.

MeSH Terms
Amino Acid Sequence Animals Cells, Cultured DNA-Binding Proteins/chemistry,genetics,metabolism Dimerization Drosophila Proteins/chemistry,genetics,metabolism Drosophila melanogaster/physiology Hedgehog Proteins/genetics,metabolism Humans Lysine/metabolism Molecular Sequence Data Proteasome Endopeptidase Complex/metabolism Protein Conformation Protein Folding Protein Structure, Tertiary RNA Interference Repressor Proteins/genetics,metabolism Signal Transduction/physiology Transcription Factors/chemistry,genetics,metabolism Ubiquitin/metabolism Zinc Fingers
Chemicals
DNA-Binding Proteins Drosophila Proteins Hedgehog Proteins Repressor Proteins Transcription Factors Ubiquitin ci protein, Drosophila Proteasome Endopeptidase Complex Lysine
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Wang Yifei
Department of Biomedical Sciences, University of Sheffield, Sheffield S10 2TN, United Kingdom.
Price Mary Ann
References (51)
51 references, click to expand
  1. RNA interference of gene expression (RNAi) in cultured Drosophila cells.
    Sci STKE. 2001 Aug 14;2001(95):pl1 PMID: 11752672
  2. Crystal structure of the Aequorea victoria green fluorescent protein.
    Science. 1996 Sep 6;273(5280):1392-5 PMID: 8703075
  3. Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain.
    Genes Dev. 2001 Apr 15;15(8):1007-20 PMID: 11316794
  4. Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1.
    Cell. 2002 Mar 22;108(6):823-35 PMID: 11955435
  5. Costal2, a novel kinesin-related protein in the Hedgehog signaling pathway.
    Cell. 1997 Jul 25;90(2):235-45 PMID: 9244298
  6. Cotranslational dimerization of the Rel homology domain of NF-kappaB1 generates p50-p105 heterodimers and is required for effective p50 production.
    EMBO J. 2000 Sep 1;19(17):4712-22 PMID: 10970863
  7. Targeting the Hedgehog pathway in cancer.
    Nat Rev Drug Discov. 2006 Dec;5(12):1026-33 PMID: 17139287
  8. 4E binding proteins inhibit the translation factor eIF4E without folded structure.
    Biochemistry. 1998 Jan 6;37(1):9-15 PMID: 9453748
  9. Hedgehog-regulated Costal2-kinase complexes control phosphorylation and proteolytic processing of Cubitus interruptus.
    Dev Cell. 2005 Feb;8(2):267-78 PMID: 15691767
  10. A conserved processing mechanism regulates the activity of transcription factors Cubitus interruptus and NF-kappaB.
    Nat Struct Mol Biol. 2005 Dec;12(12):1045-53 PMID: 16299518
  11. A ubiquitin-based tagging system for controlled modulation of protein stability.
    Nat Biotechnol. 2000 Dec;18(12):1298-302 PMID: 11101811
  12. Regulation of the Hedgehog and Wingless signalling pathways by the F-box/WD40-repeat protein Slimb.
    Nature. 1998 Jan 29;391(6666):493-6 PMID: 9461217
  13. Dorsal-ventral patterning of the spinal cord requires Gli3 transcriptional repressor activity.
    Genes Dev. 2002 Nov 15;16(22):2865-78 PMID: 12435629
  14. Cotranslational biogenesis of NF-kappaB p50 by the 26S proteasome.
    Cell. 1998 Mar 20;92(6):819-28 PMID: 9529257
  15. Roadkill attenuates Hedgehog responses through degradation of Cubitus interruptus.
    Development. 2006 May;133(10):2001-10 PMID: 16651542
  16. Regulation of Ci-SCFSlimb binding, Ci proteolysis, and hedgehog pathway activity by Ci phosphorylation.
    Dev Cell. 2007 Oct;13(4):481-95 PMID: 17925225
  17. Ubiquitin as a degradation signal.
    EMBO J. 1992 Feb;11(2):497-505 PMID: 1311250
  18. Proteolysis of cubitus interruptus in Drosophila requires phosphorylation by protein kinase A.
    Development. 1999 Oct;126(19):4331-9 PMID: 10477300
  19. Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability.
    J Mol Biol. 1993 Sep 20;233(2):305-12 PMID: 8377205
  20. Ubiquitin-metallothionein fusion protein expression in yeast. A genetic approach for analysis of ubiquitin functions.
    J Biol Chem. 1988 Nov 5;263(31):16364-71 PMID: 2846542
  21. Processing of the Drosophila hedgehog signaling effector Ci-155 to the repressor Ci-75 is mediated by direct binding to the SCF component Slimb.
    Curr Biol. 2006 Jan 10;16(1):110-6 PMID: 16386907
  22. Multiple Cos2/Ci interactions regulate Ci subcellular localization through microtubule dependent and independent mechanisms.
    Dev Biol. 2004 Apr 15;268(2):493-505 PMID: 15063184
  23. Phosphorylation by double-time/CKIepsilon and CKIalpha targets cubitus interruptus for Slimb/beta-TRCP-mediated proteolytic processing.
    Dev Cell. 2005 Dec;9(6):819-30 PMID: 16326393
  24. Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb.
    Cell. 2000 Feb 18;100(4):423-34 PMID: 10693759
  25. Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development.
    Genes Dev. 2002 Sep 15;16(18):2403-14 PMID: 12231629
  26. A novel protein-processing domain in Gli2 and Gli3 differentially blocks complete protein degradation by the proteasome.
    J Biol Chem. 2007 Apr 13;282(15):10846-52 PMID: 17283082
  27. Anteroposterior patterning in the limb and digit specification: contribution of mouse genetics.
    Dev Dyn. 2006 Sep;235(9):2337-52 PMID: 16894622
  28. Multisite protein kinase A and glycogen synthase kinase 3beta phosphorylation leads to Gli3 ubiquitination by SCFbetaTrCP.
    Mol Cell Biol. 2006 Jun;26(11):4316-26 PMID: 16705181
  29. Evidence for the direct involvement of {beta}TrCP in Gli3 protein processing.
    Proc Natl Acad Sci U S A. 2006 Jan 3;103(1):33-8 PMID: 16371461
  30. Drosophila Roc1a encodes a RING-H2 protein with a unique function in processing the Hh signal transducer Ci by the SCF E3 ubiquitin ligase.
    Dev Cell. 2002 Jun;2(6):757-70 PMID: 12062088
  31. Sonic hedgehog signaling regulates Gli2 transcriptional activity by suppressing its processing and degradation.
    Mol Cell Biol. 2006 May;26(9):3365-77 PMID: 16611981
  32. Interactions with Costal2 and suppressor of fused regulate nuclear translocation and activity of cubitus interruptus.
    Genes Dev. 2000 Nov 15;14(22):2893-905 PMID: 11090136
  33. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.
    Science. 2002 May 3;296(5569):913-6 PMID: 11988576
  34. CG15031/PPYR1 is an intrinsically unstructured protein that interacts with protein phosphatase Y.
    Arch Biochem Biophys. 2006 Jul 1;451(1):59-67 PMID: 16631104
  35. Cooperative requirement of the Gli proteins in neurogenesis.
    Development. 2005 Jul;132(14):3267-79 PMID: 15983404
  36. Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone.
    Cell. 2001 Nov 30;107(5):667-77 PMID: 11733065
  37. Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells.
    Science. 2003 Mar 28;299(5615):2039-45 PMID: 12663920
  38. Nuclear trafficking of Cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression.
    Cell. 1999 Aug 6;98(3):305-16 PMID: 10458606
  39. The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B.
    Cell. 1994 Sep 9;78(5):773-85 PMID: 8087845
  40. A glycine-rich region in NF-kappaB p105 functions as a processing signal for the generation of the p50 subunit.
    Mol Cell Biol. 1996 May;16(5):2248-54 PMID: 8628291
  41. Protein kinase A directly regulates the activity and proteolysis of cubitus interruptus.
    Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2349-54 PMID: 9482888
  42. Analysis of compositionally biased regions in sequence databases.
    Methods Enzymol. 1996;266:554-71 PMID: 8743706
  43. Hedgehog signaling in animal development: paradigms and principles.
    Genes Dev. 2001 Dec 1;15(23):3059-87 PMID: 11731473
  44. Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor.
    Cell. 1997 Jun 27;89(7):1043-53 PMID: 9215627
  45. Structural motifs involved in ubiquitin-mediated processing of the NF-kappaB precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain.
    Mol Cell Biol. 1999 May;19(5):3664-73 PMID: 10207090
  46. A hedgehog-responsive region in the Drosophila wing disc is defined by debra-mediated ubiquitination and lysosomal degradation of Ci.
    Dev Cell. 2003 Jun;4(6):917-28 PMID: 12791275
  47. Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.
    Cell. 2000 Sep 1;102(5):577-86 PMID: 11007476
  48. Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator.
    Nature. 1998 Dec 24-31;396(6713):749-53 PMID: 9874371
  49. Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus.
    Cell. 1999 Mar 19;96(6):819-31 PMID: 10102270
  50. An amphioxus Gli gene reveals conservation of midline patterning and the evolution of hedgehog signalling diversity in chordates.
    PLoS One. 2007 Sep 12;2(9):e864 PMID: 17848995
  51. A hedgehog-induced BTB protein modulates hedgehog signaling by degrading Ci/Gli transcription factor.
    Dev Cell. 2006 Jun;10(6):719-29 PMID: 16740475
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
1098-5549
DOI
10.1128/MCB.00524-08
Published
2008-09-00
Epub
2008-00-14
Pages
5555-68
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC2546926
Subset
IM
Grants
Cancer Research UK · C14503/A48 · United Kingdom
Wellcome Trust · United Kingdom
External Links
PubMed source DOI: 10.1128/MCB.00524-08 PMC Full Text View journal details
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