A conserved processing mechanism regulates the activity of transcription factors Cubitus interruptus and NF-κB

The proteasome degrades some proteins, such as transcription factors Cubitus interruptus (Ci) and NF-κB, to generate biologically active protein fragments. Here we have identified and characterized the signals in the substrate proteins that cause this processing. The minimum signal consists of a simple sequence preceding a tightly folded domain in the direction of proteasome movement. The strength of the processing signal depends primarily on the complexity of the simple sequence rather than on amino acid identity, the resistance of the folded domain to unraveling by the proteasome and the spacing between the simple sequence and folded domain. We show that two unrelated transcription factors, Ci and NF-κB, use this mechanism to undergo partial degradation by the proteasome in vivo. These findings suggest that the mechanism is conserved evolutionarily and that processing signals may be widespread in regulatory proteins.

[1]  M. Nakafuku,et al.  Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and Gli3 as primary mediators of Shh signaling. , 1999, Development.

[2]  A. Matouschek,et al.  An unstructured initiation site is required for efficient proteasome-mediated degradation , 2004, Nature Structural &Molecular Biology.

[3]  T. Endo,et al.  Latent membrane perturbation activity of a mitochondrial precursor protein is exposed by unfolding. , 1988, The EMBO journal.

[4]  S. Jentsch,et al.  Activation of a Membrane-Bound Transcription Factor by Regulated Ubiquitin/Proteasome-Dependent Processing , 2000, Cell.

[5]  B. Wang,et al.  Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus , 2002, Nature.

[6]  W. Greene,et al.  Cotranslational dimerization of the Rel homology domain of NF‐κB1 generates p50–p105 heterodimers and is required for effective p50 production , 2000, The EMBO journal.

[7]  T. Kornberg,et al.  Proteolysis That Is Inhibited by Hedgehog Targets Cubitus interruptus Protein to the Nucleus and Converts It to a Repressor , 1997, Cell.

[8]  T. Maniatis,et al.  Generation of p50 subunit of NF-kB by processing of p105 through an ATP-dependent pathway , 1991, Nature.

[9]  S. Jentsch,et al.  Taking a bite: proteasomal protein processing , 2002, Nature Cell Biology.

[10]  A. Ciechanover,et al.  The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. , 2002, Physiological reviews.

[11]  A. Ciechanover,et al.  SCFβ‐TrCP ubiquitin ligase‐mediated processing of NF‐κB p 105 requires phosphorylation of its C‐terminus by IκB kinase , 2000 .

[12]  S. Nagata,et al.  The Fused Protein Kinase Regulates Hedgehog-stimulated Transcriptional Activation in Drosophila Schneider 2 Cells* , 2001, The Journal of Biological Chemistry.

[13]  J. Tobias,et al.  Universality and structure of the N-end rule. , 1989, The Journal of biological chemistry.

[14]  A. Varshavsky,et al.  The degradation signal in a short-lived protein , 1989, Cell.

[15]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[16]  John M. Walker,et al.  The Proteomics Protocols Handbook , 2005, Humana Press.

[17]  Robert E. Cohen,et al.  Proteasomes and their kin: proteases in the machine age , 2004, Nature Reviews Molecular Cell Biology.

[18]  A. Matouschek,et al.  ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. , 2001, Molecular cell.

[19]  Tsutomu Nakamura,et al.  Systematic circular permutation of an entire protein reveals essential folding elements , 2000, Nature Structural Biology.

[20]  Lawrence Lum,et al.  The Hedgehog Response Network: Sensors, Switches, and Routers , 2004, Science.

[21]  R D Appel,et al.  Protein identification and analysis tools in the ExPASy server. , 1999, Methods in molecular biology.

[22]  T. Maniatis,et al.  Two different virus‐inducible elements are required for human beta‐interferon gene regulation. , 1989, The EMBO journal.

[23]  G. Struhl,et al.  Regulation of the Hedgehog and Wingless signalling pathways by the F-box/WD40-repeat protein Slimb , 1998, Nature.

[24]  J. Wootton,et al.  Analysis of compositionally biased regions in sequence databases. , 1996, Methods in enzymology.

[25]  D. Kalderon,et al.  Proteolysis of the Hedgehog Signaling Effector Cubitus interruptus Requires Phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1 , 2002, Cell.

[26]  S. Jentsch,et al.  Mobilization of Processed, Membrane-Tethered SPT23 Transcription Factor by CDC48UFD1/NPL4, a Ubiquitin-Selective Chaperone , 2001, Cell.

[27]  W. Greene,et al.  Cotranslational Biogenesis of NF-κB p50 by the 26S Proteasome , 1998, Cell.

[28]  Philip A Beachy,et al.  Nuclear Trafficking of Cubitus interruptus in the Transcriptional Regulation of Hedgehog Target Gene Expression , 1999, Cell.

[29]  T. Maniatis,et al.  NF-κB p105 Processing via the Ubiquitin-Proteasome Pathway* , 1998, The Journal of Biological Chemistry.

[30]  Thomas Henkel,et al.  Intramolecular masking of the nuclear location signal and dimerization domain in the precursor for the p50 NF-κB subunit , 1992, Cell.

[31]  M. Karin,et al.  Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. , 2000, Annual review of immunology.

[32]  Tom Maniatis,et al.  The ubiquitinproteasome pathway is required for processing the NF-κB1 precursor protein and the activation of NF-κB , 1994, Cell.

[33]  Mingsheng Zhang,et al.  Repeat Sequence of Epstein-Barr Virus-encoded Nuclear Antigen 1 Protein Interrupts Proteasome Substrate Processing* , 2004, Journal of Biological Chemistry.

[34]  W Keilholz,et al.  Cleavage motifs of the yeast 20S proteasome beta subunits deduced from digests of enolase 1. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Cheng-Ting Chien,et al.  Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development. , 2002, Genes & development.

[36]  A. Varshavsky,et al.  In vivo half-life of a protein is a function of its amino-terminal residue. , 1986, Science.

[37]  S. Ghosh,et al.  A glycine-rich region in NF-kappaB p105 functions as a processing signal for the generation of the p50 subunit , 1996, Molecular and cellular biology.

[38]  Stefan Imreh,et al.  A minimal glycine-alanine repeat prevents the interaction of ubiquitinated IκBα with the proteasome: a new mechanism for selective inhibition of proteolysis , 1998, Nature Medicine.

[39]  A. Ciechanover,et al.  Structural Motifs Involved in Ubiquitin-Mediated Processing of the NF-κB Precursor p105: Roles of the Glycine-Rich Region and a Downstream Ubiquitination Domain , 1999, Molecular and Cellular Biology.

[40]  Erica S. Johnson,et al.  Methotrexate Inhibits Proteolysis of Dihydrofolate Reductase by the N-end Rule Pathway (*) , 1995, The Journal of Biological Chemistry.

[41]  Robert T Sauer,et al.  Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[42]  C. Larsen,et al.  Protein Translocation Channels in the Proteasome and Other Proteases , 1997, Cell.

[43]  Konrad Basler,et al.  Hedgehog Controls Limb Development by Regulating the Activities of Distinct Transcriptional Activator and Repressor Forms of Cubitus interruptus , 1999, Cell.

[44]  A. Israël,et al.  The precursor of NF-κB p50 has IκB-like functions , 1992, Cell.

[45]  W. Greene,et al.  The generation of nfkb2 p52: mechanism and efficiency , 1999, Oncogene.

[46]  R. Goodman,et al.  Protein kinase A directly regulates the activity and proteolysis of cubitus interruptus. , 1998, Proceedings of the National Academy of Sciences of the United States of America.