SCF(beta-TRCP) and phosphorylation dependent ubiquitinationof I kappa B alpha catalyzed by Ubc3 and Ubc4.
NF kappa B is an important transcriptional regulator of multiple pro-inflammatory genes. In non-stimulated cells NF kappa B is anchored in the cytoplasm via the inhibitory protein I kappa B alpha. Following exposure to diverse pro-inflammatory signals (e.g. TNF alpha, IL1, LPS) various signal transduction cascades are initiated converging on the I kappa B kinase (IKK). IKK phosphorylates I kappa B alpha on serines 32 and 36 signaling the inhibitory protein for ubiquitin-mediated degradation. The SCF beta-TRCP complex is the ubiquitin ligase responsible for mediating phosphorylation dependent ubiquitination of I kappa B alpha. Here we reconstitute phosphorylation dependent ubiquitination of I kappa B alpha using recombinant components. Our results suggest that the cullin specificity of the SCF complex may reflect its ability to associate with Rbx1. We demonstrate specific ubiquitination of I kappa B alpha by Ubc3 and Ubc4 in a phosphorylation and SCF beta-TRCP dependent manner and that both are capable of associating with the SCF beta-TRCP complex isolated from human cells. Finally, we show that Ubc4 is in excess to Ubc3 in THP.1 cells and 19 times more efficient in catalyzing the reaction, suggesting that Ubc4 is the preferentially used Ubc in this reaction in vivo. Our results also suggest that ubiquitin is transferred directly from the Ubc to phospho-I kappa B alpha in a SCF beta-TRCP dependent reaction. Oncogene (2000) 19, 3529 - 3536
Mitotix, Inc., One Kendall Square, Building 600, Suite 622, Cambridge, Massachusetts, MA 02139, USA.
P
P
Strack
M
M
Caligiuri
M
M
Pelletier
M
M
Boisclair
A
A
Theodoras
P
P
Beer-Romero
S
S
Glass
T
T
Parsons
R A
RA
Copeland
K R
KR
Auger
P
P
Benfield
L
L
Brizuela
M
M
Rolfe
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology; D044842:Cullin Proteins; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D008025:Ligases / Q000502:physiology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D009097:Multienzyme Complexes / Q000502:physiology; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D009363:Neoplasm Proteins / Q000502:physiology; D010453:Peptide Synthases / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000502:physiology; D044786:S-Phase Kinase-Associated Proteins; D044843:SKP Cullin F-Box Protein Ligases; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins
C471644:BTRC protein, human; C503645:CUL3 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D044786:S-Phase Kinase-Associated Proteins; D014452:Ubiquitins; D044844:beta-Transducin Repeat-Containing Proteins; D000072000:NF-KappaB Inhibitor alpha; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; C063109:ubiquitin-conjugating enzyme UBC4; D064173:Anaphase-Promoting Complex-Cyclosome; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D019204:GTP-Binding Proteins; D008025:Ligases; D010453:Peptide Synthases