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
Mesh Terms:
Amino Acid Sequence, Carrier Proteins, Catalysis, Cell Cycle Proteins, Cullin Proteins, DNA, Complementary, DNA-Binding Proteins, GTP-Binding Proteins, Humans, I-kappa B Kinase, I-kappa B Proteins, Ligases, Macromolecular Substances, Molecular Sequence Data, Monocytes, Multienzyme Complexes, NF-kappa B, Neoplasm Proteins, Peptide Synthases, Phosphorylation, Protein Binding, Protein Processing, Post-Translational, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, S-Phase Kinase-Associated Proteins, SKP Cullin F-Box Protein Ligases, Signal Transduction, Tumor Cells, Cultured, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases, Ubiquitins, beta-Transducin Repeat-Containing Proteins
Amino Acid Sequence, Carrier Proteins, Catalysis, Cell Cycle Proteins, Cullin Proteins, DNA, Complementary, DNA-Binding Proteins, GTP-Binding Proteins, Humans, I-kappa B Kinase, I-kappa B Proteins, Ligases, Macromolecular Substances, Molecular Sequence Data, Monocytes, Multienzyme Complexes, NF-kappa B, Neoplasm Proteins, Peptide Synthases, Phosphorylation, Protein Binding, Protein Processing, Post-Translational, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, S-Phase Kinase-Associated Proteins, SKP Cullin F-Box Protein Ligases, Signal Transduction, Tumor Cells, Cultured, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases, Ubiquitins, beta-Transducin Repeat-Containing Proteins
Oncogene
Date: Jul. 20, 2000
PubMed ID: 10918611
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