Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U.
beta-TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor subunit of the SCF(beta-TrCP) ubiquitin ligase (E3). Surprisingly, although its two recognized substrates, IkappaB(alpha) and beta-catenin, are present in the cytoplasm, we have found that E3RS is located predominantly in the nucleus. Here we report the isolation of the ... major E3RS-associated protein, hnRNP-U, an abundant nuclear phosphoprotein. This protein occupies E3RS in a specific and stoichiometric manner, stabilizes the E3 component, and is likely responsible for its nuclear localization. hnRNP-U binding was abolished by competition with a pIkappaB(alpha) peptide, or by a specific point mutation in the E3RS WD region, indicating an E3-substrate-type interaction. However, unlike pI(kappa)Balpha, which is targeted by SCF(beta-TrCP) for degradation, the E3-bound hnRNP-U is stable and is, therefore, a pseudosubstrate. Consequently, hnRNP-U engages a highly neddylated active SCF(beta-TrCP), which dissociates in the presence of a high-affinity substrate, resulting in ubiquitination of the latter. Our study points to a novel regulatory mechanism, which secures the localization, stability, substrate binding threshold, and efficacy of a specific protein-ubiquitin ligase.
Mesh Terms:
Active Transport, Cell Nucleus, Animals, CHO Cells, Cricetinae, Cricetulus, DNA-Binding Proteins, GTP-Binding Proteins, Hela Cells, Heterogeneous-Nuclear Ribonucleoprotein U, Heterogeneous-Nuclear Ribonucleoproteins, Humans, I-kappa B Proteins, Macromolecular Substances, Mice, Models, Biological, Nuclear Proteins, Peptide Fragments, Peptide Synthases, Point Mutation, Protein Binding, Protein Interaction Mapping, Protein Processing, Post-Translational, Ribonucleoproteins, SKP Cullin F-Box Protein Ligases, Subcellular Fractions, Substrate Specificity, Ubiquitin, beta-Transducin Repeat-Containing Proteins
Active Transport, Cell Nucleus, Animals, CHO Cells, Cricetinae, Cricetulus, DNA-Binding Proteins, GTP-Binding Proteins, Hela Cells, Heterogeneous-Nuclear Ribonucleoprotein U, Heterogeneous-Nuclear Ribonucleoproteins, Humans, I-kappa B Proteins, Macromolecular Substances, Mice, Models, Biological, Nuclear Proteins, Peptide Fragments, Peptide Synthases, Point Mutation, Protein Binding, Protein Interaction Mapping, Protein Processing, Post-Translational, Ribonucleoproteins, SKP Cullin F-Box Protein Ligases, Subcellular Fractions, Substrate Specificity, Ubiquitin, beta-Transducin Repeat-Containing Proteins
Genes Dev.
Date: Feb. 15, 2002
PubMed ID: 11850407
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