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.
Davis
Matti
M
The Lautenberg Center for Immunology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.
Hatzubai
Ada
A
Andersen
Jens S
JS
Ben-Shushan
Etti
E
Fisher
Gregory Zvi
GZ
Yaron
Avraham
A
Bauskin
Asne
A
Mercurio
Frank
F
Mann
Matthias
M
Ben-Neriah
Yinon
Y
D021581:Active Transport, Cell Nucleus; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D020672:I-kappa B Proteins; D046911:Macromolecular Substances; D051379:Mice; D008954:Models, Biological; D000072000:NF-KappaB Inhibitor alpha; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010453:Peptide Synthases / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012261:Ribonucleoproteins / Q000502:physiology*; D044843:SKP Cullin F-Box Protein Ligases; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins
C471644:BTRC protein, human; C471643:Btrc protein, mouse; D004268:DNA-Binding Proteins; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D020672:I-kappa B Proteins; D046911:Macromolecular Substances; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D009687:Nuclear Proteins; D010446:Peptide Fragments; D012261:Ribonucleoproteins; D025801:Ubiquitin; D044844:beta-Transducin Repeat-Containing Proteins; D000072000:NF-KappaB Inhibitor alpha; D044843:SKP Cullin F-Box Protein Ligases; D019204:GTP-Binding Proteins; D010453:Peptide Synthases