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Genes & Dev. 16 (4): 439-451

Copyright © 2002 by Cold Spring Harbor Laboratory Press.

Vol. 16, No. 4, pp. 439-451, February 15, 2002

Pseudosubstrate regulation of the SCFbeta -TrCP ubiquitin ligase by hnRNP-U

Matti Davis,1 Ada Hatzubai,1 Jens S. Andersen,2 Etti Ben-Shushan,1 Gregory Zvi Fisher,1 Avraham Yaron,1 Asne Bauskin,3 Frank Mercurio,4 Matthias Mann,2 and Yinon Ben-Neriah1,5

1 The Lautenberg Center for Immunology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel; 2 Protein Interaction Laboratory, University of Southern Denmark, DK-5230 Odense M, Denmark; 3 Centre for Immunology, St. Vincent's Hospital and University of New South Wales, Sydney 2010, Australia; 4 Signal Research Division, Celgene Corp., San Diego, California 92121, USA

beta -TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor subunit of the SCFbeta -TrCP ubiquitin ligase (E3). Surprisingly, although its two recognized substrates, Ikappa Balpha 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 pIkappa Balpha peptide, or by a specific point mutation in the E3RS WD region, indicating an E3-substrate-type interaction. However, unlike pIkappa Balpha , which is targeted by SCFbeta -TrCP for degradation, the E3-bound hnRNP-U is stable and is, therefore, a pseudosubstrate. Consequently, hnRNP-U engages a highly neddylated active SCFbeta -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.

[Key Words: beta -TrCP/E3RS; hnRNP-U; Ikappa Balpha ; nuclear transport; ubiquitin ligase]

5 Corresponding author.

GENES & DEVELOPMENT 16:439-451 © 2002 by Cold Spring Harbor Laboratory Press  ISSN 0890-9369/02 $5.00

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