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SCFβ-TRCP and phosphorylation dependent ubiquitination of IκBα catalyzed by Ubc3 and Ubc4

Oncogene volume 19, pages 3529–3536 (2000)Cite this article

Abstract

NFκB is an important transcriptional regulator of multiple pro-inflammatory genes. In non-stimulated cells NFκB is anchored in the cytoplasm via the inhibitory protein IκBα. Following exposure to diverse pro-inflammatory signals (e.g. TNFα, IL1, LPS) various signal transduction cascades are initiated converging on the IκB kinase (IKK). IKK phosphorylates IκBα on serines 32 and 36 signaling the inhibitory protein for ubiquitin-mediated degradation. The SCFβ-TRCP complex is the ubiquitin ligase responsible for mediating phosphorylation dependent ubiquitination of IκBα. Here we reconstitute phosphorylation dependent ubiquitination of IκBα 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 IκBα by Ubc3 and Ubc4 in a phosphorylation and SCFβ-TRCP dependent manner and that both are capable of associating with the SCFβ-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-IκBα in a SCFβ-TRCP dependent reaction.

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Acknowledgements

We thank JW Harper and H Zhang for β-TRCP and Skp2 cDNAs respectively, William Lane of Harvard Microchemistry for protein sequencing and Susan Fish for IκBα/p50/p65 preparation.

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Author notes

  1. P Strack, T Parsons & M Rolfe

    Present address: Millennium Pharmaceuticals, Inc., 75 Sidney Street, Cambridge, 02139, MA, USA

  2. M Pelletier

    Present address: Biogen, 14 Cambridge Center, Cambridge, 02138, MA, USA

  3. L Brizuela

    Present address: Institute of Proteomics, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, MA, USA

  4. P Strack and M Caligiuri: P Strack and M Caligiuri contributed equally to this study

Authors and Affiliations

  1. Mitotix, Inc., One Kendall Square, Building 600, Suite 622, Cambridge, 02139, Massachusetts, MA, USA

    P Strack, M Caligiuri, M Pelletier, M Boisclair, A Theodoras, P Beer-Romero, S Glass, T Parsons, L Brizuela & M Rolfe

  2. DuPont Pharmaceuticals Company, Route 141 & Henry Clay Road, Wilmington, 19880, Delaware, DE, USA

    R A Copeland, K R Auger & P Benfield

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  1. P Strack
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Strack, P., Caligiuri, M., Pelletier, M. et al. SCFβ-TRCP and phosphorylation dependent ubiquitination of IκBα catalyzed by Ubc3 and Ubc4. Oncogene 19, 3529–3536 (2000). https://doi.org/10.1038/sj.onc.1203647

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