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

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

Correspondence to: M Caligiuri, Mitotix, Inc., One Kendall Square, Building 600, Suite 622, Cambridge, Massachusetts, MA 02139, USA

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

^bCurrent address: Biogen, 14 Cambridge Center, Cambridge, MA 02138, USA

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

^dP Strack and M Caligiuri contributed equally to this study

NFB is an important transcriptional regulator of multiple pro-inflammatory genes. In non-stimulated cells NFB is anchored in the cytoplasm via the inhibitory protein IB. Following exposure to diverse pro-inflammatory signals (e.g. TNF, IL1, LPS) various signal transduction cascades are initiated converging on the IB kinase (IKK). IKK phosphorylates IB on serines 32 and 36 signaling the inhibitory protein for ubiquitin-mediated degradation. The SCF^-TRCP complex is the ubiquitin ligase responsible for mediating phosphorylation dependent ubiquitination of IB. Here we reconstitute phosphorylation dependent ubiquitination of IB 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 IB by Ubc3 and Ubc4 in a phosphorylation and SCF^-TRCP dependent manner and that both are capable of associating with the SCF^-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-IB in a SCF^-TRCP dependent reaction. Oncogene (2000) 19, 3529-3536

IB; NFB; SCF^-TRCP; cullin; ubiquitin