SCF beta-TRCP and phosphorylation dependent ubiquitination of I kappa B alpha catalyzed by Ubc3 and Ubc4

NF kappa B is an important transcriptional regulator of multiple pro-inflam matory genes. In non-stimulated cells NF kappa B is anchored in the cytopla sm via the inhibitory protein I kappa B alpha. Following exposure to divers e proinflammatory signals (e.g. TNF alpha, IL1, LPS) various signal transdu ction cascades are initiated converging on the I kappa B kinase (IKK). IKK phosphorylates I kappa B alpha on serines 32 and 36 signaling the inhibitor y protein for ubiquitin-mediated degradation. The SCFbeta-TRCP complex is t he ubiquitin ligase responsible for mediating phosphorylation dependent ubi quitination of I kappa-B alpha. Here we reconstitute phosphorylation depend ent ubiquitination of I kappa B alpha using recombinant components. Our res ults suggest that the cullin specificity of the SCF complex may reflect its ability to associate with Rbx1, We demonstrate specific ubiquitination of I kappa B alpha by Ubc3 and Ubc4 in a phosphorylation and SCFbeta-TRCP depe ndent manner and that both are capable of associating with the SCFbeta-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 react ion, suggesting that Ubc4 is the preferentially used Ubc in this reaction i n vivo. Our results also suggest that ubiquitin is transferred directly fro m the Ubc to phospho-I kappa B alpha in a SCFbeta-TRCP dependent reaction.