The SCFβ-TRCP–ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro (original) (raw)
- Jeffrey T. Winston,
- Peter Strack,
- Peggy Beer-Romero,
- Claire Y. Chu,
- Stephen J. Elledge, and
- J. Wade Harper
- Verna & Marrs McLean Department of Biochemistry, Department of Molecular and Human Genetics, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030 USA; Mitotix, Inc., Cambridge, Massachusetts 02139 USA
Abstract
Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules such as cyclins, CKIs, p53, and IκBα. Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-κB inhibitor protein IκBα. Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of the E3–ubiquitin ligases with their substrates. One class of E3 ligases is defined by the recently described SCF complexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and theF-box protein Cdc4. These complexes recognize their substrates through modular F-box proteins in a phosphorylation-dependent manner. Here we describe a biochemical dissection of a novel mammalian SCF complex, SCFβ-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IκBα (residues 21–41) in a phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif in β-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. Endogenous IκBα–ubiquitin ligase activity cofractionates with SCFβ-TRCP. Furthermore, recombinant SCFβ-TRCPassembled in mammalian cells contains phospho-IκBα-specific ubiquitin ligase activity. Our results suggest that an SCFβ-TRCP complex functions in multiple transcriptional programs by activating the NF-κB pathway and inhibiting the β-catenin pathway.
