Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules such as cyclins, CKIs, p53, and IB␣. Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-B inhibitor protein IB␣. 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 the F-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, SCF ␤-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IB␣ (residues 21-41) in a phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif in ␤-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. Endogenous IB␣-ubiquitin ligase activity cofractionates with SCF ␤-TRCP . Furthermore, recombinant SCF ␤-TRCP assembled in mammalian cells contains phospho-IB␣-specific ubiquitin ligase activity. Our results suggest that an SCF ␤-TRCP complex functions in multiple transcriptional programs by activating the NF-B pathway and inhibiting the ␤-catenin pathway.

Ubiquitin-mediated destruction of regulatory proteins is a frequent means of controlling progression through signaling pathways [1]. F-box proteins [2] are components of modular E3 ubiquitin protein ligases called SCFs, which function in phosphorylation-dependent ubiquitination ([3] [4] [5], reviewed in [6] [7]). F-box proteins contain a carboxy-terminal domain that interacts with substrates and a 42-48 amino-acid F-box motif which binds to the protein Skp1 [2] [3] [4]. Skp1 binding links the F-box protein with a core ubiquitin ligase composed of the proteins Cdc53/Cul1, Rbx1 (also called Hrt1 and Roc1) and the E2 ubiquitin-conjugating enzyme Cdc34 [8] [9] [10] [11]. The genomes of the budding yeast Saccharomyces cerevisiae and the nematode worm Caenorhabditis elegans contain, respectively, 16 and more than 60 F-box proteins [2] [7]; in S. cerevisiae, the F-box proteins Cdc4, Grr1 and Met30 target cyclin-dependent kinase inhibitors, G1 cyclins and transcriptional regulators for ubiquitination ([3] [4] [5] [8] [10], reviewed in [6] [7]). Only four mammalian F-box proteins (Cyclin F, Skp1, beta-TRCP and NFB42) have been identified so far [2] [12]. Here, we report the identification of a family of 33 novel mammalian F-box proteins. The large number of these proteins in mammals suggests that the SCF system controls a correspondingly large number of regulatory pathways in vertebrates. Four of these proteins contain a novel conserved motif, the F-box-associated (FBA) domain, which may represent a new protein-protein interaction motif. The identification of these genes will help uncover pathways controlled by ubiquitin-mediated proteolysis in mammals.

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Stimulation of quiescent Balb/c 3T3 fibroblasts into S phase requires the synergistic action of platelet-derived growth factor (PDGF) and progression factors found in platelet-poor plasma (PPP). Traverse of the G 1 /S phase boundary and the initiation of DNA replication require functional cyclin E-cyclin-dependent kinase (Cdk) 2 and cyclin A-Cdk2 complexes; however, the mechanisms by which PDGF and PPP regulate Cdk2 activation are not known. Density-arrested fibroblasts contain low levels of cyclins E and A, and high levels of the Cdk inhibitor p27