Ubiquitin-conjugation targets numerous cellular regulators for proteasome-mediated degradation. Thus, the identi®cation of ubiquitin ligases and their physiological substrates is crucially important, especially for those cases in which aberrant levels of regulatory proteins (e.g., b-catenin, p27) result from a deregulated ubiquitination pathway. In yeast, the proteolysis of several G1 regulators is controlled by ubiquitin ligases (or SCFs) formed by three subunits: Skp1, Cul A (Cdc53), and one of many F-box proteins. Speci®c

F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 aminoacid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) [1]. Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins [2] [3]. In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs. The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1). SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins. The need for high substrate specificity and the large number of known F-box proteins in yeast and worms [2] [4] suggest the existence of a large family of mammalian F-box proteins. Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel. Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein-protein interaction modules or no recognizable motifs. We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats, Fbls, and the remaining ones Fbxs. We have further characterized representative members of these three classes of F-box proteins.

The cell cycle has been the object of extensive studies for the in vitro. [21][22][23] It was subsequently cloned and found to be able past years. A complex network of molecular interactions has to block mammalian cells in the G1 phase of the cell cycle been identified. In particular, a class of cell cycle inhibitory prowhen overexpressed. 17,18 teins has been cloned and characterized but details of the molCkis have been suggested as the products of potential antiecular mechanism of their action have yet to be resolved. These oncogenes since their function is often missing in tumor cells. ation by these kinases is involved in the process of p27 proteop27 protein is expressed at higher levels in quiescent cells lysis.than in proliferating cells. [36][37][38][39] In addition, p27 abundance Keywords: cell cycle; cyclin dependent-kinase inhibitors; p27; tumor suppressors; ubiquitination increases in cells treated with cAMP, 40 tamoxifen, 41 lovastatin, 23 or rapamycin. 37 This increase is thought to be involved in the G1 block induced by these agents. Similarly, cells The eukaryotic cell division cycle is a highly ordered series undergoing differentiation have elevated levels of p27. 42-44 of events, regulated by a number of cyclin dependent kinases Interestingly, levels of p27 mRNA do not change in any of the (Cdks), each consisting of a regulatory cyclin subunit and a analyzed conditions. catalytic serine/threonine kinase subunit (for a review, see Ref.The contrast between oscillation of p27 protein abundance 1). Several cyclin-Cdk complexes have been identified and and the constant amounts of its mRNA observed at different found to be required at different stages of the cell cycle. There cell cycle phases prompted us to determine whether p27 halfare several distinct molecular mechanisms for controlling the life was chronologically regulated by alterations of protein activity of the different Cdks: regulated synthesis and destrucstability. 45 We found that the half-life of p27 is much longer tion of the cyclin regulatory subunit; post-translational modiin quiescent than in proliferating cells. This result led us to fication of the kinase subunit by highly specific kinases and investigate the mechanisms regulating p27 degradation. 45 phosphatases, and association/dissociation with a variety of Ubiquitin is a MW 7000 protein which is covalently bound inhibitory proteins (reviewed in Ref. 2).through an isopeptide bond to a target protein, forming a polyBased on sequence homology and specificity of interaction ubiquitin tree (see Refs 46 and 47 for a review). This reaction with Cdks, two families of inhibitors (ckis) have been identis catalyzed by the ubiquitinating enzymes E1, E2s and E3s. ified in mammalian cells (for a review, see Ref.3). The first E1, also called ubiquitin activating enzyme, is the first enzyme family includes p16 (also called Ink4A, Mts1, Cdkn2 and involved in protein ubiquitination. It forms a thioester bond Cdk4i 4,5 ), p15 (also called Ink4B, Mts2 5-8 ) p18 (also called between the C-terminus...