E3 ligases confer specificity to ubiquitination by recognizing target substrates and mediating transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to substrate. The activity of most E3s is specified by a RING domain, which binds to an E2 approximately ubiquitin thioester and activates discharge of its ubiquitin cargo. E2-E3 complexes can either monoubiquitinate a substrate lysine or synthesize polyubiquitin chains assembled via different lysine residues of ubiquitin. These modifications can have diverse effects on the substrate, ranging from proteasome-dependent proteolysis to modulation of protein function, structure, assembly, and/or localization. Not surprisingly, RING E3-mediated ubiquitination can be regulated in a number of ways. RING-based E3s are specified by over 600 human genes, surpassing the 518 protein kinase genes. Accordingly, RING E3s have been linked to the control of many cellular processes and to multiple human diseases. Despite their critical importance, our knowledge of the physiological partners, biological functions, substrates, and mechanism of action for most RING E3s remains at a rudimentary stage.

Multisubunit UBIQUITIN LIGASES (E3s) that are assembled on a CULLIN scaffold were first reported seven years ago 1,2. The discovery of the archetypical cullin-RING ubiquitin ligase-SCF Cdc4-benefited from a strong foundation of genetic studies on cell division in Saccharomyces cerevisiae and Caenorhabditis elegans. The model established by SCF can now be extended to a superfamily of CULLIN-RING LIGASES (CRLS) that are found throughout eukaryotes. Together, these enzymes regulate a dazzling array of cellular and organismal processes-from glucose sensing and DNA replication to limb patterning and circadian rhythms. Although there is a great diversity of CRLs in terms of their composition and function, we propose that these enzymes can be characterized by a set of general principles that will apply to most members of the superfamily. In this review, we summarize what has been learned about SCF and other CRLs over the past seven years and, from this, we extract the key features that typify these enzymes. We also highlight the murky areas in which our understanding remains far from clear. Cullin-RING ligases are modular The cullin family. Human cells express seven different cullins (CUL1, 2, 3, 4A, 4B, 5 and 7) that each nucleate a multisubunit ubiquitin ligase (FIG. 1). In addition, at least two other proteins (the APC2 subunit of the ANAPHASE-PROMOTING COMPLEX/CYCLOSOME (APC/C) and the p53 cytoplasmic anchor protein PARC) contain a 'cullin-homology domain' 3-5. Although APC2 and PARC have ubiquitin-ligase activity, they are clearly distinct from the other cullins and are not considered here. The archetypal CRLs, which contain CUL1, are named SCF ubiquitin ligases, whereas the other CRLs have distinct subunit compositions and have been referred to by various names (TABLE 1). Cullin-RING ligases have an extended, rigid architecture. Much of what is known and inferred about the architecture of CRLs comes from protein-protein interaction studies and sequence comparisons that have been interpreted in light of three-dimensional (3D) X-ray crystal structures (for a summary of the solved structures for CRL complexes and related proteins, see online supplementary information S1 (table)). CUL1 and presumably all other cullins have a curved, yet rigid, N-terminal stalk that is comprised of three repeats of a five-helix bundle (cullin repeat (CR)1-3) and is linked to a C-terminal globular domain 6 (FIG. 2). The SKP1 adaptor binds to the N-terminal CR1 region, whereas the zinc-binding RING-H2-DOMAIN proteinwhich is known as either ROC1, RBX1 or HRT1 (REFS 7-10; and is referred to here as the 'RING' subunit)-binds 100 Å away from SKP1 and interdigitates itself with the C-terminal globular domain. SKP1 recruits substrate receptors and the RING subunit recruits the ubiquitin-conjugating enzyme (E2) to form the active ligase complex. The rigidity of the N-terminal stalk of CUL1 might juxtapose the E2 and the substrate to favour ubiquitin transfer, because a mutation that

The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex containing Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degradation. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compound, proteintargeting chimeric molecule 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the IB␣ phosphopeptide that is recognized by the F-box protein ␤-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCF ␤-TRCP , ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.

The 26S proteasome mediates degradation of ubiquitin-conjugated proteins. Although ubiquitin is recycled from proteasome substrates, the molecular basis of deubiquitination at the proteasome and its relation to substrate degradation remain unknown. The Rpn11 subunit of the proteasome lid subcomplex contains a highly conserved Jab1/MPN domain-associated metalloisopeptidase (JAMM) motif-EX(n)HXHX(10)D. Mutation of the predicted active-site histidines to alanine (rpn11AXA) was lethal and stabilized ubiquitin pathway substrates in yeast. Rpn11(AXA) mutant proteasomes assembled normally but failed to either deubiquitinate or degrade ubiquitinated Sic1 in vitro. Our findings reveal an unexpected coupling between substrate deubiquitination and degradation and suggest a unifying rationale for the presence of the lid in eukaryotic proteasomes.

Protein degradation is deployed to modulate the steady-state abundance of proteins and to switch cellular regulatory circuits from one state to another by abrupt elimination of control proteins. In eukaryotes, the bulk of the protein degradation that occurs in the cytoplasm and nucleus is carried out by the 26S proteasome. In turn, most proteins are thought to be targeted to the 26S proteasome by covalent attachment of a multiubiquitin chain. Ubiquitination of proteins requires a multienzyme system. A key component of ubiquitination pathways, the ubiquitin ligase, controls both the specificity and timing of substrate ubiquitination. This review is focused on a conserved ubiquitin ligase complex known as SCF that plays a key role in marking a variety of regulatory proteins for destruction by the 26S proteasome.

Depletion of a subset of 70K stress proteins in yeast mutants shows that they are involved in the post-translational import of precursor polypeptides into both mitochondria and the lumen of the endoplasmic reticulum. The identification of such a basic function may explain the remarkable evolutionary conservation of the gene family encoding these proteins.

Oscillations in the activity of cyclin-dependent kinases (CDKs) promote progression through the eukaryotic cell cycle. This review examines how proteolysis regulates CDK activity-by degrading CDK activators or inhibitors-and also how proteolysis may directly trigger the transition from metaphase to anaphase. Proteolysis during the cell cycle is mediated by two distinct ubiquitin-conjugation pathways. One pathway, requiring CDC34, initiates DNA replication by degrading a CDK inhibitor. The second pathway, involving a large protein complex called the anaphase-promoting complex or cyclosome, initiates chromosome segregation and exit from mitosis by degrading anaphase inhibitors and mitotic cyclins. Proteolysis therefore drives cell cycle progression not only by regulating CDK activity, but by directly influencing chromosome and spindle dynamics.

In S. cerevisiae, the G1/S transition requires Cdc4p, Cdc34p, Cdc53p, Skp1p, and the Cln/Cdc28p cyclin-dependent kinase (Cdk). These proteins are thought to promote the proteolytic inactivation of the S-phase Cdk inhibitor Sic1p. We show here that Cdc4p, Cdc53p, and Skp1p assemble into a ubiquitin ligase complex named SCFCdc4p. When mixed together, SCFCdc4p subunits, E1 enzyme, the E2 enzyme Cdc34p, and ubiquitin are sufficient to reconstitute ubiquitination of Cdk-phosphorylated Sic1p. Phosphorylated Sic1p substrate is specifically targeted for ubiquitination by binding to a Cdc4p/Skp1p subcomplex. Taken together, these data illuminate the molecular basis for the G1/S transition in budding yeast and suggest a general mechanism for phosphorylation-targeted ubiquitination in eukaryotes.