Degradation of the mammalian cyclin-dependent kinase (CDK) inhibitor p27 is required for the cellular transition from quiescence to the proliferative state. The ubiquitination and subsequent degradation of p27 depend on its phosphorylation by cyclin-CDK complexes. However, the ubiquitin-protein ligase necessary for p27 ubiquitination has not been identified. Here we show that the F-box protein SKP2 specifically recognizes p27 in a phosphorylation-dependent manner that is characteristic of an F-box-protein-substrate interaction. Furthermore, both in vivo and in vitro, SKP2 is a rate-limiting component of the machinery that ubiquitinates and degrades phosphorylated p27. Thus, p27 degradation is subject to dual control by the accumulation of both SKP2 and cyclins following mitogenic stimulation.

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitinproteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G 1 -enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G 1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G 1 extracts. Furthermore, another p27 mutant [p27(CK − )] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor.

Previous studies have indicated that the ATP-dependent 26S protease complex that degrades proteins conjugated to ubiquitin is formed by the assembly of three factors in an ATP-requiring process. We now identify one of the factors as the 20S "multicatalytic" protease, a complex of low molecular weight subunits widely distributed in eukaryotic cells. Comparison of the subunit compositions of purified 20S and 26S complexes indicates that the former is an integral part of the latter. By the use of detergent treatment to activate latent protease activity, we show that the 20S protease becomes incorporated into the 26S complex in the ATP-dependent assembly process. It thus seems that the 20S protease is the "catalytic core" of the 26S complex of the ubiquitin proteolytic pathway.

Processing of the p105 precursor to form the active subunit p50 of the NF-kB transcription factor is a unique case in which the ubiquitin system is involved in limited processing rather than in complete destruction of the target substrate. A glycine-rich region along with a downstream acidic domain have been demonstrated to be essential for processing. Here we demonstrate that following IkB kinase (IkK)-mediated phosphorylation, the C-terminal domain of p105 (residues 918±934) serves as a recognition motif for the SCF b-TrCP ubiquitin ligase. Expression of IkKb dramatically increases processing of wild-type p105, but not of p105-D918±934. Dominant-negative b-TrCP inhibits IkK-dependent processing. Furthermore, the ligase and wild-type p105 but not p105-D918±934 associate physically following phosphorylation. In vitro, SCF b-TrCP speci®cally conjugates and promotes processing of phosphorylated p105. Importantly, the TrCP recognition motif in p105 is different from that described for IkBs, b-catenin and human immunode®-ciency virus type 1 Vpu. Since p105-D918±934 is also conjugated and processed, it appears that p105 can be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. Keywords: IkB kinase (IkK)/NF-kB/p105/b-TrCP/ ubiquitin IntroductionThe NF-kB transcription factors play key roles in basic processes such as regulation of the immune and in¯am-matory responses, development and differentiation, malignant transformation and apoptosis (Baeuerle and Baltimore, 1996;Baldwin, 1996;Barnes and Karin, 1997;Ghosh et al., 1998;Foo and Nolan, 1999). The precursor molecules p105 and p100 undergo ubiquitinand proteasome-mediated limited processing to yield the respective active subunits p50 and p52 (Palombella et al., 1994;Orian et al., 1995;Betts and Nabel, 1996), which are derived from the N-terminal domain of the molecule. The C-terminal domain is degraded (Fan and Maniatis, 1991). These subunits typically heterodimerize with members of the rel family, such as p65, RelB or c-Rel, to generate the active transcription factor. In the resting cell, the heterodimer generates a ternary complex with a member of the IkB family of inhibitory proteins and is sequestered in the cytosol. Following stimulation, speci®c IkB kinases are activated (Mercurio et al., 1997;Woronicz et al., 1997;Zandi et al., 1997) and phosphorylate the protein on serine residues 32 and 36 (Brown et al., 1995). The phosphorylation leads to recognition of the molecule by the SCF b-TrCP ubiquitin ligase complex (see, for example, Yaron et al., 1998;Winston et al., 1999), polyubiquitylation on Lys21 and/or Lys22 and subsequent degradation by the 26S proteasome (Alkalay et al., 1995;Chen et al., 1995). Following degradation of IkBa, the heterodimer is translocated into the nucleus where it initiates speci®c transcription.The ubiquitin pathway is involved in the regulation of many basic cellular processes, such as cell cycle progression, differentiation and development, and the immune and in¯...

Significance The mitotic checkpoint system has an important role to ensure accurate segregation of chromosomes in mitosis. This system regulates the activity of the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) by the formation of a negatively acting Mitotic Checkpoint Complex (MCC). When the checkpoint is satisfied, MCC is disassembled, but the mechanisms of MCC disassembly are not well understood. We show here that the ATP-hydrolyzing enzyme Thyroid Receptor Interacting Protein 13 (TRIP13), along with the MCC-targeting protein p31 comet , promote the disassembly of the mitotic checkpoint complexes and the inactivation of the mitotic checkpoint. The results reveal an important molecular mechanism in the regulation of APC/C by the mitotic checkpoint.