The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Ubiquitin-mediated degradation of regulatory proteins plays important roles in the control of numerous processes, including cell-cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation. In this review we discuss recent information on functions and mechanisms of the ubiquitin system. Since the selectivity of protein degradation is determined mainly at the stage of ligation to ubiquitin, special attention is focused on what we know, and would like to know, about the mode of action of ubiquitin-protein ligation systems and about signals in proteins recognized by these systems.

The Ubiquitin System

▪ Abstract The conjugation of ubiquitin to other cellular proteins regulates a broad range of eukaryotic cell functions. The high efficiency and exquisite selectivity of ubiquitination reactions reflect the properties of enzymes known as ubiquitin-protein ligases or E3s. An E3 recognizes its substrates based on the presence of a specific ubiquitination signal, and catalyzes the formation of an isopeptide bond between a substrate (or ubiquitin) lysine residue and the C terminus of ubiquitin. Although a great deal is known about the molecular basis of E3 specificity, much less is known about molecular mechanisms of catalysis by E3s. Recent findings reveal that all known E3s utilize one of just two catalytic domains—a HECT domain or a RING finger—and crystal structures have provided the first detailed views of an active site of each type. The new findings shed light on many aspects of E3 structure, function, and mechanism, but also emphasize that key features of E3 catalysis remain to be elucidated.

Mechanisms underlying ubiquitination.

The tumour-suppressor p53 is a short-lived protein that is maintained at low, often undetectable, levels in normal cells. Stabilization of the protein in response to an activating signal, such as DNA damage, results in a rapid rise in p53 levels and subsequent inhibition of cell growth. Tight regulation of p53 function is critical for normal cell growth and development, and one mechanism by which p53 function is controlled is through interaction with the Mdm2 protein. Mdm2 inhibits p53 cell-cycle arrest and apoptic functions and we show here that interaction with Mdm2 can also result in a large reduction in p53 protein levels through enhanced proteasome-dependent degradation. Endogenous levels of Mdm2 are sufficient to regulate p53 stability, and overexpression of Mdm2 can reduce the amount of endogenous p53. Because mdm2 is transcriptionally activated by p53, this degradative pathway may contribute to the maintenance of low p53 concentrations in normal cells. Furthermore, mechanisms regulating the Mdm2-induced degradation of p53 may play a role in controlling the extent and duration of the p53 response.

Regulation of p53 stability by Mdm2

Cisplatin, carboplatin and oxaliplatin are platinum-based drugs that are widely used in cancer chemotherapy. Platinum–DNA adducts, which are formed following uptake of the drug into the nucleus of cells, activate several cellular processes that mediate the cytotoxicity of these platinum drugs. This review focuses on recently discovered cellular pathways that are activated in response to cisplatin, including those involved in regulating drug uptake, the signalling of DNA damage, cell-cycle checkpoints and arrest, DNA repair and cell death. Such knowledge of the cellular processing of cisplatin adducts with DNA provides valuable clues for the rational design of more efficient platinum-based drugs as well as the development of new therapeutic strategies.

Cellular processing of platinum anticancer drugs.

A family of cyclin-dependent kinases (CDKs) cooperatively regulates mammalian cell cycle progression (for review, see Sherr 1993). During G1 phase, D-type cyclins (D1, D2, and D3) are synthesized and assemble with either CDK4 or CDK6 in response to growth factor stimulation, thereby generating active holoenzymes that help inactivate the growth-suppressive function of the retinoblastoma protein (Rb) through its phosphorylation (for review, see Weinberg 1995). Cyclin D holoenzyme complexes also titrate CDK inhibitors, such as p27Kip1 and p21Cip1, facilitating the activation of cyclin E-CDK2 and subsequent entry into the DNA synthetic phase of the cell cycle (for review, see Sherr and Roberts 1995).

Ras-mediated pathways are important for cyclin D1 induction and its assembly with CDKs. Overexpression of activated oncogenic Ras alleles, but not wild-type Ras, initiates DNA synthesis independently of growth factor stimulation (Feramisco et al. 1984). Conversely, microinjection of antibodies that inactivate Ras or introduction of certain dominant-negative Ras alleles can block S-phase entry induced by mitogens (Mulcahy et al. 1985; Mittnacht et al. 1997; Peeper et al. 1997). Both cyclin D1 expression and assembly require the sequential activities of Raf1, mitogen-activated protein kinase-kinases (MEK1 and MEK2), and the sustained activation of extracellular signal-regulated protein kinases (ERKs; Albanese et al. 1995; Lavoie et al. 1996; Winston et al. 1996; Aktas et al. 1997; Kerkhoff and Rapp 1997; Weber et al. 1997; Cheng et al. 1998).

In turn, cyclin D1 degradation is mediated by phosphorylation-triggered, ubiquitin-dependent proteolysis (Diehl et al. 1997). Polyubiquitination of protein substrates involves the sequential action of three distinct enzymes termed E1, E2 (UBC; ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase; Ciechanover 1994; King et al. 1996). Specificity of substrate recognition is dependent on several factors including E2 and E3 selectivity (King et al. 1996; Skowyra et al. 1997; Renny-Feldman et al. 1997), recognition motifs within the target proteins themselves (Glotzer et al. 1991), and, in some cases, a requirement for phosphorylation of specific residues within the substrate (Deshaies et al. 1995; Clurman et al. 1996; Lanker et al. 1996; Won et al. 1996). Ubiquitin-dependent degradation of cyclin D1 requires phosphorylation of a specific threonine residue (Thr-286) located near the protein carboxyl terminus, and this phosphorylation is not mediated by cyclin D-dependent kinases themselves (Diehl et al. 1997). Because the kinase that phosphorylates this residue has not yet been identified, it remains unclear whether cyclin D1 proteolysis, like its synthesis and assembly, is subject to mitogen regulation.

The subcellular distribution of D-type cyclins is also likely to be regulated by cell cycle-dependent events. Cyclin D1 accumulates in the nuclei of cells during G1 phase, but once DNA replication begins, it disappears from the nucleus (Baldin et al. 1993), despite the fact that its level of synthesis does not decrease markedly during S phase (Matsushime et al. 1991). The mechanisms that regulate the periodic subcellular redistribution of cyclin D1 during the cell division cycle have also not been defined.

We now demonstrate that glycogen synthase kinase-3β (GSK-3β) catalyzes the phosphorylation of cyclin D1 on Thr-286, thereby regulating cyclin D1 turnover in response to mitogenic signals. In turn, GSK-3β-mediated phosphorylation of cyclin D1 redirects the protein from the nucleus to the cytoplasm. Our results support a model in which phosphorylation of cyclin D1 on Thr-286 by GSK-3β links processes governing cyclin D1 subcellular localization with its proteasomal degradation.

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Glycogen synthase kinase-3β regulates cyclin D1 proteolysis and subcellular localization

The p53 tumour-suppressor protein exerts antiproliferative effects, including growth arrest and apoptosis, in response to various types of stress. The activity of p53 is abrogated by mutations that occur frequently in tumours, as well as by several viral and cellular proteins. The Mdm2 oncoprotein is a potent inhibitor of p53. Mdm2 binds the transcriptional activation domain of p53 and blocks its ability to regulate target genes and to exert antiproliferative effects. On the other hand, p53 activates the expression of the mdm2 gene in an autoregulatory feedback loop. The interval between p53 activation and consequent Mdm2 accumulation defines a time window during which p53 exerts its effects. We now report that Mdm2 also promotes the rapid degradation of p53 under conditions in which p53 is otherwise stabilized. This effect of Mdm2 requires binding of p53; moreover, a small domain of p53, encompassing the Mdm2-binding site, confers Mdm2-dependent detstabilization upon heterologous proteins. Raised amounts of Mdm2 strongly repress mutant p53 accumulation in tumour-derived cells. During recovery from DNA damage, maximal Mdm2 induction coincides with rapid p53 loss. We propose that the Mdm2-promoted degradation of p53 provides a new mechanism to ensure effective termination of the p53 signal.

Anat Kazaz

Papers

Mdm2 promotes the rapid degradation of p53

c-Abl neutralizes the inhibitory effect of Mdm2 on p53.