Avram Hershko

SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27.

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 ubiquitin system

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 cell-cycle regulatory protein Cks1 is required for SCF Skp2 -mediated ubiquitinylation of p27

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded in late G1 phase by the ubiquitin pathway, allowing CDK activity to drive cells into S phase. Ubiquitinylation of p27 requires its phosphorylation at Thr 187 (refs 3, 4) and subsequent recognition by S-phase kinase associated protein 2 (Skp2; refs 5–8), a member of the F-box family of proteins that associates with Skp1, Cul-1 and ROC1/Rbx1 to form an SCF ubiquitin ligase complex. However, in vitro ligation of p27 to ubiquitin could not be reconstituted by known purified components of the SCFSkp2 complex. Here we show that the missing factor is CDK subunit 1 (Cks1), which belongs to the highly conserved Suc1/Cks family of proteins that bind to some CDKs and phosphorylated proteins and are essential for cell-cycle progression. Human Cks1, but not other members of the family, reconstitutes ubiquitin ligation of p27 in a completely purified system, binds to Skp2 and greatly increases binding of T187-phosphorylated p27 to Skp2. Our results represent the first evidence that an SCF complex requires an accessory protein for activity as well as for binding to its phosphorylated substrate.

Degradation of Cdc25A by β-TrCP during S phase and in response to DNA damage

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that β-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of β-TrCP1 and β-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that β-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of β-TrCP expression results in radioresistant DNA synthesis in response to DNA damage—a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that β-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation.

Roles of ubiquitin-mediated proteolysis in cell cycle control.

Selective degradation of cyclins, inhibitors of cyclin-dependent kinases and anaphase inhibitors is responsible for several major cell cycle transitions. The degradation of these cell cycle regulators is controlled by the action of ubiquitin-protein-ligase complexes, which target the regulators for degradation by the 26S proteasome. Recent results indicate that two types of multisubunit ubiquitin ligase complexes, which are connected to the protein kinase regulatory network of the cell cycle in different ways, are responsible for the specific and programmed degradation of many cell cycle regulators.

Inverse relation between levels of p27Kip1 and of its ubiquitin ligase subunit Skp2 in colorectal carcinomas

Previous studies have shown that low levels of p27Kip1, an inhibitor of G1 cyclin–dependent kinases, are associated with high aggressiveness and poor prognosis in a variety of cancers. Decreased levels of p27 are caused, at least in part, by acceleration of the rate of its ubiquitin‐mediated degradation. In cultured cells and cell‐free biochemical systems, it has been shown that p27 is targeted for degradation by a ubiquitin ligase complex that contains Skp2 (S‐phase kinase‐associated protein 2) as the specific substrate‐recognizing and rate‐limiting subunit. This investigation was undertaken to examine the possible relation between levels of p27 and of its specific ubiquitin ligase subunit Skp2 in human cancers.

Occurrence of a polyubiquitin structure in ubiquitin-protein conjugates

In the ubiquitin-mediated pathway for the degradation of intracellular proteins, several molecules of ubiquitin are linked to the protein substrate by amide linkages. It was noted that the number of ubiquitin-protein conjugates and their apparent molecular size are higher than expected from the number of amino groups in the protein. When the amino groups of ubiquitin were blocked by reductive methylation, it was efficiently conjugated to lysozyme, but the higher-molecular-weight conjugates were not formed. This suggests that the higher-molecular-weight conjugates with native ubiquitin contain structures in which one molecule of ubiquitin is linked to an amino group of another molecule of ubiquitin. Methylated ubiquitin stimulated protein breakdown at about one half the rate obtained with native ubiquitin, and isolated conjugates of 125I-lysozyme with methylated ubiquitin were broken down by reticulocyte extracts. These findings indicate that the formation of polyubiquitin chains is not obligatory for protein breakdown, though it may accelerate the rate of this process.

The Ubiquitin Pathway for the Degradation of Intracellular Proteins

Publisher Summary This chapter discusses the mode of action and biological functions of the ubiquitin system. Ubiquitin is a small polypeptide present in apparently all eukaryotic cells. It was originally isolated in the characterization of polypeptide hormones of the calf thymus. The sequence of ubiquitin is identical in organisms as diverse as cattle, man, toad, and insects. The extraordinary conservation of ubiquitin in evolution indicates some important cellular functions. There are two processes in which ubiquitin is known to be involved—namely, histone modification and intracellular protein breakdown. In both cases, ubiquitin is linked to protein amino groups. Though the general structure of polyubiquitin genes has been conserved in evolution, there are species differences in the number of ubiquitin repeats, number of genomic loci, and nature of the aminoacid residue(s) following the carboxyl-terminus of the last ubiquitin repeat. For direct examination of the notion that ubiquitin-protein conjugates are intermediates in protein breakdown, it is necessary to demonstrate the existence of an enzyme system that preferentially degrades proteins conjugated to ubiquitin, but not unconjugated proteins. The main enzymatic steps in the formation of ubiquitin protein conjugates have been delineated and a broad outline of the major routes of the degradation of ubiquitin-conjugated proteins has been described in the chapter. Powerful tools are now available to study the biological functions of the ubiquitin system, including specific antibodies, a temperature-sensitive mutant, microinjection techniques, and cloned genes.

The ubiquitin pathway for protein degradation.

Cellular proteins are marked for selective degradation by their ligation to the polypeptide ubiquitin. Recent studies have revealed information on the mechanisms involved in the selection of proteins for ligation to ubiquitin and on the mode of degradation of ubiquitinated proteins. Much remains to be learned about the high selectivity of this degradation pathway. Recent evidence that the cell-cycle regulatory proteins, cyclins, are degraded by the ubiquitin pathway points the way to future challenges in ubiquitin research.

p31comet promotes disassembly of the mitotic checkpoint complex in an ATP-dependent process

Accurate segregation of chromosomes in mitosis is ensured by a surveillance mechanism called the mitotic (or spindle assembly) checkpoint. It prevents sister chromatid separation until all chromosomes are correctly attached to the mitotic spindle through their kinetochores. The checkpoint acts by inhibiting the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. The activity of APC/C is inhibited by a mitotic checkpoint complex (MCC), composed of the APC/C activator Cdc20 bound to the checkpoint proteins MAD2, BubR1, and Bub3. When all kinetochores acquire bipolar attachment the checkpoint is inactivated, but the mechanisms of checkpoint inactivation are not understood. We have previously observed that hydrolyzable ATP is required for exit from checkpoint-arrested state. In this investigation we examined the possibility that ATP hydrolysis in exit from checkpoint is linked to the action of the Mad2-binding protein p31comet in this process. It is known that p31comet prevents the formation of a Mad2 dimer that it thought to be important for turning on the mitotic checkpoint. This explains how p31comet blocks the activation of the checkpoint but not how it promotes its inactivation. Using extracts from checkpoint-arrested cells and MCC isolated from such extracts, we now show that p31comet causes the disassembly of MCC and that this process requires β,γ-hydrolyzable ATP. Although p31comet binds to Mad2, it promotes the dissociation of Cdc20 from BubR1 in MCC.