I. INTRODUCTION AND OVERVIEW OF UBIQUITIN-MEDIATED PROTEOLYSIS

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Like all macromolecular components of an organism, the proteome is in a dynamic state of synthesis and degradation. During proteolysis, the peptide bonds that link amino acids are hydrolyzed, and free amino acids are released. The process is carried out by a diverse group of enzymes termed proteases. During proteolysis, the energy invested in the synthesis of the peptide bond is released. Distinct proteolytic mechanisms serve different physiological requirements and allow the organism to accommodate to changing environmental and pathophysiological conditions.

One should distinguish between destruction of "foreign" and "self" proteins. Foreign dietary proteins are degraded "outside" the body, in the lumen of the gastrointestinal tract. To avoid triggering an immune response, the epithelial lining of the digestive tract does not allow absorption of intact proteins into the body, and they are degraded to nonantigenic amino acids that are absorbed by the body and serve as building blocks for synthesis of its own proteins. Self proteins can also be classified into extracellular and intracellular; the two groups of proteins are degraded via two distinct mechanisms. Extracellular proteins such as the blood coagulation factors, immunoglobulins, albumin, cargo-carrying proteins [such as the core protein of the low-density lipoprotein (LDL)], and peptide hormones (such as insulin) are taken up via pinocytosis or receptor-mediated endocytosis. They are then carried via a series of vesicles (endosomes) that fuse with primary lysosomes where they are degraded. During this process, the extracellular proteins are never exposed to the intracellular environment (the cytosol) and remain "extracellular" (topologically) throughout. Degradation of proteins in lysosomes is not specific, and all engulfed proteins exposed to lysosomal proteases are degraded at approximately the same rate.

Several observations lead to the prediction that degradation of intracellular proteins must be carried out by completely distinct mechanisms. The process is highly specific, and different proteins have half-life times that vary from a few minutes (e.g., the tumor suppressor p53) to several days (e.g., the muscle proteins actin and myosin) and up to a few years (crystalline). Furthermore, inhibitors of lysosomal degradation, weak bases such as chloroquine, for example, do not have any effect on degradation of intracellular proteins under basal metabolic conditions. These compounds titrate the normal acidic intralysosomal pH and bring it to a point that does not allow activity of the lysosomal proteases. These findings led to the hypothesis that degradation of intracellular proteins must be carried out by a nonlysosomal proteolytic system that is endowed with a high degree of specificity toward its substrates. Also, the fact that the proteolytic enzymes and their substrates reside in the same cellular compartment predicted a requirement for tightly regulated machinery that uses metabolic energy for control. The discovery of the ubiquitin-proteasome proteolytic pathway has resolved these enigmas.

Degradation of a protein via the ubiquitin-proteasome pathway involves two discrete and successive steps: 1) tagging of the substrate by covalent attachment of multiple ubiquitin molecules and 2) degradation of the tagged protein by the 26S proteasome complex with release of free and reusable ubiquitin. This last process is mediated by ubiquitin recycling enzymes [deubiquitinating enzymes (DUBs); see sect. VIII]. Conjugation of ubiquitin, a highly evolutionarily conserved 76-residue polypeptide, to the protein substrate proceeds via a three-step cascade mechanism (Fig. 1). Initially, the ubiquitin-activating enzyme E1 activates ubiquitin in an ATP-requiring reaction to generate a high-energy thiol ester intermediate, E1-S~ubiquitin. One of several E2 enzymes [ubiquitin-carrier proteins or ubiquitin-conjugating enzymes (UBCs)] transfers the activated ubiquitin moiety from E1, via an additional high-energy thiol ester intermediate, E2-S~ubiquitin, to the substrate that is specifically bound to a member of the ubiquitin-protein ligase family, E3. There are a number of different classes of E3 enzymes (see sect. II and Figs. 1 and 2). For the HECT (homologous to the E6-AP COOH terminus) domain E3s, the ubiquitin is transferred once again from the E2 enzyme to an active site Cys residue on the E3, to generate a third high-energy thiol ester intermediate, ubiquitin~ S-E3, before its transfer to the ligase-bound substrate. RING finger-containing E3s catalyze direct transfer of the activated ubiquitin moiety to the E3-bound substrate. E3s catalyze the last step in the conjugation process: covalent attachment of ubiquitin to the substrate. The ubiquitin molecule is generally transferred to an -NH₂ group of an internal Lys residue in the substrate to generate a covalent isopeptide bond. In some cases, however, ubiquitin is conjugated to the NH₂-terminal amino group of the substrate. By successively adding activated ubiquitin moieties to internal Lys residues on the previously conjugated ubiquitin molecule, a polyubiquitin chain is synthesized (Fig. 1). The chain is recognized by the downstream 26S proteasome complex (see sect. VI). Thus E3s play a key role in the ubiquitin-mediated proteolytic cascade since they serve as the specific recognition factors of the system.

View larger version (30K): [in this window] [in a new window]   Fig. 1. The ubiquitin proteolytic pathway. 1: Activation of ubiquitin by the ubiquitin-activating enzyme E1, a ubiquitin-carrier protein, E2 (ubiquitin-conjugating enzyme, UBC), and ATP. The product of this reaction is a high-energy E2~ubiquitin thiol ester intermediate. 2: Binding of the protein substrate, via a defined recognition motif, to a specific ubiquitin-protein ligase, E3. 3: Multiple (n) cycles of conjugation of ubiquitin to the target substrate and synthesis of a polyubiquitin chain. E2 transfers the first activated ubiquitin moiety directly to the E3-bound substrate, and in following cycles, to previously conjugated ubiquitin moiety. Direct transfer of activated ubiquitin from E2 to the E3-bound substrate occurs in substrates targeted by RING finger E3s. 3': As in 3, but the activated ubiquitin moiety is transferred from E2 to a high-energy thiol intermediate on E3, before its conjugation to the E3-bound substrate or to the previously conjugated ubiquitin moiety. This reaction is catalyzed by HECT domain E3s. 4: Degradation of the ubiquitin-tagged substrate by the 26S proteasome complex with release of short peptides. 5: Ubiquitin is recycled via the activity of deubiquitinating enzymes (DUBs).

View larger version (34K): [in this window] [in a new window]   Fig. 2. The hierarchical structure of the ubiquitin system. The simplified view of the hierarchical structure of the ubiquitin conjugation machinery is that a single E1 (red) activates ubiquitin for all conjugation reactions. E1 interacts with all E2s (yellow). Typically, each E2, exemplified by E2E, interacts with several E3s (E3E, E3F, and E3G; blue). Each E3 targets several substrates (green). The interactions of the conjugating enzymes among themselves and with many of the target substrates may differ from this "classical" cascade. For example, a single E3 can interact with 2 distinct E2s (E3E, for example, interacts with E2D and E2E). Also, a single E3 (E3B, for example) can have several distinct recognition sites targeting different classes of substrates (SD,E,F, SG,H,I, SJ,K,L, and SM,N,O). Finally, some substrates can be targeted by different E3s, recognizing different motifs (SM,N,O and SP,Q,R). It should be noted that not all recognition cascades have been demonstrated experimentally, and some are still putative. Also, because of the complexity of the scheme and space constraints, only a few examples could be brought, and we could not assign distinct enzymes to defined substrates. Thus the ligase (E3C) that ubiquitinates lysozyme (SM,N,O... ) is not necessarily the same ligase that ubiquitinates, in the cell, p105 (SP,Q,R... ), as one may conclude from the scheme (see sect. III for details).

The proteasome is a large, 26S, multicatalytic protease that degrades polyubiquitinated proteins to small peptides. It is composed of two subcomplexes: a 20S core particle (CP) that carries the catalytic activity and a regulatory 19S regulatory particle (RP). The 20S CP is a barrel-shaped structure composed of four stacked rings, two identical outer -rings and two identical inner -rings (Fig. 5). The eukaryotic - and -rings are composed each of seven distinct subunits, giving the 20S complex the general structure of _1-71-71-71-7. The catalytic sites are localized to some of the -subunits. Each extremity of the 20S barrel can be capped by a 19S RP. One important function of the 19S RP is to recognize ubiquitinated proteins and other potential substrates of the proteasome. A ubiquitin-binding subunit of the 19S RP has indeed been identified; however, its importance and mode of action have not been discerned. A second function of the 19S RP is to open an orifice in the -ring that will allow entry of the substrate into the proteolytic chamber. Also, because a folded protein would not be able to fit through the narrow proteasomal channel, it is assumed that the 19S particle unfolds substrates and inserts them into the 20S CP. Both the channel opening function and the unfolding of the substrate require metabolic energy, and indeed, the 19S RP contains six different ATPase subunits. After degradation of the substrate, short peptides derived from the substrate are released, as well as reusable ubiquitin (for general schemes, see Figs. 1 and 5).

A major unresolved question is, How does the system achieve its high specificity and selectivity? Why are certain proteins extremely stable in the cell, whereas others are extremely short-lived? Why are certain proteins degraded only at a particular time point during the cell cycle or only after specific extracellular stimuli, yet they are stable under most other conditions? It appears that specificity of the ubiquitin system is determined by two distinct and unrelated groups of proteins: 1) E3s and 2) ancillary proteins. First, within the ubiquitin system, substrates must be specifically recognized by an appropriate E3 as a prerequisite to their ubiquitination. In most cases however, substrates are not recognized in a constitutive manner and are not recognized directly by the E3. In some instances, the E3 must "be switched on" by undergoing posttranslational modification to yield an active form that recognizes the substrate (Fig. 3). In many other cases, it is the substrate that must undergo a certain change that renders it susceptible for recognition (Fig. 3). The stability of additional proteins depends on association with ancillary proteins such as molecular chaperones that act as recognition elements in trans and serve as a link to the appropriate ligase. Others, such as certain transcription factors, have to dissociate from the specific DNA sequence to which they bind in order to be recognized by the system. Stability of yet other proteins depends on oligomerization. Thus, in addition to the E3s themselves, modifying enzymes (such as kinases), ancillary proteins, or DNA sequences to which substrates bind, also play an important role in the recognition process.

Ubiquitin-mediated proteolysis of a variety of cellular proteins plays an important role in many basic cellular processes. Among these are regulation of cell cycle and division, differentiation and development, involvement in the cellular response to stress and extracellular effectors, morphogenesis of neuronal networks, modulation of cell surface receptors, ion channels and the secretory pathway, DNA repair, transcriptional regulation, transcriptional silencing, long-term memory, circadian rhythms, regulation of the immune and inflammatory responses, and biogenesis of organelles. The list of cellular proteins that are targeted by ubiquitin is growing rapidly. Among them are cell cycle regulators such as cyclins, cyclin-dependent kinase inhibitors, and proteins involved in sister chromatid separation, tumor suppressors, as well as transcriptional activators and their inhibitors. Cell surface receptors and endoplasmic reticulum (ER) proteins are also targeted by the system. Finally, mutated and denatured/misfolded proteins are recognized specifically and are removed efficiently. In this capacity, the system is a key player in the cellular quality control and defense mechanisms.

With the numerous substrate proteins targeted and the multitude of processes involved, it is not surprising that aberrations in the ubiquitin system have been implicated in the pathogenesis of many inherited and acquired human pathologies. In some cases, the linkage between the proteolytic system and the resulting pathology is direct, whereas in others it is less obvious. It is impossible to cover in a single review, or even in a comprehensive monograph, all that we currently know of the ubiquitin system, its enzymatic components, ancillary proteins, modes of action, mechanisms of substrate recognition, modes of regulation, and most of all, the numerous processes it is involved in or the consequences of aberrations in its activity. We therefore decided to bring to the reader an updated view of the general components of the system, the conjugating enzymes and the proteasome, explain their mode of action, and highlight, via specific examples, several of the processes in which the system is involved. We will end by describing in greater detail the recently evolving field of the involvement of this system in pathogenesis of human diseases. For more details about the ubiquitin system, the reader is referred to the many specific reviews written on it recently. On ubiquitin ligases, see References 82, 198, 207, 333, 350, 449, 468; on proteasomes, see References 37, 124, 458; and for recent monographs on the ubiquitin system, see References 170 and 343.

	II. THE UBIQUITIN CONJUGATING MACHINERY: E1, E2, AND E3

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E1 activates ubiquitin, via a two-step intramolecular and ATP-dependent reaction, to generate a high-energy E1-thiol-ester~ubiquitin intermediate (Fig. 1). The activated ubiquitin moiety is then transferred to E2 (163). The yeast genome encodes for a single ubiquitin-activating enzyme, UBA1. Inactivation of this gene is lethal (302). The protein contains a nuclear localization signal (149, 302). The enzyme is phosphorylated, a modification that was suggested to play a role in its cell cycle-dependent nuclear localization (422). However, the physiological relevance of this modification has not been further substantiated.

E2s catalyze covalent attachment of ubiquitin to target proteins, or, when acting along with HECT domain E3s, transfer of the activated ubiquitin moiety to a high-energy E3~ubiquitin intermediate. They all share an active-site ubiquitin-binding Cys residue and are distinguished by the presence of a UBC domain required for binding of distinct E3s. In a few cases, they can also interact with the substrate (217). The physiological significance of this interaction is not known. Eleven ubiquitin conjugating enzymes (Ubc1-8, 10, 11, 13) have been identified in the yeast genome. Two additional enzymes, Ubc9 and Ubc12, are members of the UBC family, although they conjugate the ubiquitin-like proteins Smt3 and Rub1, respectively, and not ubiquitin (see sect. IX). Many more E2s have been described in higher organisms. Typically, each E2 interacts with a number of ligases, thus being involved in targeting numerous substrates (for hierarchy of the ubiquitin pathway, see Fig. 2 and below).

Yeast Ubc2/Rad6 acts along with Ubr1/E3 to target N-end rule substrates (478), but this pair can also ubiquitinate many other substrates targeted by other motifs (132, 366). Another E2, Ubc3/Cdc34, acts along with the different permutations of the SCF complex (see below), targeting mostly phosphorylated substrates (82). Ubc4 and Ubc5 are involved in targeting the bulk of short-lived/abnormal/misfolded proteins (403). Ubc1 functionally overlaps with Ubc4 and Ubc5 but appears to act primarily in the early stages of growth after germination of spores (404). These three enzymes constitute a family essential for cell growth and viability.

Ubc6 is an ER membrane-anchored E2, while Ubc7 is a membrane-associated E2 (34, 416) recruited to the ER membrane by Cue1 (35). They are involved in degradation of proteins from within the ER (23, 353), but surprisingly, also of soluble proteins such as the transcription factor MAT2 (58). Ubc8 (357) is involved in catabolite inactivation of fructose-1,6-bisphosphatase (394); however, inactivation of the gene does not lead to any detectable phenotype. Ubc10 is involved in peroxisome biogenesis (470), but the underlying mechanism(s) is still obscure. Ubc11 acts along with the cyclosome/anaphase promoting complex (APC), which is the E3 that targets cell cycle regulators (333). Ubc13, similar to Ubc2/Rad6, is a member of the error-free postreplication repair pathway in Saccharomyces cerevisiae (48). It forms a specific heteromeric complex with Mms2p, a complex that is required for assembly of polyubiquitin chains linked through Lys⁶³. Mms2p is a ubiquitin-conjugating enzyme variant (UEV) protein that resembles E2s but lacks the defining E2 active site Cys residue. A deletion mutant ubc13 yeast strain is ultraviolet (UV) sensitive, and it is possible that the noncanonical, novel polyubiquitin chains signal in DNA repair (179).

The number and variety of different E2s in mammalian species is much greater. For example, BRUCE (BIR repeat-containing ubiquitin-conjugating enzyme) is a colossal (528 kDa) E2 isolated from mice (154). It is membrane associated and localizes to the Golgi apparatus. Remarkably, in addition to being an active E2, BRUCE bears a baculovirus inhibitor of apoptosis repeat (BIR) motif, which has been identified exclusively in apoptosis inhibitors of the IAP-related protein family. The BIR motifs of IAP proteins are indispensable for their anti-cell death activity and function probably through protein-protein interaction. This suggests that BRUCE may combine properties of IAP-like proteins and E2, and it is possible that the two activities are related. Interestingly, certain ubiquitin ligases, E3s, are IAPSs and have been shown to modulate apoptosis (483a). They are RING-finger ligases (see below) that have an autoubiquitinating activity. They are degraded after apoptotic stimuli and target pro-apoptotic proteins for degradation in the nonapoptosing cell.

The terminology of the different E2 enzymes is confusing, and similar terms given to yeast and mammalian enzymes do not reflect functional or structural homology. Thus human UBCH1 (215) is not the human homolog of yeast Ubc1, but rather the homolog of yeast Ubc2/Rad6. Similarly, yeast Ubc11 is the homolog of clam E2-C, Xenopus UBCx, and human E2-H10, all four partners with the APC E3 complex (440, 441). Yeast ER Ubc6 and Ubc7 are not the homologs of human UbcH6 and UbcH7 that are soluble enzymes involved in targeting of soluble proteins in the cytosol (321).

Sites of ubiquitination vary among different substrates. For most proteins, the first ubiquitin moiety is conjugated to an -NH₂ group of an internal Lys residue. For at least three substrates, the transcription factor MyoD (45), the latent membrane protein 1 (LMP1) of the Epstein-Barr virus (EBV) (18), and the E7 oncoprotein of the human papillomavirus (HPV) (365), it has been shown that the first ubiquitin moiety is attached to the free -NH₂ terminus of the protein. In either class, additional ubiquitin moieties are then conjugated to an -NH₂ group of an internal Lys residue in the previously conjugated ubiquitin. As for the location of the Lys residues that are tagged in the target protein, no rules can be formulated. For signal-induced degradation of IB, it has been shown that the polyubiquitin chain is conjugated specifically to either Lys-21 or Lys-22 (391). The same residues can also be sumoylated, possibly protecting the inhibitor from ubiquitination and subsequent degradation (83). For p53, multiple Lys residues that reside in a limited region in the COOH-terminal domain (K372, K373, K381, and K382) are targeted by the E3 enzyme Mdm2 (317); substitution of all of them decreased ubiquitination significantly. Interestingly, the same Lys residues are also targeted by acetyl groups, and it appears that acetylation and ubiquitination play opposite roles in governing the stability of the tumor suppressor. In contrast, for cyclin B (227) and the -chain of the T cell receptor (TCR) (184), mutagenic analyses indicate that there is no specificity as for the Lys residue targeted, and no single residue serves as a specific anchor for the polyubiquitin chain.

Another hurdle that E3s must clear is their association with upstream and downstream elements of the system. Such links are important to ensure efficiency and processivity of the proteolytic process. It has been shown that RING finger E3s bind, via their RING finger domain, to their partner E2s (198, 207, 449, 500). Another important interaction could be between the ligases and the proteasome. One can assume that the binding site on the proteasome for the polyubiquitin chains attached to target substrates may be sufficient, and proteins, once conjugated, detach from the conjugation machinery and attach to the proteasome. However, a more efficient machinery could be one in which the conjugation machinery itself associates with the proteasome. It has been recently shown that two human homologs of the yeast ubiquitin-like protein Dsk2, hPLIC-1 and hPLIC-2, associate with both proteasomes and ubiquitin ligase complexes. Overexpression of hPLIC proteins interferes with cellular degradation of two unrelated ubiquitin-proteasome substrates, p53 and IB, but not of a ubiquitin-independent substrate, ODC (236). The mechanism of inhibition has not been deciphered, and it is not known whether additional proteins are also involved in linking the conjugation and proteolysis machineries. A similar mechanism has been described recently in S. cerevisiae, in which an adaptor protein, Cic1, links the proteasome with components of an SCF E3 complex (199). A linkage between ligases and E2s on one hand and proteasomes on the other hand may provide an explanation for the rapid and efficient signal- and cell cycle-induced degradation of key regulatory cellular proteins that possibly occurs in a processive manner.

Because of lack of significant homology among the ligases initially identified, it was thought that they belong to a large number of protein families. Recently, it has become clear that even though E3s are heterogeneous, they can nevertheless be classified into two major groups: HECT domain- and RING finger-containing E3s and several minor groups.

HECT domain proteins harbor a 350-amino acid residue sequence homologous to the COOH-terminal domain of the prototypical member of the family E6-AP (E6-associated protein) (190, 191). This domain contains a conserved Cys residue to which the activated ubiquitin moiety is transferred from E2 (389). The NH₂-terminal domain, which varies among the different HECT domain proteins, is probably involved in specific substrate recognition. The first enzyme described in this family, E6-AP, targets p53 for rapid degradation in the presence of the HPV oncoprotein E6 (388). In the absence of viral ancillary protein, E6-AP targets for degradation other native cellular proteins, such as Blk, a member of the Src family of kinases (146, 323). Mutations in E6-AP have been implicated in the pathogenesis of Angelman syndrome, a severe form of inherited mental and motor retardation (229). Under these conditions, the yet to be identified target substrate that accumulates is probably toxic to the developing brain cells.

NEDD4 is a HECT domain E3 that targets the kidney epithelial Na⁺ channel (1, 376), while both basal and induced degradation of the yeast uracil permease, FUR4, is mediated by the Npi1/Rsp5 HECT domain ligase (117). The transforming growth factor (TGF)- family of proteins regulates many different biological processes, including cell growth and differentiation. TGF- ligands signal across cell membrane through type I and type II serine/threonine-kinase receptors, which in turn activate the SMAD signaling pathway. Inside the cell, a receptor-regulated group of SMADs is phosphorylated by the receptor kinases. In this way, receptors for different factors are able to pass on specific signals along the pathway. Thus receptors for bone morphogenetic protein (BMP) signal via SMADs 1, 5, and 8, whereas receptors for activin and TGF- signal via SMADs 2 and 3. The SMAD proteins serve therefore as key signaling effectors for the TGF- superfamily of growth factors. Phosphorylation of SMADs leads to their association with Smad4, the "common partner" SMAD, which results in translocation of the complex into the nucleus and initiation of specific transcriptional activity. The activity of SMADs must be tightly regulated to ensure timely activity of the different proteins activated under different conditions by distinct ligands. Smurf1, a new member of the HECT family of ligases, selectively interacts with SMADs specific for the BMP pathway (502). Ectopic expression of Smurf1 inhibits the transmission of BMP signals and affects pattern formation in Xenopus. Smurf2, also a member of the HECT domain family of E3s, appears to act similarly on SMAD1, though, in high concentration, it decreases also the level of SMAD2 (497). These findings suggest that ubiquitination by Smurf2 may regulate the competence of a cell to respond to TGF-/BMP signaling through a distinct degradation pathway that is similar to, yet independent of, Smurf1.

For many years, RING finger domains were thought to play a role in dimerization of proteins. It is only recently that RING finger domain-containing proteins were identified as ubiquitin ligases, transferring ubiquitin to both heterologous substrates as well as to the RING proteins themselves (198, 207, 280, 449). RING fingers have been defined by a pattern of conserved Cys and His residues that form a cross-brace structure that probably binds two Zn cations CX₂CX_(9-39)CX_(1-3)HX_(2-3)C/HX₂CX_(4-48)CX₂C (39, 385). RING finger domains are classified into RING-HC and RING-H2, depending on whether a Cys or His occupies the fifth coordination site, respectively. At least for the HC domain, structural analysis has revealed that it has two interleaved Zinc-binding sites. RING fingers probably function to recruit the E2 component of the ubiquitination machinery. The crystal structure of c-Cbl, a RING finger ligase involved in targeting activated receptor tyrosine kinases (269), bound to a cognate E2 and a kinase peptide (representing the substrate) shows how the RING domain recruits the E2 UbcH7 (500). The E2 binds to the RING through contacts between a groove in the RING and two loops in the E2 fold of UbcH7. The structure reveals a rigid coupling between the peptide-binding and the E2-binding domains and a conserved surface channel leading from the peptide to the E2 active site, suggesting that RING E3s may function as scaffolds that position the substrate and the E2 optimally for ubiquitin transfer (500). Comparison with the HECT domain group reveals that a similar region in the E2 is recognized by a similar spatially organized cleft in the HECT E3, although the latter is clearly distinct in its primary structure from that of the RING finger domain (187).

The RING finger domain-containing E3 family is composed of two distinct groups, single and multisubunit proteins. Certain members, Mdm2 (42, 120, 280), Ubr1/E3 (250, 367), and Parkin (411), for example, are monomers or homodimers and contain both the RING finger domain and the substrate-binding/recognition site in the same molecule. Many others are part of multisubunit complexes. Among them are the APC involved in degradation of cell cycle regulators (333), the von-Hippel Lindau-Elongins B and C (VBC)-Cul2-RING finger complex (196, 276, 338) involved in the degradation of HIF1- (195, 197, 220, 299), and the Skp1-Cullin/Cdc53-F-box protein (SCF)-RING finger complexes involved in degradation of signal- and cell cycle-induced phosphorylated proteins (82). In some of these complexes, such as in SCF or VBC, the RING finger domain component, Rbx1/Hrt1/Roc1 (413, 430), is involved in E2 recruitment and assembly of other components of the complex, but not in substrate recognition. A different subunit, the F-box protein in SCF, and most probably, the pVHL subunit in VBC, carry out the substrate recognition function. In APC, the RING finger domain protein is Apc11, which has been demonstrated to bind, at least in vitro, also the cyclin Clb2, and to catalyze its ubiquitination (266). However, this experiment was carried out with recombinant Apc11, in the absence of the other complex subunits, and therefore it is not clear that this subunit is the only substrate-binding protein in the complex (see however below).

A) SPECIFIC RING FINGER COMPLEXES: SCF COMPLEXES. The SCF complexes act along with the E2 Cdc34/Ubc3 (82), and possibly with members of the UBCH5 family (131). The catalytic complex may have the following structure: {E2·Hrt1/Rbx1/Roc1·Cdc53/Cullin·Skp1·F-box protein· protein substrate}, such that the E2 is recruited by the RING finger-containing protein and the substrate is bound to the F-box protein. A fifth component in the SCF ligase complex, Sgt1, has been recently described (233); however, its role is not yet clear. The Hrt1/Rbx1/Roc1, Skp1, and members of the Cdc53/Cullin-1 family components are probably common to all SCF complexes. The specific substrate binding F-box protein is the most variable component; therefore, the different complexes are designated according to the variable F-box component [e.g., SCF^Cdc4, SCF^-TrCP (transducin repeat-containing protein), SCF^Skp2]. All SCF complexes are probably involved in targeting phosphorylated substrates. SCF^-TrCP targets phosphorylated IB (487), -catenin (234), and HIV-1-Vpu (293). The signal recognized by SCF^-TrCP in all three substrates is DS(P)GXS(P) (222), although p105 (157, 327), p100, and other, yet to be identified substrates, may utilize slightly different signals (see below). Despite the fact that Vpu undergoes phosphorylation that recruits the E3, it is the CD4 receptor that is ubiquitinated and degraded in trans after formation of a CD4·Vpu·SCF ternary complex.

SCF^Skp2 targets E2F-1 (294) and p27^Kip1 (52). Although p27^Kip1 must be phosphorylated on Thr-187 to be recognized, it has not been shown whether recognition of E2F-1 also requires phosphorylation (see below), although it is highly likely that this is the case here too. SCF^Grr4 in budding yeast (413) targets the phosphorylated form of the G1 cyclin Cln1.

Regulation of the activity of SCF complexes is mediated, in large, via timely phosphorylation of its different substrates. However, an additional layer of regulation may be operational via modulation of the level of the F-box protein component of the complex, its key substrate recognition element. It has been reported (501) that in S. cerevisiae, Cdc4, the F-box protein that is involved in targeting cell cycle regulators, is unstable, in contrast to other, commonly shared, components of the complex. Grr1, another F-box component of SCF complexes, is also unstable, and like Cdc4, targeted for degradation by the ubiquitin system. Interestingly, ubiquitination of Cdc4 is mediated by SCF^Cdc4. A dominant negative species of Cdc4 that lacks the F-box domain, and therefore cannot recruit the Skp1 component, is stable, suggesting that ubiquitination is catalyzed by Cdc4 in an intramolecular mechanism within the SCF complex. Furthermore, the mutant protein inhibited cell proliferation by interfering, most probably, in the targeting of a variety of cell cycle regulators. This interference may be due not only to the inability of the SCF^Cdc4 to catalyze ubiquitination of its own substrates because the F-box protein is mutated, but also because of the inability of other F-box proteins to assemble into active SCF complexes, as the other subunits are engaged with the stable Cdc4. Thus the finding that the F-box components are unstable suggests a mechanism of regulating SCF function through ubiquitination and proteolysis of these components. In a different study, Spiegelman et al. (419) presented evidence that -catenin/TCF signaling elevates the expression of -TrCP mRNA and protein. Induction of -TrCP expression by the -catenin/TCF pathway results in accelerated degradation of the wild-type -catenin, suggesting that the negative feedback loop regulation may control the -catenin/TCF pathway.

An interesting case involves the yeast SCF^Met30 that targets the transcription factor Met4. Surprisingly, Met4 is a relatively stable protein, and its abundance is not influenced by Met30. However, transcriptional repression of Met4 target genes correlates with SCF^Met30-dependent ubiquitination of Met4. Functionally, ubiquitinated Met4 associates with target promoters but fails to activate transcription (214). Thus it appears that ubiquitination of transcription factors that does not involve their proteolysis can be utilized to directly regulate their activities. The mechanism of ubiquitination has not been deciphered. It may be similar to Ubc13/Uev1A and TRAF6 (a RING finger protein)-mediated activation of IB kinase (IKK) that involves generation of a ubiquitin chain assembled via Lys-63, that is not involved in targeting for degradation (79). It has been shown that TRAF6, which may be a ubiquitin ligase, is ubiquitinated following cell signaling, and that ubiquitination of TRAF6 and/or of an additional, yet unidentified, protein(s) is essential for activating the TAK1·TAB1·TAB2 kinase complex. Activation of this complex is required for activation of the downstream IKK. It is not clear whether any component of the TAK1 kinase must be also ubiquitinated during this process (461).

B) VBC. The VBC-Cul2-Rbx1 complex has a structure similar to that of SCF. It contains Elongins B and C, Cullin 2, and pVHL, in addition to Hrt1/Rbx1/Roc1 (219), the same RING finger protein that is also shared by SCF complexes. The substrate binding/recognizing subunit has not been identified, but it is most probably pVHL. One known substrate targeted by this E3 is HIF1- (195, 197, 220, 299). Similar to the modularity of F-box proteins in SCF complexes, it is possible that more than one substrate recognition protein is involved in VBC·Cul2·Rbx1 E3 complexes. Thus Socs1 was reported to target the hematopoietic specific guanine nucleotide exchange factor VAV for degradation in context of Elongins B and C complex (81).

C) APC. In budding yeast, the APC/cyclosome (333) contains 11 subunits, of which, Apc11 was shown to be the RING finger domain protein, that may also carry the substrate-binding and ubiquitination functions, at least toward certain substrates (266). Apc2 carries a Cullin homology domain and therefore may be related to the Cdc53/Cullin1 component of the SCF complexes (493). Specificity of the ligase complex toward its many mitotic and possibly G₁/S transition substrates is probably determined by a set of substoichiometric regulators that associate with it, such as Cdc20 (Fizzy/CDC20/p55) and Hct1/Cdh1 (Fizzy-related/HCT1). In addition, regulation also involves phosphorylation of APC subunits [such as by Cdc5, the Polo-like kinase (Plk/Plx)] and dephosphorylation (see below). It should be noted that the activity of the APC/cyclosome is not regulated by modulation of the level of its different subunits, as they all appear to be constitutively expressed throughout the cell cycle. As for APC/cyclosome-associated E2 enzymes, APC acts along with Ubc4 (UBC4) and Ubc11 (E2-H10 in human, UBCx in Xenopus, and E2-C in clam; see above); however, it is not known whether the different E2s are specific for distinct substrates, or whether they are interchangeable. Deletion analysis in yeast suggests that Ubc11 and Ubc4/Ubc5 can substitute for one another (441). Ubiquitination of cyclins, but also of other APC substrates, such as Geminin, a Xenopus protein involved in the inhibition of DNA replication (301), is mediated by a short, 9-amino acid residue sequence, the "destruction box" (see below). APC also recognizes and ubiquitinates the inhibitors of sister chromatid separation, Pds1 (securin) in S. cerevisiae, and Cut 2 in S. pombe, via a "destruction box." These proteins are inhibitors of Esp1 (separin), the protease that upon activation (after removal of securin) is involved in cleavage of cohesin, the protein that attaches the two sister chromatids to one another. Ase1, a yeast protein required for elongation of mitotic spindle during mitosis, is also targeted by the APC/cyclosome.

A recently discovered protein is a ubiquitin chain elongation factor, E4. Koegl et al. (241) have shown that efficient multiubiquitination required for proteasomal targeting of a model ubiquitin-fusion substrate utilizes an additional conjugation factor, named E4. This protein is identical to Ufd2 (ubiquitin fusion degradation pathway), involved in targeting of chimeric model substrates with a stable ubiquitin moiety fused to their NH₂ terminus (209). In yeast, E4 binds to the ubiquitin moieties of preformed short conjugates and catalyzes ubiquitin chain elongation in conjunction with E1, E2, and E3. It thus renders them preferred substrates for proteasomal degradation. E4 defines a novel protein family that shares a modified version of the RING finger, designated as U box, that lacks the hallmark metal-chelating residues of the RING finger motif (12). Most of the signature Cys residues of the RING finger are not conserved in the U box, and the structure is probably stabilized by hydrogen bonds and salt bridges. A typical box contains the following residues: xxxxhxsxlxxphhx-shxxxxsxxxhppxxIxpxhxxxxxxxxsPxsxxxxxxxxxlxsxxxxpxxxx, where x denotes any residue, h is a hydrophobic residue, l is an aliphatic residue, and s is small, p is polar, and - is a negatively charged residue. P and I indicate Pro and Ile that appear in more than 90% of identified members of the family.

A number of U-box proteins have been shown to elongate chains dependent on E1 and E2, but independent on E3 (153). Therefore, it is possible that U-box enzymes constitute a subfamily of E3 enzymes that has the ability, together with E1 and E2, to ubiquitinate ubiquitin-protein fusions or elongate short polyubiquitin chains by mediating transfer of ubiquitin to a previously conjugated ubiquitin molecule rather than to the substrate itself, in effect elongating chains (241). An E3 would still be necessary in this scenario to attach the first ubiquitin to the substrate. In addition, they can function also as ubiquitin ligases that act independently of the action of an E3, i.e., target their substrates directly (153). It is not clear yet whether the U box, like the E3 RING finger motif, is involved in recruiting the E2 component of the conjugation machinery, or whether it binds to the short ubiquitin chain conjugated to the target protein. An interesting member of the family is CHIP, which has recently been shown to be involved in the degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and the glucocorticoid receptor (66, 303). CHIP was shown to interact, through a set of tetratricorepeat motifs, with Hsc70 and Hsp90. In the case of CFTR, it functions with Hsc70 to sense the folded state of CFTR and to target aberrant forms for proteasomal degradation by promoting their ubiquitination. It has been shown the CHIP acts as an independent ubiquitin ligase that targets both Hsc70 (204a) and also other unfolded proteins for degradation (315a). It is possible that CHIP does not have a substrate binding site, but ubiquitinates unfolded proteins that are bound to Hsp70. Thus it is the heterodimer CHIP-Hsp70 that acts as a ubiquitin ligase (75b). The U box appears to be essential for this process because overexpression of CHIP--U box inhibited the action of endogenous CHIP and blocked CFTR ubiquitination and degradation. In the case of the steroid receptor, CHIP acts along with Hsc90. It is possible that CHIP is the E4 component in the conjugation machinery of CFTR and the steroid receptor, but it can also be the direct E3 ubiquitin ligase.

	III. HIERARCHICAL STRUCTURE OF THE UBIQUITIN SYSTEM

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The ubiquitin system has a rhomboid-shaped structure, with its tips at the E1 and the proteasome and the broadest point at the recognition plane between the substrates and the E3s (Fig. 2). Thus a single E1 enzyme activates ubiquitin for all conjugation reactions and transfers it to all known E2s (161, 244, 350, 474), and a single enzyme, the proteasome, proteolyses all substrates targeted for degradation by ubiquitination (124, 458). Most E2s interact with several E3s (Fig. 2), and usually, most E3 are found to interact with several different protein substrates via similar or identical recognition motifs. However, this hierarchy is more complicated and cannot be seen simply as a pyramid structure, but rather as a complex network of overlapping interactions between multiple components (Fig. 3). For instance, specific E3s can often interact with more than one E2, and some substrates can be targeted by more than one E3. A few examples highlight the complex combinatorics of different E2-E3-substrate interactions (Fig. 2).

View larger version (27K): [in this window] [in a new window]   Fig. 3. Modes of recognition of proteolytic substrates by ubiquitin-protein ligases, E3s. Ligases are in red, and substrates are in blue. A: recognition of substrates via their NH2-terminal residue (N-end rule pathway). B: peptide-induced allosteric activation of Ubr1. C: recognition of phosphorylated substrate. D: phosphorylation of the E3 is required for its activity. E: phosphorylation of both the ligase and its substrate is required for ubiquitination. F: recognition in trans via an ancillary protein. G: the ubiquitin system degrades selectively abnormal/mutated/un- and misfolded proteins, including also defective ribosomal products, DRiPs (nascent chains that are degraded cotranslationally). EGF-R, epidermal growth factor receptor. For details, see section IV. Recognition via hydroxylated proline is not shown.

1) A single E2, E2_A, acts along with a single E3 (E3_A) to target a set of substrates (S_A,B,C... ) that share a common recognition motif. This is probably the case with E2-C/Ubc11/UBCx/UBCH-10 that acts along with the cyclosome/APC in targeting cell cycle regulators that share the destruction box recognition motif (14, 333, 441).

2) E2_B interacts with E3_B, a ligase that has three distinct recognition sites. Each of these sites recognizes a distinct targeting motif on a defined set of substrates. Thus this E3 can interact independently with three sets of substrates, S_D,E,F..., S_{G,H,I... .,} and S_J,K,L... . This is the case with mammalian E2-14 kDa (351) and its yeast homolog Ubc2 (203), which interact with mammalian E3 (366, 478) and yeast Ubr1 (287), respectively. E3/Ubr1 has two N-end rule pathway recognition sites; the first recognizes substrates with basic NH₂ termini (site I), whereas the second recognizes substrates with hydrophobic NH₂-terminal residues (site II) (250, 368). However, this ligase also has a third site, a "body" site (site III), that recognizes an as yet to be identified motif(s) residing downstream to the NH₂ terminus (133, 161, 250, 368, 454).

3) Certain substrates contain two different recognition motifs in the same molecule and can be recognized by different E3s: S_M,N,O... for example, is targeted by E2_B/E3_B and either E2_C/E3_C or E2_D/E3_C that recognize the two different sites, respectively. The model substrate lysozyme is targeted by the N-end rule E2-14kDa/E3 pair that recognizes the NH₂-terminal Lys residue. However, its complete degradation requires concomitant recognition by an additional pair of E2(s), probably a member(s) of the UBCH5 family of E2s, and an unidentified E3 that recognizes a downstream body motif (133).

4) E3_D can interact with two different E2s, E2_C and E2_D, to recognize and ubiquitinate a set of substrates (S_S,T,U... ) via a common recognition motif. The F-box protein -TrCP recognizes IB, HIV-1 Vpu, and -catenin via a general doubly phosphorylated motif (see above). SCF^-TrCP acts both with members of the UBCH5 family of E2s as well as with the UBC3/Cdc34 E2s (131, 487). A different F-box protein, Skp2, which is the substrate-recognizing subunit of the SCF^Skp2 E3 complex, targets the CDK inhibitor p27^Kip1 after a single phosphorylation on Thr-187 (52). It probably partners with the same E2s as SCF^-TrCP.

5) S_P,Q,R..., like S_M,N,O..., is recognized by two distinct E3s, E3_C and E3_D, each targeting a different motif. However, unlike E3_B that is acting with a single E2 (E2_B), both E3_C and E3_D act each with two E2s, E2_C and E2_D. p105, the precursor of the NF-B transcription factor subunit p50, is targeted for limited processing by -TrCP that recognizes a phosphorylated COOH-terminal motif similar to that of IB/-catenin/HIV-1 Vpu (157, 158, 327). An additional, yet to be identified, E3 targets an acidic domain in the middle of the molecule (328). As described above, -TrCP acts with members of the UBCH5 family and with UBC3/Cdc34 E2s, while the acidic site E3 acts probably with E2-25 kDa and members of the UBCH5 family of E2s (67) (A. Ciechanover, H. Gonen, and H. Achbert, unpublished data). The case of p105 and lysozyme may be similar to that of the S. cerevisiae MAT2 mating factor that has two degradation signals, Deg1 and Deg2. Deg1 is targeted by two E2s, Ubc6 and Ubc7 (58). Ubc4 and Ubc5 have also been implicated in targeting the mating factor, but it is not clear whether they are involved in recognition via the Deg2 motif. The E3 that acts along with the Ubc6/Ubc7 E2s has been identified as the ER/nuclear protein Doa10/Ssm4 (427a), but the ligase that acts along with Ubc4/Ubc5 has not been identified.

	IV. MODES OF SUBSTRATE RECOGNITION AND REGULATION OF THE UBIQUITIN PATHWAY

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Targeting of a protein via the ubiquitin system must involve specific binding of the protein to the appropriate ubiquitin ligase, E3. Despite recent progress in our understanding of modes of recognition and regulation of the system, it is only in a handful of cases where the E3 recognition motif has been identified precisely. In principle, recognition can be mediated via several mechanisms: either the substrate is modified so as to be recognized or not by the appropriate E3, or the activity of the E3 can be modulated. Although the number of cases is still too low to make sweeping generalizations, the mode of regulation appears to correlate with the class of E3. Ubiquitination by SCF complexes requires phosphorylation of the substrate; APC activity is modulated by the presence of ancillary substrate-binding factors and/or by phosphorylation of the complex subunits, and activity of the HECT-domain proteins may also depend on ancillary proteins that bridge between the enzyme and the substrate. E3/Ubr1 appears to be constitutively active toward certain substrates and allosterically regulated toward others.

The ubiquitin-proteasome pathway can be regulated at the level of ubiquitination or at the level of proteasome activity. Because conjugation and proteasomal degradation is required for a multitude of cellular functions, regulation must be delicately and specifically tuned. In two cases however, it was reported that general rather than specific components of the pathway could be modulated by physiological signals. One is the upregulation of the pathway that is observed during massive degradation of skeletal muscle proteins. This occurs in mammals under normal fasting, but also under pathological conditions such as cancer cachexia, severe sepsis, metabolic acidosis, or after denervation (260, 308) (see sect. XG). It occurs also during specific developmental processes, such as insect metamorphosis; the massive breakdown of larval tissue before the development of the imago is accompanied by upregulation of the ubiquitin pathway (313). A different case in which degradation can be regulated is by changing the specificity of the proteasome cleavage sites within the frame of its function in antigen presentation (37, 124, 238, 370). It was shown that in mammalian cells, three components of the 20S proteasome, two that are encoded within the MHC locus and one that is encoded by a different region, are upregulated after interferon- treatment. They replace three other proteasomal subunits that confer to the proteasome a different peptide cleavage specificity, presumably favoring the types of peptides that are better bound to the major histocompatibility complex (MHC) class I molecules of the presenting cell and the T cell receptor of the cytotoxic T cell (CTL).

There are several modes for specific substrate recognition; some of them are depicted in Figure 3.

1.  Recognition via the NH₂-terminal residue: the N-end rule pathway (Fig. 3A)

In recognition of substrates via the N-end rule pathway (454), substrates bind directly to the ligase via their NH₂-terminal residue. The E3 involved, E3/Ubr1, has two N-end rule recognition sites: site I for substrates with basic NH₂ termini and site II for substrates with hydrophobic NH₂ termini (250, 368). A third site, site III, is involved in targeting non-N-end rule substrates. (see above and below). The targeting NH₂-terminal amino acids were designated "destabilizing" residues. Most of the studies on the N-end rule pathway were carried out using model substrates, and inactivation of several enzymatic components of the pathway did not yield a clear phenotype in yeast cells. Therefore, identification of substrates that are targeted via NH₂-terminal recognition has not been easy. RGS4, a member of the RGS (regulator of G protein signaling) involved in specific G protein activation, is arginylated posttranslationally at the NH₂ terminus, a modification that renders it sensitive to Ubr1-mediated ubiquitination and rapid degradation in vitro (73). However, because a substrate from which the N-end rule targeting motif was removed was still unstable in vivo, it is not clear whether this motif acts also in the context of the intact cell. Recently, Rao et al. (360) have shown that a 33-kDa fragment generated by separin (Esp1) from the cohesin subunit SCC1 during mitosis and chromatid sister separation contains an Arg residue at the NH₂ terminus and is degraded via the N-end rule pathway. Its degradation is essential for chromosome stability. It should be noted that activation of separin results from APC-mediated degradation of its inhibitor, Pds1 (securin). Activated separin then cleaves cohesin, the "glue" protein that attaches the two sister chromatids. This process is important for proper division of the genetic material between the daughter cells and its inheritance. The fragment is probably the first bona fide substrate of the N-end rule pathway.

2.  Peptide binding-mediated allosteric activation (Fig. 3B)

Ubr1 has also a third recognition site that targets unidentified "body" motifs downstream from the NH₂-terminal residue (250, 368). Thus it can target N--acetylated proteins (132) and also the yeast G protein involved in growth regulation in response to pheromones (288, 387). The Cup9 transcription factor is a negative regulator of the di/tripeptide transporter Ptr1 gene. It is targeted by Ubr1, most probably after recognition by the body site (site III) of the enzyme. Peptides that bind to site I or II activate Ubr1 allosterically and increase the rate of catalysis of Cup9 ubiquitination and degradation. This in turn leads to induction of Ptr1 and to upregulation of peptides uptake, which reflects their increased concentration in the growth medium (447). Thus it is extremely important to discriminate between degradation following NH₂-terminal recognition of the substrate, a pathway that targets a limited subset of substrates, and recognition by E3, the N-end rule pathway ligase that recognizes in addition many more substrates via non-N-end rule signals.

3.  Phosphorylation of substrates and/or ubiquitination enzymes (Fig. 3, C-E)

Phosphorylation of substrates was shown to yield opposite effects on different substrates, and sometimes on different sites of the same substrate. An increasing number of substrates of the ubiquitin pathway are modified by phosphorylation before their ubiquitination, a modification that in certain cases at least was shown to be necessary for direct recognition of the modified protein by the appropriate E3. A nonexhaustive list includes the yeast G₁ cyclins Cln2 and Cln3, the yeast cyclin-dependent kinase (CDK) inhibitors Sic1 and Far1, the mammalian G₁ cyclins D and E, the mammalian CDK inhibitor p27^Kip1, the mammalian transcriptional regulators IB and -catenin (244), and, recently, the NF-B precursor p105 (157, 327). Strikingly, in all the instances where the E3 enzyme was identified, it turns out to be of the SCF type. Two of the best-characterized cases are those of IB and Sic1.

The mammalian transcription factor NF-B is inhibited by IB, which binds to it and sequesters it in an inactive form in the cytosol. NF-B is activated by a variety of extracellular stimuli such as cytokines, bacterial and viral products, and ionizing irradiation, among others (222). This activation is achieved by proteolysis of IB, which releases NF-B to be translocated to the nucleus. Proteolysis of IB requires its phosphorylation on two specific residues, Ser-32 and Ser-36, after which it is recognized by a specific SCF complex, SCF^-TrCP (487). Activity of SCF^-TrCP appears to be constitutive, while the IB kinases, IKK and IKK, are activated by the NF-B-inducing stimuli. Another substrate of SCF^-TrCP, -catenin, is phosphorylated by a distinct kinase, GSK3, on two serines embedded in a sequence similar to the Ser-32-Ser-36 region of IB (151). These two regions together with a third -TrCP-binding protein, HIV-1 Vpu (293), allow the definition of a consensus recognition motif for -TrCP binding: DS(P)GXS(P) [single-letter amino acid code; S(P) stands for phospho-serine,  stands for a hydrophobic residue]. This sequence by itself is sufficient for binding of the ligase, since a phosphopeptide that spans this recognition region can immobilize -TrCP and inhibit IB ubiquitination and degradation both in vitro and in vivo (486). A similar motif has recently been described for the NF-B precursor protein p105 (157, 327). After IKK-mediated phosphorylation, the target proteins are ubiquitinated by SCF^-TrCP. Unlike -TrCP, however, no consensus recognition motif has been defined yet for other F-box proteins.

Sic1 is an inhibitor of yeast B-type (Clb) cyclin/CDK complexes, but not of G₁ (Cln) cyclin/CDK complexes. Rapid degradation of Sic1 at the end of G₁ enables the initiation of DNA replication (398). In wild-type cells, Sic1 appears at the end of mitosis and disappears shortly before S phase. G₁ cyclin/CDK complexes phosphorylate Sic1 at a number of specific sites (455); the phosphorylated protein is then recognized by the SCF^Cdc4 ubiquitination complex and degraded (398, 455). Interestingly, and unlike recognition of defined phosphorylated residues by TrCP, the affinity of CDC4 to the phosphorylated Sic1 increases with the number of phosphorylated residues (317a). All available evidence indicates that Sic1 degradation is uniquely regulated by its phosphorylation, while the SCF^Cdc4 activity is constitutive: the cell cycle-specific degradation is due to the temporal expression of the G₁ cyclins that are required for Sic1 phosphorylation, and when these G₁ cyclins are ectopically expressed, Sic1 becomes constitutively unstable (21). Further support for the assumption that SCF^Cdc4 activity is constitutive comes from the observation that Gcn4, another SCF^CDC4 substrate, is constitutively degraded during the cell cycle (244, 304).

Degradation of the mammalian G1 CDK inhibitor p27^Kip1 is required for the cellular transition from quiescence to the proliferative state. Ubiquitination and subsequent degradation of p27 depend on its phosphorylation by G₁ cyclin/CDK complex at a specific residue, Thr-187, which is followed by SCF^Skp2-mediated ubiquitination (52, 445). Interestingly, both in vivo and in vitro, Skp2 is a rate-limiting component of the machinery that ubiquitinates and degrades phosphorylated p27; its expression is cell cycle regulated and peaks at the S phase (496). Thus p27 degradation is subject to dual control by the presence or absence of the specific F-box protein and of cyclin A/CDK complexes that are regulated by mitogenic stimuli. The transcription factor E2F-1 is also ubiquitinated by SCF^Skp2 (294); however, it is not known yet whether E2F-1 degradation also requires phosphorylation. Interestingly, it has been reported that many short-lived proteins contain a sequence enriched in PEST residues (Pro, Glu, Ser, Thr) (372) that has been implicated in destabilization of the proteins. Although the role of the region has never been deciphered, it appears to act via phosphorylation of its Ser/Thr residues.

Experiments with APC/cyclosome from clam oocytes demonstrated that enzyme isolated from interphase extract could be activated by the Cdc2 kinase, and conversely, enzyme isolated from mitotic extracts could be inactivated by the addition of phosphatase (252). Although it was suggested that Cdc2 can directly phosphorylate certain APC/cyclosome subunits, the kinetics of APC/cyclosome activation suggest that the enzyme subunits may not be directly activated by Cdc2 and that the kinase acts upstream to activate the ligase via modification of intermediary kinases (333). Immunoprecipates of Plk, a protein kinase related to the D. melanogaster POLO, revealed that the protein is complexed to APC1, Cdc16, and Cdc27. Furthermore, Plk was shown to be activated by Cdc2 (245). Purification of Xenopus and human APC/cyclosomes revealed that at least four subunits, APC1, CDC16, CDC23, and CDC27, are hyperphosphorylated in mitotic extracts (344). While the physiological role of subunit phosphorylation in APC is currently unclear, it appears that modification of a certain subunit(s) by specific kinase may affect its activity against a certain subset of substrates at a particular point of mitosis (see also below).

Subunit phosphorylation also appears to negatively regulate APC/cyclosome activity. Experiments in S. pombe implicate the protein kinase A (PKA) pathway as an inhibitor of APC activity. For example, addition of mammalian PKA to cyclosome fractions inhibits cyclin B ubiquitination even if the ligase was previously activated by Plk (245). Overexpression of cAMP phosphodiesterase in cut4-533, a mutant defective in APC/cyclosome complex formation, restores incorporation of the ligase subunits into high-molecular-mass complex, suggesting that subunit phosphorylation by PKA may negatively regulate the assembly process (483). Phosphatases have also been implicated in APC/cyclosome activation. Type I protein phosphatases (PP1) are required for the onset of anaphase in many eukaryotes (171), and an APC6 (cut9-665) mutant is synthetically lethal with mutations in dis2+ that encodes a catalytic subunit of PP1 (481). Thus it appears that the APC/cyclosome is positively and negatively regulated by an intricate network of kinases and phosphatases, and each acts probably on a specific subunit(s) and modulates the activity of the enzyme toward a specific substrate and at a specific point along the mitotic process.

Targeting of the epidermal growth factor receptor (EGF-R) involves initial autophosphorylation at a previously identified lysosome-targeting motif that leads to recruitment of the RING finger ligase c-Cbl/Sli-1. This is followed by Tyr phosphorylation of the ligase at a site adjacent to the RING finger domain that allows receptor ubiquitination and subsequent routing to the lysosome for degradation (269).

Phosphorylation can also inhibit ubiquitination as has been described in several instances. Degradation of the protooncogene c-mos by the ubiquitin pathway is inhibited by phosphorylation on Ser-3 (319, 320). Interestingly, activation of c-mos leads to phosphorylation and stabilization of c-fos, another substrate of the ubiquitin pathway. Although c-mos is a serine/threonine kinase, it probably does not phosphorylate c-fos directly. Rather, it is thought to act via activation of the MEK1/ERK pathway (325). c-Jun also appears to be stabilized by phosphorylation after its association with the signalosome complex (see below) (318, 401). Another example is that of the anti-apoptotic protein Bcl-2: dephosphorylation of Bcl-2 upon apoptotic stimuli renders it susceptible to degradation by the ubiquitin pathway (89).

4.  Recognition in trans (Fig. 3F)

In several cases, the target protein is not recognized directly by the ligase, but rather in trans, following binding to an ancillary protein. Viruses were found to exploit the ubiquitin system by targeting cellular substrates that may interfere with propagation of the virus. In some instances, the viral protein functions as a bridging protein between the E3 and the substrate. The prototype of such an interaction is the HECT domain ligase E6-AP that acts along with the HPV protein E6 to target p53. E6 binds both p53 and E6-AP, and formation of this ternary complex results in the ubiquitination and degradation of p53. This activity of E6 can account, at least partially, for the oncogenicity of the strains of papillomaviruses that express it (388). A second instance of a viral protein-mediated ubiquitination of an endogenous substrate is that of the degradation of the T-cell CD4 receptor. As mentioned above, the Vpu protein of the HIV-1 virus is recognized, after phosphorylation, by the F-box protein -TrCP. Vpu also binds to the CD4 receptor of the T cells infected by the virus; this binding leads to ubiquitination and degradation of CD4 by the SCF^-TrCP complex (293). This case is interesting in particular as the phosphorylated protein Vpu to which the E3 binds is not ubiquitinated and remains stable. It is the CD4 protein that is not phosphorylated that is targeted for degradation. The problem is obviously how a substrate to which the ligase is not bound is targeted in trans. In a different case, molecular chaperones appear to facilitate ubiquitination and degradation of certain proteins. This has been shown for several soluble model proteins (29, 262), and not surprisingly also for membrane proteins. Thus the chaperone BiP was demonstrated recently to bind to a mutant Prion protein and to mediate its degradation by the proteasome (205). Similarly, mutated CFTR and the glucocorticoid receptor are degraded after complex formation with the cochaperone and E3 CHIP and the chaperones Hsc70 and Hsc90, respectively (see above).

5.  Abnormal/mutated/misfolded proteins (Fig. 3G)

The ubiquitin system selectively and efficiently degrades denatured/misfolded proteins that arise as a result of mutations, immaturation, or posttranslational environmental stress (62, 162, 377, 403). It can also degrade cotranslationally nascent peptide chains that do not attain native structure due to errors in translation or in posttranslational processes necessary for proper folding. Cotranslational degradation is an extensive process, and ~30% of nascent chains are degraded and do not mature to native proteins (393, 448). The degraded chains were designated DRiPs (defective ribosomal products). It should be noted, however, that these products have not been shown to be defective though premature/incomplete polypeptide chains (such as are generated in the presence of puromycin) are known to be preferred substrates for the ubiquitin system. The nature of the signals and the identity of the E3(s) involved are still mysterious. It is possible that exposure of hydrophobic domains that are normally buried in protein-protein interaction surfaces or within the protein core, serve as recognition signals for immature, nascent, and otherwise abnormal and misfolded proteins. It is possible that since nascent chains associate with chaperones prior to completion of synthesis of the entire polypeptide chain, the E3 CHIP may be involved in their targeting (see above). An interesting case involves the oxygen-induced ubiquitination of HIF- by the pVHL ligase complex, where the ligase recognizes specifically a hydroxylated Pro residue (Pro564) generated in the target protein under hyperoxia. In this case, however, although the modification renders the protein "abnormal" by generating an oxidized amino acid derivative, it is a specific and regulated change, catalyzed by a specific enzyme, prolyl hydroxylase (47a), that targets for degradation, under high oxygen pressure, a transcription factor that operates normally during normoxia or hypoxia (see above).

6.  Recognition via specific sequences (not shown in Fig. 3)

Ubiquitination of mitotic cyclins is mediated by a small NH₂-terminal motif known as the "destruction box." The minimal motif is nine residues long, and it has the following consensus sequence: R-A/T-A-L-G-X-I/V-G/T-N (absolutely conserved residues are in boldface) (482). The function of the destruction box is not clear, because it is not phosphorylated or ubiquitinated. It may serve, however, as a binding site for the ligase subunit of the APC/cyclosome complex. Destruction boxes may not be the only determinants of cyclin stability. The boxes of cyclin A and B are not interchangeable, and indestructible cyclin constructs that contain a destruction box derived from a different cyclin can still be polyubiquitinated (239). Thus, although ubiquitination is probably necessary for cyclin destruction, it may not be sufficient.

7.  Regulation by ubiquitin-like proteins (not shown in Fig. 3)

Both enzymes and substrates of the ubiquitin system have been found to be modified by ubiquitin-like proteins (UBL; see below). In the case of enzymes, it affects their activity or stability. In the case of substrates, it affects their availability to the ubiquitination/degradation machinery.

The UBL NEDD8 was shown to modify each of the five mammalian Cullins, Cul1-5. The unique yeast Cullin, Cdc53, is modified by Rub1, the yeast homolog of NEDD8 (488). NEDD8 conjugation to the Cul1 component of SCF^Skp2 was shown to be important for the activity of the enzyme in catalyzing ubiquitination of the CDK inhibitor p27^Kip1 (354). The mechanism(s) that underlie the NEDD8 conjugation-induced activation of the SCF^Skp2 complex is still obscure but it may be important for recruitment of the E2 enzyme (223a). Modification of Mdm2 by SUMO