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Article

Ubiquitin-Dependent Signaling: The Role of Ubiquitination in the Response of Cells to Their Environment.^{1 ,2}

Department of Biochemistry, Emory University School of Medicine

	ABSTRACT

TOP ABSTRACT INTRODUCTION Targeting Functions of the... Conjugation and Deconjugation of... Role of Ubiquitination in... Proteolysis to Repair Cellular... Checkpoint Regulation: Repair... Summary. REFERENCES

 
The response of a cell to its external environment requires rapid and significant alteration of protein amount, localization and/or function. This regulation involves a complex combination of processes that control synthesis, localization and degradation. All of these processes must be properly regulated and are often interrelated. Intracellular proteolysis is largely accomplished by the ubiquitin-dependent system and has been shown to be required for growth control, cell cycle regulation, receptor function, development and the stress response. Substrates subject to regulated degradation by this system include cyclins and cyclin-dependent kinase inhibitors, tumor suppressors, transcription factors and cell surface receptors. In addition, proteins that are damaged by oxidation or that are improperly folded or localized are substrates whose degradation by this system often leads to antigen presentation on the surface of the cell in the context of Class I major histocompatibility complex molecules. A very large body of work in the last fifteen years has shown that degradation by this system requires the covalent attachment of a small protein called ubiquitin and that this modification serves to direct target proteins for degradation by a 26S proteolytic particle, the proteasome. Thus, the attachment of the ubiquitin domain is of vital importance in regulating normal growth and differentiation, as well as in defending against cellular damage caused by xenobiotics, environmental insults, infection and mutation. This review focuses on the role of ubiquitination in the cellular signaling pathways that deal with these external influences.

KEY WORDS: • ubiquitin • proteasome • receptor • stress-response • apoptosis

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Targeting Functions of the... Conjugation and Deconjugation of... Role of Ubiquitination in... Proteolysis to Repair Cellular... Checkpoint Regulation: Repair... Summary. REFERENCES

 
There has been a huge expansion in our understanding of protein degradation in the last few years, largely due to the elucidation of the ubiquitin-dependent proteolysis pathway. It is now clear that ubiquitin serves primarily as a targeting signal and that this pathway influences all cellular functions. Many recent reviews have summarized different aspects of the system (Bonifacino & Weissman 1998 , D'Andrea & Pellman 1998 , Hershko & Ciechanover 1998 , Kopito 1999 , Orlowski 1999 , Scheffner 1998 , Schwartz & Ciechanover 1999 , Strous & Govers 1999 , Wilkinson 1998 ). This contribution will describe some of the most recent studies. Many of these involve "classical" signal transduction pathways, as well as recent understanding of the response to cellular damage, including apoptosis. This review will be focused on the cells' response to the external environment. Because of space limitations, I will refer to recent reviews and will generally confine the other citations to the most recent or informative contributions. A modest effort by the reader should quickly reveal details of the systems discussed, as well as earlier contributions that are uncited, but important.

	Targeting Functions of the Ubiquitin Domain.

TOP ABSTRACT INTRODUCTION Targeting Functions of the... Conjugation and Deconjugation of... Role of Ubiquitination in... Proteolysis to Repair Cellular... Checkpoint Regulation: Repair... Summary. REFERENCES

 
Ubiquitin targets proteins for proteolysis. It has recently become clear, that protein degradation can be thought of as a specialized case of protein localization. The major mechanism of targeting proteins for degradation by the proteasome involves the covalent attachment of ubiquitin to a side-chain lysine on the target protein (Hershko & Ciechanover 1998 ). Proteolytic substrates contain a polyubiquitin chain formed by the conjugation of a second ubiquitin to the K48⁴ residue of the first ubiquitin, and subsequent elongation of this polyubiquitin chain (Pickart 1997 ). The proteasome consists of the 20S core protease and a 19S regulator complex (sometimes called PA700). PA700 contains six ATPases, ubiquitin binding subunit(s) and a deubiquitinating enzyme and is thought to endow the 20S proteasome with specificity for ubiquitinated proteins (Glickman et al. 1998 , Tanaka 1998 ). While the attachment of K48-linked polyubiquitin chains targets proteins for degradation, polyubiquitin chains can also be synthesized with linkages between the C-terminus of ubiquitin and K6 and K11, K29, and K63 on the adjacent ubiquitin (Hofmann & Pickart 1999). The precise role of these linkages is unknown, although there is biochemical and genetic evidence that K29-linkages are necessary for degradation of ubiquitin-fusion proteins (Koegl et al. 1999 ) and K63-linkages are vital in regulating DNA repair (Hofmann & Pickart 1999) and ubiquitination of cell surface receptors (Hicke 1999 , Strous & Govers 1999 ).

Ubiquitin-like proteins also act as targeting signals.

The ubiquitin system is the prototype for a newly recognized family of pathways that result in the covalent modification of target proteins (Hodges et al. 1998 , Li & Hochstrasser 1999 ). These systems all use the same regulatory logic. A protein containing the ubiquitin-like domain and containing a glycine at the C-terminus is covalently attached to target proteins by at least five distinguishable systems. These systems use different ubiquitin-like proteins, different enzymes and act on different substrates, but share a common chemistry (Tanaka et al. 1998 ). Covalent attachment of ubiquitin-like domains has been implicated in the localization of proteins to the proteasome and/or the endosome/lysosome pathways (ubiquitin), the nucleus (SUMO-1/Smt3p), to the cytoskeleton (ISG15), to autophagic vesicles (Apg12p) and in the recruitment of proteins to macromolecular complexes (NEDD8/Rub1p). A number of other proteins contain a ubiquitin-like domains but are not conjugated to other proteins (Tanaka et al. 1998 ). Thus, ubiquitin-domains serve as versatile targeting signals used in a number of cellular processes.

	Conjugation and Deconjugation of the Ubiquitin Domain.

TOP ABSTRACT INTRODUCTION Targeting Functions of the... Conjugation and Deconjugation of... Role of Ubiquitination in... Proteolysis to Repair Cellular... Checkpoint Regulation: Repair... Summary. REFERENCES

 
To conjugate the ubiquitin domain, it is first adenylylated by an activating enzyme (E1), then transferred to a conjugating enzyme (E2) as a thiol ester, and finally attached to a target protein by a ligase (E3)(Hershko & Ciechanover 1998 , Schwartz & Ciechanover 1999 ). For ubiquitin there are at least three families of ligase enzymes: single chain proteins such as E6AP containing a motif called the HECT domain (homology to E6AP C-terminus), multienzyme complexes such as the SCF ligases (in yeast consisting of Skp1p, Cdc53p, Roc1p and an F-box adapter protein) and the more complex multisubunit anaphase promoting complex/cyclosome APC/C). A K48-linked polyubiquitin chain is assembled and targets the protein for localization to the proteasome and subsequent proteolysis (Hershko & Ciechanover 1998 , Pickart 1997 , Tanaka 1998 ). Once the attached protein has been degraded, the polyubiquitin chain is removed and disassembled by the action of a series of processing proteases called deubiquitinating enzymes (D'Andrea & Pellman 1998 , Wilkinson 1998 ).

The conjugation of the ubiquitin domain is a reversible process and the removal of the ubiquitin domain is catalyzed by deubiquitinating enzymes (DUBs). These enzymes catalyze the hydrolysis of the peptide bond at G76 of ubiquitin. There are at least 17 proteins in yeast that are related to known deubiquitinating enzymes. Many more are known to exist in higher eukaryotes. Why are there so many? One possibility is that these enzymes have specificity for the type of linkage in the polyubiquitin chains (i.e., K29, K48, or K63-linked chains may be cleaved by different DUBs). They may also exhibit specificity for different ubiquitin domains, although a new family of deconjugating proteins specific for Smt3p has been identified (Li & Hochstrasser 1999 ). Thus, there may be so many of these enzymes because of the number of different ubiquitin domains and the variety in the way in which they can be polymerized.

This pathway of reversible modification resembles phosphorylation in many respects. The enzymes that catalyze the deconjugation of ubiquitin-like proteins from target proteins then could serve the same regulatory roles as do phosphatases. These enzymes would be proteases with specificity for cleavage of the isopeptide bond formed upon modification, or with specificity for cleaving the peptide bond of the pro-proteins to yield the mature ubiquitin-like protein. A number of examples of processes regulated by DUBs are now known (D'Andrea & Pellman 1998 , Wilkinson 1998 ).

	Role of Ubiquitination in Regulating Signal Transduction.

TOP ABSTRACT INTRODUCTION Targeting Functions of the... Conjugation and Deconjugation of... Role of Ubiquitination in... Proteolysis to Repair Cellular... Checkpoint Regulation: Repair... Summary. REFERENCES

 
Receptor internalization and degradation. Several permeases, transporters and cell surface receptors are internalized and degraded in a ligand-dependent and ubiquitin-dependent manner (Bonifacino & Weissman 1998 , Hicke 1999 , Strous & Govers 1999 ). Most often the degradation occurs in acidic compartments, although infrequently it requires the proteasome. It is thought that the ligand engagement triggers either exposure of the ligase binding site(s) or phosphorylation sites. Phosphorylation by the appropriate kinase then recruits the conjugation machinery. Once modified, the ubiquitinated protein participates in some unknown way in endocytosis.

There are several steps in the internalization/degradation pathway that may be regulated by ubiquitination. First, it has been suggested that in some cases it is not the receptor, but another nonreceptor protein that requires ubiquitination. A candidate protein is Eps15, a protein that becomes phosphorylated, ubiquitinated, and localized to the clathrin-coated pits upon ligand engagement of the growth hormone or epidermal growth factor receptors (van Delft et al. 1997 ). Second, some receptors such as the PDGF and CSF-1 receptors induce the ubiquitination of a tyrosine kinase regulator, c-cbl (Wang et al. 1999 ). This protein has recently been shown to participate in the endosomal sorting pathway by binding to and enhancing the ubiquitination of the cytoplasmic domain of the endocytosed receptors (Levkowitz et al. 1998 ). Such ubiquitinated receptors are localized to the lysosome for degradation, while those that are not ubiquitinated are recycled to the cell surface.

Upstream signal transduction events.

The classical phosphoinositide signaling system utilizes a heterotrimeric G-protein coupled receptor to activate phospholipase C, release Ca²⁺ and activate protein kinase C. Monoubiquitination of Ca²⁺- calmodulin greatly reduces its activation of a target kinase without causing the degradation of calmodulin (Laub et al. 1998 ). In yeast, the G-protein coupled receptor itself is ubiquitinated and degraded (Hicke 1999 ). Similarly, there is evidence that the inositol triphosphate receptors in the ER (Oberdorf et al. 1999 ) and a variety of protein kinase C isoforms (Lu et al. 1998 ) are substrates for ubiquitin-dependent degradation upon activation of this pathway.

Tyrosine kinase activated receptor signaling is also modulated by ubiquitination. Signaling by these receptors is through both the Jak/STAT and the Ras/Map Kinase pathways. Signaling by IL-3 is prolonged in the presence of proteasome inhibitors (Callus & Mathey-Prevot 1998 ) and signaling by a number of other pathways requires an intact ubiquitin pathway (Bonifacino & Weissman 1998 , Hicke 1999 , Strous & Govers 1999 ). The reversal of ubiquitination is also important. In drosophila, proper eye development requires intercellular communication that is disrupted in mutants of faf, a deubiquitinating enzyme. This mutation interacts genetically with the receptor tyrosine kinase/Ras/MAP Kinase signaling pathway (Li et al. 1997 ) and the mammalian homolog of this DUB has been shown to bind to AF-6, a Ras target involved in cell-cell adhesion (Taya et al. 1998 ). Cytokine-induced signaling events have also been shown to induce a family of mammalian deubiquitinating enzymes involved in the regulation of growth (D'Andrea & Pellman 1998 ). These DUBs were shown to be immediate early gene products produced upon cytokine stimulation, with each mechanistically distinct cytokine inducing a different isozyme of this deubiquitinating enzyme. The JAK2 pathway is involved in transducing the signal and overexpression of these enzymes results in suppression of growth. It seems likely that the action of these DUBs is to deubiquitinate the cytoplasmic tail of the internalized receptor to allow its return to the cell surface. This "up-regulation" would be necessary to halt receptor degradation and allow the cell to respond to the next bolus of cytokines that might be encountered.

Downstream signal transduction events.

More than a dozen transcription factors are modulated by ubiquitination ((Hershko & Ciechanover 1998 ) and references below). Ubiquitination has effects on the rate of transcription factor generation including; those of the NFB, Wnt/Wingless and Hedgehog signaling pathways (Maniatis 1999 ), the hypoxia-induced factor Hif1 (Huang et al. 1998 ), and the unfolded protein response factor Hac1p (Kaufman 1999 ). The ubiquitin system has also been shown to affect the half-life of the transcription factors Sp1 (Su et al. 1999 ) fos, jun, myc, myb, myoD (Ciechanover et al. 1999 ), the estrogen receptor, (Nawaz et al. 1999 ) bcl-6 (Niu et al. 1998 ), ß-catenin (Easwaran et al. 1999 ), ATF2 (Fuchs & Ronai 1999 ) and tramtrack (Hu & Fearon 1999 ). In the case of STAT1(Callus & Mathey-Prevot 1998 ), HAND (Yamagishi et al. 1999 ), and heat-shock factors (Mathew et al. 1998 ) the effects of ubiquitination are not known in detail. A few examples of the role of ubiquitin in transcription factor function are discussed below.

Two well-studied signaling pathways respond by triggering ubiquitin-dependent proteolysis events that generate the active form of the transcription factor. The best studied of these is the NFB pathway that is activated in response to a variety of signals including cytokines, growth factors, oxidants, UV irradiation, infections, and stress (Maniatis 1999 ). TNF action is mediated by NIK (NFB inducing kinase) while the HTLV-1 protein Tax activates through the MEKK1 pathway. Both kinases have been shown to phosphorylate IKK (IB kinase) activating it to phosphorylate two specific serines on IB in the inactive IB-NFB complex. The phosphorylated inhibitor dissociates from the complex and is ubiquitinated by ß-TrCP, an analogue of Slimb involved in the Wnt/Wingless pathway in drosophila. IB is then degraded by an ubiquitin-dependent pathway and the NFB released is transported to the nucleus where it activates a large number of genes. Another regulator of transcription, ß-catenin is also a substrate for ß-TrCP ubiquitin ligase. ß-catenin is a short-lived multifunctional protein that participates in cadherin-mediated cell-cell adhesion and in the transduction of the Wnt growth factor signal that regulates development. When ß-catenin is allowed to accumulate it enters the nucleus and binds to the high mobility group box transcription factor LEF (lymphocyte-enhancer binding factor) and directly regulates gene transcription (Easwaran et al. 1999 ).

A third transcription factor, Hif-1 (hypoxia-inducible transcription factor 1) is constitutively synthesized and rapidly degraded by an ubiquitin-dependent process in many cells (Huang et al. 1998 ). When the oxygen concentration drops, Hif-1 becomes stable and is transported into the nucleus where it associates with ARNT (aryl hydrocarbon receptor nuclear transporter) and forms the active heterodimeric transcription factor that induces the expression of erythropoietin, vascular endothelial growth factor, and glycolytic enzymes. Hif-1 is a nonheme iron protein and oxygen binding at this center is thought to be the signal for ubiquitination by a multienzyme complex containing the elongin BC complex, Cul2, and the von Hippel-Lindau tumor suppressor protein (Lonergan et al. 1998 , Maxwell et al. 1999 ). Mutations in the latter protein lead to a variety of highly vascularized tumors in humans. It should be noted that elongin C is a homolog of Skp1 and elongin B contains a ubiquitin-like domain suggesting a similarity to the structure and function of the Skp1/Cdc53/F-box class of ubiquitin ligases.

In most other cases, the exact nature of the regulation is not known, although it is common that the transcription factor itself is degraded by a ubiquitin-dependent pathway and that the ubiquitination is modulated by ligand binding or heterodimerization.

	Proteolysis to Repair Cellular Damage.

TOP ABSTRACT INTRODUCTION Targeting Functions of the... Conjugation and Deconjugation of... Role of Ubiquitination in... Proteolysis to Repair Cellular... Checkpoint Regulation: Repair... Summary. REFERENCES

 
Stress, viral infection, oxidative and chemical damage, or the production of defective proteins also trigger vital cellular response. These can be thought of as rescue operations, either to repair the damage, or to limit its toxicity to the cell.

ER quality control.

The degradation of abnormal ER and secretory proteins has been shown to be ubiquitin-dependent in yeast and mammalian cells (Plemper & Wolf 1999 ). Yeast proteins resident to or traversing the ER and that are misfolded or inappropriately glycosylated undergo retrograde transport to the cytoplasm in a reaction that requires the Sec61p transporter and other ER membrane proteins. The protein is then ubiquitinated in a process requiring Cue1p, Ubc6p and Ubc7p and is then degraded by the cytoplasmic proteasome. A significant pool of proteasomes has been shown to be perinuclear, suggesting that the machinery for this ubiquitination and degradation may be localized at the ER membrane (Enenkel et al. 1999 ). Thus, export and degradation may be tightly coupled.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular signaling pathway from the ER to the nucleus (Kaufman 1999 ), termed the unfolded protein response (UPR). In yeast, the production of abnormal proteins in the lumen of the ER results in the dimerization of Ire1p, a transmembrane signaling molecule. Dimerization activates the endonuclease activity of Ire1p and this activity initiates an unusual cytoplasmic splicing reaction that results in higher levels of translation of the Hac1 message (Kaufman 1999 ). Hac1p is normally kept at low levels by inefficient translation of the message and ubiquitin-dependent proteolysis of the protein. The increased levels of Hac1p bind to the UPR response element to induce the synthesis of a variety of ER chaperones and the enzymes necessary to expand the ER compartment to deal with the damaged proteins. The pathway is similar, but more complicated, in mammalian cells.

The aggresome is a site for degradation of abnormal proteins.

When the cell is unable to deal with large amounts of damaged or denatured proteins, these proteins appear to accumulate as inclusions at the centrosome/microtubule organizing center (Anton et al. 1999 , Wigley et al. 1999 ). This structure has been called the aggresome (Johnston et al. 1998 ). Recent evidence suggests that the abnormal proteins associate with the microtubules and are transported to the centrosome. A significant fraction of the proteins are ubiquitinated and other components of the ubiquitin pathway are also sequestered there, including the ubiquitin binding protein p62, ubiquitin, the proteasome, Hsp70 and Hsp90. It is not known if ubiquitination occurs before or after aggregation but it is tempting to speculate that ubiquitination may be a signal to dock to the microtubules and be transported to the site(s) where proteasomes are abundant. The presence of the heat shock proteins (Hsp) suggests that active refolding may also be an activity of this structure or required for overall proteolysis. Clearly though, this structure is intimately involved in degrading and perhaps refolding damaged proteins that accumulate in the cytoplasm.

	Checkpoint Regulation: Repair vs. Apoptosis.

TOP ABSTRACT INTRODUCTION Targeting Functions of the... Conjugation and Deconjugation of... Role of Ubiquitination in... Proteolysis to Repair Cellular... Checkpoint Regulation: Repair... Summary. REFERENCES

 
DNA damage and a number of other extracellular signals modulate steps regulating progression through the cell cycle. These checkpoints are necessary to assure that damage and recombination events are fully resolved before replication of the genome. In general, the cell responds to this damage by either repairing it, or undergoing apoptosis (Orlowski 1999 ). The classic example is that of p53 (Amundson et al. 1998 , Scheffner 1998 ). The amount of this unstable transcription factor is increased by various cellular insults and is thought to activate the transcription of genes involved in pausing the cell cycle. Some oncogenic viruses escape this regulation, at least in part, by increasing the rate of ubiquitin-dependent degradation of this regulator (Scheffner 1998 ). Very recently it has been shown that the ataxia-telangiectasia related protein ATR mediates the DNA-dependent phosphorylation of p53 (Lakin et al. 1999 ). This inhibits binding of p53 to Mdm2, a protein that is thought to target p53 for ubiquitin-dependent degradation. Thus, the signal from damaged DNA may be transduced to this transcription factor via phosphorylation to mount the cellular response. In yeast, DNA damage also prevents the metaphase to anaphase transition, in part by regulating the levels of the E3 ligase APC/C (the anaphase promoting complex, also known as the cyclosome) (Tinker-Kulberg & Morgan 1999 ). This multienzyme complex is responsible for ubiquitinating a variety of cyclins and the anaphase inhibitor Pds1p. A number of proteins are involved in regulating this transition by modulating the activity of the APC/C. These have been collectively termed the mitotic exit network.

When attempts to repair the damage are unsuccessful the cells undergo apoptosis. There seem to be multiple ways to trigger this pathway, including damage to the genome and signaling from the plasma membrane receptors such as those for TNF or FAS. Proteasome inhibitors have been variously reported to activate or inhibit apoptosis, although it appears that some of the differences are due to the proliferative state of the cells used (Drexler 1998 ) and the levels of inhibitor employed (Lin et al. 1998 ). The ubiquitin-like protein SUMO-1 and its E2 conjugating enzyme (Ubc9) have been reported to bind to the intracellular domain of the TNF-R1 and Fas/APO1 receptors (Liou & Liou 1999 ) and influence apoptosis Finally, several substrates for ubiquitin-mediated proteolysis also regulate apoptosis, notably: p53 (Amundson et al. 1998 , Scheffner 1998 ), NFB/IB (Maniatis 1999 ), bcl2 (Dimmeler et al. 1999 ), bax, mdm2(Chang et al. 1998 ), c-myc and E2F-1 (Hengstschlager et al. 1999 ).

	Summary.

TOP ABSTRACT INTRODUCTION Targeting Functions of the... Conjugation and Deconjugation of... Role of Ubiquitination in... Proteolysis to Repair Cellular... Checkpoint Regulation: Repair... Summary. REFERENCES

 
The themes emphasized in this work are that the ubiquitin domain is a targeting signal and that a very wide variety of cellular processes are regulated by ubiquitination events. The three major ways in which a protein can be recognized and ubiquitinated are through damage events or misfolding, via constitutively active ubiqutination signals, or via inducible modifications of the protein such as phosphorylation or cofactor binding. These proteolytic events can destroy active proteins, degrade inhibitors to generate active proteins, generate the active protein via proteolysis, or via translocation to the proper cellular compartment. In the coming years, many more roles of this pathway will come to light and its impact and importance is already beginning to rival that of the kinase/phosphatase cascades.

Address correspondence to: 4017 Rollins Research Building, Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322-3050

	FOOTNOTES

 
¹ This work was supported by grants from the National Institutes of Health GM30308, HD35576 and RR11418.

² Manuscript received 8 September 1999

⁴ Amino acids are shown using the one-letter code. Proteins from yeast end with the suffix p. Ubiquitin-like proteins and their accession numbers are: SUMO-1, Swiss:Q93068; Smt3p, Swiss:Q12306; ISG15, Swiss:P05161; Apg12p, PIR:S46093; Nedd8, Swiss:Q15843; Rub1p, Swiss:Q03919. Other abbreviations are: APC/C, an E3 ubiquitin ligase complex known as the Anaphase promoting complex or the cyclosome; ß-TrCP, beta-transducin repeat-containing protein; UPR, unfolded protein response; DUBs, deubiquitinating enzymes; E1, ubiquitin activating enzyme; E2, ubiquitin conjugating enzyme; E3, ubiquitin protein ligase; E6AP, the E3 ubiquitin ligase known as E6 associated protein; ER, endoplasmic reticulum; HECT-domain, homology to E6AP C-terminus; Hsp, heat shock protein; IKK, IB kinase; IL-3, interleukin 3; NIK, NFB inducing kinase; PA700, the 19S regulator complex of the proteasome. SCF, an E3 ubiquitin ligase complex containing Skp1p, Cdc53p, Roc1p, and an F-box containing protein.

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TOP ABSTRACT INTRODUCTION Targeting Functions of the... Conjugation and Deconjugation of... Role of Ubiquitination in... Proteolysis to Repair Cellular... Checkpoint Regulation: Repair... Summary. REFERENCES

 

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