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Opinion TRENDS in Cell Biology Vol.13 No.1 January 2003 7 PHD domains and E3 ubiquitin ligases: viruses make the connection Laurent Coscoy and Don Ganem Departments of Microbiology and Medicine, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143-0414, USA PHD domains constitute a widely distributed subfamily of zinc fingers whose biochemical functions have been unclear until now. Recently, several PHD-containing viral proteins have been identified that promote immune evasion by downregulating proteins that govern immune recognition. Studies show that these viral regulators lead to ubiquitination of their targets by functioning as E3 ubiquitin ligases – an activity that requires the PHD motif. These are the first examples linking the PHD domain to E3 activity, but the recent discovery of PHD-dependent E3 activity in the cellular kinase MEKK1 and the close structural relation of PHD domains to RING fingers hint that many other PHD proteins might share this activity. The plant homeodomain (PHD) motif is a sequence encoding a specialized form of zinc finger that was first recognized in an Arabidopsis homeobox protein by Schindler et al. [1] nearly a decade ago. Since that time, related motifs have been identified in over 400 eukaryotic proteins, many of which are nuclear DNA-binding proteins with known or suspected roles in regulating chromatin organization or gene expression [2]. But a clear picture of the exact biochemical roles of the PHD domain has remained elusive. Although early speculations about PHD function centered around possible roles in directly binding DNA [3], more recent views have emphasized the idea that PHD domains, like the related LIM and RING finger domains, are probably involved in protein – protein interactions [4,5]. Despite these speculations, the full repertoire of biological processes that might be controlled by PHDmediated interactions has been largely unexplored. Our view of the magnitude and diversity of that repertoire has undergone considerable expansion in the past year, with the discovery of several new PHD-containing proteins that are targeted to cellular membranes [6,7,14] and the cytosol [8]. This new work reveals that these proteins are charter members of a novel class of E3 ubiquitin ligases. These E3 ligases control the trafficking and/or degradation of target proteins involved in different cellular functions outside the nucleus and raise the possibility that other PHD-containing proteins might also function as E3 ligases. Here we discuss the events that led to these advances and consider their broader implications. Corresponding author: Don Ganem ([email protected]). Viruses open the door The story begins with studies that were aimed at a different issue – namely, the evasion of host immunity by viral infection. Many viruses have evolved strategies for evading T-cell-mediated host immunity by preventing the surface display of major histocompatibility complex (MHC) class I molecules [9,10]. Class I molecules are normally involved in presenting antigenic peptides derived from the proteolysis of viral proteins to cytotoxic T lymphocytes (CTLs) expressing T cell receptors (TCRs) specific for such peptides. As outlined in Fig. 1, the antigen presentation pathway involves the import into the endoplasmic reticulum (ER) of peptides generated by the proteasome in the cytoplasm. The imported peptides are bound by the peptide-binding groove of assembling MHC class I chains in the ER; in fact, only chains that have bound peptide are capable of stable assembly in this organelle [11,12]. After export from the ER, vesicular transport delivers the peptide– MHC complexes to the plasma membrane, where they can be recognized by CTLs expressing the appropriate TCR. Recognition reaction results in a polarized delivery of cytotoxic materials to the target (virus-infected) cell, leading to its destruction. This is a central mechanism of host defense against many viruses including those of the herpesvirus family – a fact that is strongly attested by the increased severity of herpesviral infections in individuals with defects in T cell function. Accordingly, in the course of their evolution herpesviruses have acquired several functions aimed at reducing the surface display of MHC class I molecules and thereby (partially) protecting themselves from the actions of host CTLs [9,10]. Some of these viral functions bind and retain MHC class I chains in the ER, some lead to their ERassociated degradation and others block the import of peptides into the ER, thereby preventing the maturation and egress of MHC class I molecules. Kaposi’s sarcoma-associated virus (KSHV) is a recently identified herpesvirus that is causally linked to the development of Kaposi’s sarcoma – a common neoplasm of individuals suffering from AIDS [13]. To define KSHV genes involved in the evasion of host immunity, we screened a large collection of viral genes for their ability to downregulate surface MHC class I molecules in cultured cells after being delivered by retroviral vectors [6]. This resulted in the identification of two viral genes, termed K3 and K5, that each specifically downregulate human MHC http://ticb.trends.com 0962-8924/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(02)00005-3 Opinion 8 TRENDS in Cell Biology Vol.13 No.1 January 2003 + MIR1, MIR2 Peptide b2-micro globuline Golgi Lysosome MHC-I heavy chain MK3 Properly assembled MHC-I protein TAP pump Endoplasmic reticulum (ER) Proteasome Nucleus TRENDS in Cell Biology Fig. 1. The MHC class I antigen presentation pathway. Either self or foreign antigens are ubiquitinated and degraded into small peptides by the proteasome. These peptides, after being imported into the ER by transporter associated with antigen processing (TAP), are loaded onto newly synthesized MHC class I molecules associated with b2microglobulin. Proper assembly of this complex is required for both its transport to the cell surface and its stable expression. The KSHV MIR proteins enhance endocytosis of MHC class I chains from the plasma membrane, after which they are directed to the lysosome by the multivesicular body (MVB) pathway. The related MK3 protein of MHV68 promotes the degradation of ER-associated MHC class I chains by cytosolic proteasomes. class I molecules without affecting the surface display of many other polypeptides; similar results have been obtained by others [7,14,15]. Although not detectably related to previously identified viral or host genes, K3 and K5 were found to encode proteins that share 40% identity. The resulting gene products are now called, respectively, modulator of immune recognition (MIR)-1 and MIR2. The sequence organization of the MIR proteins predicted an amino (N)terminal PHD domain [16], two putative transmembrane domains and a short conserved region just distal to the second transmembrane domain. Confocal microscopy showed that the bulk of these chains are located in the ER [6,14,17], and biochemical analyses indicated that the PHD motif and the conserved region element are arranged on the cytosolic side of the membrane [18] (Fig. 2). As noted above, although many viruses encode molecules that lead to ER retention or ER-associated MHC-I B7.2 ICAM-I MIR1 or MIR2 Cytosol K E2 K Ub Endocytosis TRENDS in Cell Biology Fig. 2. Model of MIR target recognition and ubiquitination. Transmembrane and juxtamembrane regions of MIR and their target (MHC class I) chains mediate an interaction in the plane of the membrane; this juxtaposes the N-terminal PHD domain of the MIR (with its associated E2 activity) to the cytosolic tail of the target, thereby facilitating ubiquitination (Ub) of its substrate lysine (K) residues. The ubiquitinated intracytoplasmic region of the targets are then recognized by the endocytic machinery, which promotes their removal from the cell surface and their subsequent routing to, and degradation by, the lysosome. http://ticb.trends.com degradation of MHC class I molecules [9,10], it soon became clear that this was not the mechanism by which the KSHV MIR proteins function. MHC class I chains in MIR-expressing cells exit the ER and arrive at the plasma membrane with normal kinetics, but are subsequently internalized into vesicular structures in a dynamindependent fashion, which strongly suggests an upregulation of MHC class I endocytosis [6,14]. After internalization, MHC class I chains in MIR-expressing cells undergo degradation in the lysosome, as indicated by the inhibition of this degradation by chloroquin, bafilomycin and other inhibitors of endolysosomal acidification [6]. Although distinct from the host pathways targeted by other viral regulators of MHC class I, this pathway strongly recalls features of the regulated endocytosis of growth factor receptors, such as the growth hormone and epidermal growth factor receptors (EGFRs), in the presence of their ligands – a pathway that also involves endocytosis and lysosomal degradation [19]. In those cases, activated receptors recruit kinases that phosphorylate tyrosines in the cytosolic tails of the receptors, thereby recruiting E3 ubiquitin ligases (for EGFR, for example, the E3 RING-finger protein known as c-Cbl) [20,21]. These ligases result in ubiquitination of the receptor cytosolic tail, triggering endocytosis and, ultimately, delivery of the endosomal contents to the lysosome. Notably, in yeast the internalization of many different proteins and the subsequent delivery of their endosomes to the lysosome-like vacuole are steps that are also controlled by ubiquitination ([19,22,23] and see below). The involvement of ubiquitin in the regulation of endocytosis, in conjunction with the known structural similarity of PHD domains to RING fingers [24,25] – which are prominently linked to E3 activity [26] – raised the possibility that the PHD domains in MIR proteins might have an analogous E3-like function. Indeed, it was discovered that MHC class I chains undergo ubiquitination in the presence of MIR1 or MIR2, and that mutational ablation of the two lysine residues in the MHC class I cytosolic tail blocks both ubiquitin addition and MHC class I downregulation [27,28]. In addition, fusion proteins containing the MIR2 PHD domain can undergo autoubiquitination in vitro in the presence of E1, E2, ubiquitin Opinion TRENDS in Cell Biology and ATP – a signature of proteins with E3 ubiquitin ligase activity [27]. This ligase activity is inactivated by mutation of the zinc-coordinating residues of the PHD domain – mutations that also inactivate the downregulation of MHC class I molecules in vivo [14,18]. Thus, KSHV MIR1 and MIR2 define a new family of membrane-bound E3 ubiquitin ligases that rely on PHD domains rather than on RING fingers. …MIR1 and MIR2 define a new family of membrane-bound E3 ubiquitin ligases that rely on PHD domains rather than on RING fingers The resulting ubiquitinated MHC class I chains not only undergo enhanced endocytosis but are also directed preferentially to lysosomes. In both yeast and mammalian cells, the latter trafficking steps are usually accomplished through the multivesicular body (MVB). This structure is formed by invagination of the late-endosome membrane to generate internal vesicles into which proteins destined for the lysosome (such as MHC class I) are sorted. This sorting requires ubiquitination of the target protein and the function of Vps23, which is part of a multisubunit complex called endosomal complex required for transport (ESCRT)1 involved in the recognition and disposition of ubiquitinated target proteins in the endosome [23,29,30]. Although all the details of the regulation of sorting into the MVB are still being worked out, it is clear that events broadly similar to those described in yeast are occurring in higher cells. For example, TSG101, the mammalian homolog of Vps23, is known to function in late endosome sorting in mammalian cells [31]. Recent elegant studies of Hewitt et al. [28] show that siRNA-mediated depletion of TSG101 also blocks MIR-mediated MHC class I degradation, which strongly suggests that the same pathway is used for the disposal of endocytosed MHC class I chains. But many questions remain unanswered in MIRmediated regulation. For example, is all of the control by MIRs exerted at the initial (dynamin-dependent) internalization step, or are downstream events in the MVB pathway also being directly regulated? The complexity of the downstream events is underscored by the recent findings of Lorenzo et al. [32], which show that proteasome inhibitors, although not affecting the MIR-mediated removal of MHC class I from the cell surface, impair one or more distal steps in the delivery of the target chains to the lysosome. This inhibition is probably indirect, but its exact mechanism awaits clarification. How do MIR proteins recognize their targets? RING finger E3 ligases function by recognizing ubiquitinactivated E2 ubiquitin-conjugating enzymes and delivering them to their targets, usually by direct interaction between the E3 ligase and its target. Clearly, in the MIRs the PHD domain recognizes and recruits the E2 enzyme [27], as does the homologous RING finger in classical E3 ligases. But what accounts for the target recognition? http://ticb.trends.com Vol.13 No.1 January 2003 9 Central to deciphering this is the fact that MHC class I is not the only target of MIR regulation. MIR2, but not MIR1, can also downregulate two other molecules involved in immune recognition: B7.2 and ICAM-I [33,34]. B7.2 is a costimulatory signaling molecule involved in activating helper T cells; ICAM-1 is an intercellular adhesion molecule that functions at the immunological synapse. By making chimeras of MIR1 and MIR2 and looking for the ability of the resulting protein to downregulate these MIR2-specific targets, Sanchez et al. [18] showed that the transmembrane domains of MIR are the key to target selectivity. In addition, similar studies using chimeras of human and mouse MHC class I proteins (human but not mouse MHC class I chains are downregulated by MIR2) suggest that the transmembrane domain of the target proteins is the site of MIR recognition [27]. Figure 2 summarizes these relationships in a model of how MIR proteins function in target recognition and ubiquitination. In this model, the transmembrane domains of MIR proteins interact with those of the target proteins; whether this binding is direct or indirect (i.e. involves additional cellular cofactors) is not yet known. This interaction juxtaposes the appropriate E2 (bound to the N-terminal PHD of the MIR) to the cytosolic tail of the target, which can then be ubiquitinated. But a conundrum remains. The bulk of the MIR chains accumulate in the ER, yet the functional data clearly indicate that this MIR – MHC interaction results in internalization at the plasma membrane. This conundrum could be reconciled if MHC chains undergo ubiquitination in the ER, and this modification marks them for subsequent internalization once they reach the plasma membrane. This model has been rendered unlikely, however, by experiments that show that cell-surface MHC class I chains made in the absence of MIRs can be endocytosed on subsequent MIR expression (L. Coscoy and D. Ganem, unpublished). More likely, a small subset of MIR proteins can escape the ER to act distally in the vesicular pathway. Consistent with this, direct co-immunoprecipitation studies show that MIR proteins can be detected in complexes with MHC chains, and that the ubiquitinated MHC chains found in these complexes are resistant to endonuclease H [28], suggesting that the interaction occurs primarily in a postER compartment. The functional diversity of herpesviral homologs of MIR KSHV is not the only herpesvirus to encode MIR-like proteins. Stevenson et al. [7] examined the immune evasion functions of a murine herpesvirus, MHV68, and identified a single gene, MK3, that encodes a homolog of MIR1. Like MIR1, this protein downregulates the surface display of MHC class I chains and possess an N-terminal PHD domain and two transmembrane domains. It, too, is primarily localized in the ER [7] and shares a similar transmembrane orientation [35]. In striking contrast to MIR1, however, MK3 acts by promoting the ER-associated degradation of MHC class I chains. This degradation is strongly blocked by proteasome-specific inhibitors and takes place in the cytosol [35]. MK3 protein interacts directly with MHC class I chains in Opinion 10 TRENDS in Cell Biology Vol.13 No.1 January 2003 the ER, leading to ubiquitination of the ER-associated chains in vivo. Like the MIR proteins, MK3 does not require its PHD domain to bind to its target; however, mutation of the PHD domain completely ablates ubiquitination [35]. Although it was not shown directly that MK3 can catalyze ubiquitin transfer in vitro, in the context of the work on KSHV MIR proteins there would seem to be little doubt that it can. These two classes of viral E3 ligases thus show us a remarkable functional diversity. How is it that two proteins that are related in sequence with similar domain structures, localized to the same organelle and capable of promoting ubiquitination of their (common) targets can have such markedly different processes for the disposition of their targets? Clearly, there must be additional levels of regulation superimposed on the system. For example, KSHV MIR chains might be associated with other host proteins in the ER that negatively regulate their ability to promote ubiquitination in this organelle. Alternatively, the MIR proteins might lack the ability to interact with an ER-affiliated E2 enzyme that is recognized by MK3. A third possibility is that MIRs might require additional positive regulators that are themselves localized to more distal compartments. And there’s more The sequences of other viral genomes indicate that transmembrane proteins related to the MIRs are present in a considerable of large DNA viruses, mostly belonging to the herpesvirus and poxvirus families. Figure 3a shows the alignment of the PHD regions of these virally encoded MIR homologs. (Note that all of these proteins also have two transmembrane domains [not shown] that are located carboxy (C)-terminal to the PHD, just as in the MIRs, which further affirms their grouping with the MIRs and MK3 as a distinct subfamily of PHD proteins). Little is known about most of these proteins, but it would be surprising if some of them were not involved similarly in immune evasion. Indeed, a report has already been published implicating a MIR homolog in myxoma virus, a poxvirus of rabbits, in MHC class I downregulation [36]. Close inspection of the primary sequence of the viral MIR-like molecules suggests that these PHD regions differ subtly but recognizably from canonical PHD elements, especially in the spacing of zinc-coordinating cysteines 3 and 4, and in the conservation of a tryptophan residue between cysteines 6 and 7 (Fig. 3b). These features are not shared with the bulk of conventional PHD-containing proteins (Fig. 3b), but the functional significance of these differences remains to be explored. Although all of these examples are from viruses, like most discoveries in virology they probably presage similar findings in host proteins. In fact, a study has already reported that MEKK1, a cellular mitogen-activated protein (MAP) kinase kinase kinase that phosphorylates several different MEKs, contains a PHD domain and has E3 activity. The N-terminal PHD domain of MEKK1 is well separated from its C-terminal kinase domain. Activation of MEKK1 by osmotic shock leads to activation of a MAP kinase cascade that results in prompt phosphorylation of extracellular-signal-regulated kinases 1 and 2 (ERK1/2). The ERK1/2 chains are degraded after their activation – a reaction that is associated with polyubiquitination and (a) Homology – Consensus KSHV MIR1 KSHV MIR2 BHV4 pBo5 BHV4 pBo4 MHV68 MK3 Sheep LAP/PHD Swine ORF C7L YLD ORF 5L LSD ORF E3L Myx ORF m153R Fib gp153R + CWICKDEEGVEK-NYCNCKGELKVVHKECLEEWINTS--RNKSCKICNTPY 10 20 30 40 50 CWICNEELGNERFRACGCTGELENVHRSCLSTWLTIS--RNTACQICGVVY CWICREEVGNEGIHPCACTGELDVVHPQCLSTWLTVS--RNTACQMCRVIY CWICRDGESLPEARYCNCYGDLQYCHEECLKTWISMS--GEKKCKFCQTPY CWICKGSEGIIDVKYCHCIGDLQYVHSECLVHWIRVS--GTKQCKFCQYTY CWICHQPEGPLK-RFCGCKGSCAVSHQDCLRGWLETS--RRQTCALCGTPY CWICKDEYNVSA-NFCNCKNEFKIVHKNCLEEWINFS--HDTKCKICNGKY CWICKDDYSIEK-NYCNCKNEYKVVHDECMKKWIQYC--RERSCKLCNKEY CWICNDVCDERN-NFCGCNEEYKVVHIKCMQLWINYS--KKKECNLCKTKY CWICKDEYNVST-NFCNCKNEFKIVHKNCLEEWINFS--HNTKCKICNGKY CWICKEACDIVP-NYCKCRGDNKIVHKECLEEWINTDVVKNKSCAICESPY CWICKESCDVVP-NYCKCRGDNKIVHKECLEEWINTDTVKNKSCAICETPY (b) Viral PHDs C--W--I--C-X(10-11)-C---X----C---X6---Φ-H-X2-C-Φ-X2-W-X3-S/D-X(4-6)-C-X2-C MEEK1 C--P--I--C----X12---C---X3---C---X3---Φ-H-X2-C-Φ-X2-W-X3-C----X12---C-X2-C PHD C-X(1-2)-C--X(7-21)-C-X(2-4)-C-X(3-4)-Φ-H-X2-C-Φ----X(9-43)---W--X--C-X2-C RING C-X(1-2)-C--X(9-39)-C-X(1-3)-H-X(1-2)-Φ-C-X2-C-Φ----X(3-47)---------C-X2-C TRENDS in Cell Biology Fig. 3. Viral and cellular PHD motifs. (a) Sequence alignment of the PHD regions of predicted proteins found in other viral genomes. The consensus sequence of the related PHD domains is shown on top; details of the alignment are given below. BHV4, bovine herpesvirus 4; Fib: rabbit fibroma virus; KSHV, Kaposi’s sarcoma-associated herpesvirus; LSD, lumpy skin disease virus; MHV68: murine herpesvirus 68; myx: myxoma virus; Sheep, sheeppox virus; Swine, swinepox virus; YLD, yaba-like disease virus. (b) Cardinal features of the zinc-coordinating regions of viral MIR-like PHD motifs (first row), the MEKK1 PHD domain (second row), canonical PHD domains (third row) and classical RING-finger domains (fourth row). http://ticb.trends.com Opinion TRENDS in Cell Biology blocked by proteasome inhibitors. The ability of MEKK1 to associate with ERKs, coupled with its clear involvement of ubiquitin-mediated proteolysis, suggests that MEKK1 might be the E3 ligase that directs this proteolysis. This hypothesis has been validated by direct biochemical experiments that unambiguously show that this protein indeed possesses E3 ligase activity for the ERKs [8]. This activity is PHD-dependent, as mutations in the core of this domain abolish E3 function. Notably, when the PHD domain of MEKK1 is aligned with other PHD motifs, it seems to lack the characteristic C3 – C4 spacing of the viral MIR-like proteins, but it conserves the tryptophan residue found in the viral family (Fig. 3b). Concluding remarks The MIR and MK3 proteins represent the pioneer members of a class of membrane-bound PHD-containing proteins that regulate the accumulation and trafficking of target membrane proteins by acting as E3 ubiquitin ligases. The association of E3 activity with PHD domains strongly recalls the association between E3 activity and RING fingers, to which PHDs are closely related structurally. We speculate that, as for RING finger-containing proteins, additional PHD-containing proteins not previously suspected of being E3 ligases will be found to have this activity. The findings with MEKK1 accord with this notion and, more importantly, suggest that E3 ligase activity will not be limited to PHD-containing proteins that are membrane-bound, nor will the functional roles of these proteins be restricted to governing the trafficking of membrane or secretory proteins. Rather, it seems likely that E3 ligases of this family will be found in many cellular compartments and mediate different functions in the cell economy. A larger question – and one for which no firm answer is available as yet – is what fraction of PHD-containing proteins are E3 ligases? It is possible, of course, that the MIRs and their relatives are special cases that are not representative of PHD proteins as a whole. 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