Download PHD domains and E3 ubiquitin ligases: viruses make the connection

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Vol.13 No.1 January 2003
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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
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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.
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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
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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?
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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
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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
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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).
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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. But if RINGfinger proteins are any guide, then E3 activity might be
ultimately found in a substantial proportion of PHD
proteins. Time will tell.
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