From the Department of Molecular and Experimental
Medicine, The Scripps Research Institute, La Jolla, California
92037 and § The Cleveland Clinic Foundation,
Cleveland, Ohio 44195
Received for publication, August 18, 2002, and in revised form, February 7, 2003
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ABSTRACT |
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ISG15 is a ubiquitin-like protein that conjugates
to numerous proteins in cells treated with interferon or
lipopolysaccharide. Dysregulation of protein ISG15 modification
(ISGylation) in mice leads to decreased life expectancy, brain cell
injury, and hypersensitivity to interferon. Although ISG15 was
identified more than two decades ago, the exact biochemical and
physiological functions of ISG15-modification remain unknown, and the
proteins targeted by ISG15 have not been identified. The major purpose
of this work was to identify ISG15 targets among well characterized
proteins that could be used as models for biological studies. We
purified ISGylated proteins from human thymus by immunoaffinity
chromatography and analyzed ISG15 conjugates by a high-throughput
Western blot screen (PowerBlotTM). We found that
three key regulators of signal transduction, phospholipase C ISG15 is one of the most strongly induced genes after interferon
(IFN)1 treatment (1, 2) and
is also significantly induced by viral infection (3) and
lipopolysaccharide (LPS) (4). The sequence of ISG15 protein was noted
to possess significant homology to a diubiquitin sequence, accounting
for its cross-reactivity with some anti-ubiquitin antibodies (3).
Several reports demonstrate that ISG15 is released by various cell
types and can act as a cytokine leading to proliferation of NK cells
(5). It has also been demonstrated that ISG15 is induced in the uterine
endometrium during early pregnancy and was suggested to play a
significant role in embryo implantation (6). ISG15 sequences are absent in yeast, nematode (Caenorhabditis), plant
(Arabidopsis), and insect (Drosophila) indicating
that the ISG15 conjugation system is restricted to higher animals with
evolved IFN signaling.
Most remarkably, ISG15 was found to be conjugated to intracellular
proteins via an isopeptide bond in a manner similar to ubiquitin (Ub)
and other Ub-like proteins (Ubls) such as SUMO and Nedd8 (7).
Conjugation of Ubls involves a three-step mechanism whereby specific
enzymes (or enzyme complexes) activate and covalently link Ubls to
their substrates (8, 9). ISG15 conjugation occurs via a similar but
distinct pathway compared with Ub conjugation (10), and an activating
enzyme for ISG15 has recently been rediscovered as specific to ISG15
and not Ub (11). Similar to modification by other Ubls, the conjugation
of ISG15 is reversible and is accomplished by a highly specific
protease UBP43 (12). Based on sequence homology, UBP43 belongs to a
family of Ub-specific proteases and was initially cloned in our
laboratory from leukemogenic AML-ETO knock-in mice (13, 14).
UBP43 has since been cloned independently by other groups
(15-17). UBP43 is induced by IFN, LPS, and viral infection
and, similarly to the ISG15 gene, is regulated via p38 MAPK
pathway and IFN regulatory factor 3 (18). A
UBP43 A major reason for the undefined biochemical function of
ISG15 conjugation is the lack of known ISG15 targets that may serve as
experimental models. Only one target, serpin 2a, has recently been
reported, but its exact function is not known (23). In this study we
used a large-scale Western blot-based screening process to identify new
targets of ISG15 modification from a group of well characterized signal
transduction proteins.
Here we report the first systematic attempt to establish the identity
of ISGylated proteins. We show that several key regulators of signal
transduction (namely, PLC Immobilization of Antibodies and Purification of ISGylated
Proteins--
Hybridoma (clone 2.1) (24) that produces monoclonal
mouse anti-human ISG15 antibodies was cultured in
Hybridoma-serum-free medium (Invitrogen, Carlsbad, CA). Cells
were removed by centrifugation at 2000 × g for 5 min,
and the supernatants were filtered through a 0.22-µm filter to remove
debris. IgGs were purified on a protein G column (Amersham
Biosciences) according to manufacturer's instructions. Eluted
IgGs were dialyzed against coupling buffer (0.2 M
Na2HPO4, 0.2 M NaCl, pH 8.5). Six
mg of purified IgGs were mixed with 1 ml of glyoxal-activated agarose
(Active Motif, Carlsbad, CA) and NaBH3CN (Sigma) to a final
concentration of 50 mM. Coupling proceeded overnight at
24 °C with constant rocking. Coupled resin was removed, and the
supernatant was allowed to couple with a fresh batch of glyoxal-activated agarose. Success of immobilization was determined by
measuring protein concentration in the buffer before and after coupling
and was estimated to be 2.6 and 1.6 mg of IgG per ml of resin after
first and second coupling, respectively. Unreacted sites on coupled
resin were blocked with 10 mM ethanolamine, pH 8.0 (2 h at
24 °C). IgG-resin was washed sequentially with 10 volumes of each of
the following: PBS, 1% Triton X-100 in PBS, PBS, 2 M NaCl
in PBS, and PBS and stored in PBS containing 0.01% thimerosal (Sigma)
at 4 °C. Immediately before use in immunoaffinity purification of
ISG15 conjugates the IgG-resin was washed with 10 volumes of 0.1 M glycine, pH 2.5.
Thymus samples were obtained (with ethical approval) from children
(aged 1-10 years) during routine cardiac surgery. Nine grams of tissue
were macerated with a razor blade and homogenized using a tissue
homogenizer in 25 ml of RIPA buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.1% w/v SDS, 1% v/v
Triton X-100, 0.5% w/v sodium deoxycholate, 2 mM
phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma,
product number P2714)). The slurry was sonicated (four 30-s pulses),
and insoluble material was removed by 10-min centrifugation at
18,000 × g. This extraction procedure yielded 500 mg
of total protein. Supernatant was stored at High-throughput Western Blot Screening and Data
Analysis--
Primary screening (pay per service) was performed at BD
Biosciences Transduction Laboratories (Lexington, KY) using the
PowerBlotTM assay. Briefly, the purified ISG15 conjugates
were separated on six high-resolution gradient gels and transferred
onto nitrocellulose membranes. Each membrane was divided into 40 lanes
by applying a chamber-forming grid. To each chamber a mixture of mAbs
was added (1-8 mAbs per mixture; identities of mAbs and location with respect to lanes and templates are available from BD Biosciences Transduction Laboratories). After 1 h of incubation at room
temperature the chambers were rinsed and incubated with secondary
antibodies under the same conditions (Alexa 680-labeled goat anti-mouse
IgGs; Molecular Probes, Eugene, OR). The images were acquired with an infrared scanner (Li-Cor, Lincoln, NE). The bands were identified, and
molecular masses were assessed using specialized software by the
service. Each observed band, where possible, was manually matched
against the expected molecular mass of a protein recognized by an
individual antibody in the mixture. The computer-generated data were
scrutinized in our laboratory by careful manual analysis and were found
to be more than 90% accurate.
Immunoprecipitations and Western Blots--
To verify the
results of the primary screen we performed immunoprecipitations (IPs)
with additional controls as well as reciprocal IPs. Anti-Stat1 (rabbit
polyclonal, antipeptide) antibodies were from Santa Cruz Biotechnology
(Santa Cruz, CA). Anti-ERK1, anti-PLC
With the exception of anti-ERK1 (lysate was prepared in denaturing
conditions as recommended by the manufacturer of mAbs), all lysates
were prepared and IPs were performed in RIPA buffer. Protein
A-Sepharose was used with polyclonal antibodies, and protein G-Sepharose was used with mAbs. Western blotting was performed as
previously described (12).
Cell Culture--
Wild type and
UBP43 Primary Screening by High-throughput Western
Blot--
ISGylated proteins purified from human thymus by
immunoaffinity chromatography were submitted for analysis by BD
Transduction Laboratories. The proteins in this assay separated on
SDS-PAGE and transferred onto nitrocellulose membrane, and the membrane is divided into multiple lanes. Each individual lane is incubated with
a blend of several specifically selected monoclonal antibodies that
recognize proteins of non-overlapping molecular weights (see "Materials and Methods" for further details). The assay uses 860 individual mAbs of which 710 cross-react with human proteins. Some of
the mAbs were different clones recognizing the same protein or
phosphorylated versions of the same protein. Therefore, 645 individual
proteins can be detected by this technology in various human tissues
and cells. Computer-assisted and subsequent manual examination of
detected signals revealed 149 bands. Based on the confidence with which
the identity of the protein could be deduced we separated the bands
into three groups. Group one (72 bands) included proteins that (a)
matched very well the expected molecular mass of unmodified proteins
and (b) were unlikely to be the result of a smaller protein that has
been modified by ISG15. Proteins of this group most likely were
nonspecifically bound to IgG-resin (either agarose itself or IgGs) but
also might have been co-purified with ISGylated protein(s). Group one
was treated as irrelevant and is not discussed in this study. Group two
(73 bands) incorporated the bands of ambiguous identity. Apparent
molecular masses of group two bands corresponded to an expected
molecular mass of an unmodified protein but also may correspond to a
molecular mass of another protein modified by one or two molecules of
ISG15. The proteins that belong to this group are currently being
validated in our laboratory.
Molecular masses of group 3 proteins (three bands or groups of bands)
did not correspond to any protein and were considered as the candidates
likely to be ISG15-modified. Fig. 1 shows
three fragments of respective Western blot panels. PLC Verification with Individual Antibodies--
To validate the
results of high-throughput Western blot analysis we performed IPs with
anti-ISG15 mAb and analyzed the immunocomplexes by Western blot with
anti-PLC Analysis of ISG15 Conjugates in UBP43 Knock-out Mice--
Our
laboratory has developed a knock-out mouse model in which a gene coding
for the ISG15-specific protease UBP43 is deleted (19). Most tissues of
UBP43 Proteasome Inhibitors Do Not Increase Amount of ISG15
Conjugates--
Attachment of monoubiquitin to its target protein
serves to modulate activity or location (26). However, the major
function of ubiquitin is the formation of polyubiquitin chains and
localization of targeted proteins to proteasomes for subsequent
degradation (27, 28). Loeb and Haas (7) point out that pulse studies by
Knight and Cordova (29) indicated rapid turnover of both free and
conjugated ISG15. It is not known whether conjugated ISG15 may act as
Ub and direct the conjugated proteins for proteasomal degradation.
Although reports point to the evidence (or absence of such) against it,
no experimental data have been presented (7, 23).
UBP43 A protein modified by ISG15 acquires two new characteristics:
gains affinity to anti-ISG15 antibodies and migrates more slowly on
SDS-PAGE. We used these features to our advantage and identified candidate proteins modified by ISG15. High throughput immunoblotting service was originally developed for comparative analysis of two or
more samples (e.g. protein extracts from normal and diseased tissues) with the intent to identify proteins whose levels are altered.
Our work proves that this method is a useful tool for identification of
post-translationally modified proteins in samples enriched for the
modified species. Nevertheless, we have not utilized this technology to
full potential, and there are several ways to improve the experimental
scheme. First, although postsurgical thymus appeared to be a good
source of ISGylated proteins, the amount of ISG15 conjugates in this
tissue was approximately three times lower than in IFN-treated A549 or
U937 cells.2 Second, a
significant amount of free ISG15 that is present in cells and tissues
strongly binds to immobilized anti-ISG15 antibodies, occupies valuable
sites, and decreases efficiency of purification of ISG15 conjugates.
Free ISG15, however, can be efficiently removed by gel filtration
chromatography on Sephadex G-50.2 Third, false-positives
detected in the primary screening step, which resulted from nonspecific
binding to IgG-resin, can be eliminated if simultaneous immunoaffinity
purification is performed on immobilized anti-ISG15 and isotype control
IgG. Proteins absorbed to both resins can be then analyzed on parallel
blots, and nonspecific bands can be identified. Fourth, the sensitivity
of the method could be increased more than 10-fold since in our
conditions only 0.5 µg of total protein was loaded per lane, while,
the technology permits loading of up to 7.5 µg of total protein per lane.
All four proteins reported in this study are known to be major players
in signal transduction and are the subjects of intensive investigation
by numerous laboratories. The PLC isozymes hydrolyze phosphatidylinositol bisphosphate thus modulating both calcium and
protein kinase C-regulated pathways (30). PLC The Jak family of receptor-associated protein kinases is directly
involved in response to IFN and other cytokines (33). Jak1 is rapidly
phosphorylated in response to IFN and is required for the
phosphorylation of the transcription factor Stat1 (25). We demonstrate
that Stat1 is also modified by ISG15 in poly(I-C) or IFN The family of kinases known as ERKs or MAPKs are activated after cell
stimulation by a variety of hormones and growth factors. Numerous
proteins represent the downstream effectors for the active ERK and
implicate it in the control of cell proliferation, differentiation, as
well as carcinogenesis and inflammation (37, 38).
Several proteins in our report (PLC Coordinated induction of ISG15, UBP43, and
UBE1L suggests that ISG15 conjugation is a dynamic and
highly controlled process. ISG15-activating enzyme UBE1L was found to
be absent in 14 different lung cancer cell lines suggesting that
decrease of ISG15 conjugation may contribute to carcinogenesis (39).
Certain viruses can specifically block conjugation or synthesis of
ISG15 (11) possibly in an attempt to suppress host cell suicide
and inflammatory response. Dysregulation of ISGylation due to
deletion of UBP43 leads to decreased life expectancy, brain
cell injury, hypersensitivity to interferon, and apoptosis in
hematopoietic tissues (19, 20). These reports combined with the fact
that IFN suppresses cellular proliferation indicate that the balance of
ISG15 conjugation is important for the control of cell differentiation
and growth suppression. Our current report indicates that ISGylation
may be directly involved in the regulation of several signal
transduction pathways in organisms challenged with IFN elicitors. It is
possible that upon IFN stimulation the cell fine tunes the degree of
response via ISGylation of Jak1 and Stat1, crucial players in the IFN
pathway. Moreover, other signaling pathways may also be directly
affected by means of ISGylation of respective critical regulators
(e.g. PLC1, Jak1,
and ERK1 are modified by ISG15. In addition to that, we demonstrate
that transcription factor Stat1, an immediate substrate of Jak1 kinase,
is also ISGylated. Using whole cell protein extracts and phospholipase
C
1 as an example we demonstrate that ISG15 conjugates are not
accumulated in cells treated with specific inhibitors of proteasomes.
Our work suggests a role for ISG15 in the regulation of multiple signal
transduction pathways and offers attractive models to further elucidate
the biochemical function of ISGylation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mouse model generated in our
laboratory exhibits a massive accumulation of ISG15 conjugates in
various tissues, which leads, either directly or indirectly, to
decreased life expectancy, brain cell injury (19), and hypersensitivity
to IFN stimulation (20). Although the role of Ub-, Nedd8-, and
SUMO-modification has been assessed in numerous studies (8, 21, 22),
the exact biochemical and physiological role of ISGylation has not been studied.
1, Jak1, Stat1, and ERK1) are targeted by
ISG15. Our data describes valid methodology for identification of new
ISG15 targets, offers attractive models to study the function of
ISGylation, and suggests a biological role for ISG15 modification in
regulation of multiple signal transduction pathways.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C and passed
through a 0.4-µm filter immediately before use in immunoaffinity
purification. In a series of pilot experiments we established that
certain ISG15 conjugates would bind to IgG-resin inefficiently at pH
8.0 but bound efficiently at pH 7.0. The addition of ethylene glycol
improved conditions for binding of an overlapping but distinct set of
ISG15 conjugates. Combination of pH 7.0 with ethylene glycol, on the
other hand, resulted in massive binding of certain nonspecific
proteins. We therefore adopted the following two-step protocol.
IgG-resin (0.5 ml) was mixed with thymic protein extract (250 mg of
total protein, at a final concentration of 3 mg/ml) in RIPA
buffer, pH 7.0, final volume 83 ml. Binding of ISG15 conjugates was
carried out overnight at 4 °C. The resin was then washed three times
with 20 volumes of RIPA buffer, pH 7.0, and stored at
20 °C. The
protein extract was adjusted to pH 8.0, and ethylene glycol was added
to a final concentration of 25% v/v. Fresh IgG-resin was added, and
binding was carried out as above. The resin was washed three times with
20 volumes of RIPA buffer, pH 8.0, and 25% ethylene glycol and stored
at
20 °C. Bound proteins resulting from both steps were eluted
from IgG-resins by the addition of 2 volumes of SDS-PAGE loading buffer and a 5-min incubation at 90 °C. The ISG15 conjugates from both binding steps (pH 8.0 and pH 7.0, 25% ethylene glycol) were pooled and
analyzed by high-throughput Western blot.
1, and anti-Jak1 mAbs were
purchased from BD Biosciences Transduction Laboratories. Rabbit
anti-human ISG15 polyclonal antibodies were previously described (24).
Rabbit anti-mouse ISG15 polyclonal antibodies were generated as
follows. Murine ISG17 (pro-ISG15) was PCR-amplified (primers
TGGAATTCTTAGGCACACTGGTCCCC and AATTCGATTCTGGATCCGCCTGGGACC) from
a cDNA library (a kind gift of Drs. M. Robek and S. Uprichard, The
Scripps Research Institute) prepared from hepatitis B virus -infected hepatocytes and cloned into pGEM-T Easy vector
(Promega, Madison, WI). The correct sequence was verified by
sequencing, the insert excised with BamHI and
SalI restriction endonucleases and recloned into respective
sites of pQE-30 vector (Qiagen, Valencia, CA) to yield
His6-tagged ISG17. His6-ISG17 was then
expressed in Escherichia coli and purified on
nickel-nitrilotriacetic acid resin (Qiagen) as recommended by the
manufacturer. Rabbit sera were generated by Covance (Denver, PA).
Anti-ISG15-specific IgGs were immunoaffinity-purified on immobilized
His6-ISG15 and depleted against immobilized Ub (Sigma) to
remove any cross-reacting IgGs. His6-ISG15 and Ub were
immobilized on glyoxal-activated agarose as described above for mAbs.
/
MEFs (murine embryonic fibroblasts)
were generated from a litter of embryos on embryonic day 12.5. Briefly, embryonic torsos were minced and trypsinized for 30 min at 37 °C.
Cells were harvested, resuspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, and plated on 10-cm
dishes. MEFs (9 × 105) were plated on 60-mm plates
and replated every 3 days for over 20 passages until they were
immortalized. Where indicated MEFs were treated with 200 units/ml of
IFN
, 5 µM lactacystin, and 10 µM MG132
(Calbiochem, La Jolla, CA). Thymocytes and bone marrow cells were
isolated from 4-6 week-old mice and were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 100 units/ml penicillin
and 100 µg/ml streptomycin (Invitrogen). Bone marrow cultures were
also supplemented with the following growth factors: 10 ng/ml
interleukin-3, 10 ng/ml interleukin-6, and 100 ng/ml stem cell factor
(PeproTech, Rocky Hill, NJ).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 was
represented by four bands migrating in the range of 145-185 kDa
(fragment A), whereas ERK1 and Jak1 were represented by single
bands of 57 and 145 kDa, respectively.
The molecular masses of these bands (with the exception of the 145-kDa
band recognized by PLC
1, which may be a product of proteolysis; see
Figs. 2, 3 and "Discussion") were in
good correlation with expected shifts in mobility upon addition of one
or more molecules of ISG15.
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Fig. 1.
High throughput immunoblot identification of
ISGylated proteins. IGS15 conjugates purified by
immunoaffinity chromatography on anti-ISG15 IgG-resin were screened
with 860 monoclonal antibodies at BD Biosciences Transduction
Laboratories. Relevant fragments of high throughput immunoblot
templates are shown. A, template D, lanes 5-7;
B, template F, lanes 38-40; C,
template E, lanes 8-10. Expected positions of indicated
proteins (middle lanes) are shown with arrows,
and observed conjugates are shown with asterisks. Positions
of molecular mass markers and heavy (HC) and light
(LC) chains of immunoglobulin are shown.
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Fig. 2.
ISG15-modified PLC 1,
ERK1, and Jak1 are detected in reciprocal immunoprecipitations.
A, proteins were precipitated with nonspecific isotype
control mAbs (N) or with specific anti-ISG15 mAb
(S). Ten micrograms of whole cell lysate were loaded
(L) to locate positions of unmodified proteins (shown with
arrows). Panels were probed with anti-PLC
1, anti-Jak1, or
anti-ERK1 monoclonal antibodies. B, proteins were
precipitated with nonspecific isotype control mAbs (N) or
with specific (S) anti-PLC
1, anti-Jak1, or anti-ERK1
monoclonal antibodies. Panels were probed with rabbit anti-human ISG15
antibodies (left) and reprobed with the antibodies used for
IP (right). Positions of unmodified proteins are shown with
arrows, and positions of conjugates are shown with
asterisks. Positions of molecular mass markers and
immunoglobulin heavy chain (HC) are shown on the
left.
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Fig. 3.
PLC 1 and ERK1 are
modified by ISG15 in murine embryonic fibroblasts. IGS15
conjugates were immunoprecipitated from untreated or IFN
-induced
UBP43+/+ and IFN
-induced
UBP43
/
MEFs. Immunocomplexes (lanes
1-3) and cell lysates (lane 4) were subjected to
Western blot with anti-PLC
1 and anti-ERK1 mAbs. Positions of
unmodified proteins are shown with arrows, and positions of
conjugates are shown with asterisks. Positions of molecular
mass standards and location of IgG heavy chain (HC) are
indicated on the left.
1, anti-Jak1, and anti-ERK1 mAbs. Bands identical to those
observed on high throughput immunoblotting images were detected only
when specific anti-ISG15 mAb but not when an isotype control IgG were
used for IP (Fig. 2A). We consequently performed reciprocal
IPs with individual mAbs against PLC
1, Jak1, and ERK1 (Fig.
2B). Immunocomplexes were analyzed by Western blot with
polyclonal rabbit anti-human ISG15 antibodies, and again, the signals
corresponding to ISGylated proteins were detected. These results
strongly suggest that PLC
1, Jak1, and ERK1 are ISGylated proteins.
The anti-ERK1 mAbs (BD Biosciences Transduction Laboratories, catalog
number 610030) recognize both the 44- and 42-kDa bands that may
represent ERK1 and ERK2, respectively. It is therefore not excluded
that ISG15-modified protein identified in our work may be ERK2.
/
mice have a massive increase of ISG15
conjugates, relative to wild type. This difference can be further
increased following dosing with LPS or poly(I-C). We used MEFs derived
from UBP43
/
and
UBP43+/+ mice to assess the relevance of our
findings in human tissue to the murine model. MEFs of both genotypes
were incubated with IFN
for 24 h. ISGylated proteins were
immunoprecipitated with purified rabbit anti-mouse ISG15 antibodies,
and immunocomplexes were analyzed by Western blot. The results
presented in Fig. 3 demonstrate that murine PLC
1 and ERK1 are also
modified by ISG15. The modification, however, was only obvious after
IFN
treatment and, as expected, was stronger in the
UBP43
/
cells. Interestingly, the band of 145 kDa that was detectable with anti-PLC
1 in immunoprecipitates from
human thymus was also present in MEFs and was likely to be a product of
specific proteolysis of ISGylated PLC
1. We were not able to detect
ISGylation of Jak1 in MEFs; however, the modification was detectable in
murine thymocytes (not shown) suggesting that the set of ISGylated
proteins may be cell-specific. In a separate paper we report that
Stat1, a transcription factor and substrate of Jak1, remains activated for a longer period of time in UBP43
/
cells
(20). In many Western blot experiments, where
UBP43
/
animals or isolated cells were
challenged with Jak-Stat activators we observed the appearance of
higher molecular mass bands recognized by Stat1 antibodies (Fig.
4A). We hypothesized that
these high molecular mass bands were Stat1 molecules conjugated by
ISG15. Reciprocal immunoprecipitations and Western blotting with rabbit polyclonal antibodies against Stat1 and ISG15 revealed the presence of
up to five specific bands that are likely to be Stat1-ISG15 conjugates
(Fig. 4B). The two bands (Stat1
/
, 91 and 84 kDa) corresponding to unmodified Stat1 are non-specifically recognized by
ISG15 antibodies due to the overload of Stat1 from immunoprecipitation using Stat1 antibodies. These two bands also appeared on the membrane with Ponceau S staining (Fig. 4B, left panels).
Noticeably, the same bands corresponding to unmodified Stat1
/
are
detectable with anti-Stat1 in the proteins immunoprecipitated with
anti-ISG15 (Fig. 4B, right panels). Stat1 is
known to form homodimers (25), and the copurification of unmodified
molecules may be caused by protein-protein interaction between
ISGylated and unmodified Stat1. In addition, other proteins that
interact with Stat1 to form ISGF3 and other complexes may be ISGylated
and cause the copurification of unmodified Stat1. To answer the
question whether Stat1 is ISGylated in human thymus we performed
Western blot with anti-Stat1 on purified ISG15 conjugates, the same
preparation that was used for high throughput immunoblotting (Fig.
4C). A banding pattern similar to that observed in murine
T-cells (Fig. 4B) was detected.
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Fig. 4.
Stat1 is modified by ISG15 in murine and
human tissues. A, total cell lysates of indicated
tissues were resolved on SDS-PAGE and Western blotted with anti-Stat1
antibodies. Isolated bone marrow cells were treated in vitro
with 100 units/ml of IFN for 1 or 24 h. Thymi were
excised from mice injected with poly(I-C) 1 or 24 h prior to
sacrifice. B, reciprocal immunoprecipitations from
IFN
-treated UBP43+/+ and
UBP43
/
thymocytes were performed using
rabbit anti-Stat1 or anti-ISG15 antibodies and then probed with
anti-ISG15 or anti-Stat1, respectively. Proteins were precipitated with
nonspecific rabbit IgGs (N) or with specific antibodies
(S). Ten micrograms of whole cell lysate were loaded
(L) to locate positions of unmodified proteins. The Ponceau
S staining shows the large amount of Stat1 in the immunoprecipitates
(marked by asterisks). C, ISG15 conjugates
immunoaffinity-purified on anti-human ISG15 mAb resin from human thymus
(same as in Fig. 1) and probed with polyclonal anti-ISG15 or
anti-Stat1.
/
cells, due to the absence of ISG15
deconjugation, appear to be an excellent model to analyze the relation
between Ub, ISG15, and the proteasome. We treated
UBP43+/+ and UBP43
/
cells, uninduced or IFN
-induced, with a highly specific proteasome inhibitor lactacystin. Western blot revealed no difference in the
amount of ISG15 conjugates between lactacystin-treated and untreated
samples, while as expected, an increase in the amount of ubiquitinated
proteins was observed (Fig.
5A). Noticeably, the level of
ISGylation had no detectable effect on the appearance of Ub conjugates
in either UBP43
/
or
UBP43+/+ cells treated with IFN
. To confirm
this observation we immunoprecipitated PLC
1 from MEFs treated with a
proteasome inhibitor and probed immunocomplexes with anti-ISG15
antibodies (Fig. 5B). Consistent with the data of total
protein analysis we did not observe any increase in the amount of
ISGylated PLC
1 upon inhibition of proteasomes. These results
demonstrate that ISGylated proteins are not degraded by proteasomes and
suggest that ISGylation does not prevent conjugation of
polyubiquitin.
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Fig. 5.
Proteasome inhibitors do not affect the level
of ISG15 modification. A,
UBP43+/+ and UBP43 /
MEFs were incubated with or without IFN
for 18 h and were
treated with lactacystin at a final concentration of 5 µM
for 3 h. An equal volume of Me2SO (vehicle) was added
to control samples. Cell lysates (10 µg of total protein) were
resolved on 8-18% minigel, and Western blot was performed with rabbit
anti-mouse ISG15. Membrane was stripped and reprobed with anti-Ub
serum. B, UBP43+/+ (not treated with
IFN) and UBP43
/
(incubated with IFN
for
18 h) MEFs were treated with MG132 at a final concentration of 10 µM for 3 h. An equal volume of Me2SO
(vehicle) was added to control samples. PLC
1 was immunoprecipitated,
and immunocomplexes were resolved on 7% minigel. The membrane was
probed with anti-ISG15 and, after stripping, was reprobed with
anti-PLC
1. Positions of unmodified PLC
1 are shown with
arrows and positions of conjugates are shown with
asterisks. Cell lysates (10 µg of total protein) were also
resolved on an identical minigel to assess amount of ISG15 and Ub
conjugates. Western blot was performed with rabbit anti-mouse ISG15,
and after stripping, the membrane was reprobed with anti-Ub
serum.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 is essential for
growth factor-induced cell motility, mitogenesis (31), normal growth
and development, carcinogenesis, and cellular transformation (32).
-treated
murine cells and found that Stat1 is also modified in human thymus
(Fig. 4C). High-throughput immunoblotting failed to detect
this, possibly because of lower sensitivity of the antibodies used by
the service. Jak1-deficient mice exhibit perinatal lethality (34),
whereas Stat1 knock-out mice, although viable, lack all the
physiological functions associated with the IFNs and display a
remarkable sensitivity to viral infections and other pathological agents (35, 36).
1, Stat1) consistently show more
than one modified band (Figs. 1-4). Hamerman et al. (23) observed two modified bands of Serpin 2a. The Majority of polyUb chains
are formed by conjugation of new Ub molecules to the Lys-48 on the
previous Ub. In its C-terminal Ub-like domain, ISG15 does have a Lys
residue conserved at the position corresponding to Lys-48 of Ub (3).
Therefore, the possibility that short polyISG15 chains (two to five
ISG15 molecules) are formed remains open. Alternatively, modification
of more than one lysine on the same protein may take place. Formation
of polyUb on substrate proteins, however, does not stop after addition
of a few Ub molecules but results in formation of long chains. This
does not seem to be the case with ISGylated proteins,
suggesting that conjugation of single ISG15 molecules to several
lysines on the same protein is more likely. Although, the biochemical
function of ISG15-modification remains unknown the results of our
experiments with proteasome inhibitors indicate that ISG15 conjugates
are not degraded by proteasomes and ISG15 does not protect targeted
proteins from ubiquitination and subsequent degradation. The proteins
thus far known to be modified by ISG15 (Serpin 2a, ERK1, PLC
1, Jak1,
and Stat1) have diverse biochemical functions. It is reasonable to assume that the major role of ISG15 conjugation is IFN or other signal-induced modulation of such characteristics of the protein as
solubility, stability, localization, etc. In this respect the role of
ISG15 may be similar to polyubiquitin, which conjugates to a vast
number of diverse proteins, yet the consequences of binding are the
same: acquired affinity to a proteasomal subunit and degradation. Loeb
and Haas (7) noticed that the data of Knight and Cordova (29) indicated
rapid turnover of both free and conjugated ISG15. In most of our
experiments we were able to observe fragments of PLC
1, Jak1, and
ERK1 (Figs. 1 and 2B) as well as Stat1 (data not shown). It
is tempting to speculate that ISG15 conjugation promotes degradation of
targeted proteins via a pathway alternative to proteasomal.
However, it is also possible that protein ISGylation plays a
specific role in regulating enzymatic or DNA binding activity of target
proteins similar to other ubiquitin-like modifiers.
1, ERK1) to orchestrate overall growth,
differentiation, and survival.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. K. Ritchie for critical reading of the manuscript, Dr. L. Crisa and K. Allin for helpful technical suggestions, and Drs. M. Robek and S. Uprichard for providing reagents. We also thank Drs. D. Bichell and E. Breisch and the nurses of the Children's Hospital Cardiac Surgery team (San Diego, CA) for supplying the pediatric thymic tissue for the studies.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant CA79849 and American Cancer Society Grant LBC-99438. The Stein Endowment Fund partially supported the Departmental Molecular Biology Service Laboratory for DNA Sequencing and Oligonucleotide Synthesis.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Leukemia and Lymphoma Society Scholar. To whom correspondence should be addressed: MEM-L51, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-9558; Fax: 858-784-9593; E-mail: dzhang@scripps.edu.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M208435200
2 M. Malakhov and D.-E. Zhang, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
IFN, interferon;
ERK, extracellular signal-regulated kinase;
IP(s), immunoprecipitation(s);
ISG15, interferon-stimulated gene 15 kDa;
Jak, Janus family protein tyrosine kinase;
LPS, lipopolysaccharide;
mAb(s), monoclonal antibody(ies);
MEF, murine embryonic fibroblast;
PLC1, phospholipase C
1;
Stat, signal transducers and activators of
transcription;
Ub, ubiquitin;
Ubl(s), ubiquitin-like protein(s);
MAPK, mitogen-activated protein kinase;
PBS, phosphate-buffered saline.
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