Molecular Cell, Vol. 2, 233±239, August, 1998, Copyright 1998 by Cell Press
SUMO-1 Modification of I?B?
Inhibits NF-?B Activation
lysine in the target protein. Proteins destined for degra-
dation via the proteasome are coupled to multiple cop-
ies of ubiquitin (Ciechanover and Schwartz, 1998). In
the case of I?B?, Ubch5 is the E2 (Chen et al., 1996),
but ubiquitination requires the presence of a ubiquitin
protein ligase activity that recognizes I?B? phosphory-
lated on residues S32 and S36 (Chen et al., 1996; Yaron
et al., 1997).
Whereas addition of multiple copies of ubiquitin tar-
gets proteins fordegradation, additionofa singleubiqui-
tin does not target the protein fordegradation but alters
the properties of the modified protein (Bradbury, 1992).
Likewise, covalent attachmentof other ubiquitin-related
molecules does not resultindegradationof the modified
protein. Recently a small ubiquitin-like protein variously
known as sentrin, GMP1, SUMO-1, UBL1, and PIC1 has
been found covalently linked to Ran GTPase activating
protein 1 (RanGAP1) and associated with a variety of
other proteins (Boddy et al., 1996; Matunis et al., 1996;
Shen et al., 1996; Kamitani et al., 1997; Mahajan et al.,
1997).SUMO-1and Smt3p, ayeasthomolog of SUMO-1,
are conjugated to target proteins by a pathway that is
distinct from, but analogous to, ubiquitin conjugation.
A separate E1-like enzyme is responsible for SUMO-1
modification (Desterro et al., 1997), and in yeast the
enzyme responsible for Smt3p activation is a hetero-
dimer of Uba2p and Aos1p (J ohnson et al., 1997). In
both yeast and human cells, the E2-like SUMO-1/Smt3p
conjugating activity is the product of the Ubc9 gene
(Desterro et al., 1997; J ohnson and Blobel, 1997; Saitoh
et al., 1998; Schwarz et al., 1998), which is required for
cell cycle progression in yeast (Seufert et al., 1995).
As we had previously isolated Ubch9as an I?B? inter-
acting proteininayeasttwo-hybrid screen,we searched
for physiological situations in which I?B? is modified by
SUMO-1. In a number of cell types, I?B? is modified by
SUMO-1 to a form that is resistant to TNF?-induced
proteolysis. Thus, overexpression of SUMO-1 inhibits
signal-induced activation of NF-?B. Mutational analysis
revealed that SUMO-1 is linked to I?B? primarily on
residue K21, which is alsoutilized forubiquitin modifica-
tion. However, unlike ubiquitin modification, which re-
quires phosphorylation of S32 and S36, this is not the
case with SUMO-1. Thus, by blocking ubiquitination,
SUMO-1 modification creates a privileged pool of I?B?
that is resistant to signal-induced degradation.
J oana M. P. Desterro, Manuel S. Rodriguez,
and Ronald T. Hay*
School of Biomedical Science
University of St. Andrews
St. Andrews, Fife KY169AL
Activation of NF-?B is achieved by ubiquitination and
proteasome-mediated degradation of I?B?. We have
detected modified I?B?, conjugated to the small ubiq-
uitin-like protein SUMO-1, which is resistant to signal-
induced degradation. In the presence of an E1 SUMO-
1-activating enzyme, Ubch9 conjugated SUMO-1 to
I?B? primarily on K21, whichis also utilized forubiqui-
tin modification. Thus, SUMO-1-modified I?B? cannot
be ubiquitinated and is resistant to proteasome-medi-
transcription. Unlike ubiquitin modification, which re-
quires phosphorylation ofS32 andS36, SUMO-1modi-
fication of I?B? is inhibited by phosphorylation. Thus,
while ubiquitination targets proteins for rapid degra-
dation, SUMO-1 modification acts antagonistically to
generate proteins resistant to degradation.
In unstimulated cells, the transcription factor NF-?B is
held inthe cytoplasmin aninactive state by I?B inhibitor
proteins. Activation of NF-?B is mediated by signal-
induced degradation of I?B?, which allows the active
transcriptionfactorto translocateintothe nucleus.Bind-
ing of NF-?B to its DNA recognition sites activates tran-
scriptionfrom responsivegenes.As part ofan autoregu-
latory loop, NF-?B activates transcription of the I?B?
gene (Baldwin, 1996). Newly synthesized I?B? accu-
mulates transiently in the nucleus, where it negatively
regulates NF-?B-dependent transcription (Arenzana-
Seisdedos et al., 1995) and mediates transport of the
NF-?B/I?B complex back to the cytoplasm (Arenzana-
Seisdedos etal., 1997). Signal-induced phosphorylation
of I?B? on S32 and S36 is mediated by an I?B kinase
complex whichcontains two subunits that are both nec-
essary for NF-?B activation (Verma and Stevenson,
1997).Specific inhibitionof the proteolytic activity of the
proteasome prevents NF-?B activationand results inthe
accumulation of ubiquitinated forms of I?B?, indicating
that I?B? is targeted for degradation by a phosphoryla-
tion-dependent ubiquitination process (Baldwin, 1996).
Ubiquitin addition involves E1, E2, and E3 enzymes
and results in formation of an isopeptide bond between
the C terminus of ubiquitin and the ?-amino group of
Detection of a Modified Form of I?B? Resistant
to Signal-Induced Degradation
Based on a previously reported (Desterro et al., 1997)
yeast two-hybrid screen, using the N terminus of I?B?
as ªbait,º a strong and highly specific interaction had
been suggested between I?B? and Ubch9. Although
Ubch9 is homologous to ubiquitinconjugating enzymes,
the substrate forthis enzyme is notubiquitin but SUMO-1
(Desterro et al., 1997). While ubiquitinated I?B? can be
detected inthe presence of proteasome inhibitors, there
*To whom correspondence should be addressed (e-mail: rth@
Figure 1. A Modified Form of I?B?, Present in Different Cell Lines,
Is Resistant to Signal-Induced Degradation
(A) Extracts (20 ?g protein) of different cell lines, prepared either by
standard NP40 lysis or by direct lysis in SDS buffer, were fraction-
ated by SDS-PAGE, and I?B? protein was analyzed by Western
blotting using the I?B? antibody C21.
(B) Extracts lysed in SDS were analyzed as in (A) using a rabbit
polyclonal anti-I?B? antibody.
(C) 293 cells were exposed to 10 ng/ml of TNF? for the indicated
times. Extracts were lysed in SDS, separated by SDS-PAGE, and
proteins analyzed by Western blotting using the C21 antibody.
Positions of prestained molecular weight markers, I?B?, and the
modified form of the protein (I?B?*) are indicated.
Figure 2. SUMO-1 Is Conjugated to I?B? In Vivo
(A) SDS lysates (300 ?g protein) from different cell lines were incu-
bated with either rabbit anti-I?B? IgG cross-linked to protein
A±agarose beads or identically prepared IgG (PI). Immunoprecipi-
tates werefractionated bySDS-PAGE and analyzed byECL Western
blotting with anti-SUMO-1 mAb 21C7.
(B) COS7 cells were transfected with the indicated plasmids, and
after36hrof expressionSDS lysates were prepared.SUMO-1 exog-
enous expression was analyzed by Western blotting with the HA
tag±specific mAb 12CA5.
(C)SDS celllysatesfrom transfections indicatedin(B)were immuno-
precipitated with anti-I?B? proteinA beads and immunoprecipitates
analyzed by Western blotting with the HA tag±specific mAb 12CA5.
(D) After ECL development, the blot used in (C) above was stripped
and reprobed with the anti-SUMO-1 mAb 21C7.
(E) The same blot was again stripped and reprobed with SV5 PK
tag mAb to detect exogenously expressed and immunoprecipitated
Molecular weight markers and the positions of I?B? and its SUMO-
1-conjugated form (I?B?-SUMO-1) are indicated.
have beenno reports of SUMO-1-modified I?B?. A num-
berof celllines were therefore surveyedforthe presence
of a more slowly migrating form of I?B? that would be
consistent with SUMO-1 modification. Western blotting
of cell extracts prepared using standard NP40 lysis pro-
cedures failed to show any SUMO-1-modified I?B? (Fig-
ure 1A). However, whencells were lysed directly, a more
slowly migrating form of I?B? consistent with SUMO-1
modification (I?B?*) was detected in substantial amounts
in 293, COS7, and HeLa cells with smaller amounts in
J urkat cells (Figure 1A). To rule out the possibility that
the more slowly migrating form of I?B? detected in Fig-
ure 1A was a peculiarity of the antibody preparation,
anotherwell-characterized I?B?antibody raised against
recombinant I?B? (Mellits et al., 1993; Arenzana-Seis-
dedos et al., 1995) was also employed. COS7, 293, and
J urkat cells revealed the same slowly migrating species
(I?B?*) when Western blot analysis was conducted with
the antibodyto recombinantI?B? (Figure 1B).As SUMO-
1-modified proteins do not appearto be targets forpro-
teasome-mediated degradation, the fate of the modified
form of I?B?was determined aftertreatment of 293cells
withTNF?.Whilethe unmodifiedformof I?B?undergoes
signal-induced phosphorylation and degradation as ex-
pected, the modified form of I?B? appears to be resis-
tant to TNF-induced degradation (Figure 1C).
with the antibody raised against recombinant I?B? and
associated proteins analyzed by Western blotting with
the SUMO-1-specific mouse mAb 21C7 (Matunis et al.,
1996). In HeLa, J urkat, COS7, and 293 cells, I?B? anti-
bodies immunoprecipitated a polypeptide of about 55
kDa, recognized by the SUMO-1-specific mAb, which
is consistent with addition of a single molecule of
SUMO-1 to I?B?. A more slowly migrating immunoreac-
tive species was also detected with the SUMO-1-spe-
cific mAb. This species remains unidentified and failed
to react with antibodies to NF-?B family members (data
not shown). Under identical conditions, no SUMO-1 re-
active species were immunoprecipitated withthe preim-
mune serum (Figure 2A).
To confirm these findings, pcDNA3 expression plas-
mids containing cDNAs for HA-tagged SUMO-1, I?B?-
ctag, and Ubch9 were transfected in various combina-
tions into COS7 cells and SDS lysates prepared as
SUMO-1 Is Conjugated to I?B? In Vivo
To demonstrate directly that I?B? was conjugated to
SUMO-1, SDS cell lysates were immunoprecipitated
SUMO-1 Modification of I?B?
Figure 3. SUMO-1 Expression Inhibits NF-?B-Dependent Transcription
COS7 cells were cotransfected for 48 hrwith the NF-?B-dependent reporter 3EnhConALuc, and either control pcDNA3 plasmid (empty vector)
or one of the indicated expression plasmids. Transfected cells were induced with TNF? (10 ng/ml), IL1 (10 ng/ml), or okadaic acid (OKA, 75
nM) for the last 8 hr in culture, and luciferase activity was measured in cell lysates. Luciferase activity (RLU/?g of protein) is expressed as
fold activation relative to the activity in untreated cells. The same experiment was performed using a different NF-?B-dependent reporter
vector (HIV-LTR) and an equivalent control vector lacking NF-?B binding sites (??B HIV-LTR). These experiments were performed on three
separate occasions with similar results.
described. When HA-SUMO-1 alone is exogenously ex-
pressed, a substantial amount of unconjugated protein
is detected along with a series of higher molecular
weight species that presumably represent conjugation
to a range of polypeptides (Figure 2B). When I?B?-ctag
is cotransfected, less free HA-SUMO-1 is detected, and
this amount is further reduced by the additional pres-
ence ofplasmid expressing Ubch9(Figure 2B). Todetect
I?B?-SUMO-1 conjugates, SDS cell lysates were sub-
jected to immunoprecipitation with I?B? antibodies and
the associated proteins analyzed by Western blotting
using either antibodies to HA, ctag, or SUMO-1. When
I?B?-ctag and HA-SUMO-1 are cotransfected, either in
the presence orabsence of Ubch9, conjugates between
I?B?-ctag and SUMO-1 are detected using antibodies
specific forHA (Figure 2C), SUMO-1 (Figure 2D), orctag
(Figure 2E). Thus, SUMO-1 modification of I?B? is de-
tected on endogenous, as well as exogenously ex-
luciferase reporter (3enh conA luc) was cotransfected
into COS7 cells along with expression plasmids for
SUMO-1, Ubch9, orI?B?, and reporteractivity was mea-
sured after treatment of the cells with the NF-?B activa-
tors TNF?, IL-1?, or okadaic acid. When the empty vec-
tor is cotransfected with the ?B reporter, substantial
transcriptional activation is obtained with TNF (9.8-fold),
IL-1 (98-fold), and okadaic acid (85-fold). As expected,
cotransfectionof the reporterwitha plasmid expressing
I?B? reduced TNF-, IL-1-, and okadaic acid±induced
activation. When the reporter was cotransfected with a
plasmid expressing SUMO-1, or the SUMO-1 conjugat-
ing enzyme Ubch9, the extent of activation was also
reduced with TNF, IL-1, and okadaic acid. Cotransfec-
tion of the reporter plasmid with combinations of plas-
mids expressing I?B?, SUMO-1, and Ubch9 demon-
strated a more complete inhibition of transcriptional
activation than either plasmid alone. Thus, the com-
bination of I?B? and SUMO-1 expression results in an
80% inhibitionofTNF-induced transcriptionalactivation
whereas expression of either I?B? or SUMO-1 alone
only gives about 50% inhibition. Reporter activity from
a plasmid lacking NF-?B binding sites (conA luc) was
not increased by TNF, IL-1, or okadaic acid, and the
basal levelofreporteractivity was unchanged by exoge-
nous expression of I?B?, SUMO-1, or Ubch9 (data not
shown). To investigate the specificity of the SUMO-1-
mediated inhibition of NF-?B-dependent transcription,
SUMO-1 Expression Inhibits
As it was demonstrated that SUMO-1-modified I?B?
was resistant to signal-induced degradation and that
exogenously supplied SUMO-1 could be conjugated to
I?B?, it was of interest to determine the effect of ex-
pressed SUMO-1 on signal-induced activation of NF-
?B. To measure NF-?B activation, an NF-?B-dependent
Figure 4. Requirements for SUMO-1 Conju-
gation of I?B? In Vitro
(A) In vitro expressed I?B? 256 (aa 1±256)
presence (?)orabsence (?)ofdifferent com-
ponents as indicated in the figure. Reaction
products were fractionated by SDS-PAGE,
and the dried gel was analyzed by phos-
phorimaging.The positions ofI?B?,the phos-
phorylated form of I?B? (I?B?-P), and I?B?-
SUMO are indicated.
(B) Invitro expressedI?B? (W/T, K21R, K22R,
and K21,22R) labeled with
incubated with recombinant p65, Ubch9,
SUMOGG, and HeLa FrII.4 containing SUMO
E1 activity. Reaction products were analyzed
as in (A).
(C) In vitro expressed and
wild-type I?B? (W/T) and mutants
S32,36A; S32,36D; and S32,36E) were as-
sayed for SUMO-1 conjugation in vitro as in
(B). Assays of I?B? WT and 1±256 were con-
ducted either in the presence (?) or absence
(?) of recombinant p65.
35S-Met was incubated in the
(D) The N terminus of I?B? (1±70) fused to GST (GST-I?B?N) was phosphorylated in vitro with purified I?B kinase in the presence of ?-32P-
(E) Sites of SUMO-1 conjugation. Comparison of amino acid sequences containing the residues in RanGAP1 and I?B? that serve as acceptor
sites for SUMO-1 modification.
35S-Met labeled in vitro expressed I?B?1±256and32P-labeled GST-I?B?N were assayed for SUMO-1 conjugation in vitro as in (B).
an alternative reporter plasmid and an expression plas-
mid for ubiquitin were employed. While activation of the
HIV-LTR by TNF and IL-1 is almost entirely dependent
on NF-?B, activation of the HIV-LTR by okadaic acid
has both NF-?B-dependent and -independent compo-
nents. Thus, ??B HIV-LTR can still be activated by oka-
daic acid (Vlach et al., 1995). Like the 3enh conA luc
reporter, the HIV LTR is activated by TNF (12-fold), IL-1
(35-fold), and okadaic acid (11-fold), and the activation
is reduced by cotransfection of plasmids expressing
I?B? or SUMO-1. Cotransfection of a plasmid express-
ing ubiquitin has little inhibitory effect. Cotransfection
of the combination of I?B? and SUMO-1 substantially
inhibits transcriptional activationmediated by TNF, IL-1,
or okadaic acid whereas cotransfection of both I?B?
and ubiquitin expressionplasmids has a relatively minor
effect on transcriptional activity. The ??B HIV LTR re-
porter was activated by neither TNF nor IL-1 but was
activated approximately 3-fold by okadaic acid. Co-
transfection of the ??B HIV LTR reporter with I?B?,
SUMO-1, ubiquitin, orcombinations of I?B? and SUMO-1
or I?B? and ubiquitin had no substantial effect on basal
activity and did not influence the ?B-independent oka-
daic acid±induced activation of the reporter (Figure 3).
Thus, SUMO-1 appears to inhibit specifically NF-?B-
dependent transcriptional activation.
these components and was dependent on the addition
of ATP (Figure 4A). Other HeLa fractions such as II.1
failed to substitute for fractions II.4, and SUMO-1 was
only fully active ifitwas truncated by fouraminoacids at
the C terminus to exposeG97. Infact, some conjugation
activity was observed with the full-length SUMO-1, but
itis thought that this is due to the presence of aSUMO-1
C-terminal hydrolase in the column fractions, which
cleaves the C terminus of SUMO-1afterG97 to generate
the active form of the protein (Figure 4A).
Prior to signal-induced degradation, I?B? is ubiquiti-
nated on residues K21 and K22 and to a lesser extent
on residue K38 and K47 (Scherer et al., 1995; Baldi et
al., 1996; Rodriguez et al., 1996). To determine the sites
at which I?B? is modified by SUMO-1, the previously
described (Rodriguez et al., 1996) series of molecules
containing K-to-R changes in I?B? was employed. The
K21R mutant of I?B? is severely compromised as a
substrate for SUMO-1 modification in vitro, while this is
less so for the K22R mutant. When both lysines are
changed to arginine(K21R, K22R)SUMO-1 modification
is abolished (Figure 4B). Thus, the majorsite of SUMO-1
modification is K21, which is also used to conjugate
As ubiquitination of I?B? is dependent on signal-
induced phosphorylation of S32 and S36, we investi-
gated the role of these residues in SUMO-1 modifica-
tion. The S32A, S36A mutant of I?B? that is resistant to
signal-induced phosphorylation is efficiently modified
by SUMO-1, as is the S32D, S36D mutant. However,
SUMO-1 modification of the S32E, S36E mutant, which
most closely mimics phosphorylated I?B?, is severely
compromised (Figure 4C). To demonstrate that phos-
phorylated I?B? is nota substrate forSUMO-1modifica-
tion,residues 1±70 ofI?B?fused to GST were phosphor-
ylated in vitro with purified I?B kinase (Regnier et al.,
1997) in the presence of ?-32P-ATP.32P-GST-I?B?N was
Requirements for SUMO-1 Conjugation
to I?B? In Vitro
To investigate the biochemical requirements for SUMO-1
modification of I?B?, an in vitro system that could
accurately recapitulate the invivo phenomenon was de-
translation was used as substrate in the presence of
recombinant SUMO-1, recombinant Ubch 9, and a frac-
tion from HeLa cells (Desterro et al., 1997). SUMO-1
modification of I?B? required the presence of each of
35S-Met-labeled I?B? generated by in vitro
SUMO-1 Modification of I?B?
recovered and tested for its ability to act as a sub-
strate forSUMO-1 conjugationinvitro.Underconditions
where unlabeled GST-I?B?N was efficiently conjugated
to SUMO-1 (data not shown), the labeled material was
not conjugated to SUMO-1.
lated I?B?256was efficiently modified in the same assay
A mutant ofI?B? containing aminoacids 1±256, which
is resistant to signal-induced degradation (Kroll et al.,
1997), was efficiently modified by SUMO-1 in vitro, indi-
cating that the C-terminal region of the protein (amino
acids 257±317) is dispensable for SUMO-1 modification
(Figure 4C). While most assays contained recombinant
NF-?B p65, its omission was without consequence for
SUMO-1 modification of I?B? (Figure 4C).
a binding site for NUP358, a nucleoporin associated
with the cytoplasmic fibers of the nuclear pore complex
(Matunis etal., 1998).The onlyother reportedsubstrates
for SUMO-1 modification are the nuclear dot (ND) asso-
ciated proteins PML and Sp100, and inthis case SUMO-1
modification appears to regulate the subnuclear parti-
tioning of these proteins (Sternsdorf et al., 1997; Muller
et al., 1998). However, as mutation of K21 and K22 does
not appear to affect the ability of I?B? to accumulate in
the cell nucleus (Zabel et al., 1993), it is unlikely that
SUMO-1 modification has a majorrole in nuclear import
Although targeting of protein for ubiquitination ap-
pears to be highly specific, it is often the case that
multiple lysine residues can act as acceptor sites with
modification of either residue being sufficient to target
the protein for degradation (Hou et al., 1994; King et al.,
1996). In contrast, SUMO-1 modification appears to be
highly specific. I?B? appears to be modified by SUMO-1
predominantly on K21 (Figure 4) while RanGAP1 is con-
jugated to SUMO-1 solely via K526 (Mahajan et al.,
1998). Comparison of the sequences surrounding the
acceptor lysines inI?B? and RanGAP1reveals a striking
similarity (Figure 4E), which suggests that this sequence
may representa recognition site forthe SUMO-1 conju-
gation machinery. It is likely that this recognition is
achieved by Ubch9, as an interaction between Ubch9
and the N terminus of I?B? was detected in a yeast two-
hybrid screen (Desterro et al., 1997).
While signal-induced ubiquitination of I?B? requires
phosphorylation of S32 and S36, this is not the case for
SUMO-1 modification, as an S32A, S36A mutant is more
tein. In contrast, an S32E, S36E mutant or I?B? phos-
phorylated in vitro on S32 and S36 are poor substrates
forSUMO-1conjugation. Thus, SUMO-1acts antagonis-
tically to ubiquitination: while multiubiquitinationof I?B?
targets the proteinfordestruction,SUMO-1modification
creates a pool of I?B? that is resistant to degradation.
This function of SUMO is rathersimilar to that observed
when mutations are introduced into ubiquitin in the ly-
sine residues that are used formultiubiquitination. K29R
and K48R mutants in ubiquitin generate proteins that
can be conjugated to substrates but that cannot form
multiubiquitin chains. As such, the modified proteins are
resistant to degradation (J ohnsonet al., 1995). Although
only a few substrates for SUMO-1 modification have
been identified, it is evident that many cellular proteins
are modified in such a fashion. The balance between
ubiquitination and this activity of SUMO-1 may be a
general mechanism for controlling the level of critical
proteins within the cell.
Inuninducedcells, the transcriptionfactorNF-?B is held
in an inactive state by its inhibitor I?B?. After exposure
of cells to activators such as IL-1? or TNF?, the I?B?
inhibitor is marked by phosphorylation and targeted for
degradation by ubiquitination.Inany particularcell type,
the amount of active NF-?B released is therefore deter-
minedsimply by theamountof NF-?B boundtoinducibly
degradable forms ofI?B. Herewe demonstratethat I?B?
is modified by SUMO-1 (Figures 1 and 2) on K21, which
is also used for ubiquitin conjugation (Figure 4). Thus,
SUMO-1-modified I?B? cannot be ubiquitinated and is
therefore resistant to proteasome-mediated degradation.
This creates a ªprivilegedº pool of NF-?B/I?B?-SUMO-1
complexes that do not respond to signal induction (Fig-
ure 1). As a consequence, exogenous expression of
SUMO-1 has a strong inhibitory effect onNF-?B-depen-
dent transcription. Inhibition by SUMO-1 appears to be
specific to NF-?B-dependenttranscription as other pro-
moters are not affected. As the amount of SUMO-1-
modified I?B? appears to vary between different cell
types, this may provide a mechanism by which the cell
can precisely regulate the quantity of NF-?B available
fortranscriptional activation. The existence of hydrolases
thatcleave the bond betweenthe C terminus of SUMO-1
and the lysine to which it is conjugated has been re-
ported (Matunis et al., 1996; Mahajanet al., 1997; Muller
et al., 1998), and the activity of these enzymes is such
that the detection of I?B?-SUMO-1 conjugates is diffi-
cult unless special precautions are taken to inactivate
these enzymes quickly (Figure 1). Western blot analysis
of endogenous SUMO-1 indicates that there are many
cellular proteins that are conjugated to SUMO-1, and
as no freeSUMO-1 was detected, this implies that virtu-
ally all of the endogenous SUMO-1 is conjugated to
proteins (Matunis et al., 1996; data not shown). Thus,
SUMO-1islimiting,suggesting that SUMO-1deconjuga-
tionis requiredtorelease freeSUMO-1forfurthermodifi-
WhileSUMO-1modificationof I?B?canserveto block
signal-induced ubiquitination and thus degradation of
I?B?, SUMO-1modificationof RanGAP1serves to direct
the modified protein to the nuclear pore complex (Ma-
tunis et al., 1996, 1998; Mahajan et al., 1997, 1998).
SUMO-1 modification of RanGAP1 creates, orexposes,
Human recombinant TNF? was provided by the MRC ADP reagent
program. Human recombinant Interleukin 1? (IL1) was purchased
from Sigma, and Okadaic acid (Oka)was obtainedfrom Calbiochem.
C-21 (Santa Cruz) is a rabbit polyclonal antibody raised against a
peptide (amino acids 297±317)corresponding to the carboxyl termi-
nus of I?B?. A rabbit polyclonal antibody raised against purified
human recombinant I?B? was described previously (Mellits et al.,
1993). 21C7 (Matunis et al., 1996) is a mAb that recognizes GMP1
(SUMO-1) and was purchased from Zymed. The SV5 Pk tag mAb
(Hanke et al., 1992) was used to detect I?B? ctag (Rodriguez et al.,
1996). HA-SUMO-1 was detected using mAb 12CA5, which recog-
nizes YPYDVPDYA from influenza HA, obtained from Babco.
This work was supported bythe Medical ResearchCouncil, Biotech-
nology and Biological Sciences Research Council(BBSRC), and the
European Union Concerted Action BIOMED II (ROCIO II project).
J . M. P. D. was also supported by J NICT-Praxis XXI (Portugal). We
would like to thank Alex Houston for DNA sequencing, R. E. Randall
for the Pk tag antibody, Ellis J affray for purified recombinant pro-
teins,J ill Thomsonforproviding the Ubch9cDNA, and Lesley Thom-
son for purified I?B kinase.
Cell Culture, Transfection, and Reporter Assays
HeLa, COS7, and human embryo kidney 293 cell lines were main-
tained in exponential growth in Dulbecco's modified Eagle's me-
dium, containing 10% fetal calf serum. Plasmid DNAs (1 ?g±2 ?g)
were transfected for 14 hr in subconfluent cells seeded in 25 cm3
flasks using Lipofectamine. After transfection, cells were trypsin-
ized, and aliquots were seeded into six-well plates and cultured for
an additional 36 hr. Cells from a single transfection were incubated
for 8 hr with medium containing IL-1? (10 ng/ml), TNF? (10 ng/ml),
okadaic acid (75 nM), or control medium and processed for lucifer-
ase reporter activity as described (Rodriguez et al., 1996).
Received April 1, 1998; revised May 26, 1998.
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Cell extracts were prepared either as described (Roff et al., 1996)
or in SDS sample buffer (5% SDS, 0.15 M Tris HCl [pH 6.7], 30%
glycerol) diluted 1:3inRIPA buffer(25mM Tris [pH 8.2], 50 mMNaCl,
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containing 10 mM iodoacetamide and complete protease inhibitor
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(J affray et al., 1995). Horseradish peroxidase±conjugated anti-
mouse IgG and anti-rabbit IgG (Amersham) were used as secondary
antibodies. After ECL detection, Western blots were stripped as
described (Roff et al., 1996).
IgG froma preimmune rabbit serumorfromanti-I?B? rabbit antisera
were covalently cross-linked to proteinA±Sepharose beads (Sigma)
using dimethylpimelimidate as described (Arenzana-Seisdedos et
al., 1995). Cells were lysed in SDS and diluted 1:10 in PBS/0.5%
NP40 plus complete protease inhibitors before incubation for 3 hr
at 20?C with the antibody-coated beads. The beads were collected,
washed five times with ice-cold PBS/0.5% NP40 plus complete
protease inhibitor cocktail, and the antigen±antibody complexes
were recovered by boiling in SDS sample buffer.
DNA encoding the HA peptide (YPYDVPDYA) was inserted into
pcDNA3using KpnIand BamHIcloning sites and the following oligo-
C TACGCCCTTTCCCC TTG-3? and 5?-GATC CAAGGGAAAGCCGT
SUMO-1 cDNA (Desterro etal., 1997) was inserted into the BamHI
cloning site of the constructed pcDNA3/HA-Nvector. I?B? ctag and
mutants in pcDNA3, ConALuc, 3 EnhConALuc, HIV1LTRLuc, and
??BHIV1LTRLuc reporter vectors and the gene for Ubch9 inserted
into pcDNA3and pGEX-2T were as previously described (Rodriguez
et al., 1996; Desterro et al., 1997). The DNA sequence of all plasmid
inserts was determined by automated DNA sequencing (ABI377).
In Vitro SUMO-1 Conjugation Assay
35S-Met-labeled in vitro transcribed/translated (Promega, wheat
germ) I?B? proteins (0.5 ?l) were incubated with 3 ?l of HeLa cell
fraction (frII.4) containing SUMO-1 E1 activity (Desterro et al., 1997)
in a 10 ?l reaction including an ATP regenerating system (50 mM
Tris [pH7.6], 5 mMMgCl2, 2mMATP, 10mMcreatinephosphate, 3.5
U/mlof creatinekinase, and 0.6U/mlof inorganic pyrophosphatase),
SUMO-1 (200 ?g/ml), Ubch9 (50 ?g/ml), and 2.5 ?g/ml of recombi-
nant p65. Reactions were incubated at 37?C for 2 hr. After terminat-
ing the reactions with SDS sample buffercontaining mercaptoetha-
nol, reaction products were fractionated by SDS-PAGE (8.5%) and
the dried gels analyzed by phosphorimaging (Fujix BAS 1500, Mac-
BAS software). SUMO and Ubch9 were expressed and purified as
reported (Desterro et al., 1997).
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