IκB kinase α phosphorylation of TRAF4 downregulates innate immune signaling.
ABSTRACT Despite their homology, IκB kinase α (IKKα) and IKKβ have divergent roles in NF-κB signaling. IKKβ strongly activates NF-κB while IKKα can downregulate NF-κB under certain circumstances. Given this, identifying independent substrates for these kinases could help delineate their divergent roles. Peptide substrate array technology followed by bioinformatic screening identified TRAF4 as a substrate for IKKα. Like IKKα, TRAF4 is atypical within its family because it is the only TRAF family member to negatively regulate innate immune signaling. IKKα's phosphorylation of serine-426 on TRAF4 was required for this negative regulation. Binding to the Crohn's disease susceptibility protein, NOD2, is required for TRAF4 phosphorylation and subsequent inhibition of NOD2 signaling. Structurally, serine-426 resides within an exaggerated β-bulge in TRAF4 that is not present in the other TRAF proteins, and phosphorylation of this site provides a structural basis for the atypical function of TRAF4 and its atypical role in NOD2 signaling.
- [show abstract] [hide abstract]
ABSTRACT: The cytosolic sensors Nod1 and Nod2 and Toll-like receptors (TLRs) activate defense signaling pathways in response to microbial stimuli. However, the role of Nod1 and Nod2 and their interplay with TLRs during systemic bacterial infection remains poorly understood. Here, we report that macrophages or mice made insensitive to TLRs by previous exposure to microbial ligands remained responsive to Nod1 and Nod2 stimulation. Furthermore, Nod1- and Nod2-mediated signaling and gene expression are enhanced in TLR-tolerant macrophages. Further analyses revealed that innate immune responses induced by bacterial infection relied on Nod1 and Nod2 and their adaptor RICK in macrophages pretreated with TLR ligands but not in naive macrophages. In addition, bacterial clearance upon systemic infection with L. monocytogenes was critically dependent on Nod1 and Nod2 when mice were previously stimulated with lipopolysaccharide or E. coli. Thus, Nod1 and Nod2 are important for microbial recognition and host defense after TLR stimulation.Immunity 03/2008; 28(2):246-57. · 19.80 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The eukaryotic cell division cycle is characterized by a sequence of orderly and highly regulated events resulting in the duplication and separation of all cellular material into two newly formed daughter cells. Protein phosphorylation by cyclin-dependent kinases (CDKs) drives this cycle. To gain further insight into how phosphorylation regulates the cell cycle, we sought to identify proteins whose phosphorylation is cell cycle regulated. Using stable isotope labeling along with a two-step strategy for phosphopeptide enrichment and high mass accuracy mass spectrometry, we examined protein phosphorylation in a human cell line arrested in the G(1) and mitotic phases of the cell cycle. We report the identification of >14,000 different phosphorylation events, more than half of which, to our knowledge, have not been described in the literature, along with relative quantitative data for the majority of these sites. We observed >1,000 proteins with increased phosphorylation in mitosis including many known cell cycle regulators. The majority of sites on regulated phosphopeptides lie in [S/T]P motifs, the minimum required sequence for CDKs, suggesting that many of the proteins may be CDK substrates. Analysis of non-proline site-containing phosphopeptides identified two unique motifs that suggest there are at least two undiscovered mitotic kinases.Proceedings of the National Academy of Sciences 08/2008; 105(31):10762-7. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Recent investigations have elucidated the cytokine-induced NF-kappaB activation pathway. IkappaB kinase (IKK) phosphorylates inhibitors of NF-kappaB (IkappaBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-kappaB. We have examined the possibility that IKK can phosphorylate the p65 NF-kappaB subunit as well as IkappaB in the cytokine-induced NF-kappaB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-alpha in a time dependent manner similar to IkappaB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKbeta efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-kappaB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-kappaB.IkappaB complex.Journal of Biological Chemistry 11/1999; 274(43):30353-6. · 4.65 Impact Factor
I?B Kinase ? Phosphorylation of TRAF4 Downregulates Innate
Jill M. Marinis,aJessica E. Hutti,bCraig R. Homer,eBrian A. Cobb,aLewis C. Cantley,c,dChristine McDonald,eand Derek W. Abbotta
Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USAa; Lineberger Comprehensive Cancer Center and Department of
Biology, University of North Carolina, Chapel Hill, North Carolina, USAb; Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USAc; Division of
Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USAd; and Department of Pathobiology, Lerner Research Institute, Cleveland Clinic
Foundation, Cleveland, Ohio, USAe
autoinflammatory disorders (11, 44). In no pathway is this more
evident than in the inflammatory disease-associated NOD2 sig-
naling pathway. The CARD15 gene, which encodes NOD2, was
granulomatous inflammatory disorder of the gastrointestinal
osis (EOS) and Blau syndrome (3, 8, 23). Like Crohn’s disease,
inflammation, only at different anatomic locations in the body.
Since both gain-of-function and loss-of-function mutations and
polymorphisms in NOD2 cause granulomatous inflammation,
nation of NOD2 signaling. When this balance is disrupted, in-
flammatory disease results.
series of ubiquitination and phosphorylation events that ulti-
mately allow a coordinated cytokine response (6, 26, 45). This
response is largely mediated through NF-?B and, by extension,
losome consists of the kinases IKK? and IKK? held together by
the scaffolding protein IKK? (NEMO, for NF-?B essential modi-
fier). IKK? and IKK? are highly homologous kinases (9, 11). Of
the two, IKK? has been much better studied. Upon activation,
IKK? phosphorylates the NF-?B inhibitory protein, I?B?, to tar-
array technology outlined the preferred amino acid phosphoryla-
direct much of the positive NF-?B signaling in the cell.
myriad of diseases, including both immunodeficiencies and
In contrast, much less is known about IKK?. Fewer substrates
have been identified, and of these substrates, most are also phos-
phorylated by IKK?, suggesting a possible redundancy between
these two kinases. However, this redundancy has been challenged
recently by genetic mouse models suggesting that IKK? has a
stances, IKK? may actually help limit NF-?B activation (28, 29).
viable but have exacerbated inflammation at a variety of mucosal
surfaces. In these mice, IKK? inactivation results in greatly in-
creased expression of proinflammatory and antiapoptotic genes
(28). This negative regulatory role of IKK? has also recently been
shown to be crucial for signaling in the A20 ubiquitin-editing
complex. IKK?, but not IKK?, phosphorylates the ubiquitin
binding protein TAX1BP1. This phosphorylation is essential for
the formation of the A20 ubiquitin-editing complex and is re-
quired for proper termination of tumor necrosis factor (TNF)-
and interleukin-1 (IL-1)-dependent NF-?B signaling (43). Given
the enigmatic nature of IKK? and the surprising role that it plays
in negatively regulating NF-?B signaling, the identification of
In this work, we perform peptide substrate array analysis on
IKK? and identify the atypical TRAF family member protein,
TRAF4, as a novel substrate for IKK?. Members of the TNF re-
acterized as downstream adaptor molecules in a variety of proin-
Received 20 January 2012 Returned for modification 10 February 2012
Accepted 18 April 2012
Published ahead of print 30 April 2012
Address correspondence to Derek W. Abbott, firstname.lastname@example.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
July 2012 Volume 32 Number 13Molecular and Cellular Biology p. 2479–2489mcb.asm.org
that work to positively regulate inflammatory signaling down-
stream of agonists like TNF, lipopolysaccharide (LPS), or IL-1,
TRAF4 negatively regulates innate immune signaling. TRAF4
binds directly to the Nod-like receptor (NLR) family member,
NOD2, to inhibit NF-?B activation (34). Additionally, TRAF4
also binds to TRAF6 and TRIF to dampen Toll-like receptor
reason TRAF4 functions so much differently than the other
TRAFs is unknown. In this work, we identify an IKK?-inducible
phosphorylation site on TRAF4 that is required for its inhibitory
not present in the other TRAF family members. Phosphorylation
of this ?-bulge increases TRAF4 stability and activity to allow
activity. Thus, in this work, we generate proteomic tools to help
identify IKK? substrates. We identify a phosphorylation site on
TRAF4 that helps explain TRAF4’s unique function among the
TRAF family proteins as well as identifying a novel mechanism of
an innate immune signaling pathway.
MATERIALS AND METHODS
5% fetal bovine serum (FBS; HyClone). RAW264.7 macrophages and
HCT116 cells were maintained in DMEM containing 10% FBS (Hy-
followed by neomycin selection (300 ?g/ml; InvivoGen) in their respec-
tive media. Clones (?1,000) were pooled. Calcium phosphate precipita-
tion transfections in HEK293T cells were carried out as previously de-
EGTA, 1 mM EGTA, 2.5 mM sodium orthophosphate, 1 mM ?-glycero-
phosphate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM NaVO4,
Protein G Sepharose beads (Invitrogen) were added to lysates, and IPs
were washed five times in lysis buffer prior to Western blotting. Western
blotting was completed on nitrocellulose membranes (Bio-Rad) as previ-
ously described (2).
Antibodies, plasmids, and reagents. RIP2, Omni, and TRAF4 anti-
bodies were purchased from Santa Cruz Technology. Glutathione
chased from Cell Signaling Technology. Antihemagglutinin (anti-HA;
HA-11) was purchased from Covance. Phospho-IKK motif antibody was
generated as previously described (19). NTAP-TRAF4, Omni-TRAF4,
and NotI sites of pEBG. The GST-IKK? K44A, Omni-S426A TRAF4,
NTAP-S426A TRAF4, and Omni-S426D TRAF4 mutants were generated
by QuikChange site-directed mutagenesis (Stratagene). Point mutations
containing an N-terminal GST tag was purchased from Millipore. A 5=
Flag-fused retroviral TRAF4 was generated as previously described for
generation of stable macrophages and HT29 cells (34). Flag beads (M2)
(MDP) and LPS were obtained from Invivogen. IL-1? was purchased
from R&D Systems.
Positional scanning peptide library assay. Recombinant GST-IKK?
48). Briefly, the kinase buffer contained 50 mM Tris (pH 7.5), 12 mM
[?-32P]ATP per reaction mix. Kinase reaction mixes were incubated at
30°C for 2 h before being spotted on streptavidin-coated nitrocellulose.
After extensive washing, including washes in 1% SDS and 2 M NaCl,
(PSSM) plots were generated by the quantification of relative32P incor-
For example, proline at the ?5 position was quantitated and ranked
against the average signal of the 21 amino acids at that position.
In vitro kinase assays. NTAP-TRAF4 and NTAP-S426A TRAF4 were
isolated from HEK293T cell lysates using streptavidin-agarose as de-
scribed above. Recombinant full-length human GST-IKK? (150 ng; Mil-
dithiothreitol (DTT), 1 mM Na3VO4, 10 mM MgCl2, and 100 ?M ATP.
Reaction mixes were incubated at 30°C for 30 min. The reaction was
stopped by the addition of 2? SDS-PAGE sample buffer, followed by
alized by autoradiography.
Luciferase reporter gene assay. HEK293T cells were plated in tripli-
cate at 4 ? 104cells/well. Reporter plasmids and expression constructs
Quantitative reverse transcription-PCR (RT-PCR). Total RNA was
extracted using an RNeasy kit (Qiagen) according to the manufacturer’s in-
structions from the cell lines indicated in the text and figure legends. DNA
synthesis was performed using Qiagen’s Quantitect reverse transcription kit
using primers against mouse TNF-? (forward, 5=-GGTGCCTATGTCTCA
GCCTCTT-3=; reverse, 5=-GCCATAGAACTGATGAGAGGGAG-3=),
MIP2? (forward, 5=-CCACCAACCACCAGGCTACAGGGGC-3=; re-
verse, 5=-AGGCTCCTCCTTTCCAGGTCAGTTAGC-3=), and glyceral-
dehyde-3-phosphate dehydrogenase (GAPDH) (forward, 5=-AGGCCGG
TGCTGAGTATGTC-3=; reverse, 5=-TGCCTGCTTCACCACCTTCT-
3=), along with the iQ SYBR green Supermix (Bio-Rad) and detection
using a Bio-Rad iCycler. Data shown are normalized to GAPDH.
Salmonella infection and gentamicin protection assay. Salmonella
day, Salmonella cells were diluted 1:8 and allowed to grow for another 45
min until they reached exponential growth phase. Salmonella was diluted
in DMEM lacking antibiotics, and cells were infected at a multiplicity of
infection (MOI) of 10:1. After 30 min, Salmonella-containing medium
was replaced with fresh medium containing gentamicin sulfate (50 ?g/
ml). After 1 h, lysates were placed on ice for 5 min in 1% Triton X-100 in
LB agar plates and incubated overnight at 30°C. CFU were counted the
Statistics. Data are presented as means, with error bars representing
standard error of the mean (SEM) of at least three different experiments
5, software. Significance was determined by P values of ?0.05 after one-
way analysis of variance (ANOVA).
protein structure homology modeling was done using SWISS-MODEL
Marinis et al.
mcb.asm.orgMolecular and Cellular Biology
an E-value of 2.9E?43 (39) and Q mean Z-score of ?0.143. DeepView-
Swiss-PdbViewer was used for visualization of the model structure.
Determination of IKK? phosphorylation motif and novel sub-
strates. Many innate immune signaling pathways converge at the
level of the IKK signalosome, the core of which consists of the
catalytic subunits IKK? and IKK? and the regulatory scaffolding
protein IKK? (NEMO) (9, 11). IKK?, but not IKK?, is required
IKK? is instead emerging as a negative regulator of canonical
NF-?B activation (28, 29, 43). To elucidate the kinase-dependent
mechanisms by which IKK? inhibits NF-?B, it is important to
identify novel substrates of IKK? that are not also substrates of
IKK?. To this end, we performed a proteomic and bioinformatic
screen to identify novel substrates for IKK?. Positional scanning
peptide array technology was used to identify the preferred phos-
phorylation motif for IKK?. Biotinylated peptide libraries were
subjected to kinase assays using recombinant GST-IKK? and [?-
32P]ATP. The libraries consisted of peptides that have a centrally
acids on either side, except for one position which is fixed to a
single amino acid (17). Phosphothreonine or phosphotyrosine
phosphorylation events within the preferred substrate motif for
IKK?. Peptides were captured on a streptavidin-coated mem-
each amino acid at each position relative to the phosphorylation
(K44M IKK?) was utilized. This analysis revealed that IKK?, but
not kinase-dead K44M IKK?, displayed both positive and nega-
tive sequence specificity at several positions relative to the central
phosphoacceptor (Fig. 1A). Selection preferences for IKK? are
preferred at the ?2 and ?3 positions, and branched hydropho-
bics are preferred at the ?1 position. IKK? has a preference for
?4 positions. In fact, the elevated phosphothreonine at the ?4
position is consistent with whole-cell mass spectrometry experi-
ments that suggest threonine at ?4 is phosphorylated in TRAF4
(http://www.phosida.de) (16, 38). PSSM plots show the relative
for bioinformatic searching of potential substrates (Fig. 1B).
There is a strongly preferred phosphorylation sequence between
IKK? (19) and IKK?, which is not unexpected given the high
degree of homology within their kinase domains.
After determining this preferred phosphorylation motif for
IKK?, we conducted a tiered bioinformatic screen to narrow our
strates. Given our lab’s long-term interest in NOD2 signaling, we
known to bind to NOD2 or known to proximally affect NOD2
signaling (45). Twenty-four of these proteins were subjected to
Phosphosite (www.phosphosite.org) and Phosida (www.phosida
.de) searches to identify proteins that contain phosphorylation
of five independent instances of mass spectrometry identification
Scansite (http://scansite.mit.edu/) matrix search using IKK?’s
phosphorylation motif further narrowed the data set to five pro-
teins that passed the first two filters and also contained the pre-
ferred IKK? phosphorylation motif (Fig. 1C). Of these proteins,
we focused on TRAF4. Similar to the atypical nature of IKK?,
(25). Unlike the other six TRAF family members, TRAF4 plays a
negative regulatory role in NF-?B activation (34, 47). Since S426
within the predicted IKK? phosphorylation motif of TRAF4 is
also conserved across species (Fig. 1D), we sought to validate
TRAF4 as an IKK? substrate.
IKK? phosphorylates TRAF4 at S426. To determine if IKK?
in fact phosphorylates TRAF4 and to determine the specificity of
this phosphorylation motif compared to other IKK family mem-
bers, we cotransfected HEK293T cells with Omni-TRAF4 and the
IKK family members IKK?, IKK?, and IKKε. Using an antibody
that immunoprecipitates proteins containing a phosphorylated
IKK motif (19), phospho-TRAF4 was immunoprecipitated in the
presence of IKK? but not in the presence of IKK? or IKKε (Fig.
2A). To verify that IKK? phosphorylation occurred at S426, the
point mutation S426A was introduced into TRAF4. IKK? phos-
fected with GST-IKK? and Omni-S426A TRAF4 (Fig. 2B). In or-
der to verify that TRAF4 could be phosphorylated directly by
IKK? at S426, Omni-tagged TRAF4 or S426A TRAF4 was immu-
nant full-length human GST-IKK? and [?-32P]ATP. We found
that IKK? could phosphorylate TRAF4 but not S426A TRAF4 in
vitro (Fig. 2C). Taken together, these data strongly suggest that
IKK? phosphorylates TRAF4 at S426.
Innate immune activation induces phosphorylation of en-
dogenous TRAF4. Endogenous TRAF4 phosphorylation was de-
tected in a time- and stimulus-dependent manner. RAW264.7
between 15 and 30 min after agonist addition (Fig. 3A and B).
that upon treatment with IL-1 or LPS, a variety of kinases in ad-
databases such as Phosphosite or Phosida show at least eight in-
that upon treatment with agonists, which activate a variety of ki-
the double banding represents multiphosphorylated TRAF4 (for
instance, a potential example would include phospho-S426 alone
or phospho-T422/phospho-S426 double phosphorylation; both
of these bands could be visualized by this motif antibody). In all,
phosphorylation of S426 has been identified in over five indepen-
dent mass spectrometry studies (7, 16, 38); phosphorylation of
S426 was identified as a potential substrate of IKK? by peptide
substrate array technology, and in both in vitro overexpression
systems (Fig. 2) and in response to IKK? agonists in endogenous
systems (Fig. 3), TRAF4 is phosphorylated.
Because TRAF4 is a NOD2 binding protein and negatively af-
which is upstream of the IKK signalosome (1, 20), also induced
TRAF4 phosphorylation. RAW264.7 macrophages were stimu-
lated with the NOD2 agonist, muramyl dipeptide (MDP), for 45,
IKK? Phosphorylates TRAF4 To Inhibit Immune Signaling
July 2012 Volume 32 Number 13mcb.asm.org 2481
lular cytosolic sensor, NOD2, is activated (45). Similar to the re-
sults of IL-1? and LPS stimulation, endogenous TRAF4 phos-
phorylation was detected following MDP-induced NF-?B (Fig.
3C). To ensure that TRAF4 phosphorylation following MDP
stimulation occurs at S426, RAW264.7 macrophages were stably
transduced with a Flag-TRAF4 retrovirus or a Flag-S426A TRAF4
retrovirus. Stable expression of TRAF4 increased both basal and
MDP-induced TRAF4 phosphorylation, while neither basal nor
stimulus-dependent TRAF4 phosphorylation was detected in
macrophages stably expressing S426A TRAF4 (Fig. 3D).
IKK? phosphorylation of TRAF4 following NOD2 activa-
tion requires NOD2 binding. Since MDP activation of NOD2
induced TRAF4 phosphorylation, we suspected that NOD2 over-
pressing functional IKK?. Overexpression of NOD2 in HEK293T
cells provided a model system that we could manipulate to deter-
mine if NOD2-induced TRAF4 phosphorylation was dependent
on IKK?. HA-NOD2 cotransfection with NTAP-TRAF4 was suf-
ficient for TRAF4 phosphorylation. Importantly, the presence of
the kinase-dead GST-K44A IKK? was able to inhibit NOD2-in-
FIG 1 Determination of IKK? preferred phosphorylation motif and target substrates. (A) Kinase assays were performed on 198 peptide libraries with recom-
binant IKK? or, as a control, kinase-dead IKK?. The general sequence for these libraries was Y-A-X-X-X-Z-X-S/T-X-X-X-X-A-G-K-K-biotin (Z, fixed amino
acid; X, equimolar mixture of amino acids) with a fixed amino acid at the indicated position. Relative incorporation of32P was measured after the individual
degenerate phosphorylated peptides were captured on a streptavidin-coated membrane. While IKK? (upper panel) gave a strong, consistent motif, no motif
could be identified using a kinase-inactive (K44A) IKK?. (B) PSSM plot of positive and negative amino acid selections for IKK?. There is a general, strong
acids. (C) Bioinformatics strategies were used to decipher potential substrates of IKK? downstream in the NOD2 signaling pathway. Phosphosite and Phosida
mass spectrometry-verified phosphorylation databases were searched with proteins known to either bind NOD2 or proximally affect NOD2 signaling. Phos-
sites identified more than five times in mass spectrometry databases (13 total phosphorylation sites). These sites were then subjected to Scansite analysis, using
the IKK? preferential phosphorylation motif identified in a matrix. Of the 13 potential sites identified, 5 of these passed Scansite screening as potential IKK?
phosphorylation sites. (D) The potential IKK? phosphorylation sites identified were analyzed for evolutionary conservation. TRAF4 showed the highest degree
of evolutionary conservation.
Marinis et al.
mcb.asm.orgMolecular and Cellular Biology
sion in IKK??/?murine embryonic fibroblasts (MEFs) failed to
induce TRAF4 phosphorylation (Fig. 4B, left panel). As a control,
NOD2-induced phosphorylation of TRAF4 in wild-type (WT)
MEFs was included (Fig. 4B, right panel). We next sought to de-
dent on NOD2 activity and/or TRAF4 binding to NOD2. We first
compared the ability of wild-type NOD2 or the most common
Crohn’s disease-associated variant, L1007insC NOD2, to phos-
tation that introduces a premature stop codon leading to the pro-
duction of a truncated protein lacking the last 33 amino acids in
the leucine rich repeat region (36). The L1007insC is a loss-of-
function mutation in that it has decreased capacity for IKK signa-
losome activation and subsequent NF-?B activation (27, 36, 49).
We have previously shown that TRAF4 maintains binding to
L1007insC NOD2 (34). However, cotransfection of HEK293T
cells with Omni-TRAF4 and HA-L1007insC failed to induce
TRAF4 phosphorylation (Fig. 4C) suggesting that NOD2 activity
is necessary for TRAF4 phosphorylation. Collectively, these find-
ings suggest that in addition to activating IKK? for I?B? phos-
phorylation, NOD2 activation induces downstream IKK? activa-
tion to promote TRAF4 phosphorylation at S426.
The TRAF4 binding motif of NOD2 has been mapped to a
GLEE motif at the N terminus of the NOD domain of NOD2
(amino acids 277 to 280). Mutation of the two glutamates to ala-
nines, EE279AA, prevents TRAF4 binding to NOD2 and prevents
binding is required for NOD2-induced TRAF4 phosphorylation,
upon cotransfection experiments (Fig. 4D). Since the EE279AA
NOD2 mutant has increased basal NF-?B-inducing activity, this
result suggests that IKK signalosome and NF-?B activation is not
sufficient for NOD2-induced phosphorylation of TRAF4 (34).
These findings suggest a mechanism whereby NOD2 both acti-
it as a substrate.
TRAF4 phosphorylation increases TRAF4 stability and ac-
tivity. Given that the TRAF family members have been shown to
be regulated by proteasomal-mediated degradation (22, 30, 31),
the effect of phosphorylation on TRAF4 stability and activity was
tested. The half-life of TRAF4 was measured in transfected
HEK293T cells treated with the protein biosynthesis inhibitor cy-
cloheximide in the absence or presence of IKK? or K44A IKK?.
of cycloheximide treatment. Cotransfection of IKK? increases
TRAF4 expression while the kinase-inactive K44A IKK? did not
(Fig. 5A). Similarly, HT29 cells that stably express WT TRAF4 or
S426A TRAF4 were treated with cycloheximide. In the stably
transduced cell lines, WT TRAF4 was not degraded as rapidly;
however, the half-life of S426A TRAF4 was markedly decreased
(Fig. 5B). These results indicate that phosphorylation at S426 by
IKK? increases the stability of TRAF4. The substrate for TRAF4
remains unknown; however, since TRAF4 is an E3 ubiquitin li-
gase, its autoubiquitination can be used as a surrogate measure of
FIG 2 IKK? phosphorylates TRAF4 at S426. (A) Omni-TRAF4 was cotransfected into HEK293T cells with GST-IKK?, IKK?, or IKKε. Proteins containing a
phosphorylated IKK (p-IKK) motif were immunoprecipitated and subjected to Western blotting for TRAF4. Only IKK? induced TRAF4 phosphorylation. (B)
the S426A TRAF4 did not. (C) In vitro kinase assays were performed using recombinant full-length GST-IKK? and purified NTAP-TRAF4 or NTAP-S426A
TRAF4 from transfected HEK293T cells. GST-IKK? induced phosphorylation of TRAF4 but not S426A TRAF4 as shown by incorporation of [?-32P]ATP. rbt,
rabbit; ms, mouse; gt, goat.
IKK? Phosphorylates TRAF4 To Inhibit Immune Signaling
July 2012 Volume 32 Number 13mcb.asm.org 2483
tion with S426A TRAF4 failed to induce autoubiquitination (Fig.
5C). Furthermore, cotransfection of K44A IKK? inhibited basal
levels of TRAF4 autoubiquitination (Fig. 5C). Taken together,
these results support a model whereby IKK? phosphorylation at
S426 increases the stability and activity of TRAF4.
S426 lies in a structural domain unique to TRAF4. TRAF4 is
an atypical member of the TRAF family of proteins. Unlike other
TRAF proteins, TRAF4 has not been found to bind to classical
TNF receptors (25); instead, TRAF4 is the only TRAF family
member to inhibit TLR and NLR signal transduction pathways
(34, 47). To determine if phosphorylation of S426 could contrib-
ute to the novel function of TRAF4, we examined whether this
residue contributed to the unique structure of TRAF4 relative to
of the TRAF domain, which contains S426 and which confers
substrate binding, has not been examined. Given this, we first
been determined (4, 39, 52, 53). Residues of the ?-sheets that
constitute the antiparallel ?-sandwich of the TRAF2 TRAF do-
main are indicated in arrows above the corresponding TRAF4
residues (Fig. 6A). Of note, S426 resides within an amino acid
stretch that is not present in any of the other TRAFs (Fig. 6A, red
box) and structurally resides in an extended ?-bulge found be-
tween ?-sheets 6 and 7 that is unique to TRAF4 (Fig. 6B). Impor-
tantly, S426 is placed prominently within this exaggerated loop
erties relative to the other TRAF family members.
To test whether TRAF4 phosphorylation of this unique
?-bulge augments TRAF4 function, as would be predicted from
the stability and activity results, NF-?B luciferase reporter assays
were conducted in HEK293T cells cotransfected with NOD2 and
either Omni-TRAF4 or Omni-S426A TRAF4. TRAF4 was able to
inhibit MDP-induced NF-?B reporter activity in a dose-depen-
result. RAW264.7 macrophages stably expressing TRAF4 had
dampened NF-?B responses, indicated by decreased I?B? phos-
phorylation, while macrophages expressing S426A TRAF4 main-
tained a response similar to empty vector-transduced macro-
phages treated with MDP (Fig. 6D). These results suggest that
as a negative regulator. If this is true, we might expect a potential
phosphomimetic S426D TRAF4 to maintain or enhance the in-
hibitory role of TRAF4. In fact, RAW264.7 macrophages stably
tivation (Fig. 6D).
To then determine if TRAF4 phosphorylation and inhibition
of NF-?B signaling had a physiological impact, we examined
mRNA levels of two genes regulated by NF-?B, TNF-?, and
MIP2?. These genes were chosen because expression of these cy-
tokines is increased in macrophages derived from kinase-inactive
IKK? knock-in mice (28). RAW264.7 macrophages stably ex-
(Fig. 7B) both basally and following stimulation with MDP com-
pared to cells expressing empty vector. In contrast, expression of
S426A TRAF4 failed to decrease expression of TNF-? and MIP2?
FIG 3 Endogenous TRAF4 is phosphorylated in both a time- and stimulus-dependent manner. (A to C) RAW264.7 Macrophages were stimulated with IL-1?
(A), LPS (B), or MDP (C) for the indicated times. Lysates were subjected to immunoprecipitation with a phospho-IKK motif antibody. Endogenous phosphor-
WT TRAF4 or S426A TRAF6. After neomycin selection, MDP-inducible TRAF4 phosphorylation was detected in macrophages expressing WT TRAF4 but not
in macrophages expressing the S426A TRAF4.
Marinis et al.
mcb.asm.orgMolecular and Cellular Biology
(Fig. 7A and B).
NOD2 activation plays an important role in bacterial clear-
ance, and this has been studied using the intracellular Gram-neg-
ative bacterium Salmonella as a model. MDP stimulation during
Salmonella infection enhances bacterial clearance in a NOD2-de-
pendent manner (12, 13, 35, 51), and TRAF4 has been shown to
FIG 4 NOD2 activation and binding to TRAF4 induce TRAF4 phosphorylation, and this is dependent on IKK?. (A) HA-NOD2 was cotransfected into
this phosphorylation was lost when the kinase-dead K44A IKK? was present. (B) IKK??/?MEFs were cotransfected with Omni-TRAF4 or HA-NOD2 (left
phosphorylation, while the K44A IKK? failed to do so. NOD2 induced TRAF4 phosphorylation in wild-type MEFs (right panel). (C) HEK293T cells were
cotransfected with Omni-TRAF4 and HA-NOD2 or HA-L1007insC NOD2. Both IKK? and NOD2 induced TRAF4 phosphorylation, while the L1007insC
NOD2 failed to do so. (D) To determine if TRAF4 binding is required for NOD2-induced phosphorylation, HEK293T cells were cotransfected with Omni-
TRAF4 and HA-NOD2 or HA-EE279AA NOD2, which has previously been shown to be unable to bind TRAF4 (34). Wild-type NOD2 induced TRAF4
phosphorylation while the EE279AA NOD2 did not, suggesting that binding of NOD2 to TRAF4 is important for S426 phosphorylation.
FIG5 IKK? phosphorylation of TRAF4 at S426 increases TRAF4 stability and activity. (A and B) Cycloheximide time courses were performed to assess TRAF4
stability in HEK293T cells that were transfected with TRAF4 alone or in the presence of IKK? or K44A IKK? (A) and in HT29 cells stably expressing TRAF4 or
S426A TRAF4 (B). Cells were treated with cycloheximide for indicated times. Lysates were standardized for total protein concentration and expression levels of
TRAF4 were detected by Western blotting. IKK?, but not K44A IKK?, increased TRAF4 expression and stability (A). S426A TRAF4 had decreased stability in
stable HT29 cells (B). (C) Autoubiquitination and activity of TRAF4 were detected in HEK293T cells transfected with Omni-TRAF4, HA-ubiquitin, and either
IKK? or K44A IKK?. IKK? but not K44A IKK? increased autoubiquitination of TRAF4. IKK? did not increase autoubiquitination of S426A TRAF4.
IKK? Phosphorylates TRAF4 To Inhibit Immune Signaling
July 2012 Volume 32 Number 13mcb.asm.org 2485
inhibit NOD2-enhanced Salmonella killing by a mechanism de-
pendent on a physical interaction of TRAF4 with NOD2 (34). We
therefore tested whether TRAF4 phosphorylation was critical for
inhibition of NOD2 antibacterial activity. Gentamicin protection
assays were performed in HCT116 cells transfected with either
the S426D TRAF4 mutant inhibited MDP-induced Salmonella
clearance, while the S426A TRAF4 mutant did not affect NOD2
antibacterial activity (Fig. 7C). Similar results were observed in
gentamicin protection assays performed in RAW264.7 macro-
MDP-enhanced Salmonella clearance was inhibited by TRAF4
and S426D TRAF4 but not affected by S426A TRAF4 (Fig. 7D).
Collectively, these results indicate that TRAF4 contains a func-
between ?-sheets 6 and 7 unique to this TRAF family member,
which is required for TRAF4 inhibition of the NOD2 signaling
IKK signalosome, IKK? and IKK?, have opposing in vivo roles.
it has been difficult to differentiate their respective substrates de-
be the major kinase mediating activation of NF-?B while IKK?
of TRAF4 with other TRAF family members reveals that S426 is within an amino acid stretch that is unique to TRAF4 between strands ?6 and ?7. This stretch
adds 6 amino acid residues (boxed in red) to the linker between ?6 and ?7. Asterisk, single, fully conserved residue; colon, conservation between groups of
TRAF2 crystal structure (39, 53). The left panel shows ribbon diagram of the TRAF2 TRAF domain crystal structure while the right panel shows the predicted
in a stimulus-dependent manner, RAW264.7 macrophages retrovirally transduced to stably express TRAF4, S426A TRAF4, or S426D TRAF4 were subjected to
MDP stimulation. NF-?B activation was assessed by Western blotting lysates for I?B? phosphorylation. Macrophages expressing TRAF4 or the phospho-
mimetic S426D TRAF4 had reduced NF-?B activation while the S426A-expressing macrophages maintained intact signaling.
Marinis et al.
mcb.asm.org Molecular and Cellular Biology
substrates for IKK? using a proteomic/bioinformatic approach.
To this end, we performed peptide substrate array analysis fol-
lowed by bioinformatic screening to identify the E3 ubiquitin li-
IKK?, but not IKK?, phosphorylates TRAF4 at S426 following
showed that IKK? phosphorylation of TRAF4 is required for
inhibit NOD2-induced NF-?B activation (Fig. 6) and Salmonella
killing (Fig. 7). NOD2-induced phosphorylation is dependent on
IKK? activity as neither kinase-inactive K44A IKK? nor cells ge-
netically deficient for IKK? allow NOD2-induced phosphoryla-
tion of TRAF4 (Fig. 3). NOD2-induced IKK? phosphorylation of
TRAF4 was also dependent on NOD2 activation as the loss-of-
function Crohn’s disease-associated variant, L1007insC, had a
markedly decreased ability to induce TRAF4 phosphorylation
TRAF4, as a novel substrate of IKK? and suggest that phosphor-
ylation by IKK? plays a key role in the negative regulation of the
NOD2 signaling pathway.
signaling and bacterial clearance is consistent with our previous
findings that there is increased bacterial killing and, correspond-
ingly, decreased amounts of intracellular Salmonella bacteria
when endogenous TRAF4 expression is inhibited by small inter-
studies showing increased bacterial clearance in the absence of
IKK? or in the presence of a catalytically inactive IKK? (28, 29).
ance of bacteria and elevated levels of NF-?B target genes (28).
FIG 7 TRAF4 phosphorylation is required for inhibition of NOD2-induced target genes and Salmonella killing. (A and B) RAW264.7 macrophages stably
expressing TRAF4, S426A TRAF4, or S426D TRAF4 were stimulated with MDP for 4 h. RNA was isolated and subjected to quantitative RT-PCR for TNF-? (A)
or MIP2 (B) mRNA. Macrophages expressing TRAF4 or the phosphomimetic TRAF4 inhibited MDP-induced TNF-? and MIP2 mRNA levels while S426A-
(C). TRAF4 and the phosphomimetic S426D TRAF4 inhibited MDP-induced Salmonella killing while the S426A TRAF4 maintained MDP-induced Salmonella
(D), while S426A TRAF4-expressing macrophages maintained MDP-induced Salmonella killing. Data are represented as mean ? SEM.
IKK? Phosphorylates TRAF4 To Inhibit Immune Signaling
July 2012 Volume 32 Number 13mcb.asm.org 2487
Similarly, knock-in mice harboring a kinase-inactive IKK? also
have elevated NF-?B target genes and bacterial clearance (29).
Increased bacterial clearance could be beneficial to the host; how-
ever, the importance of having negative regulators to counterbal-
the kinase-inactive IKK? knock-in mice. While the kinase-inac-
tive IKK? mice are viable and able to clear a bacterial challenge,
they have unchecked and exacerbated inflammation, which ulti-
mately increases their mortality rates (28). In humans, loss of a
negative regulator can also contribute to disease pathogenesis
and/or severity, and this may help explain the inflammation seen
tualize how Crohn’s disease-associated NOD2 alleles can lose the
ability to activate NF-?B but still give rise to a disease character-
ized by inflammation. Our results suggest the possibility that in
addition to loss of acute NF-?B activity, the loss-of-function
NOD2 allele, L1007insC, also loses the ability to activate negative
regulators of NF-?B, such as IKK? and TRAF4. Loss of TRAF4
phosphorylation could uncouple the coordination of signaling
events that occur following NOD2 activation and could exacer-
bate inflammatory disease.
TRAF4 itself is highly conserved, with 97% identity between
mouse and human orthologues. Evolutionarily, the only TRAF
proteins described in Danio rerio are TRAF4a and TRAF4b (77%
and 68% identity with human TRAF4). Of the two, TRAF4a is
more homologous to TRAF4, while TRAF4b more closely resem-
bles TRAF6. This high degree of evolutionary conservation, cou-
pled with the fact that TRAF4 and TRAF6 precursor genes have
arisen earliest during evolution, implies that TRAF4 serves a very
important biological role (24, 25). In fact, there are unique struc-
tural elements in TRAF4 that are not present in any of the other
TRAFs. IKK?’s TRAF4 phosphorylation site lies in a 6-amino-
acid insertion that is not present in any of the other TRAFs. This
gests a functionally relevant evolutionary divergence of TRAF4.
to TRAF4 by creating an exaggerated ?-bulge that connects
?-sheets 6 and 7 within the TRAF domain (Fig. 6). This unique
?-bulge, with its corresponding phosphorylation, allows TRAF4
to then function as a negative regulator of NOD2 signaling. In
light of our previous findings showing that the binding of TRAF4
to NOD2 is required for inhibition of NOD2 signaling, we pro-
pose a model here whereby both the initial binding of TRAF4 to
NOD2 and the subsequent phosphorylation of TRAF4 by IKK?
are required for it to function as a negative regulator of NOD2
signaling. This is an induced proximity model in which the bind-
ing of TRAF4 to NOD2 serves to nucleate TRAF4 with the IKK
signaling complex for subsequent phosphorylation of TRAF4 by
from our previous study showing that MDP-induced TRAF4
(34), while time courses from the present study (Fig. 3C) show
MDP-induced phosphorylation occurring slightly later, between
45 and 90 min. The requirement for TRAF4 to bind NOD2 pre-
ceding its phosphorylation is supported by data in which the
phosphorylation (Fig. 4D). Thus, the unique ?-bulge structural
feature, with its phosphorylation by a negative regulator of NF-
?B, could confer TRAF4’s ability to function differently than the
other TRAF proteins.
function of TRAF4. We highlight the importance of TRAF4’s
structural divergence from the other TRAFs as TRAF4 contains a
phosphorylation site for the atypical IKK family member, IKK?.
Phosphorylation of this unique site confers TRAF4’s ability to act
negatively regulate a key signaling pathway in inflammatory dis-
This work was supported by NIH research grants R01GM86550-01
R01GM056203 (L.C.C.) and a Burroughs Wellcome Career Award for
Biomedical Scientists (10061206.01 to D.W.A.).
We thank George Dubyak (Case Western Reserve University, Cleve-
land, OH) and George Stark and XiaoXia Li (Cleveland Clinic Founda-
tion, Cleveland, OH) for helpful comments on the project.
1. Abbott DW, Wilkins A, Asara JM, Cantley LC. 2004. The Crohn’s
of a novel site on NEMO. Curr. Biol. 14:2217–2227.
2. Abbott DW, et al. 2007. Coordinated regulation of Toll-like receptor and
NOD2 signaling by K63-linked polyubiquitin chains. Mol. Cell. Biol. 27:
3. Casanova JL, Abel L. 2009. Revisiting Crohn’s disease as a primary im-
munodeficiency of macrophages. J. Exp. Med. 206:1839–1843.
4. Chung JY, Lu M, Yin Q, Lin SC, Wu H. 2007. Molecular basis for the
unique specificity of TRAF6. Adv. Exp. Med. Biol. 597:122–130.
5. Chung JY, Park YC, Ye H, Wu H. 2002. All TRAFs are not created equal:
common and distinct molecular mechanisms of TRAF-mediated signal
transduction. J. Cell Sci. 115:679–688.
6. Coll RC, O’Neill LA. 2010. New insights into the regulation of signalling
by toll-like receptors and nod-like receptors. J. Innate Immun. 2:406–
7. Dephoure N, et al. 2008. A quantitative atlas of mitotic phosphorylation.
Proc. Natl. Acad. Sci. U. S. A. 105:10762–10767.
8. Fritz JH, Ferrero RL, Philpott DJ, Girardin SE. 2006. Nod-like proteins
in immunity, inflammation and disease. Nat. Immunol. 7:1250–1257.
9. Ghosh S, Karin M. 2002. Missing pieces in the NF-?B puzzle. Cell
10. Hacker H, Karin M. 2006. Regulation and function of IKK and IKK-
related kinases. Sci. STKE 2006:re13. doi:10.1126/stke.3572006re13.
11. Hayden MS, Ghosh S. 2008. Shared principles in NF-?B signaling. Cell
12. Hisamatsu T, et al. 2003. CARD15/NOD2 functions as an antibacterial
factor in human intestinal epithelial cells. Gastroenterology 124:993–
13. Homer CR, Richmond AL, Rebert NA, Achkar JP, McDonald C. 2010.
ATG16L1 and NOD2 interact in an autophagy-dependent, anti-bacterial
pathway implicated in Crohn’s disease pathogenesis. Gastroenterology
14. Hu MC, et al. 2004. IkappaB kinase promotes tumorigenesis through
inhibition of forkhead FOXO3a. Cell 117:225–237.
15. Hugot JP, et al. 2001. Association of NOD2 leucine-rich repeat variants
with susceptibility to Crohn’s disease. Nature 411:599–603.
16. Hutchins AP, Robson P. 2009. Unraveling the human embryonic stem
cell phosphoproteome. Cell Stem Cell 5:126–128.
17. Hutti JE, et al. 2004. A rapid method for determining protein kinase
phosphorylation specificity. Nat. Methods 1:27–29.
18. Hutti JE, et al. 2009. Phosphorylation of the tumor suppressor CYLD by
the breast cancer oncogene IKKε promotes cell transformation. Mol. Cell
19. Hutti JE, et al. 2007. IkappaB kinase beta phosphorylates the K63 deu-
biquitinase A20 to cause feedback inhibition of the NF-?B pathway. Mol.
Cell. Biol. 27:7451–7461.
Marinis et al.
mcb.asm.org Molecular and Cellular Biology
21. Inohara N, et al. 2003. Host recognition of bacterial muramyl dipeptide
22. Kalkan T, Iwasaki Y, Park CY, Thomsen GH. 2009. Tumor necrosis
factor-receptor-associated factor-4 is a positive regulator of transforming
23. Kanazawa N, et al. 2005. Early-onset sarcoidosis and CARD15 mutations
with constitutive nuclear factor-kappaB activation: common genetic eti-
ology with Blau syndrome. Blood 105:1195–1197.
24. Kedinger V, et al. 2005. Spatial and temporal distribution of the traf4
genes during zebrafish development. Gene Expr. Patterns 5:545–552.
Med. Biol. 597:60–71.
26. Kim YG, et al. 2008. The cytosolic sensors Nod1 and Nod2 are critical for
ligands. Immunity 28:246–257.
27. Kobayashi K, et al. 2002. RICK/Rip2/CARDIAK mediates signalling for
receptors of the innate and adaptive immune systems. Nature 416:194–
28. Lawrence T, Bebien M, Liu GY, Nizet V, Karin M. 2005. IKK? limits
macrophage NF-?B activation and contributes to the resolution of in-
flammation. Nature 434:1138–1143.
29. Li Q, et al. 2005. Enhanced NF-?B activation and cellular function in
macrophages lacking I?B kinase 1 (IKK1). Proc. Natl. Acad. Sci. U. S. A.
30. Li S, et al. 2010. Ubiquitin ligase Smurf1 targets TRAF family proteins for
ubiquitination and degradation. Mol. Cell. Biochem. 338:11–17.
31. Li X, Yang Y, Ashwell JD. 2002. TNF-RII and c-IAP1 mediate ubiquiti-
nation and degradation of TRAF2. Nature 416:345–347.
32. Li ZW, et al. 1999. The IKK?subunit of I?B kinase (IKK) is essential for
33. Luo JL, et al. 2007. Nuclear cytokine-activated IKKalpha controls pros-
tate cancer metastasis by repressing Maspin. Nature 446:690–694.
34. Marinis JM, Homer CR, McDonald C, Abbott DW. 2011. A novel motif
in the Crohn’s disease susceptibility protein, NOD2, allows TRAF4 to
down-regulate innate immune responses. J. Biol. Chem. 286:1938–1950.
35. Oehlers SH, et al. 2011. The inflammatory bowel disease (IBD) suscep-
tibility genes NOD1 and NOD2 have conserved anti-bacterial roles in
zebrafish. Dis. Model. Mech. 4:832–841.
36. Ogura Y, et al. 2001. A frameshift mutation in NOD2 associated with
susceptibility to Crohn’s disease. Nature 411:603–606.
37. Ogura Y, et al. 2001. Nod2, a Nod1/Apaf-1 family member that is re-
38. Olsen JV, et al. 2010. Quantitative phosphoproteomics reveals wide-
spread full phosphorylation site occupancy during mitosis. Sci. Signal.
39. Park YC, Burkitt V, Villa AR, Tong L, Wu H. 1999. Structural basis for
self-association and receptor recognition of human TRAF2. Nature 398:
modulates NIK stability through IKK?-mediated phosphorylation. Sci.
Signal. 3:ra41. doi:10.1126/scisignal.2000778.
41. Reiley W, Zhang M, Wu X, Granger E, Sun SC. 2005. Regulation of the
deubiquitinating enzyme CYLD by I?B kinase gamma-dependent phos-
phorylation. Mol. Cell. Biol. 25:3886–3895.
42. Sakurai H, Chiba H, Miyoshi H, Sugita T, Toriumi W. 1999. I?B kinases
phosphorylate NF-?B p65 subunit on serine 536 in the transactivation
domain. J. Biol. Chem. 274:30353–30356.
43. Shembade N, Pujari R, Harhaj NS, Abbott DW, Harhaj EW. 2011. The
phorylating the regulatory molecule TAX1BP1. Nat. Immunol. 12:834–
44. Smahi A, et al. 2002. The NF-?B signalling pathway in human diseases:
from incontinentia pigmenti to ectodermal dysplasias and immune-
deficiency syndromes. Hum. Mol. Genet. 11:2371–2375.
45. Strober W, Watanabe T. 2011. NOD2, an intracellular innate immune
sensor involved in host defense and Crohn’s disease. Mucosal Immunol.
46. Takeda K, et al. 1999. Limb and skin abnormalities in mice lacking IKK?.
47. Takeshita F, et al. 2005. TRAF4 acts as a silencer in TLR-mediated sig-
naling through the association with TRAF6 and TRIF. Eur. J. Immunol.
48. Turk BE, Hutti JE, Cantley LC. 2006. Determining protein kinase sub-
strate specificity by parallel solution-phase assay of large numbers of pep-
tide substrates. Nat. Protoc. 1:375–379.
49. Watanabe T, et al. 2006. Nucleotide binding oligomerization domain 2
specific colitis. Immunity 25:473–485.
50. Wegener E, et al. 2006. Essential role for I?B kinase beta in remodeling
Carma1-Bcl10-Malt1 complexes upon T cell activation. Mol. Cell 23:
51. Yamamoto-Furusho JK, Barnich N, Hisamatsu T, Podolsky DK. 2010.
MDP-NOD2 stimulation induces HNP-1 secretion, which contributes to
NOD2 antibacterial function. Inflamm. Bowel Dis. 16:736–742.
52. Ye H, et al. 2002. Distinct molecular mechanism for initiating TRAF6
signalling. Nature 418:443–447.
53. Ye H, Park YC, Kreishman M, Kieff E, Wu H. 1999. The structural basis
for the recognition of diverse receptor sequences by TRAF2. Mol. Cell
IKK? Phosphorylates TRAF4 To Inhibit Immune Signaling
July 2012 Volume 32 Number 13 mcb.asm.org 2489