See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/7356272

Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor

signaling by mediating Mal degradation

Article  in  Nature Immunology · March 2006

DOI: 10.1038/ni1299 · Source: PubMed

CITATIONS READS

416 168

10 authors, including:

Sarah L Doyle Pearl Gray

Trinity College Dublin 16 PUBLICATIONS   2,374 CITATIONS   
31 PUBLICATIONS   2,449 CITATIONS   
SEE PROFILE
SEE PROFILE

Peter J Crack
University of Melbourne
89 PUBLICATIONS   3,452 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

HBV Immune Responses View project

Investigating the role of metabolites secreted by the parasite T.b. brucei in innate immune evasion. View project

All content following this page was uploaded by Ashley Mansell on 23 March 2014.

The user has requested enhancement of the downloaded file.

© 2006 Nature Publishing Group http://www.nature.com/natureimmunology ARTICLES

Suppressor of cytokine signaling 1 negatively

regulates Toll-like receptor signaling by mediating
Mal degradation
Ashley Mansell1, Rosealee Smith1, Sarah L Doyle2, Pearl Gray2, Jennifer E Fenner1, Peter J Crack1,
Sandra E Nicholson3, Douglas J Hilton3, Luke A J O’Neill2,4 & Paul J Hertzog1,4

Toll-like receptor (TLR) signals that initiate innate immune responses to pathogens must be tightly regulated to prevent
excessive inflammatory damage to the host. The adaptor protein Mal is specifically involved in signaling via TLR2 and TLR4.
We demonstrate here that after TLR2 and TLR4 stimulation Mal becomes phosphorylated by Bruton’s tyrosine kinase (Btk)
and then interacts with SOCS-1, which results in Mal polyubiquitination and subsequent degradation. Removal of SOCS-1
regulation potentiates Mal-dependent p65 phosphorylation and transactivation of NF-jB, leading to amplified inflammatory
responses. These data identify a target of SOCS-1 that regulates TLR signaling via a mechanism distinct from an autocrine
cytokine response. The transient activation of Mal and subsequent SOCS-1–mediated degradation is a rapid and selective
means of limiting primary innate immune response.

Bacterial cell wall components are potent activators of the innate
immune system, initiating a proinflammatory response via their
recognition by the Toll-like receptor family of pattern recognition
receptors1. All members of the TLR family signal inflammation via a
conserved pathway2,3. This pathway is initiated by a conserved
cytosolic protein sequence termed the Toll–interleukin 1 receptor
(TIR) domain, which activates signaling mediators including inter-
leukin 1–associated kinase (IRAK)-1 and IRAK-4, TRAF6, MAP
kinases and IkB kinases and leads to activation of the prototypic
inflammatory transcription factor, NF-kB. There are now five
identified cytosolic TIR-containing adaptor proteins MyD88, Mal
(MyD88-adaptor like, also called TIRAP), TRIF (TIR domain–
containing adaptor that induces interferon-b, also called TICAM-1),
TRAM (TRIF-related adaptor molecule, also called TICAM-2) and
SARM (sterile a and armadillo motif–containing protein), whose
function is unknown4.
In addition to the conserved NF-kB activation pathway, there are
distinct signaling pathways initiated by individual TLRs that are
dependent upon recruitment of specific adaptor proteins. Although
nearly all TLRs recruit MyD88, only some recruit Mal, Tram and TRIF,
giving rise to specificity in signaling. TRIF is critical for signaling by
lipopolysaccharide (LPS) via TLR4 and for signaling by poly(I)poly
(C) via TLR3, whereas TRAM is required for TLR4 signaling only5,6.
Notably, TRIF can induce the expression of IFN-b in response to
TLR4 and TLR3 ligands. Although no specific role for Mal was
originally described in TLR2 and TLR4 responses independent from
MyD88, the importance of Mal in host response was demonstrated by
the total absence of proinflammatory cytokine production during
either TLR2 or TLR4 signaling7,8. A new interaction between Mal and
TRAF6 was recently reported that results in regulation of serine
phosphorylation of the p65 subunit of NF-kB that renders NF-kB
active as a transcription factor9. This interaction could explain the lack
of NF-kB–dependent gene expression in Mal-deficient cells, despite
NF-kB nuclear translocation. Thus, Mal is necessary to initiate the
transcriptional activation of proinflammatory genes.
Although the TLR-mediated inflammatory response is critical for
protecting the host against pathogenic bacteria, dysfunctional inflam-
matory responses may lead to both acute and chronic diseases that
clinically manifest as potentially fatal or severe sepsis, septic shock or
autoimmune disease. Therefore, the intensity and duration of TLR
responses must be tightly controlled.
Several studies have described negative regulation of TLR path-
ways10. Triad3A targets some TLRs for ubiquitination and proteolytic
degradation11, whereas the TIR-containing proteins ST2 and Single
immunoglobulin IL-1–related (SIGGIR) also act as negative regulators
of signaling by sequestering MyD88 via homotypic TIR-TIR inter-
actions12. A splice variant of MyD88 (MyD88s), lacking the amino-
terminal death domain, negatively regulates signaling by its inability to
bind the death domain of IRAKs13, and degradation of MyD88
induced by TLR-generated transforming growth factor (TGF)-b also

1Centre for Functional Genomics and Human Disease, Monash Institute of Medical Research, Monash University, Melbourne, Victoria, Australia. 2School of Biochemistry

and Immunology, Trinity College, Dublin 2, Ireland. 3Walter and Eliza Hall Institute, Parkville, Victoria, Australia. 4These authors contributed equally to this work.
Correspondence should be addressed to P.J.H. (paul.hertzog@med.monash.edu.au).
Received 4 August 2005; accepted 7 December 2005; published online 15 January 2006; doi:10.1038/ni1299

148 VOLUME 7 NUMBER 2 FEBRUARY 2006 NATURE IMMUNOLOGY


LPS (1 µg/ml)
a α-Mal
α-IκBα
Pam3Cys (1 µg/ml)
α-Mal
α-IκBα
Loxoribine (500 µM)
α-Mal
α-IκBα
CpG-DNA (1 mM)
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
α-Mal
α-IκBα
LPS (1 µg/ml)
α-MyD88
Time (min) 0 5 10 15 30 60
b LPS (1 µg/ml) + + + + + +
Control
MG132
Time (min) 0 5 10 15 30 60
decreases prolonged signaling14. Additional regulation of TLR signal-
ing has been described by the kinase inactive IRAK-M15. Also, the
TLR4 homolog RP105 can specifically inhibit the TLR4 signaling
complex by binding its microbial ligand16.
Suppressor of cytokine signaling (SOCS)-1 was discovered as a
cytokine inducible intracellular negative regulator that inhibits cyto-
kine signaling by interaction with the JAK-STAT signaling cascade17–20.
SOCS-1–deficient (Socs1–/–) mice die as neonates as a result of
excessive inflammation, which can be rescued by crossing to IFN-g–
deficient (Ifng–/–) mice21. SOCS-1 is also induced by TLR activation22.
Notably, Socs1–/–Ifng–/– mice are hypersensitive to LPS-induced shock
compared to wild-type littermates23. Socs1–/– mice are also unable to
mount LPS tolerance and SOCS-1–deficient macrophages produce
increased amounts of inflammatory cytokines such as IL-6 and TNF
in response to LPS23,24. Together, these results indicate that in
addition to a central role in adaptive immune responses downstream
of cytokine signaling, SOCS-1 is also able to negatively regulate TLR
responses. Although clearly independent of the well-characterized
effects of SOCS-1 on IFN-g signaling, the mechanism of this inter-
action was unclear.
In this study we report and characterize a mechanism of degrada-
tion of Mal by SOCS-1 that occurs upon signaling by TLRs 2 and 4.
We found that Mal associates with SOCS-1, which acts as an E3
ligase25,26 to mediate polyubiquitination of Mal on two N-terminal
lysines; the polyubiquitination of Mal results in its degradation via the
26S proteasome. We also found that Mal undergoes tyrosine
phosphorylation via activity of Btk, which is consistent with the
requirement for tyrosine phosphorylation for targets of SOCS-1
activity. Perturbed regulation of these events potentiated Mal-depen-
dent NF-kB transactivation and prolonged proinflammatory response.
Targeted degradation of Mal by SOCS-1 therefore regulates TLR
activation of NF-kB, causing instead rapid (and temporary) refrac-
toriness to prolonged TLR2 and TLR4 signaling and chronic inflam-
matory responses, which is consistent with the increased susceptibility
phenotype of Socs1–/– mice to chronic LPS-induced inflammation.
RESULTS
Mal undergoes TLR2 and TLR4–induced degradation
Mal was originally described as a MyD88 homolog because of high
homology of carboxy-terminal TIR domains. Whereas MyD88 has a
NATURE IMMUNOLOGY VOLUME 7 NUMBER 2 FEBRUARY 2006
ARTICLES
Figure 1 Mal contains a PEST domain which targets it for TLR-mediated
degradation. (a) THP-1 cells (1  106) in 1.0 ml of cultured medium
were stimulated (time, below lanes) with LPS, Pam3Cys, Loxoribane and
phosphorothioate-modified CpG-DNA 2006, lysed and equal protein
concentrations were analyzed for Mal degradation by immunoblot using
polyclonal antibody against Mal. Activation of TLR pathways by the
indicated ligands was assayed by immunoblotting for IkBa degradation,
and cells stimulated with LPS were also assayed for MyD88 degradation
by immunoblot analysis using antibodies against MyD88 (lower panel).
(b) THP-1 cells (1  106) in 1.0 ml of cultured medium were either
untreated (top) or treated (bottom) with 20 mM of the 26S proteasome
inhibitor MG132 for 45 min before stimulation with LPS for the indicated
amounts of time. Cellular lysates were assayed for Mal degradation as
described above.
well-defined N-terminal death domain that homotypically recruits
IRAK1 and IRAK4 via their respective death domains, the N terminus
of Mal had no known function. Sequence analysis of the N terminus
of both human and mouse Mal identified a putative proline, glutamic
acid, serine and threonine domain (commonly called a PEST) within
the first 84 amino acids proximal to the TIR domain (Supplementary
Fig. 1 online and http://www. emb1.bcc.univie.ac.at/embnet/tools/bio/
PESTfind). Many short-lived proteins (such as IkBa in NF-kB signal-
ing) contain PEST domains that undergo phosphorylation, polyubi-
quitination of lysine residues and targeted degradation via the 26S
proteasome27. Therefore, to investigate if the PEST domain of Mal
targets it for degradation, we stimulated human monocyte THP-1 cells
with various TLR ligands and assayed for Mal degradation. Immuno-
blot analysis demonstrated that stimulation of both TLR4 (by LPS) and
TLR2 (by Pam3Cys) rapidly induce Mal degradation within 15 min
after stimulation. Stimulation of TLR7 (by Loxoribine) and TLR9 (by
CpGDNA), however, were unable to induce the same effect (Fig. 1a),
consistent with the specific role of Mal in TLR2 and TLR4 signaling.
Notably, although IkBa was degraded in response to all TLR ligands, as
expected, MyD88, which lacks a PEST domain, failed to undergo either
LPS- (Fig. 1a) or Pam3Cys- (data not shown) mediated degradation.
Finally, the 26S proteasome inhibitor MG132 blocked LPS (Fig. 1b)
and Pam3Cys (data not shown) degradation of Mal in THP-1 cells.
Together, these results indicated that Mal, but not MyD88, specifically
undergoes selective targeted degradation via the 26S proteasome after
stimulation of TLR2 and TLR4, but not TLR7 and TLR9.
Mal interacts with SOCS-1 to induce Mal degradation
We next investigated what proteins were mediating the degradation of
Mal. SOCS-1 negatively regulates cytokine signaling by targeting
signaling mediators for proteasomal degradation28, and SOCS-1
negatively regulates TLR4 signaling23,24, although a target for
SOCS-1 activity has not been identified. We hypothesized that
SOCS-1 interacts with Mal, ubiquitinates it and targets it
for degradation. We confirmed this interaction by ectopic coexpres-
sion of Flag-tagged SOCS-1 and hemagglutinin (HA)-tagged Mal in
HEK293T cells, and subsequent immunoprecipitation of SOCS-1
(Fig. 2a). The resulting coimmunoprecipitation of Mal with SOCS-1
suggests a mechanism by which Mal may undergo degradation.
Next we determined whether SOCS-1 could induce Mal degrada-
tion. By cotransfecting HEK293T cells with a constant amount of HA-
tagged Mal either in the absence or presence of increasing amounts of
SOCS-1, we found that SOCS-1 was, in a dose-dependent manner,
able to induce the degradation of HA-tagged Mal (Fig. 2b, left). In
contrast, we observed only a marginal effect on MyD88 at the highest
concentration of SOCS-1 (Fig. 2b, right). These results are consistent
with our earlier findings of Mal, but not MyD88, degradation in
149
 ARTICLES

Figure 2 Mal interacts with SOCS-1 and

undergoes SOCS-1–mediated degradation. a b
Mal-HA (ng) 200 200 200 200 200 200 – –
Mal-HA + + + SOCS-1 (ng) 0 200 400 600 800 1,000 0 1,000
(a) HEK293T cells were cotransfected with Flag–SOCS-1 + – + MyD88-Flag (ng) – – – – – – 200 1,000
equivalent amounts of Flag-tagged SOCS-1 and IP: anti-Flag + – –
HA-tagged Mal expression plasmids, incubated
for 24 h and then lysed. Cellular lysates IB: anti-HA
were probed with anti-M2-agarose beads to
immunoprecipitate Flag–SOCS-1 (IP: anti-Flag). IB: anti-HA IB: anti-Flag
1 2 3
Mal was assayed for SOCS-1 association by
immunoblot with anti-HA (IB: anti-HA). Lane 3, c Mal-HA (ng) 200 200 200 200 d
lysates of transfected cells. (b) HEK293T cells SOCS-1 (ng) 0 1,000 200 200 LPS (1 µg/ml) + + + + + +
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology

SOCS-1 ∆SB-Flag (ng) 0 0 1,000 0

were cotransfected with HA-tagged Mal and SOCS-1 ∆SH2-Flag (ng) 0 0 0 1,000 Ifng –/–/Socs-1 +/+
SOCS-1 as shown. Immunoblot analysis with
anti-HA demonstrated a dose-dependent IB: anti-HA
degradation of HA-Mal (left). In contrast, Ifng –/–/Socs-1 –/–
Flag-tagged MyD88 did not undergo a similar IB: anti-Flag Time (min) 0 5 10 15 30 60
degree of degradation as observed with Mal at
the highest dose of SOCS-1 (right). (c) SOCS-1
specifically promotes Mal degradation. HEK293T cells were cotransfected with HA-tagged Mal and either wild-type SOCS-1, SOCS-1-DSB or SOCS-1-DSH2
as indicated, and analyzed for their ability to promote degradation of Mal. Whereas wild-type SOCS-1 fully induced Mal degradation, both mutant forms of
SOCS-1 did not. (d) BMMs from Ifng–/– and Socs1–/–Ifng–/– mice were seeded at 1  106 in six-well culture plates 24 h before stimulation with LPS for
indicated amounts of time. Cells were lysed and equal protein samples were analyzed for Mal degradation by immunoblot analysis using anti-Mal. Whereas
IFN-g-deficient BMMs underwent a time-dependent LPS-induced Mal degradation (top), the SOCS-1–deficient BMMs did not (bottom).

response to LPS. To further test the specificity of SOCS-1 on Mal after stimulation (data not shown). Together, the studies on SOCS-1
degradation, we next overexpressed two mutated forms of SOCS-1: overexpression and deficiency demonstrated a direct role for SOCS-1
SOCS-1-cSH2, which has a point mutation within the SH2 binding in mediating Mal degradation.
domain abrogating target recognition, and SOCS-1-DSB, which has a
point mutation within the SOCS box that abrogates possible recruit- SOCS-1 mediates Mal polyubiquitination
ment of the ubiquitin machinery26,29. We observed that both mutant SOCS-1 has E3 ligase activity that results in polyubiquitination of
forms of SOCS-1 were unable to induce the degradation of Mal at a target proteins and subsequent degradation by the proteasome.
dose at which wild-type SOCS-1 could (Fig. 2c). Because we demonstrated a role for SOCS-1 in mediating Mal
As these results clearly show a role for SOCS-1 in mediating the degradation, we wanted to determine whether the mechanism of
degradation of Mal, we examined whether endogenous Mal could this activity was by SOCS-1 polyubiquitination of Mal. We coex-
undergo TLR-mediated degradation in SOCS-1–deficient cells. pressed Flag-tagged Mal with HA-tagged ubiquitin either in the
Because lethal SOCS-1 deficiency can be rescued by crossbreeding presence or absence of SOCS-1 in HEK293T cells and incubated
with IFNg-deficient mice21, we used 6–8-week-old bone marrow– them for 18 h. We then incubated the cells for 4 h with or without
derived macrophages (BMM) from Socs1–/–Ifng–/– mice, which we MG132, and analyzed total cell lysates by immunoprecipitation and
compared to BMM from Ifng–/– control mice. We found that LPS-
stimulated BMM demonstrated a time-dependent degradation of Mal
corresponding to that previously demonstrated in human THP-1 cells. Mal-Flag + + + – + + + – MyD88-Flag + + + –
Mal, however, did not undergo LPS-induced degradation in SOCS-1
a SOCS-1-Myc + + – + + + – + SOCS-1–Myc + – + +
Ub-HA – + + + – + + – Ub-HA – + + +
deficient macrophages (Fig. 2d). A similar result was also found in MG132 – – – – + + + + MG132 + + + +

Pam3Cys-stimulated Socs1–/– macrophages (data not shown), which IP: anti-Flag

IP: anti-Flag
demonstrated that the TLR-mediated degradation of Mal is inde- IB: anti-HA IB: anti-HA
pendent of the autocrine effects of IFN-g. Consistent with previous
studies demonstrating SOCS-1 induction in response to LPS22, Mal

we found that SOCS-1 is rapidly induced by LPS within 15–30 min WCL: α-HA
MyD88

IP: anti-Flag
IP: anti-Flag
Figure 3 SOCS-1 specifically promotes polyubiquitination of Mal. IB: anti-Flag
IB: anti-Flag
(a) HEK293T cells were transfected with expression vectors as indicated
and treated with 20 mM of MG132 where indicated for 4 h before lysis. b c Mal-Flag + + + + +
Cellular lysates were immunoprecipitated with anti-Flag-agarose beads and Mal-Flag + + + + + – Ub-HA + + + + +
SOCS-1 + + – – – + MG132 + + + + +
immunobloted with anti-HA for the detection of polyubiquitinated Mal or SOCS-1-∆SB Pam3Cys – + + + +
– – – + – –
MyD88, respectively. (b) HEK293T cells were transfected with Flag-tagged SOCS-1-∆SH2 – – – – + – Time (min) 0 5 10 20 30
Mal, plus HA-tagged ubiquitin in conjunction with Myc-tagged SOCS-1, Ubiquitin-HA – + + + + +
MG132 + + + + + +
SOCS-1-DSB or SOCS-1-DSH2 as indicated. Cells were treated with 20 mM
MG132 for 4 h before lysis. Cellular lysates were immunoprecipitated with
anti-Flag agarose beads and immunobloted with anti-HA for the detection
IP: anti-Flag
of polyubiquitinated Mal. (c) Cells were made responsive to Pam3Cys by IP: anti-Flag IB: anti-HA
IB: anti-HA
cotransfecting HEK293T cells with 100 ng TLR2 in conjunction with
Flag-Mal and HA-ubiquitin, incubated for 24 h and pretreated for 4 h
with 20 mM MG132 before stimulation with 100 ng/ml of Pam3Cys. TLR2-
induced complexing of HA-ubquitin to Flag-Mal was assayed as described IP: anti-Flag
IB: anti-Flag
above. IP, immunoprecipitiation; IB, immunoblot; WCL, whole cell lysates.

150 VOLUME 7 NUMBER 2 FEBRUARY 2006 NATURE IMMUNOLOGY


LPS (1 µg/ml)
b LPS (1 µg/ml)
+ + + + + + + + + +
a induced degradation of Mal. (a,b) Tyrosine phosphorylation as a prerequisite
Untreated Untreated
Staurosporine
Genestien
Time (min) 0 5 10 15 30 60 Time (min) 0 5 10 15 30 60
c LPS (1 µg/ml) + + + + + + d LPS (1 µg/ml) + +
Untreated Wild type
LFM-13A
xid
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
Time (min) 0 5 10 15 30 60 Time (min) 0 5
immunoblotting. Although pretreatment of cells with MG132 is
normally required to visualize polyubiquitination of the proteins
(Fig. 3a), we were able to detect a basal amount of HA-tagged
polyubiquitin complexed to Mal that was substantially enhanced
upon addition of SOCS-1 (Fig. 3a). In contrast, although we also
detected a basal amount of HA-tagged ubiquitin complexed with
MyD88, the addition of SOCS-1 did not increase this amount
(Fig. 3a). That result indicated that SOCS-1 specifically mediates
polyubiquitination of Mal but not MyD88, which is consistent with
the TLR-mediated degradation of the former but not the latter.
Having demonstrated SOCS-1–mediated Mal polyubiquitination,
we next investigated several SOCS-1 mutants (SOCS-1-DSH2 and
SOCS-1-DSB that cannot bind via the SH2 domain or recruit the
ubiquitin machinery to the target, respectively26,29) for their ability to
polyubiquitinate Mal. Although wild-type SOCS-1 induced polyubi-
quitination of Mal above basal amounts, both SOCS-1-DSB and
SOCS-1-DSH2 were unable to induce the same effect, as the amount
of ubiquitin incorporation was similar to that observed in untreated
cells (Fig. 3b). Those results demonstrated polyubiquitination of Mal
occurs by recognition of Mal via the SOCS-1 SH2 domain and
subsequent recruitment of the ubiquitin machinery via the SOCS-1
SOCS box.
Finally, to determine whether Mal polyubiquitination is a TLR-
induced effect, we overexpressed TLR2 in HEK293T, to induce
Pam3Cys responsiveness, and then coexpressed HA-tagged ubiquitin
and Flag-tagged Mal for 18 h. Then we pretreated the cells with
MG132 for 4 h before stimulating them with 100 ng/ml of Pam3Cys.
We then lysed the cells, immunoprecipitated the HA-ubiquitin-Mal
complexes and analyzed them by immunoblotting. In contrast to
unstimulated cells, Pam3Cys stimulation was able to induce rapid
polyubiquitination of Mal (Fig. 3c), indicating that TLR2 activation
induces SOCS-1 polyubiquitination of Mal before Mal degradation.
Mal degradation requires tyrosine phosphorylation
Because the above data suggested that the SH2 domain of SOCS-1 was
necessary for Mal interaction, we investigated whether phosphoryla-
tion of Mal was a requirement for SOCS-1–mediated degradation of
Mal. We treated THP-1 cells with LPS, either in the presence or
absence of staurosporine, a broad-spectrum kinase inhibitor. Whereas
untreated cells demonstrated LPS-induced Mal degradation in a
time-dependent fashion (Fig. 1b), we found that cells pretreated
with staurosporine failed to undergo LPS-mediated degradation
(Fig. 4a). Because previous results show SOCS-1 targets tyrosine-
phosphorylated proteins for degradation28, we next stimulated cells
with LPS either in the presence or absence of the phospho-tyrosine
kinase inhibitor genestein to determine if tyrosine phosphorylation
was required for degradation of Mal. Consistent with the
results obtained with staurosporine, we found that genestein
inhibited LPS-induced degradation of Mal (Fig. 4b). These results
NATURE IMMUNOLOGY VOLUME 7 NUMBER 2 FEBRUARY 2006
ARTICLES
Figure 4 Tyrosine phosphorylation by Btk is required to facilitate TLR-
+ +
for Mal degradation was determined by pretreating cells with 20 nM
Staurosoporine (a) and 20 mM Genestein (b) for 40 min before treatment
with LPS. Cell lysates were analyzed by immunoblot for Mal degradation.
(c) The requirement for tyrosine phosophorylation by Btk for Mal degradation
+ in response to LPS was determined by stimulating cells either in the
presence or absence of the Btk inhibitor LFM-A13 (50 mM) for 45 min, and
the subsequent cell lysates immunobloted with anti-Mal. (d) Splenocytes
from xid mice (1  106/ml) were stimulated with 1 mg/ml of LPS for
indicated amounts of time, and Mal degradation was compared by
10 immunoblot with that in wild-type splenocytes.
demonstrated that tyrosine phosphorylation is required for Mal
degradation, which is consistent with a mechanism involving
SOCS-1 recognition through its SH2 domain and Mal containing
tyrosine phosphorylation.
Recently, we demonstrated that Mal undergoes TLR-induced
tyrosine phosphorylation mediated by Btk (P.G. and L.A.J.O.,
unpublished data). Therefore, we next wanted to determine if Btk-
induced tyrosine phosphorylation was required for Mal degradation.
We found that cells pretreated with the specific Btk inhibitor LFM-
A13 inhibited LPS-induced Mal degradation (Fig. 4c). To investigate
the role of Btk in Mal degradation, we used splenocytes derived from
X-linked immunodeficiency (xid) mice that have a kinase inactivating
mutation in Btk30. Consistent with the results obtained with LFM-
A13, splenocytes derived from the xid mice did not have Mal
degradation in response to LPS stimulation (compared to the degra-
dation observed in wild-type splenocytes; Fig. 4d).
Together, the above results clearly demonstrated that Mal tyrosine
phosphorylation by Btk is required for subsequent SOCS-1–mediated
degradation of Mal, which is consistent with previous reports demon-
strating that SOCS-1 recognizes tyrosine phosphorylated residues in
proteins to be targeted for degradation.
Reduced Mal degradation potentiates TLR signaling
Polyubiquitination of proteins and subsequent degradation by the 26S
proteasome is a powerful means of modulating protein function31.
Studies of several polyubiquitinated and degraded signaling proteins,
such as IkBa, have shown the specific covalent attachment of
ubiquitin to lysine residues (reviewed in ref. 27). Mal contains eight
lysine residues that are conserved between human and mouse, of
which three pairs of lysines are located within the N-terminal region at
positions Lys15,16; Lys31,32 and Lys34,35. To evaluate the potential
function of those lysines in the polyubiquitination of Mal, we mutated
specific lysine pairs (Fig. 5a) and cotransfected HEK293T cells with
HA-tagged ubiquitin and either Flag-tagged wild-type Mal or one of
the Mal lysine mutants (Mal 15/16: Mal KK15,16RR; Mal 2xKK: Mal
KK31,32,34,35RR; Mal 3xKK15,16,31,32,34,35RR; see Fig. 5a) in
either the presence or absence of SOCS-1. After 24 h, we pretreated
the cells with MG132 for 4 h to allow visualization of the complexes
and then immunoprecipitated Mal from cellular lysates with anti-Flag
and performed immunoblot analysis to detect HA-ubiquitin-Mal
complexes. Consistent with our earlier findings, SOCS-1 was able to
potentiate the basal amount of Mal polyubiquitination (Fig. 5b).
SOCS-1, however, was unable to increase polyubiquitination of
both the Mal 15/16 and Mal 3xKK mutants (Fig. 5b). Conversely,
the Mal mutant Mal 2xKK, which does not have lysines 15 and 16
mutated to arginines, was polyubiquitinated via SOCS-1, at an
amount comparable to wild-type Mal (Fig. 5b). These results demon-
strated the importance of targeted polyubiquitination of Mal at
residues Lys15,16 for subsequent degradation and regulation.
151
 ARTICLES

KK Figure 5 Mal undergoes polyubiquitination on Lys 15 and 16 by SOCS-1,

a KK
34,35
the dysregulation of which induces a potentiated NF-kB response. (a) A
KK 31,32
15,16 schematic diagram illustrates the human and mouse conserved lysines
within the N-terminal domain of Mal. (b) HEK293T cells were cotransected
PEST domain TIR domain with 100 ng HA-ubiquitin with 800 ng of either Flag-tagged wild-type Mal,
2xKK Mal 15/16, Mal 2xKK or Mal 3xKK as indicated in the presence or absence
3xKK of 100 ng SOCS-1. Mal was immunoprecipitated with anti-Flag beads, and
polyubiquitinated complexes were immunobloted with anti-HA. Both wild-
b Mal-Flag – – + + – – – – – –
type Mal and Mal 2xKK underwent SOCS-1-mediated polyubiquitination,
SOCS-1-Myc – + – + – + – + – +
Mal 15/16 – – – – + + – – – – whereas Mal 15/16 and Mal 3xKK did not undergo a potentiated
Mal 2xKK – – – – – – + + – –
polyubiquitination above basal amounts. (c) HEK293 cells transfected
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology

Mal 3xKK – – – – – – – – + +
Ubiquitin-HA + + + + + + + + + + with the kB-luciferase (left) or p65-Gal4, Gal4-luciferase (right) reporter
MG132 + + + + + + + + + +
plasmids, and Mal vectors (wedges indicate 0.5, 1.0 and 2.0 ng/ml) show
that Mal protein that is unable to undergo polyubiquitination induces
potentiated NF-kB–dependent luciferase expression. Data are presented
IP: anti-Flag
IB: anti-HA as mean ± s.e.m.

IP: α-Flag Phosphorylation of serine 536 on the p65 subunit of NF-kB (termed
IB: α-Flag
transactivation) critically regulates NF-kB–dependent gene expression,
Lane 1 2 3 4 5 6 7 8 9 10
producing transcriptionally active NF-kB33. Previous work suggested
c 35 4 Mal mediated TLR2- and TLR4-induced p65 transactivation, and
30 SOCS-1 negatively regulated Mal signaling9. Therefore, we investigated
3
Fold stimulation

the impact of dysregulated Mal degradation upon p65 phosphoryla-

Fold stimulation

25
20
15
10
5 p65Ser536 phosphorylation (Fig. 6a). Those results demonstrated a
0 0
NS Mal Mal15/16 Mal2xKK Mal3xKK
Whereas the above results suggested a mechanism by which poly-
ubiquitination occurs in Mal, we next sought to confirm the effect of
reduced degradation of Mal on the activation of NF-kB9,32 by
comparing the functions of overexpressed wild-type Mal and the
lysine mutants that cannot undergo SOCS-1–mediated regulation.
Whereas wild-type Mal induced a dose-dependent NF-kB activa-
tion by a maximum of tenfold (in a dose-dependent manner;
Fig. 5c, left), both the Mal 15/16 and Mal 3xKK mutants induced a
much greater increase (up to 25) in the NF-kB response. Conversely,
but anticipated from our previous results, the Mal 2xKK mutant
stimulated NF-kB activation in a manner comparable to that observed
with wild-type Mal. Together, the above results further illustrated the
importance of lysines 15 and 16 for targeting Mal for degradation,
thereby reducing ‘downstream’ signaling that stimulates NF-kB–
dependent pathways of activation.
We recently reported that one downstream function for Mal may be
the mediation of NF-kB transactivation by TLR2 and 4 (ref. 9). As
SOCS-1–mediated regulation of Mal may therefore be critical in
negatively regulating NF-kB transactivation to control inflammatory
responses, we next investigated NF-kB transactivation as a readout of
dysregulated Mal function. Consistent with previous results, Mal was
able to weakly drive p65 transactivation, as assayed using the p65-
Gal4-luciferase cotransfection assay, causing an increase in activation
by a factor of B1.5; in contrast, both Mal 15/16 and Mal 3xKK
demonstrated increased transactivation-dependent luciferase expres-
sion, increasing activation by a factor of 2.5 and 3.5, respectively
(Fig. 5c, right). And as expected, the Mal 2xKK mutant stimulated NF-
kB transactivation comparably to that observed with wild-type Mal.
The above results demonstrated the biological importance of SOCS-1
negative regulation of TLR signaling: disrupted Mal polyubiquitination
and subsequent degradation results in continued Mal-mediated signal-
ing that potentiates NF-kB–dependent gene expression.
2
tion. We found that LPS induced a transient phosphorylation of
p65Ser536 in wild-type mouse embryonic fibroblasts (MEFs), whereas
1 Mal-deficient MEFs displayed a complete absence of LPS-mediated
clear role for Mal in LPS-mediated phosphorylation of p65Ser536.
NS Mal Mal15/16 Mal2xKK Mal3xKK We next investigated the effect of SOCS-1 deficiency upon TLR4-
and TLR2-mediated p65 phosphorylation. Using macrophages derived
from IFN-g–deficient mice, we observed an LPS-dependent increase in
p65 phosphorylation in a time-dependent manner, with maximal
phosphorylation occurring after 5 min of LPS stimulation but then
decreasing after 10 min (Fig. 6b). In contrast, macrophages derived
from mice deficient in both SOCS-1 and IFN-g (Socs1–/–Ifng–/–)
displayed a prolonged p65 phosphorylation profile, with maximal
phosphorylation occurring at 5–30 min after stimulation. Similar
results were also observed in Pam3Cys-stimulated SOCS-1–deficient
macrophages (data not shown). These results clearly illustrated that
the absence of SOCS-1 allows a potentiated phosphorylation and
transactivation of NF-kB, thereby causing NF-kB–dependent gene
transcription to proceed longer than in wild-type cells. Notably, we
also found that phosphorylation of p38 Map kinase was unaltered in
both LPS-stimulated (Fig. 6b, bottom) and Pam3Cys-stimulated (data
not shown) SOCS-1-deficient macrophages—a result that reinforces
the specificity for Mal-dependent pathways (neither Mal or SOCS-1
has any previously described role in TLR-mediated activation of p38).
Therefore, enhanced NF-kB transactivation may explain the enhanced
proinflammatory response previously described in LPS-stimulated
SOCS-1–deficient mice.
Finally, because Mal acts as an adaptor protein for both TLR4 and
TLR2 signaling pathways, we investigated whether TLR2 could induce
a similar enhanced proinflammatory response in the absence of
SOCS-1, as previously reported with TLR4 activation. We observed
a clear potentiation of both IL-6 and TNF expression in macrophages
deficient in both SOCS-1 and IFN-g in response to both Pam3Cys and
LPS (Fig. 6c) compared to cytokine production in IFN-g–deficient
macrophages alone, consistent with reports showing a potentiated
LPS-induced proinflammatory response in SOCS-1–deficient
macrophages23,24. Thus SOCS-1–mediated degradation of Mal and its
subsequent transactivation of NF-kB inhibits sustained proinflam-
matory responses to TLR2 and TLR4 activation (Supplementary
Fig. 2 online).

152 VOLUME 7 NUMBER 2 FEBRUARY 2006 NATURE IMMUNOLOGY


a b
LPS
WT Mal –/–
p65-phospho
p65-total
Time (min) 0 15 30 60 0 15 30 60
c 5,000
Ifng –/–/Socs-1 –/–
Ifng –/–
TNFα (pg/ml)
4,000
IL-6 (pg/ml)
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
3,000
2,000
1,000
0 0.1 10 100 1,000
PamCys (nM)
4,000
TNFα (pg/ml)
IL-6 (pg/ml)
3,000
2,000
1,000
0 0.1 1 10 1,000
LPS (ng)
Figure 6 SOCS-1 negative regulation of Mal controls the transient
transactivation of NF-kB. (a) MEF cells (1  106) derived from Mal-
deficient and wild-type embryos were stimulated for indicated times with
1 mg/ml of LPS, lysed and analyzed by immunoblot analysis using specific
anti-p65Ser536 polyclonal to determine LPS-induced phosphorylation of
p65. Membranes were subsequently reprobed with anti-p65 to determine
total p65 concentrations. (b) BMM (1  106 cells) were incubated for
24 h before stimulation with 1 mg/ml of LPS and Pam3Cys, respectively, for
indicated times. Cells were lysed and separated by SDS-PAGE, transferred
to nitrocellulose and immunoblotted with antibodies to phosphorylated p65
(p65-phospho), total p65 (p65-total), phosphorylated p38 (p38-phospho)
and total p38 (p38-total). (c) Resident peritoneal macrophages from
Ifng–/– mice were compared in their responsiveness to 12 h stimulation
with Pam3Cys and LPS to Socs1–/– Ifng–/– mice. Expression of the NF-kB–
dependent inflammatory cytokines TNF and IL-6 were determined by ELISA.
Data are representative of three independent experiments using pooled
macrophages from at least two littermates for each genotype and are
presented as mean ± s.e.m.
DISCUSSION
Here we demonstrated that the TIR-containing TLR adaptor protein
Mal undergoes rapid degradation within 15 to 30 min after activation
of TLR2 or TLR4. Consistent with the specificity of Mal for these two
TLRs and the specificity of this effect, activation of TLR7 or TLR9 did
not induce Mal degradation. The degradation of Mal also represents
an important difference from another TLR adaptor, MyD88, which
does not undergo rapid degradation after TLR activation. Degradation
of essential signaling molecules is well recognized as an important
means of regulating, or more specifically limiting, signal transduction.
Together with the previous demonstration that Mal is necessary for
TLR activation of NF-kB, our results imply that the transient contri-
bution of Mal to NF-kB transactivation may be a critical regulatory
step in TLR2- and TLR4-mediated NF-kB activation. Given these data
and previous work showing selective binding of TRAF6 by Mal, but
not MyD88, that facilitates the subsequent activation of p65 phos-
phorylation, a picture is emerging of an important and specific role
for Mal in TLR signaling that has not been evident from comparative
studies of the Mal- and MyD88-deficient mice.
The observation that SOCS-1–dependent Mal degradation
required the SH2 domain of SOCS-1 implied a role for tyrosine
NATURE IMMUNOLOGY VOLUME 7
ARTICLES
LPS phosphorylation of Mal in this process. We therefore proceeded to
Ifng –/– Ifng –/–/Socs-1 –/– demonstrate that tyrosine phosphorylation of Mal was necessary for
p65-phospho the SOCS-1–mediated degradation; we showed that Btk is necessary
p65-total for tyrosine phosphorylation of Mal and that mice with a null
Time (min) 0 5 10 15 30 60 0 5 10 15 3060
mutation in the gene that encodes Btk did not exhibit LPS-induced
p38-phospho
degradation of Mal. Btk is also required for p65 phosphorylation34.
p38-total
Thus, Btk phosophorylation of Mal may initiate downstream signal-
ing, including phosophorylation of p65, before subsequent degrada-
1,200
tion of Mal via recruitment of SOCS-1. Notably, our finding that p38
900 phosphorylation is unaltered in SOCS-1–deficient macrophages is
600 consistent with earlier studies that demonstrated IkBa and p42/p44
300 phosphorylation were unaltered in SOCS-1–deficient cells in response
0 to LPS22,35. Although these earlier results implied no direct role for
0 0.1 10 100 1,000
PamCys (nM) SOCS-1 in TLR signaling, our results validate that SOCS-1 regulates
a different TLR pathway regulated by Mal and leading to NF-kB
750 transcriptional regulation, rather than either the canonical pathway
550
mediated by MyD88 or the p38 pathway. These findings are con-
sistent with data showing Mal-deficient macrophages display
350 normal NF-kB translocation in response to LPS but fail to initiate a
150 proinflammatory response7,8.
0 0.1 1 10 1,000 Our findings that SOCS-1 induces polyubiquitination of Mal on
LPS (ng)
specific residues, together with the requirement of tyrosine phosphory-
lation of Mal to facilitate degradation, are similar to the previously
characterized mechanism of SOCS-1 negative regulation of cytokine
signaling20,36–38. Dysregulation of the negative control of Mal signaling
manifests as a potentiated proinflammatory response via activation of
either TLR2 or TLR4. Notably, the data presented here also identify
the direct role of Mal in mediating phosphorylation of the p65
subunit of NF-kB, indicating a critical role for Mal in regulating the
transcriptional activity of NF-kB. In SOCS-1–deficient cells, accord-
ingly, p65 phosphorylation is potentiated in response to TLR stimula-
tion by LPS or Pam3Cys, which is consistent with an elevated
proinflammatory response owing to prolonged Mal signaling. Our
results demonstrate that tight regulation of Mal, by transient activa-
tion followed by rapid degradation after TLR stimulation, is an
essential control mechanism for preventing chronic inflammatory
signaling and, potentially, septic shock.
Our study provides an explanation for why SOCS-1–deficient mice
are more susceptible to LPS-induced septic shock. Indeed, the results
presented indicate that SOCS-1 can regulate primary TLR signaling
events, such as Mal degradation. This precedes SOCS-1 regulation of
secondary signaling events, such as interferon signaling21,39 (reviewed
in refs. 20,36–38). But the demonstration that SOCS-1 negatively
regulates IL-6 and TNF production in response to the activation of
TLR2 (which does not induce IFN production) clearly shows that the
latter is not necessary for the SOCS-1–mediated effects on signaling by
TLR2 and TLR4 for production of these proinflammatory cytokines.
Notably, the initial description of SOCS-1 as a negative regulator of
TLR signaling indicated a role for SOCS-1 in TLR9 signal transduc-
tion23. But because we clearly demonstrated that activation of TLR9
does not result in the degradation of Mal, we suggest that this previous
work may in fact be due to SOCS-1 regulation of TLR9-induced IFN
production, as suggested elsewhere22,35.
The negative regulation of TLR signaling by SOCS-1–mediated
degradation of Mal is distinct from other mechanisms of negative
regulation of TLR signaling because of its specificity for certain TLRs
(TLR2 and TLR4), its rapidity of onset, and primary TLR signaling. In
contrast, the key signaling kinase IRAK-1 undergoes rapid degradation
after stimulation and is proposed to be the moderator of LPS-
tolerance40,41. IRAK-1, however, is involved in all TIR-mediated
signaling encompassing both the TLR and IL-1 receptor signal
NUMBER 2 FEBRUARY 2006 153
 ARTICLES
transduction pathways; this then, would appear to be a generic mode
of signal regulation. Other negative regulators such as ST2, SIGGIR,
MyD88s and kinase inactive IRAK-M are induced after TLR activation
and require delayed protein expression to exert their negative regula-
tory effects. Similarly, the recent discovery that MyD88 undergoes
degradation as a means to inhibit signaling is dependent upon the
expression of TGF-b, which mediates that effect. Notably, the targeted
removal of Mal specifically regulates pathways involving Mal, that is,
TLR2 and TLR4 signaling, and would not impact upon other TLR-
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
dependent responses, leaving TLRs such as TLR3 or TLR9 to respond
normally. The specificity of Mal for TLRs 2 and 4 may also explain
crosstolerance of TLR2 and 4 signaling.
Our results might also explain previous conflicting data on the
mechanism of SOCS-1 regulation of TLR signaling. Whereas some
researchers have reported the coimmunoprecipitation of ectopically
expressed IRAK-1 and SOCS-1 (ref. 24), others were unable to identify
an endogenous association between SOCS-1 and IRAK-1, TRAF6, IKK
or TAK-1 (ref. 23). We have found that Mal interacts with endogenous
IRAK-1 after LPS stimulation (A.M. and A. Dunne, unpublished data)
and thus the IRAK-1–SOC-1 association may require Mal as a
bridging protein.
In conclusion, in addition to SOCS-1 controlling adaptive immune
responses by negatively regulating cytokine signaling, we have demon-
strated that SOCS-1 can also directly suppress Mal, a key mediator
of TLR4 and TLR2 signaling, to modulate the innate immune
response. SOCS-1 specifically modulates the proinflammatory
response by targeting Mal for programmed degradation, thereby
regulating downstream pathways such as Mal-mediated phosphoryla-
tion of the p65 subunit of NF-kB and thus expression of NF-kB–
dependent genes. Previous findings that TLR ligands rapidly induce
SOCS proteins combined with the demonstration of SOCS-1–
mediated degradation of Mal reveals a mechanism for the rapid
negative feedback control of TLR signaling. This feedback mechanism
complements the previously described regulatory mechanism for
preventing TLR-induced inflammation by the delayed expression of
TLR-inhibitory proteins such as ST2, MyD88s and IRAK-M. In
combination, the much more rapid mechanism of shutting down
TLR signaling via degradation of Mal and the relatively more delayed
regulatory mechanism of induced expression of direct negative reg-
ulators efficiently renders TLR signaling refractory to continued
stimulation, which therefore protects the host from excessive, or
inappropriate, inflammatory responses that can manifest clinically as
autoimmunity, chronic inflammation or infectious diseases. Further
studies of the functions of Mal in proinflammatory response and the
control of Mal by SOCS-1 may lead to the development of therapeutic
approaches to limit inflammatory responses associated with TLR-
mediated disease.
METHODS
Reagents, cell culture and transient transfections. We incubated human
monocyte THP-1 cells in RPMI 1640 supplemented with 10% FBS (Invitro-
gen), 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and
maintained them in a 37 1C humidified atmosphere of 5% CO2. We counted
the cells, centrifuged them and resuspended them at 2  106 viable cells/ml
(viability determined by trypan blue exclusion) in conditioned medium before
plating into 24-well plates (BD Pharmingen). We stimulated the cells with
indicated amounts of LPS (repurified K235 Escherichia coli; Sigma), Pam3Cys
(EMC microcollections), Loxoribine (Invivogen) and phosphorothioate-
modified CpG-DNA 2006 (Geneworks) for the indicated periods of time. We
centrifuged the cells, lysed them (1% Nonident P-40, 10% SDS, 5% sodium
deoxycholate) and subjected equal amounts of protein to 10% SDS-PAGE,
transferred to nitrocellulose and immunobloted to detect Mal using polyclonal
154
antibody against Mal (Pearl1; LAJ O’Neill). Anti-IkBa, anti–phospho-
p65Ser536, anti-p65, anti–phospho-p38 and anti-p38 were purchased from
Cell Signaling and anti-MyD88 was obtained from eBioscience. The protea-
some inhibitor MG132, genestein, staurosporine and LFM 13A were purchased
from Calbiochem.
We incubated human embryonic kidney cell lines HEK293 and HEK293T in
DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine,
100 U/ml of penicillin, 100 mg/ml of streptomycin and maintained them in a
37 1C humidified atmosphere of 5% and 10% CO2, respectively.
We seeded HEK293 cells (2  104) in 96-well plates 24 h before transfection.
We performed transfections with Genejuice transfection reagent (Novagen)
according to the manufacturer’s instructions, with indicated concentrations
of vectors (0.5–2 ng) and kB-luciferase (Stratagene). We determined
NF-kB–dependent gene expression using the 5 kB-luciferase reporter con-
struct (Stratagene). Using the PathDetect transient transfection kit (Stratagene),
cotransfection of pFR-luciferase in combination with Gal4-p65(1–551) (gift of
L. Schmitz), was used to analyze p65 transactivation. We used the RSV
b-galactosidase construct to normalize for transfection efficiency and an
appropriate empty vector plasmid to maintain a constant amount of DNA.
We grew the cells for 24 h after transfection, lysed them (passive lysis buffer;
Promega) and assayed them for luciferase and b-galactosidase activity using
FLUOstar Optima (BMG Technologies) after incubation in luciferase assay
reagent (Promega) or b-galactosidase assay reagent. We corrected the lumines-
cence readings for b-galactosidase activity and expressed them as factor
increases over nonstimulated control values.
Cell cultures from gene-deficient mice. We obtained bone marrow–derived
macrophages from age-matched Ifng–/– and Socs1–/–Ifng–/– mice as previously
described42. Peritoneal macrophages were obtained from 6–8-week-old Ifng–/–
and Socs1–/–Ifng–/– by peritoneal lavage with 5 ml of cold phosphate-buffered
saline. We resuspended the macrophages in DMEM (containing 10% FBS) and
seeded into a 24-well plate at 1  106 cells/ml. After stimulation with LPS and
Pam3Cys for 12 h, we determined the production of IL-6 and TNF using the
respective ELISA kits (BD Biosciences). We obtained splenocytes from CBA/
CaOlaHsd and CBA/CaOlaHsd-xid mice as previously described24, and Mal-
deficient mouse embryonic fibroblasts were a gift of K. Fitzgerald. These studies
were approved by the Monash University Monash Medical Centre A Animal
Ethics Committee (Melbourne, Australia).
Immunoprecipitation analysis. Immunoprecipitation of Mal and SOCS-1
was performed as previously described32. Briefly, we seeded 2  106 HEK293T
cells 24 h before cotransfection with 1.25 mg of both HA-tagged Mal and
Flag-tagged SOCS-1 using Genejuice (Novagen). We incubated the cells for
24 h, lysed them with lysis buffer (50 mM Tris (pH 7.4), 1.0% Triton X-100,
150 mM NaCl, 1 mM EDTA, 2 mM Na3VO4, 10 mM NaF, 1 mM PMSF
and protease cocktail inhibitor mixture; Roche). We cleared the cell lysates by
centrifugation (9,000g, 10 min, 4 1C), precleared with protein G–Sepharose
beads for 30 min, 4 1C and Flag–SOCS-1 immune complexes were immuno-
precipitated from supernatants using anti-Flag M2-agarose (Sigma) conjugated
beads for 2 h at 4 1C. We washed the beads 3 with lysis buffer and eluted
the complexes by boiling beads in 5 volumes SDS-PAGE sample buffer.
We fractionated the proteins on 15% SDS-PAGE, transferred them to nitro-
cellulose membrane and performed immunoblot analysis using anti-HA
(Rockland to detect Mal-HA. Anti-Flag M2-HRP conjugated antibody was
purchased from Sigma.
Ubiquitin assay. HEK293T cells were transfected as described above with
800 ng of Flag-Mal, 100 ng of HA-ubiquitin (gift of R. Hay), and, where noted,
with 100 ng of the relevant Myc-tagged SOCS-1 expression plasmid11. After
incubation for an additional 24 h, we pretreated cells with 20 mM MG132
for 4 h, lysed the cells and precleared the cellular lysates with protein
G–Sepharose beads before immunoprecipitation of Flag-Mal complexes using
anti-M2 agarose beads. We washed the recipitated immunocomplexes 3 with
lysis buffer and analyzed them by SDS-PAGE and immunoblotting with
anti-HA to detect ubiquitinated Mal complexes. Flag-tagged MyD88 was a gift
from J. Tschopp.
Note: Supplementary information is available on the Nature Immunology website.
VOLUME 7 NUMBER 2 FEBRUARY 2006 NATURE IMMUNOLOGY

ACKNOWLEDGMENTS
We thank T. Lang, J. Kiu and K. Palmer for technical assistance. Supported by
the National Health and Medical Research Council (334023 to A.M., 236866 to
P.C. and 284220 to P.J.H.), Science Foundation Ireland and the Cooperative
Research Centre for Oral Health Sciences (R.S.).
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/natureimmunology/
Reprints and permissions information is available online at http://npg.nature.com/
© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
reprintsandpermissions/
1. Takeda, K., Kaisho, T. & Akira, S. Toll-like receptors. Annu. Rev. Immunol. Vol. 21
(2003).
2. Dunne, A. & O’Neill, L.A. Adaptor usage and Toll-like receptor signaling specificity.
FEBS Lett. 579, 3330–3335 (2005).
3. Akira, S. & Takeda, K. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499–511
(2004).
4. O’Neill, L.A., Fitzgerald, K.A. & Bowie, A.G. The Toll-IL-1 receptor adaptor family grows
to five members. Trends Immunol. 24, 286–290 (2003).
5. Oshiumi, H. et al. TIR-containing adapter molecule (TICAM)-2, a bridging adapter
recruiting to toll-like receptor 4 TICAM-1 that induces interferon-beta. J. Biol. Chem.
278, 49751–49762 (2003).
6. Fitzgerald, K.A. et al. LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll
adapters TRAM and TRIF. J. Exp. Med. 198, 1043–1055 (2003).
7. Horng, T., Barton, G.M., Flavell, R.A. & Medzhitov, R. The adaptor molecule
TIRAP provides signalling specificity for Toll-like receptors. Nature 420, 329–333
(2002).
8. Yamamoto, M. et al. Essential role for TIRAP in activation of the signalling cascade
shared by TLR2 and TLR4. Nature 420, 324–329 (2002).
9. Mansell, A., Brint, E., Gould, J.A., O’Neill, L.A. & Hertzog, P.J. Mal interacts with TNF
receptor associated factor (TRAF)-6 to mediate NF-kB activation by Toll-like receptor
(TLR)-2 and TLR4. J. Biol. Chem. 279, 37227–37230 (2004).
10. Liew, F.Y., Xu, D., Brint, E.K. & O’Neill, L.A. Negative regulation of toll-like receptor-
mediated immune responses. Nat. Rev. Immunol. 5, 446–458 (2005).
11. Chuang, T.H. & Ulevitch, R.J. Triad3A, an E3 ubiquitin-protein ligase regulating Toll-
like receptors. Nat. Immunol. 5, 495–502 (2004).
12. Wald, D. et al. SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor
signaling. Nat. Immunol. 4, 920–927 (2003).
13. Janssens, S., Burns, K., Vercammen, E., Tschopp, J. & Beyaert, R. MyD88S, a splice
variant of MyD88, differentially modulates NF-kappaB- and AP-1-dependent gene
expression. FEBS Lett. 548, 103–107 (2003).
14. Naiki, Y. et al. Transforming growth factor-beta differentially inhibits MyD88-depen-
dent, but not TRAM- and TRIF-dependent, lipopolysaccharide-induced TLR4 signaling.
J. Biol. Chem. 280, 5491–5495 (2005).
15. Kobayashi, K. et al. IRAK-M is a negative regulator of Toll-like receptor signaling. Cell
110, 191–202 (2002).
16. Divanovic, S. et al. Negative regulation of Toll-like receptor 4 signaling by the Toll-like
receptor homolog RP105. Nat. Immunol. 6, 571–578 (2005).
17. Naka, T. et al. Structure and function of a new STAT-induced STAT inhibitor. Nature
387, 924–929 (1997).
18. Endo, T.A. et al. A new protein containing an SH2 domain that inhibits JAK kinases.
Nature 387, 921–924 (1997).
19. Starr, R. et al. A family of cytokine-inducible inhibitors of signalling. Nature 387, 917–
921 (1997).
NATURE IMMUNOLOGY VOLUME 7 NUMBER 2 FEBRUARY 2006
View publication stats
ARTICLES
20. Alexander, W.S. & Hilton, D.J. The role of suppressors of cytokine signaling (SOCS)
proteins in regulation of the immune response. Annu. Rev. Immunol. 22, 503–529
(2004).
21. Alexander, W.S. et al. SOCS1 is a critical inhibitor of interferon gamma signaling and
prevents the potentially fatal neonatal actions of this cytokine. Cell 98, 597–608
(1999).
22. Baetz, A., Frey, M., Heeg, K. & Dalpke, A.H. Suppressor of cytokine signaling (SOCS)
proteins indirectly regulate toll-like receptor signaling in innate immune cells. J. Biol.
Chem. 279, 54708–54715 (2004).
23. Kinjyo, I. et al. SOCS1/JAB is a negative regulator of LPS-induced macrophage
activation. Immunity 17, 583–591 (2002).
24. Nakagawa, R. et al. SOCS-1 participates in negative regulation of LPS responses.
Immunity 17, 677–687 (2002).
25. Kamizono, S. et al. The SOCS box of SOCS-1 accelerates ubiquitin-dependent
proteolysis of TEL-JAK2. J. Biol. Chem. 276, 12530–12538 (2001).
26. Zhang, J.G. et al. The conserved SOCS box motif in suppressors of cytokine signaling
binds to elongins B and C and may couple bound proteins to proteasomal degradation.
Proc. Natl. Acad. Sci. USA 96, 2071–2076 (1999).
27. Rechsteiner, M. & Rogers, S.W. PEST sequences and regulation by proteolysis. Trends
Biochem. Sci. 21, 267–271 (1996).
28. Kile, B.T. et al. The SOCS box: a tale of destruction and degradation. Trends Biochem.
Sci. 27, 235–241 (2002).
29. Nicholson, S.E. et al. Mutational analyses of the SOCS proteins suggest a dual domain
requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction.
EMBO J. 18, 375–385 (1999).
30. Rawlings, D.J. et al. Mutation of unique region of Bruton’s tyrosine kinase in
immunodeficient XID mice. Science 261, 358–361 (1993).
31. Glickman, M.H. & Ciechanover, A. The ubiquitin-proteasome proteolytic pathway:
destruction for the sake of construction. Physiol. Rev. 82, 373–428 (2002).
32. Fitzgerald, K.A. et al. Mal (MyD88-adapter-like) is required for Toll-like receptor-4
signal transduction. Nature 413, 78–83 (2001).
33. Vermeulen, L., De Wilde, G., Notebaert, S., Vanden Berghe, W. & Haegeman, G.
Regulation of the transcriptional activity of the nuclear factor-kappaB p65 subunit.
Biochem. Pharmacol. 64, 963–970 (2002).
34. Doyle, S.L., Jefferies, C.A. & O’Neill, L.A. Bruton’s tyrosine kinase is involved in p65-
mediated transactivation and phosphorylation of p65 on serine 536 during NFkappaB
activation by lipopolysaccharide. J. Biol. Chem. 280, 23496–23501 (2005).
35. Gingras, S., Parganas, E., de Pauw, A., Ihle, J.N. & Murray, P.J. Re-examination of the
role of suppressor of cytokine signaling 1 (SOCS1) in the regulation of toll-like receptor
signaling. J. Biol. Chem. 279, 54702–54707 (2004).
36. Inagaki-Ohara, K., Hanada, T. & Yoshimura, A. Negative regulation of cytokine signaling
and inflammatory diseases. Curr. Opin. Pharmacol. 3, 435–442 (2003).
37. Yoshimura, A., Ohishi, H.M., Aki, D. & Hanada, T. Regulation of TLR signaling and
inflammation by SOCS family proteins. J. Leukoc. Biol. 75, 422–427 (2004).
38. Naka, T., Fujimoto, M. & Kishimoto, T. Negative regulation of cytokine signaling: STAT-
induced STAT inhibitor. Trends Biochem. Sci. 24, 394–398 (1999).
39. Fenner, J.E. et al. Suppressor of cytokine signaling 1 regulates the immune response to
infection by a unique inhibition of type I interferon activity. Nat. Immunol. 7, 33–39
(2005).
40. Noubir, S., Hmama, Z. & Reiner, N.E. Dual receptors and distinct pathways mediate
interleukin-1 receptor-associated kinase degradation in response to lipopolysaccharide.
Involvement of CD14/TLR4, CR3, and phosphatidylinositol 3-kinase. J. Biol. Chem.
279, 25189–25195 (2004).
41. Yamin, T.T. & Miller, D.K. The interleukin-1 receptor-associated kinase is degraded by
proteasomes following its phosphorylation. J. Biol. Chem. 272, 21540–21547
(1997).
42. Sweet, M.J. et al. A novel pathway regulating lipopolysaccharide-induced shock by
ST2/T1 via inhibition of Toll-like receptor 4 expression. J. Immunol. 166, 6633–6639
(2001).
155