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T Cell Receptor Signals to NF-κB Are Transmitted by a Cytosolic p62-Bcl10-Malt1-IKK Signalosome


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Antigen-mediated stimulation of the T cell receptor (TCR) triggers activation of nuclear factor κB (NF-κB), a key transcriptional regulator of T cell proliferation and effector cell differentiation. TCR signaling to NF-κB requires both the Carma1-Bcl10-Malt1 (CBM) complex and the inhibitor of κB (IκB) kinase (IKK) complex; however, the molecular mechanisms connecting the CBM complex to activation of IKK are incompletely defined. We found that the active IKK complex is a component of a TCR-dependent cytosolic Bcl10-Malt1 signalosome containing the adaptor protein p62, which forms in effector T cells. Phosphorylated IκBα and NF-κB were transiently recruited to this signalosome before NF-κB translocated to the nucleus. Inhibiting the activity of the kinase TAK1 or IKK blocked the phosphorylation of IKK, but not the formation of p62-Bcl10-Malt1 clusters, suggesting that activation of IKK occurs after signalosome assembly. Furthermore, analysis of T cells from p62-deficient mice demonstrated that the p62-dependent clustering of signaling components stimulated activation of NF-κB in effector T cells. Thus, TCR-stimulated activation of NF-κB requires the assembly of cytosolic p62-Bcl10-Malt1-IKK signalosomes, which may ensure highly regulated activation of NF-κB in response to TCR engagement.
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(325), ra45. [DOI: 10.1126/scisignal.2004882] 7Science Signaling
R. Latoche and Brian C. Schaefer (13 May 2014)
Suman Paul, Maria K. Traver, Anuj K. Kashyap, Michael A. Washington, Joseph
p62-Bcl10-Malt1-IKK Signalosome
T Cell Receptor Signals to NF-{kappa}B Are Transmitted by a Cytosolic
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T Cell Receptor Signals to NF-kB Are Transmitted
by a Cytosolic p62-Bcl10-Malt1-IKK Signalosome
Suman Paul,
* Maria K. Traver,
Anuj K. Kashyap,
Michael A. Washington,
Joseph R. Latoche,
Brian C. Schaefer
Antigen-mediated stimulation of the T cell receptor (TCR) triggers activation of nuclear factor kB(NF-kB),
a key transcriptional regulator of T cell proliferation and effector cell differentiation. TCR signaling to
NF-kB requires both the Carma1-Bcl10-Malt1 (CBM) complex and the inhibitor of kB(IkB) kinase (IKK)
complex; however, the molecular mechanisms connecting the CBM complex to activation of IKK are
incompletely defined. We found that the active IKK complex is a component of a TCR-dependent
cytosolic Bcl10-Malt1 signalosome containing the adaptor protein p62, which forms in effector T cells.
Phosphorylated IkBaand NF-kB were transiently recruited to this signalosome before NF-kBtrans-
located to the nucleus. Inhibiting the activity of the kinase TAK1 or IKK blocked the phosphorylation
of IKK, but not the formation of p62-Bcl10-Malt1 clusters, suggesting that activation of IKK occurs
after signalosome assembly. Furthermore, analysis of T cells from p62-deficient mice demonstrated
that the p62-dependent clustering of signaling components stimulated activation of NF-kB in effec-
tor T cells. Thus, TCR-stimulated activation of NF-kB requires the assembly of cytosolic p62-Bcl10-
Malt1-IKK signalosomes, which may ensure highly regulated activation of NF-kB in response to TCR
Specific engagement of antigen-bound major histocompatibility
complex proteins on the surface of antigen-presenting cells (APCs)
by the T cell receptor (TCR) initiates a signaling cascade that activates
nuclear factor kB (NF-kB), a critical transcriptional regulator of T cell
proliferation and differentiation programs (1). Early TCR-proximal
signals lead to activation of protein kinase C q(PKCq), which phos-
phorylates the large adaptor protein Carma1 to stimulate its association
with a preexisting complex of the small adaptor Bcl10 and the protease
Malt1 to form the CBMcomplex. Assembly of the CBM complex is
followed by activation of the inhibitor of kB(IkB) kinase (IKK), which
is composed of the serine and threonine kinases IKKaand IKKband a
noncatalytic regulatory subunit (IKKg). Activated IKK phosphorylates
the NF-kB inhibitor, IkBa, leading to the polyubiquitination and deg-
radation of IkBa, thereby freeing NF-kB to translocate to the nucleus
to activate target genes. Whereas it is known that the constituents of the
CBM complex are required for the activation of IKK, the molecular
mechanism by which CBM proteins interact with IKK remains incom-
pletely understood (2).
Accumulating data from our group and others suggest that signaling
from the CBM complex to IKK involves a progressive series of steps,
which incorporate discrete membrane-proximal and cytosolic signaling
platforms. Biochemical studies have shown that PKCqand Carma1 are
located on lipid rafts in activated T cells (3), together with a fraction
of Bcl10, Malt1, and IKK (4,5). However, the IkBaNF-kB complex
is present only in the cytosol. Thus, current data do not explain how
membrane-associated upstream signal transducers transmit activating
signals to the cytosolic IkBaNF-kBcomplex(5). A study suggests that
the early CBM complex matures to form the Bcl10-Malt1 complex and
that this latter complex inducibly interacts with IkBa(6). Additionally, im-
aging studies from our group identified TCR-induced cytosolic clusters
of Bcl10 and Malt1, called POLKADOTS (79), which are sites of enriched
interactions between Bcl10 and the E3 ubiquitin ligase TRAF6 [tumor
necrosis factor (TNF) receptorassociated factor 6] (8). The presence of
these signaling clusters is highly correlated with the extent of nuclear
translocation of NF-kB(10), suggesting a role for POLKADOTS in acti-
vating NF-kB.
POLKADOTS are de novo cytoplasmic aggregates that require
TCR-dependent Lys
)mediated polyubiquitination of Bcl10. A
structural study demonstrated that Bcl10 forms filamentous structures in
the presence of the active form of Carma1 and that the ability to form fila-
ments correlates with the ability to activate NF-kB(11). Our data suggest
that K
-linked polyubiquitination of Bcl10 causes these filaments to cluster
around preexisting aggregates (speckles) of the ubiquitin-binding adaptor
protein, p62 (also known as SQSTM-1), to form the POLKADOTS
structures. Knockdown of p62 blocks both the formation of POLKADOTS
and the activation of NF-kB(7). These data are consistent with a study of
mice, which showed that p62 contributes to TCR-dependent IKK
activation and T cell differentiation (12). Together, these biochemical,
imaging, and genetic data are consistent with the hypothesis that p62,
Bcl10, and Malt1 form a cytosolic complex (the POLKADOTSsignalosome)
that is responsible for transducing activating signals to IKK and NF-kB.
Here, we sought to provide direct evidence that TCR-induced cyto-
solic POLKADOTS clusters direct the activation of NF-kB. Our data
demonstrate that after TCR stimulation, activated IKK localized to
cytosolic POLKADOTS, and not to the membrane-associated CBM
Department of Microbiology and Immunology, Uniformed Services Universi-
ty, Bethesda, MD 20814, USA.
Center for Neuroscience and Regenerative
Medicine, Uniformed Services University, Bethesda, MD 20814, USA.
*Present address: Department of Internal Medicine, The University of Toledo
Medical Center, 3000 Arlington Avenue, Toledo, OH 43614, USA.
Present address: Howard Hughes Medical Institute, Janelia Farm Research
Campus, Molecular Biology Shared Resource, 19700 Helix Drive, Ashburn, VA
20147, USA.
Present address: Department of Clinical Investigation, Tripler Army Medical
Center, 1 Jarrett White Road, Honolulu, HI 96818, USA.
§Present address: Integrative Cardiac and Metabolic Health Program, Wind-
ber Research Institute, 620 7th Street, Windber, PA 15963, USA.
||Corresponding author. E-mail:
RESEARCH ARTICLE 13 May 2014 Vol 7 Issue 325 ra45 1
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complex. Phosphorylated IkBaand NF-kB were also transiently present
at the POLKADOTS structures, before the nuclear translocation of NF-kB
occurred. Finally, genetic data demonstrated that cluster formation and
NF-kB activation were highly dependent on p62 abundance. These data
establish that TCR signaling drives the assembly of cytosolic p62-Bcl10-
Malt1-IKK clusters or signalosomes that direct the activation of NF-kB.
Cytosolic Bcl10-p62 clusters colocalize with phosphorylated
IKK and TRAF6 in response to TCR stimulation
In experiments with primary effector T cells and the murine T helper 2
2) clone, D10, we previously demonstrated that stimulation with
cognate antigen or anti-CD3 antibody stimulates the formation of cy-
tosolic aggregates containing Bcl10 and Malt1. These structures, which
we have named POLKADOTS, coalesce around preexisting speckles
of the adaptor protein p62 in a manner dependent on the TCR-
dependent, K
-linked polyubiquitination of Bcl10 (7,8). To ascertain
the subcellular location of key mediators in the TCRNF-kBpathway
with respect to the cytosolic Bcl10 clusters, we stimulated D10 cells
with antigen-loaded CH12 cells, which are an I-A
expressing murine
B cell line. We then used confocal microscopy to image cyan fluores-
cent protein (CFP)tagged PKCq(PKCq-CFP), yellow fluorescent
protein (YFP)tagged Bcl10 (Bcl10-YFP), and endogenous Carma1
and p62. At 10 and 20 min after stimulation of the D10 cells, PKCq
and Carma1 translocated to the immunological synapse (Fig. 1A), con-
sistent with previous f indings (3). Whereas some conjugates exhibited
prominent colocalization between Bcl10 and PKCqin D10 cells at the
immunological synapse, as we previously reported (9), all conjugates
(defined as D10-CH12 couples with PKCqtranslocation at the immu-
nological synapse) exhibited localization of Bcl10 within cytosolic
aggregates that colocalized with p62 (7) in the antigen-activated cells
Our previous fluorescence resonance energy transfer (FRET) analy-
ses demonstrated that Bcl10 is closely associated with TRAF6 in the
POLKADOTS structures (8). Because TRAF6 is involved in recruiting
and ubiquitinating IKK (1,13), and because phosphorylated IKK
(pIKK) is detected solely in the cytosol (5), we hypothesized that IKK is
activated upon association with the cytosolic Bcl10-Malt1-p62containing
POLKADOTS structures. Staining with an antibody that specifically rec-
ognizes pIKKa/b(IKKaand IKKbphosphorylated at residues Ser
and Ser
) revealed no apparent specific signal in unstimulated cells
(Fig. 1C), although nonspecific staining of the plasma membrane was
observed in all cells. At 20 min and 2 hours after stimulation, we observed
that pIKKa/bcolocalized with the cytosolic Bcl10 clusters, but not with
PKCqat the immunological synapse (Fig. 1, C and D). Total IKKbwas
present in cytosolic aggregates both before and after stimulation with
antigen (Fig. 1D). Close inspection of the imaging data revealed that
pIKKa/bstaining was consistently adjacent to, but rarely overlapping with,
total IKKbstaining (Fig. 1D and fig. S1A), suggesting that the pIKKa/b
at POLKADOTS was distinct from the pool of IKKbaggregates found in
T cells before stimulation. The lack of overlap between pIKKa/band
total IKKbstaining was further confirmed by staining with a distinct IKKb
monoclonal antibody (fig. S1B). Staining with an anti-TRAF6 antibody
also showed the TCR-induced redistribution of TRAF6 from a diffuse and
primarily cytoplasmic localization in unstimulated cells to a punctate
distribution that overlapped with Bcl10 and Malt1 in POLKADOTS
20 min after TCR stimulation with anti-CD3 antibody (Fig. 1E), with less
prominent colocalization at the 2-hour time point (fig. S1C).
To confirm these data in primary T cells, we stimulated in vitro dif-
ferentiated CD4
2 effector cells with anti-CD3 and anti-CD28 anti-
bodies (anti-CD3/28), followed by staining for endogenous Bcl10, p62,
and pIKK proteins. At 20 min after stimulation, endogenous Bcl10
clusters were colocalized with p62 speckles, and pIKK was specifically
localized to these p62-Bcl10 aggregates (Fig. 1F). Thus, the phenom-
enon of TCR-dependent colocalization of pIKK and cytosolic Bcl10
clusters was not only observed in D10 cells that overexpressed Bcl10
protein but also occurred at clusters of endogenous Bcl10 in primary
effector T cells. Together, these data suggest that IKK is recruited to
and phosphorylated at the POLKADOTS structures shortly after TCR
Activation of NF-kB occurs at the POLKADOTS signalosome
Once activated, the IKK holoenzyme phosphorylates IkBa, targeting
IkBafor proteasomal degradation, thereby enabling the nuclear trans-
location of NF-kB. Because of the rapid kinetics of proteasomal deg-
radation, pIkBais abundant only for a short period of time after TCR
stimulation. Accordingly, at 10 min after stimulation of D10 cells with
anti-CD3 antibody, we detected pIkBathat colocalized with Bcl10,
Malt1, and pIKK (Fig. 2A). The pIkBasignal was undetectable by
20 min after stimulation, presumably because of its rapid proteasomal
degradation, consistent with a previous report of IkBadegradation ki-
netics in D10 cells and primary T cells (7). These data suggest that IkBa
is transiently recruited to the POLKADOTS structures, where it is phos-
phorylated by IKK, which stimulates its proteasomal degradation.
The NF-kB family consists of five proteins that exist as homo- or
heterodimers (14), with RelA(p65)-p50 being the most common acti-
vating heterodimer. In unstimulated T cells, NF-kB resides in the cytosol,
bound to IkBa. The degradation of IkBaunmasks a nuclear localization
signal, which enables NF-kB to enter the nucleus, where it promotes the
transcription of target genes. Because we detected pIkBacolocalized with
Bcl10-Malt1 clusters, we hypothesized that RelA should be present contem-
poraneously. Imaging analysis revealed a homogeneous cytosolic distribu-
tion of RelA before T cell stimulation (Fig. 2B). After 10 min of stimulation,
either with antigen-loaded APCs or with anti-CD3 antibody, we observed
that RelA colocalized with cytosolic Bcl10-Malt1 aggregates, but was not
yet translocated to the nucleus (Fig. 2, B and C). By 20 min after stimula-
tion, RelA was no longer colocalized with the POLKADOTS structures, but
was enriched in the nucleus, consistent with our previous observations that
IkBadegradation and NF-kB activation occur between 10 and 20 min after
stimulation of the TCR on D10 cells (7,10). Thus, RelA is transiently present
at the POLKADOTS structures before it undergoes nuclear translocation.
The inhibition of either IKK or TAK1 blocks IKK
phosphorylation at cytosolic Bcl10 signalosomes,
causing reduced RelA activation
To further establish a mechanistic link between the IKK phosphoryl-
ation observed at the p62-Bcl10-Malt1 clusters (POLKADOTS) and
the nuclear translocation of RelA, we treated D10 cells with BAY
11-7082 (BAY 11) or 5Z-7-oxozeaenol (5 oxo). BAY 11 is an inhibitor
of IKK, which we used because a previous study suggests that trans-
autophosphorylation of IKKbparticipates in its activation (15). 5 oxo
is a specific inhibitor of TAK1, a kinase identified as an essential ac-
tivator of IKK phosphorylation in the TCRNF-kB pathway (16). After
stimulation with anti-CD3 antibodies, D10 cells treated with either
BAY 11 or 5 oxo showed reduced phosphorylation of IKKa/bwithout
having altered TCR-dependent phosphorylation of extracellular signal
regulated kinases 1 and 2 (ERK1/2) (Fig. 3A), consistent with a previous
study (16).
RESEARCH ARTICLE 13 May 2014 Vol 7 Issue 325 ra45 2
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Confocal microscopy analysis of antigen-stimulated D10 cells revealed
that although inhibition of either IKK or TAK1 did not affect the trans-
location of PKCqto the immunological synapse or the formation of
Bcl10 clusters (Fig. 3B), BAY 11and 5 oxotreated D10 cells exhibited
decreased pIKKa/bstaining at Bcl10 clusters compared to those in vehicle-
treated cells (Fig. 3B). These data also demonstrated that the observed anti-
pIKK plasma membrane staining is nonspecific because it was not affected
by BAY 11 or 5 oxo (Fig. 3B and fig. S2A). Additionally, both BAY 11 and
5 oxo blocked the nuclear translocation of RelA in D10 cells stimulated with
antigen-loaded APCs or anti-CD3 antibody (Fig. 3, C and D), without
apparently altering the translocation of PKCqto the immunological synapse
(Fig. 3C) or the formation of Bcl10 clusters (Fig. 3, C and D). Unstimulated,
5oxotreated D10 cells exhibited no change in the distribution of PKCq-
CFP, Bcl10-YFP, or endogenous RelA as compared to cells treated with
vehicle [dimethyl sulfoxide (DMSO)] alone (fig. S2, B and C). Quantifica-
tion of microscopy data demonstrated a statistically significant reduction
in the ratio of nuclear to cytosolic RelA in BAY 11or 5 oxotreated
cells after stimulation with either antigen-loaded APCs or anti-CD3 anti-
body (Fig. 3E). Together, these observations suggest that inhibition of
IKK or TAK1 activity prevents the efficient TCR-dependent phospho-
rylation of IKK. These data furthermore demonstrate that phosphoryla-
tion of IKK and activation of RelA are downstream of the translocation
of PKCqto the immunological synapse and the clustering of Bcl10 in
Loss of p62 blocks Bcl10 clustering, IKK phosphorylation,
and RelA nuclear translocation in effector T cells
Studies suggest that p62 is required for the activation of NF-kB in dif-
ferentiated effector T cells, but not in naïve T cells (12), which may
reflect the fact that naïve cells have little or no p62 (17). Indeed, upon
stimulation of wild-type primary CD4
T cells, we observed that p62
was low in abundance in primary CD4
T cells and that there was a
substantial increase in its abundance at 24 and 48 hours after stimula-
tion with anti-CD3 and anti-CD28 antibodies (Fig. 4A), consistent with
Fig. 1. Colocalization of pIKKa/band TRAF6 with cytosolic Bcl10-p62
clusters after TCR stimulation. (Ato C) D10 cells transduced with retro-
viruses encoding PKCq-CFP and Bcl10-YFP were stimulated for the indicated
times by CH12 cells that were either not loaded (No antigen) or loaded with
conalbumin (+Antigen). Cells were then analyzed by confocal microscopy.
Fluorescent proteins and antibody stains are indicated above each image.
(Dand E) D10 cells transduced with retroviruses encoding Bcl10-CFP and
Malt1-YFP were left untreated or were stimulated for the indicated times with (D) CH12 cells that were either not loaded (No antigen) or loaded with conalbumin
(+Antigen)or (E) anti-CD3 antibody.Cells were then stained withantibodies against theindicated proteins before being analyzed by confocalmicroscopy.
(F) Primary mouse naïve CD4
T cells were differentiated in vitro intoT
2 cells and then were incubated on coverslips coated with anti-CD3 and anti-CD28
antibodies. Cells were then stained with antibodies against the indicated proteins and analyzed by confocal microscopy. All images are representative of
three or four independent experiments. Scale bars, 10 mm.
RESEARCH ARTICLE 13 May 2014 Vol 7 Issue 325 ra45 3
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previous studies (12,17). Moreover, we observed similar clustering of
Bcl10 and pIKKa/bbeneath the capped TCR of naïve wild-type and
T cells, further suggesting that p62 does not play an essential
role in the TCR-dependent activation of NF-kBinnaïveTcells(Fig.4B).
Two observations suggest that the TCR-dependent activation of NF-
kB changes from being p62-independent in naïve T cells to being p62-
dependent in effector T cells (2). First, whereas naïve p62
cells exhibit normal IKK phosphorylation in response to anti-CD3/28
antibodies, by 24 hours after stimulation [by which time naïve CD4
cells have differentiated into interleukin-2 (IL-2)producing effector T
cells], p62
T cells fail to activate IKK (12). Second, the nuclear
translocation of RelA in response to stimulation with anti-CD3 antibody
is blocked in D10 cells, in which p62 is knocked down (7). The impli-
cation of these observations is that differentiated T cells require one or
more functions of p62 for the activation of IKK and NF-kB.
To establish a mechanistic relationship between p62 abundance and
the activation of NF-kB in effector T cells, we assessed the extent of
IKK activation in the presence and absence of p62. Western blotting
analysis demonstrated minimal IKK phosphorylation in p62
cells compared to that in wild-type T
2 cells in response to anti-
CD3/CD28 antibodies (Fig. 4C). In addition, the stimulation of wild-
type or p62
2 cells with APCs loaded with SEB (Staphylococcus
enterotoxin B) resulted in no difference in the translocation of PKCqto
the plasma membrane (Fig. 4, D and E), indicating that membrane-
proximal TCRNF-kB signaling events were not affected by the loss
of p62. However, cytosolic Bcl10 clustering was substantially impaired
in p62
T cells compared to that in wild-type T cells in response to
stimulation with antigen-loaded APCs or anti-CD3/28 antibodies (Fig. 4,
E to G), consistent with our previous observations of reduced formation
of POLKADOTS in p62-silenced D10 cells (7). We speculate that the
small population of cells with residual Bcl10 clustering may reflect the
activity of Nbr1, a protein functionally similar to p62 (17), which co-
localizes with p62 speckles in T effector cells (7). Alternatively, the
Carma1-dependent aggregation of Bcl10 (11) may inefficiently lead to
the production of large cytosolic Bcl10 clusters in the absence of p62.
Finally, the percentage of p62
2 cells that exhibited nuclear trans-
location of RelAwas statistically significantly reduced compared to thatof
wild-type T
2 cells in response to stimulation with anti-CD3/28 antibo-
dies (Fig. 4,F and H), consistent with the observed reduction in IKK phos-
phorylation. Furthermore, those p62
T cells that exhibited nuclear
translocation of RelA were the same cells that formed Bcl10-containing
POLKADOTS (Fig. 4I). Moreover, the small population of responding
cells exhibited the same degree of RelA nuclear translocationas that
of wild-type cells, as demonstrated by equivalent ratios of nuclear to
cytosolic RelA (Fig. 4J). These data are consistent with our finding that
the activation of NF-kB is digital; that is, that varying the intensityof TCR
stimulation results in variations in the percentage of cells that respond,
whereas the extent of activation of NF-kB on a per-cell basis is invariant
in the responding population (10). These data suggest that p62 markedly
enhances the efficiency of NF-kB signal transduction, increasing the per-
centage of effector T cells that successfully activate NF-kBinresponseto
TCR stimulation. In the absence of p62, the activation of NF-kBisvery
inefficient, likely reflecting the near absence of cytosolic clustering of
Here, we showed that the stimulation of T cells led to the rapid redistribu-
tion of PKCqand Carma1 to the immunological synapse. At early times
after stimulation of the TCR, Bcl10 was recruited to both the immuno-
logical synapse and p62 speckles. Notably, the pIKK complex was en-
riched at these cytosolic p62-Bcl10-Malt1 aggregates (POLKADOTS),
but not at the immunological synapse. TRAF6 was also enriched within
the POLKADOTS structures. Cytosolic RelA and pIkBaalso transiently
Fig. 2. pIkBaand RelA exhibit transient colocalization with cytosolic
Bcl10-containing signalosomes before translocating to the nucleus. (A)
D10 cells transduced with retroviruses encoding Bcl10-CFP and
Malt1-YFP were left untreated or were stimulated with anti-CD3 antibo-
dies. Cells were then stained with antibodies against the indicated pro-
teins and analyzed by confocal microscopy. (B) D10 cells transduced
with retroviruses encoding PKCq-CFP and Bcl10-YFP were stimulated
for the indicated times with CH12 cells that were either unloaded (No
antigen) or loaded with conalbumin (+Antigen). Cells were then stained
with anti-RelA and analyzed by confocal microscopy. (C) D10 cells
transduced with retroviruses encoding Bcl10-CFP and Malt1-YFP were
left untreated or were stimulated with anti-CD3 antibodies. Cells were
then stained with anti-RelA and analyzed by confocal microscopy. Inset
images below the 10-min time points are magnified views of the areas
marked by white rectangles. All of the images are representative of at
least four independent experiments. Scale bars, 10 mm.
RESEARCH ARTICLE 13 May 2014 Vol 7 Issue 325 ra45 4
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colocalized with POLKADOTS clusters, which was followed by the
persistent nuclear translocation of RelA. These data are consistent with
the interpretation that IkBaNF-kB complexes are recruited to the
POLKADOTS signalosome, where activated IKK then phosphorylates
IkBa. In additional experiments, we showed that inhibition of TAK1 and
IKK activity had the predicted effects of blocking the TCR-dependent
phosphorylation of IKK and the nuclear translocation of RelA, but
had no effect on Bcl10 clustering to form the POLKADOTS signalosome.
Thus, Bcl10 clustering is upstream of IKK activation. Finally, p62
cells exhibited inefficient Bcl10 clustering, IKK phosphorylation, and
RelA nuclear translocation; however, they showed normal TCR-proximal
signaling, suggesting that p62 plays a key role in the activation of the
IKK complex in effector T cells. In contrast, naïve T cells from wild-type
and p62
mice exhibited equivalent clustering of both Bcl10 and
pIKKa/bbeneath the cross-linked TCR, supporting a previous study that
suggested that p62 does not play an essential role in TCR-dependent
NF-kB signaling in naïve T cells (12). Together, these observations provide
evidence that TCR signals direct the assembly of a p62-Bcl10-Malt1-
TRAF6-IKK signalosome (the POLKADOTS signalosome), which stim-
ulates the activation of NF-kBineffectorTcells.
These findings establish a molecular mechanism that connects the CBM
complex to the activation of NF-kB in effector T cells. Specifically, we
showed that early, transient formation of the membrane-proximal CBM
complex was followed by the assembly of the cytosolic POLKADOTS sig-
nalosome. A lingering question not directly resolved by our experiments
is whether Carma1 is contained within the POLKADOTS signalosome.
Although our antibody staining data suggest that Carma1 is enriched at
the plasma membrane, and not at the POLKADOTS signalosome, other
evidence suggests that POLKADOTS aggregates contain small amounts
of Carma1. For example, structural studies show that activated Carma1 is
required to initiate the Bcl10 polymerization process. Thus, although the
resulting filaments are dominated by Bcl10, they also contain a few mole-
cules of activated Carma1 at one end (11). Additionally, our previous FRET
studies provided evidence that Carma1 is enriched within POLKADOTS
structures (8), even though colocalization within POLKADOTS was not
apparent by microscopy. We thus propose that Carma1 is activated at the
plasma membrane by PKCq-mediated phosphorylation. The resulting change
in the conformation of Carma1 triggers the nucleation ofBcl10 filaments.
The TCR-dependent, K
-linked polyubiquitination of Bcl10 also occurs
in concordance with filament formation, leading to capture of filamentsby
cytosolic p62 speckles. The p62-dependent aggregation of filaments cre-
ates a large network of K
-linked polyubiquitin chains, which recruit IKK
and the TAK1/TAB complex, ultimately resulting in the activation of
TAK1 and IKK (18).
An unexpected observation in these studies relates to the distribution
pattern of total IKKb. In unstimulated D10 cells, IKKbwas primarily in
cytoplasmic aggregates. After stimulation of cells with antigen, these
aggregates persisted and were found to be proximal to, but rarely
overlapping with, pIKKa/bat the POLKADOTS signalosome. The finding
of aggregates of IKKbin unstimulated cells is consistent with a recent study
Fig. 3. Inhibition of TAK1 or IKK blocks IKK phosphorylation and RelA
activation, but not Bcl10 clustering. (A) D10 cells were treated with DMSO
(control), BAY 11, or 5 oxo before being stimulated with anti-CD3 antibody for
20 min. Cells were then lysed and analyzed by Western blotting with antibo-
dies against the indicated proteins. Migration of molecular mass markers
(kD) is indicated at the left. Data are representative of three independent
experiments. (Bto D) D10 cells transduced with retroviruses encoding
PKCq-CFP and Bcl10-YFP were treated with DMSO (control), BAY 11, or 5
oxo, and were stimulated with (B and C) antigen-loaded CH12 cells (APCs)
or (D) anti-CD3 antibody. Cells were then stained with antibodies against the
indicated proteins and were analyzed by confocal microscopy. See fig. S2
for control images. DRAQ5 was used to stain nuclear DNA in (D). (E)Ratios
of the abundances of nuclear and cytosolic RelA protein were quantified in
cells from the experiments shown in (C) and (D). At least 20 cells were quan-
tified in each group. Data are means ± SEM. *P< 0.05. Images in (B) to (D)
are representative of three independent experiments. Scale bars, 10 mm.
RESEARCH ARTICLE 13 May 2014 Vol 7 Issue 325 ra45 5
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that reported that IKKg-IKKbexists in a very highmolecular weight form
in mouse embryonic fibroblasts and human embryonic kidney 293T cells
both before and after stimulation with TNF-a(15). The lack of correlation
between the detection of total IKKband pIKKa/bcould be explained by
several alternative models, including direct or indirect interference of
binding of anti-IKKbby anti-pIKKa/b. However, because two distinct
monoclonal antibodies against IKKbyielded indistinguishable data, we be-
lieve it is unlikely that antibody interference accounts for our observations.
Fig. 4. p62 is required for efficient cytosolic Bcl10 clustering, IKK phos-
phorylation, and RelA nuclear translocation in primary effector T cells. (A)
Naïve CD4
T cells isolated from wild-type (WT) C57BL/6 mice were stimu-
lated with plate-bound anti-CD3 and anti-CD28 antibodies, lysed, and then
analyzed by Western blotting with antibodies against the indicated proteins.
(B) Lymph node cells isolated from WT or p62
mice were incubated on ice
with biotinylated anti-CD3 antibody, which was followed by streptavidin-
induced cross-linking on ice (control) or at 37°C (for 20 min). The cells were
then fixed and stained with antibodies against the indicated proteins. (C)Pri-
mary T
2 cells derived from WT or p62
mice were stimulated with plate-
bound anti-CD3 and anti-CD28 antibodies, lysed, and analyzed by Western
blotting with antibodies against the indicated proteins. (A and C) Migration
of molecular mass markers (kD) is indicated at the left. Blots are represent-
ative of two independent experiments. (Dand E)T
2 cells derived from WT
or p62
mice were stimulated withSEB-loaded CHb cells (APCs), stained
with the antibodies against the indicated proteins, and then analyzed by con-
focal microscopy. Note that the p62 speckles in (D) are present only in the
CHb cells. (F)T
2 cells derived from WT or p62
mice were stimulated
on coverslips coated with anti-CD3 and anti-CD28 antibodies for 20 min,
and stained with antibodies against the indicated proteins and DAPI
(4,6-diamidino-2-phenylindole; to visualize nuclei). Cells were then ana-
lyzed by confocal microscopy. (Gto I) Data from the experiment in (F) were
quantified. Graphs show the percentages of cells that exhibited (G) cyto-
solic Bcl10 clusters (POLKADOTS) alone, (H) nuclear RelA alone, or (I) both.
(J) Graph showing the ratios of the staining intensities of nuclear and cyto-
solic RelA in anti-CD3and anti-CD28stimulated WT and p62
2 cells
that did or did not exhibit cytosolic POLKADOTS. At least 20 cells were
quantified in each group. Data in bar graphs are means ± SEM. *P< 0.05.
Data in each panel are representative of two independent experiments.
Scale bars, 5 mm (for B) or 10 mm (for D to F).
RESEARCH ARTICLE 13 May 2014 Vol 7 Issue 325 ra45 6
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Rather, we propose that the amount of pIKKa/bthat translocates to
POLKADOTS may be small in comparison to the amount of total IKKb
in the cytosolic aggregate pool, leading to undetectable anti-IKKbstaining
Our data suggest that the TCR-dependent activation of NF-kB
occurs at the POLKADOTS signalosome, and not at the cytosolic face
of the TCR, where PKCqand Carma1 are enriched. By providing a
potential molecular mechanism for the cytosolic activation of IKK
and NF-kB, these data may answer the question of how TCR signals
are transduced from the membrane-associated CBM complex to the
cytosolic IkBaNF-kB complex. The assembly of distinct cytosolic
signalosomes is emerging as a common, and perhaps essential, mecha-
nism for transmitting activating signals to the IKK complex. Activating
oligomers have been proposed as key intermediates upstream of IKK in
the TNF-areceptor and the Toll-like receptor and IL-1 receptor signaling
pathways (19), as well as in the retinoic acidinducible gene-I (RIG-I)
mitochondrial antiviral signaling protein (MAVS) pathway (20), which
induces NF-kB activation in response to viral RNA. In the case of TCR
signaling to NF-kB, activated Carma1 induces the formation of oligo-
meric filaments of Bcl10 (11). Our data suggest that these filamentous
polymers of Bcl10 are aggregated at the POLKADOTS signalosome,
creating an IKK-activating signaling superstructure in effector T cells.
Our data show that efficient activation of IKK in effector T cells re-
quires p62 and the POLKADOTS signalosome because the absence of
p62 led to poor Bcl10 clustering and minimal activation of IKK. How,
then, does NF-kB activation occur in naïve T cells, which have a low
abundance of p62 and activate NF-kB in a p62-independent manner
(12)? Although there are no data yet to directly answer this question, it
is clear that the CBM complex can activate NF-kB in naïve T cells with-
out participation of the POLKADOTS signalosome. This difference in
signaling architecture may reflect distinctions in the responses of naïve
and effector cells to TCR-activating ligands. In this regard, a study dem-
onstrated that naïve T cells require at least 20 hours of sustained signal-
ing to enter the cell cycle, whereas sustained TCR signaling in effector
T cells triggers apoptosis (21). Although there are probably several un-
derlying mechanisms that account for these differences between naïve
and effector cells (2), part of the explanation may be related to p62
playing a dual role in NF-kB activation (2,7). Specifically, p62 is re-
quired both for the formation of the POLKADOTS signalosome, which
activates IKK, and for limiting the activation of NF-kB through the se-
lective autophagy of Bcl10 (7). Thus, the architecture of the NF-kB signal-
ing pathway in naïve T cells may be optimized to enable the continuous
activation of NF-kB that is required to promote cell cycle entry, whereas
the switch to a reliance on p62 and the POLKADOTS signalosome in dif-
ferentiated cells may both increase the efficiency of signal transmission
and enable precise regulation of signal intensity and duration, preventing
the deleterious consequences of sustained signaling to NF-kB, such as
apoptosis, senescence, or both (22).
Because our data suggest that the POLKADOTS signalosome is a
key regulator of NF-kB activation that may be unique to effector T cells,
we believe that this structure is an attractive target for the development of
immunomodulatory drugs. Although several NF-kB signaling pathways
involve p62 (23), current data suggest that the p62-Bcl10 interaction is
unique to effector T cells. The POLKADOTS signalosome is therefore
likely to incorporate structurally unique features that may enable the
rational design of inhibitors to block signal transmission to IKK and
NF-kB. Through enabling highly specific inhibition of effector T cell
activation, such drugs could have much use in the treatment of auto-
immune diseases or other pathologies that are characterized by the un-
desired activation of effector T cells. It will therefore be important to
define in molecular detail the composition and properties of this key
signaling superstructure.
Mice and the differentiation of T
2 cells
Tissues were harvested from 6- to 12-week-old C57BL/6 wild-type mice
(National Cancer Institute) and p62
mice. The p62
strain (from ES cell
clone EPD0162_1_G07) was obtained from the National Institutes of
Health (NIH)supported KOMP (Knockout Mouse Project) Repository,
generated by the CSD consortium for the NIH-funded KOMP, with
established methodology (24). Harvesting of organs and the purification
and in vitro differentiation of T
2 cells were performed as described previ-
ously (7,25). Briefly, CD4
T cells were isolated from lymph nodes from
C57BL/6 wild-type or p62
mice by negative sorting with a CD4 isolation
kit (Invitrogen). To differentiate naïve cells into T
2 cells, naïve CD4
cells were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies
in the presence of IL-2, IL-4, antiinterferon-g(IFN-g) antibody, and anti
IL-12 antibody for 4 days, which was followed by 2 days of culture in IL-2
containing medium to increase cell numbers. Animal experiments were
approved by the Uniformed Services University (USU) Animal Care and
Use Committee.
Primary antibodies used in this study were as follows: rabbit anti-Carma1
(Enzo, ALX-210-903), mouse anti-Bcl10 (Santa Cruz Biotechnology,
sc-5273), rabbit anti-p62 (Sigma, P0067), mouse anti-tubulin (Santa Cruz
Biotechnology, sc-5286), rabbit anti-PKCq(Cell Signaling Technology),
mouse anti-PKCq(Enzo), anti-pERK1/2 (Cell Signaling Technology,
4370), mouse antiGAPDH (glyceraldehyde-3-phosphate dehydrogenase)
(Santa Cruz Biotechnology, sc-32233), mouse anti-tubulin (Santa Cruz Bio-
technology, sc-5286), rabbit anti-pIKKa/b(Cell Signaling Technology,
2694), mouse anti-IKKb[Imgenex, clone 10A9B6 (used in Fig. 1D) and
clone 10AG2 (used in fig. S1B)], mouse anti-TRAF6 (Santa Cruz Bio-
technology, sc-8409), mouse anti-pIkBa(Cell Signaling Technology,
9246), and rabbit anti-RelA (Santa Cruz Biotechnology, sc-372). Secondary
antibodies included anti-rabbit and anti-mouse immunoglobulin G1 (IgG1)
labeled with Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 (Mo-
lecular Probes). Alexa Fluor 647conjugated streptavidin (Molecular
Probes) was used to cross-link anti-CD3 antibodies to induce TCR capping.
DRAQ5 (Cell Signaling Technology) or DAPI [which is present in ProLong
Gold mounting medium (Invitrogen)] was used to mark the nucleus.
Cells, microscopy, and Western blotting
The D10 T
2 cell clone and B cell lines (CH12 and CHb) were main-
tained in Eagles Hams Amino Acids medium with and without IL-2,
respectively, as previously described (9). D10 cell lines expressing PKCq,
Bcl10, and Malt1 proteins fused to fluorescent proteins were previously
described (8,9). Formation of T cellB cell conjugates was performed as
previously described (7,9). For experiments with SEB (a gift from C.
Ventura and A. OBrien), CHb cells were loaded with SEB (10 mg/ml) over-
night, and then were centrifuged at 2000gfor 30 s with an equal number
of primary T
2 cells. Stimulation of cells with anti-CD3 antibody alone or
together with anti-CD28 antibody was performed as previously described
(7). Capping of TCR on naïve T cells involved the use of biotinylated anti-
CD3 antibody and Alexa Fluor 647conjugated streptavidin, as previously
described (3). For inhibitor experiments, T cells were incubated with2 mM
5 oxo (Enzo) or DMSO (vehicle) 30 min before being stimulated, or with
BAY 11 (5 mg/ml, Calbiochem) 5 min after initiation of stimulation. For
RESEARCH ARTICLE 13 May 2014 Vol 7 Issue 325 ra45 7
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confocal microscopic analysis, cells were fixed and incubated with anti-
bodies as previously described (8), and were visualized with a 40× 1.4
numerical aperture oil objective, a Zeiss 710 NLO microscope, and Zen
software. Cell stimulation, harvesting, and Western blotting analysis were
performed as previously described (7,8).
Quantification of microscopy data
Raw data exported from Zeiss Zen software were analyzed with ImageJ
software (U.S. NIH). For quantification of RelA nuclear occupancy, a
region of interest (ROI) was drawn around the nucleus and cytoplasm
of individual cells, using DRAQ5 or DAPI staining to define the nuclear
periphery and the differential interference contrast image to define the plas-
ma membrane (cytoplasm periphery). The mean pixel intensity of anti-RelA
fluorescence within the nuclear and cytosolic ROIs was obtained for each
cell and used to derive the nuclear/cytosolic RelA ratio. At least 20 cells
were examined in each group. For quantifying POLKADOTS, cells with
2 Bcl10 clusters were scored as positive for POLKADOTS formation.
Cells were scored as positive for nuclear RelA based on the presence of
overlap between RelA and DRAQ5 (or DAPI) fluorescence. At least 20 cells
were counted in each group. GraphPad Prism 6.0 software was used to plot
data and calculate errors and statistics.
Statistical analysis
Where indicated in the figure legends, Pvalues for the differences in
means were calculated with an unpaired, two-tailed Studentsttest.
Fig. S1. Distribution of total IKKb, pIKKa/b, and TRAF6 in stimulated D10 cells.
Fig. S2. Subcellular distribution of PKCq,Bcl10,pIKKa/b, and RelA in unstimulated
DMSO-or 5 oxotreated D10 cells.
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Acknowledgments: We thank A. Snow for antibodies and a critical reading of the manu-
script. Funding: This work was supported by grants from the U.S. NIH (Al057481 to B.C.S.),
the Center for Neuroscience and Regenerative Medicine (to B.C.S.), and predoctoral
fellowships (to S.P.) from the American Heart Association (10PRE3150039) and the Henry
M. Jackson Foundation. Author contributions: S.P. and B.C.S. designed the experiments
and wrote the manuscript; J.R.L. maintained and genotyped the mice; and S.P., M.K.T., A.K.K.,
and M.A.W. performed experiments and collected data. The views expressed are those of
the authors and do not necessarily reflect those of the USU or the Department of Defense.
Competing interests: The authors declare that they have no competing interests.
Submitted 4 November 2013
Accepted 25 April 2014
Final Publication 13 May 2014
Citation: S. Paul, M. K. Traver, A. K. Kashyap, M. A. Washington, J. R. Latoche,
B. C. Schaefer, T cell receptor signals to NF-kB are transmitted by a cytosolic p62-
Bcl10-Malt1-IKK signalosome. Sci. Signal. 7, ra45 (2014).
RESEARCH ARTICLE 13 May 2014 Vol 7 Issue 325 ra45 8
on May 14, 2014 stke.sciencemag.orgDownloaded from
Supplementary Materials for
T Cell Receptor Signals to NF-κ
κB Are Transmitted by a Cytosolic p62-
Bcl10-Malt1-IKK Signalosome
Suman Paul, Maria K. Traver, Anuj K. Kashyap, Michael A. Washington,
Joseph R. Latoche, Brian C. Schaefer*
*Corresponding author. E-mail:
Published 13 May 2014, Sci. Signal. 7, ra45 (2014)
DOI: 10.1126/scisignal.2004882
The PDF file includes:
Fig. S1. Distribution of total IKKβ, pIKKα/β, and TRAF6 in stimulated D10 cells.
Fig. S2. Subcellular distribution of PKCθ, Bcl10, pIKKα/β, and RelA in
unstimulated DMSO-or 5 oxo–treated D10 cells.
Fig. S1. Distribution of total IKKβ
β, pIKKα
β, and TRAF6 in stimulated D10 cells. (A)
Magnified regions of the anti-IKKβ and anti-pIKKα/β staining in D10 cell–APC conjugates
depicted in Fig. 1D. Note that the staining is in close juxtaposition, but with little overlap. (B)
The experiment in Fig. 1D was repeated with the substitution of a distinct anti-IKKβ
monoclonal antibody. (C) Colocalization of POLKADOTS (marked by Malt1-YFP) and anti-
TRAF6 staining in D10 cells 2 hours after stimulation with anti-CD3 antibody. At this time
point, most of the Bcl10 protein is degraded and no longer detectable in POLKADOTS. Note
that there are regions of overlap, but that most of the TRAF6 staining is outside of the
POLKADOTS structures. All images are representative of three or four independent
experiments. Scale bar: 10 µm.
Fig. S2. Subcellular distribution of PKCθ
θ, Bcl10, pIKKα
β, and RelA in unstimulated
DMSO- or 5 oxo–treated D10 cells. (A to C) D10 cells transduced with (A and B)
retroviruses encoding PKCθ-CFP and Bcl10-YFP or (C) a retrovirus encoding Bcl10-YFP
alone were treated with DMSO (control) or 5Z-7-oxozeaenol, and were incubated with (A and
B) CH12 cells (with no antigen) or (C) with no stimulation (no anti-CD3). Cells were then
stained with (A) anti-pIKKα/β or (B and C) anti-RelA before being analyzed by confocal
microscopy. (C) DRAQ5 was used to stain nuclear DNA. Images are representative of three
independent experiments. Scale bar: 10 µm.
... NFKB is an important transcription factor in immunity and has major roles in T-cell development and functional divergence of a number of helper T-cell subsets [15]. Autophagy has been reported in mice to facilitate NFKB signaling by formation of a SQSTM1-dependent "POLKADOTS" molecular complex after TR-dependent activation of PRKCQ (protein kinase C theta), which phosphorylates a large adapter protein CARD11/CARMA1 (caspase recruitment domain family member 11) with a small adapter protein BCL10 (BCL10 immune signaling adaptor) and a protease MALT1 (MALT1 paracaspase) [16]. However, less is known about how this pathway participates in human disease. ...
... T cells obtained from the patient did not appear to exhibit evidence of immunological exhaustion ( Figure S2(b)). We therefore posited that deficits in T-cell receptor signaling mediated through the NFKB pathway, previously associated with autophagic function in mice and human cells [16,28], may have contributed to the patient's preponderance of naive T-cells ( Table 1). As shown in Figure S4(a), siRNA knockdown of ATG9A in Jurkat T cells resulted in significantly decreased expression of ATG9A mRNA and protein, and accumulation of SQSTM1 ( Figure S4(a,b)). ...
Abbreviations: BCL2: BCL2 apoptosis regulator; BCL10: BCL10 immune signaling adaptor; CARD11: caspase recruitment domain family member 11; CBM: CARD11-BCL10-MALT1; CR2: complement C3d receptor 2; EBNA: Epstein Barr nuclear antigen; EBV: Epstein-Barr virus; FCGR3A; Fc gamma receptor IIIa; GLILD: granulomatous-lymphocytic interstitial lung disease; HV: healthy volunteer; IKBKB/IKB kinase: inhibitor of nuclear factor kappa B kinase subunit beta; IL2RA: interleukin 2 receptor subunit alpha; MALT1: MALT1 paracaspase; MS4A1: membrane spanning 4-domain A1; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH: transcription factor; NCAM1: neural cell adhesion molecule 1; NFKB: nuclear factor kappa B; NIAID: National Institute of Allergy and Infectious Diseases; NK: natural killer; PTPRC: protein tyrosine phosphatase receptor type C; SELL: selectin L; PBMCs: peripheral blood mononuclear cells; TR: T cell receptor; Tregs: regulatory T cells; WT: wild-type.
... Importantly, TXB2 is a stable TxA2-derived metabolite that has been shown to promote the development of psoriatic dermatitis [83]. In addition to the above, the promotion of skin inflammation through NFκB-mediated metabolic pathways was previously suggested, since elevated levels of LTB4 and PGJ2 have been found in the lymphocytes of patients with psoriasis vulgaris [84]. ...
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Psoriasis is the most common autoimmune disease, yet its pathophysiology is not fully understood. It is now believed that psoriasis is caused by the increased activation of immune cells, especially Th1 lymphocytes. However, in psoriasis, immune cells interfere with the metabolism of keratinocytes, leading to their increased activation. Therefore, the pathophysiology of psoriasis is currently associated with the overproduction of ROS, which are involved in the activation of immune cells and keratinocytes as well as the modulation of various signaling pathways within them. Nevertheless, ROS modulate the immune system by also boosting the increasing generation of various lipid mediators, such as products of lipid peroxidation as well as endocannabinoids and prostaglandins. In psoriasis, the excessive generation of ROS and lipid mediators is observed in different immune cells, such as granulocytes, dendritic cells, and lymphocytes. All of the above may be activated by ROS and lipid mediators, which leads to inflammation. Nevertheless, ROS and lipid mediators regulate lymphocyte differentiation in favor of Th1 and may also interact directly with keratinocytes, which is also observed in psoriasis. Thus, the analysis of the influence of oxidative stress and its consequences for metabolic changes, including lipidomic ones, in psoriasis may be of diagnostic and therapeutic importance.
... Source data are provided as a Source data file. See also Supplementary Fig. 8. activation (PDCD1, MME (CD10), TOX2, ZNF683 (Hobit)) 85,86 , (ii) involved in T cell co-stimulation (CD27 (TNFRSF7), CD28, TNFRSF4 (OX40L), TNFRSF9 (4-1BB), TNFRSF18 (GITR)) 53,87-89 , and (iii) in TCR signaling (LAT2, PTPN6) [90][91][92][93] (Fig. 7B). These observations are consistent with an important role for strong TCR signaling in mouse type 1 γδ development 53,57,[94][95][96] . ...
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Developmental thymic waves of innate-like and adaptive-like γδ T cells have been described, but the current understanding of γδ T cell development is mainly limited to mouse models. Here, we combine single cell (sc) RNA gene expression and sc γδ T cell receptor (TCR) sequencing on fetal and pediatric γδ thymocytes in order to understand the ontogeny of human γδ T cells. Mature fetal γδ thymocytes (both the Vγ9Vδ2 and nonVγ9Vδ2 subsets) are committed to either a type 1, a type 3 or a type 2-like effector fate displaying a wave-like pattern depending on gestation age, and are enriched for public CDR3 features upon maturation. Strikingly, these effector modules express different CDR3 sequences and follow distinct developmental trajectories. In contrast, the pediatric thymus generates only a small effector subset that is highly biased towards Vγ9Vδ2 TCR usage and shows a mixed type 1/type 3 effector profile. Thus, our combined dataset of gene expression and detailed TCR information at the single-cell level identifies distinct functional thymic programming of γδ T cell immunity in human. Knowledge about the ontogeny of T cells in the thymus relies heavily on mouse studies because of difficulty to obtain human material. Here the authors perform a single cell analysis of thymocytes from human fetal and paediatric thymic samples to characterise the development of human γδ T cells in the thymus.
... NF-κB is a key family of regulators of inflammation and the immune response that is composed of five members combined into homo or heterodimers. The activation of NF-κB in T cells requires the assembly of the cytoplasmic p62-Bcl10-Malt1-IKK signalosome to ensure that NF-κB can stably enter the nucleus and activate in response to T cell receptors (Paul et al., 2014). More interesting, NF-κB translocates to the nucleus and induces the expression of target genes, including p62. ...
p62 (also known as SQSTM1) is widely used as a predictor of autophagic flux, a process that allows the degradation of harmful and unnecessary components through lysosomes to maintain protein homeostasis in cells. p62 is also a stress-induced scaffold protein that resists oxidative stress. The multiple domains in its structure allow it to be connected with a variety of vital signalling pathways, autophagy and the ubiquitin proteasome system (UPS), allowing p62 to play important roles in cell proliferation, apoptosis and survival. Recent studies have shown that p62 is also directly or indirectly involved in the ageing process. In this review, we summarize in detail the process by which p62 regulates ageing from multiple ageing-related signs with the aim of providing new insight for the study of p62 in ageing.
Background: N6-methyladenosine (m⁶A) is the most abundant modification in eukaryotic mRNA. However, its role in non-small cell lung cancer (NSCLC) has not been completely elucidated. Objective: To explore whether methyltransferase like 3 (METTL3) in cancer associated fibroblasts (CAFs) affects the secretion of IL-18, which drives NSCLC cells to regulate PD-L1-mediated immunosuppression via the nuclear factor kappa B (NF-κB) pathway. Methods: Histopathological features of NSCLC tissues were identified by H&E and IHC staining. The levels of m⁶A writers (METTL3), IL-18 and NF-κB pathway related genes were assessed. The quantity of CD8+ T cells was evaluated by flow cytometry (FCM). The direct binding relationship between METTL3 and IL-18 mRNA was detected by RIP assay and RNA pulldown and confirmed by dual – luciferase reporter assay. The level of RNA m⁶A was detected by RNA m⁶A dot blot and meRIP assays. A heterotopic implantation model of NSCLC was established in NOD-SCID mice for further explore the effect of CAF derived METTL3 on immunosuppression of NSCLC in vivo. Results: Our results illustrated that METTL3 was down-regulated in CAFs, and CAF derived METTL3 alleviated PD-L1-mediated immunosuppression of NSCLC through IL-18. Subsequently, we found that IL-18 was main effector of CAF-derived METTL3 against immunosuppression of NSCLC, and IL-18 accelerated immunosuppression of NSCLC by driving NF-κB pathway. In vivo, METTL3 knockdown-derived CAFs accelerated immunosuppression of NSCLC. Conclusion: IL-18 served as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway.
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Type 1 paracaspases originated in the Ediacaran geological period before the last common ancestor of bilaterians and cnidarians (Planulozoa). Cnidarians have several paralog type 1 paracaspases, type 2 paracaspases, and a homolog of Bcl10. Notably in bilaterians, lineages like nematodes and insects lack Bcl10 whereas other lineages such as vertebrates, hemichordates, annelids and mollusks have a Bcl10 homolog. A survey of invertebrate CARD-coiled-coil (CC) domain homologs of CARMA/CARD9 revealed such homologs only in species with Bcl10, indicating an ancient co-evolution of the entire CARD-CC/Bcl10/MALT1-like paracaspase (CBM) complex. Furthermore, vertebrate-like Syk/Zap70 tyrosine kinase homologs with the ITAM-binding SH2 domain were found in invertebrate organisms with CARD-CC/Bcl10, indicating that this pathway might be the original user of the CBM complex. We also established that the downstream signaling proteins TRAF2 and TRAF6 are functionally conserved in Cnidaria. There also seems to be a correlation where invertebrates with CARD-CC and Bcl10 have type 1 paracaspases which are more similar to the paracaspases found in vertebrates. A proposed evolutionary scenario includes at least two ancestral type 1 paracaspase paralogs in the planulozoan last common ancestor, where at least one paralog usually is dependent on CARD-CC/Bcl10 for its function. Functional analyses of invertebrate type 1 paracaspases and Bcl10 homologs support this scenario and indicate an ancient origin of the CARD-CC/Bcl10/paracaspase signaling complex. Results from cnidarians, nematodes and mice also suggest an ancient neuronal role for the type 1 paracaspases.
T cell signaling is characterized by the diverse enrichment of receptors and signaling intermediates at particular subcellular regions of the T cell at specific times, resulting in complex spatiotemporal signaling distributions. These signaling distributions control the flow of information through the T cell signaling network and thus govern the efficiency of cellular activation. Here we discuss principal cellular structures driving the organization of T cell signaling including membrane topology, vesicular trafficking, cytoskeletal structures and protein complexes.
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Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.
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Antigen stimulation of T cell receptor (TCR) signaling to nuclear factor (NF)-κB is required for T cell proliferation and differentiation of effector cells. The TCR-to-NF-κB pathway is generally viewed as a linear sequence of events in which TCR engagement triggers a cytoplasmic cascade of protein-protein interactions and post-translational modifications, ultimately culminating in the nuclear translocation of NF-κB. However, recent findings suggest a more complex picture in which distinct signalosomes, previously unrecognized proteins, and newly identified regulatory mechanisms play key roles in signal transmission. In this review, we evaluate recent data and suggest areas of future emphasis in the study of this important pathway.
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The adaptor protein Bcl10 is a critically important mediator of T cell receptor (TCR)-to-NF-κB signaling. Bcl10 degradation is a poorly understood biological phenomenon suggested to reduce TCR activation of NF-κB. Here we have shown that TCR engagement triggers the degradation of Bcl10 in primary effector T cells but not in naive T cells. TCR engagement promoted K63 polyubiquitination of Bcl10, causing Bcl10 association with the autophagy adaptor p62. Paradoxically, p62 binding was required for both Bcl10 signaling to NF-κB and gradual degradation of Bcl10 by autophagy. Bcl10 autophagy was highly selective, as shown by the fact that it spared Malt1, a direct Bcl10 binding partner. Blockade of Bcl10 autophagy enhanced TCR activation of NF-κB. Together, these data demonstrate that selective autophagy of Bcl10 is a pathway-intrinsic homeostatic mechanism that modulates TCR signaling to NF-κB in effector T cells. This homeostatic process may protect T cells from adverse consequences of unrestrained NF-κB activation, such as cellular senescence.
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In response to viral infection, RIG-I-like RNA helicases bind to viral RNA and activate the mitochondrial protein MAVS, which in turn activates the transcription factors IRF3 and NF-κB to induce type I interferons. [corrected] We have previously shown that RIG-I binds to unanchored lysine-63 (K63) polyubiquitin chains and that this binding is important for MAVS activation; however, the mechanism underlying MAVS activation is not understood. Here, we show that viral infection induces the formation of very large MAVS aggregates, which potently activate IRF3 in the cytosol. We find that a fraction of recombinant MAVS protein forms fibrils that are capable of activating IRF3. Remarkably, the MAVS fibrils behave like prions and effectively convert endogenous MAVS into functional aggregates. We also show that, in the presence of K63 ubiquitin chains, RIG-I catalyzes the conversion of MAVS on the mitochondrial membrane to prion-like aggregates. These results suggest that a prion-like conformational switch of MAVS activates and propagates the antiviral signaling cascade.
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Activation of I-κB kinases (IKKs) and NF-κB by the human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, is thought to promote cell proliferation and transformation. Paradoxically, expression of Tax in most cells leads to drastic up-regulation of cyclin-dependent kinase inhibitors, p21(CIP1/WAF1) and p27(KIP1), which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21(CIP1/WAF1)-/p27(KIP1)-mediated senescence constitutes a checkpoint against IKK/NF-κB hyper-activation. Senescence induced by Tax in HeLa cells is attenuated by mutations in Tax that reduce IKK/NF-κB activation and prevented by blocking NF-κB using a degradation-resistant mutant of I-κBα despite constitutive IKK activation. Small hairpin RNA-mediated knockdown indicates that RelA induces this senescence program by acting upstream of the anaphase promoting complex and RelB to stabilize p27(KIP1) protein and p21(CIP1/WAF1) mRNA respectively. Finally, we show that down-regulation of NF-κB by the HTLV-1 anti-sense protein, HBZ, delay or prevent the onset of Tax-induced senescence. We propose that the balance between Tax and HBZ expression determines the outcome of HTLV-1 infection. Robust HTLV-1 replication and elevated Tax expression drive IKK/NF-κB hyper-activation and trigger senescence. HBZ, however, modulates Tax-mediated viral replication and NF-κB activation, thus allowing HTLV-1-infected cells to proliferate, persist, and evolve. Finally, inactivation of the senescence checkpoint can facilitate persistent NF-κB activation and leukemogenesis.
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TCR-mediated activation of the transcription factor NF-κB is required for T cell proliferation, survival, and effector differentiation. Although this pathway is the subject of intense study, it is not known whether TCR signaling to NF-κB is digital (switch-like) or analog in nature. Through analysis of the phosphorylation and degradation of IκBα and the nuclear translocation and phosphorylation of the NF-κB subunit RelA, we show that TCR-directed NF-κB activation is digital. Furthermore, digitization occurs well upstream of the IκB kinase complex, as protein kinase C translocation to the immunologic synapse and activation-associated aggregation of Bcl10 and Malt1 also demonstrate both digital behavior and high correlation with RelA nuclear translocation. Thus, similar to the TCR-to-MAPK signaling cascade, analog Ag inputs are converted to digital activation outputs to NF-κB at an early step downstream of TCR ligation.
The CARMA1/Bcl10/MALT1 (CBM) signalosome mediates antigen receptor-induced NF-κB signaling to regulate multiple lymphocyte functions. While CARMA1 and Bcl10 contain caspase recruitment domains (CARDs), MALT1 is a paracaspase with structural similarity to caspases. Here we show that the reconstituted CBM signalosome is a helical filamentous assembly in which substoichiometric CARMA1 nucleates Bcl10 filaments. Bcl10 filament formation is a highly cooperative process whose threshold is sensitized by oligomerized CARMA1 upon receptor activation. In cells, both cotransfected CARMA1/Bcl10 complex and the endogenous CBM signalosome are filamentous morphologically. Combining crystallography, nuclear magnetic resonance, and electron microscopy, we reveal the structure of the Bcl10 CARD filament and the mode of interaction between CARMA1 and Bcl10. Structure-guided mutagenesis confirmed the observed interfaces in Bcl10 filament assembly and MALT1 activation in vitro and NF-κB activation in cells. These data support a paradigm of nucleation-induced signal transduction with threshold response due to cooperativity and signal amplification by polymerization.
NF-κB (nuclear factor kappa B) family transcription factors are master regulators of immune and inflammatory processes in response to both injury and infection. In the latent state, NF-κBs are sequestered in the cytosol by their inhibitor IκB (inhibitor of NF-κB) proteins. Upon stimulations of innate immune receptors such as Toll-like receptors and cytokine receptors such as those in the TNF (tumor necrosis factor) receptor superfamily, a series of membrane proximal events lead to the activation of the IKK (IκB kinase). Phosphorylation of IκBs results in their proteasomal degradation and the release of NF-κB for nuclear translocation and activation of gene transcription. Here, we review the plethora of structural studies in these NF-κB activation pathways, including the TRAF (TNF receptor-associated factor) proteins, IKK, NF-κB, ubiquitin ligases, and deubiquitinating enzymes. Although these structures only provide snapshots of isolated processes, an emerging picture is that these signaling cascades coalesce into large oligomeric signaling complexes, or signalosomes, for signal propagation. Expected final online publication date for the Annual Review of Biophysics Volume 42 is May 06, 2013. Please see for revised estimates.
A role for polyubiquitination in the activation of inhibitor of NF-κB (IκB) kinase (IKK) through a proteasome-independent mechanism was first reported in 1996, but the physiological significance of this finding was not clear until 2000 when TRAF6 was found to be a ubiquitin E3 ligase that catalyzes lysine-63 (K63) polyubiquitination. Since then, several proteins known to regulate IKK have been linked to the ubiquitin pathway. These include the deubiquitination enzymes CYLD and A20 that inhibit IKK, and the ubiquitin binding proteins NEMO and TAB2 which are the regulatory subunits of IKK and TAK1 kinase complexes, respectively. Now accumulating evidence strongly supports a central role of K63 polyubiquitination in IKK activation by multiple immune and inflammatory pathways. Interestingly, recent research suggests that some alternative ubiquitin chains such as linear or K11 ubiquitin chains may also play a role in certain pathways such as the TNF pathway. Here I present a historical narrative of the discovery of the role of ubiquitin in IKK activation, review recent advances in understanding the role and mechanism of ubiquitin-mediated IKK activation, and raise some questions to be resolved in future research.
Allergic airway inflammation is a disease in which T helper 2 (Th2) cells have a critical function. The molecular mechanisms controlling Th2 differentiation and function are of paramount importance in biology and immunology. Recently, a network of PB1-containing adapters and kinases has been shown to be essential in this process owing to its function in regulating cell polarity and the activation of critical transcription factors. Here, we show in vivo data showing that T-cell-specific NBR1-deficient mice show impaired lung inflammation and have defective Th2 differentiation ex vivo with alterations in T-cell polarity and the selective inhibition of Gata3 and nuclear factor of activated T c1 activation. These results establish NBR1 as a novel PB1 adapter in Th2 differentiation and asthma.