The myristoylation of TRIF-related adaptor molecule
is essential for Toll-like receptor 4 signal transduction
Daniel C. Rowe*, Anne F. McGettrick†, Eicke Latz*, Brian G. Monks*, Nicholas J. Gay‡, Masahiro Yamamoto§,
Shizuo Akira§, Luke A. O’Neill†, Katherine A. Fitzgerald*¶, and Douglas T. Golenbock*¶?
*Division of Infectious Disease and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605;†Department
of Biochemistry, Trinity College, Dublin 2, Ireland;‡Department of Biochemistry, University of Cambridge, Cambridge CB2 1YP, United Kingdom; and
§Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
Edited by Tadatsugu Taniguchi, University of Tokyo, Tokyo, Japan, and approved February 21, 2006 (received for review November 18, 2005)
TRIF-related adaptor molecule (TRAM) is the fourth Toll/IL-1 resis-
tance domain-containing adaptor to be described that participates
in Toll-like receptor (TLR) signaling. TRAM functions exclusively in
the TLR4 pathway. Here we show by confocal microscopy that
TRAM is localized in the plasma membrane and the Golgi appara-
tus, where it colocalizes with TLR4. Membrane localization of
TRAM is the result of myristoylation because mutation of a pre-
dicted myristoylation site in TRAM (TRAM-G2A) brought about
dissociation of TRAM from the membrane and its relocation to the
cytosol. Further, TRAM, but not TRAM-G2A, was radiolabeled with
[3H]myristate in vivo. Unlike wild-type TRAM, overexpression of
TRAM-G2A failed to elicit either IFN regulatory factor 3 or NF-?B
signaling. Moreover, TRAM-G2A was unable to reconstitute LPS
responses in bone marrow-derived macrophages from TRAM-
deficient mice. These observations provide clear evidence that the
myristoylation of TRAM targets it to the plasma membrane, where
it is essential for LPS responses through the TLR4 signal transduc-
tion pathway, and suggest a hitherto unappreciated manner in
which LPS responses can be regulated.
innate immunity ? lipopolysaccharide
a complex immune response designed to eliminate invading
pathogens. A key structural motif involved in the signal trans-
duction of TLRs is the Toll?IL-1 resistance (TIR) domain (for
review, see ref. 1). TIR domains can be found in the cytoplasmic
portions of all TLRs and the IL-1 receptor family as well as a
third subgroup of TIR domain-containing adaptor proteins. The
initial events of TLR signal transduction are thought to involve
the recruitment of pertinent adaptor molecules, which in turn
provide a scaffold to enable the recruitment and activation of
additional signaling molecules. To date, four adaptor molecules
have been associated with TLR signaling: myeloid differentia-
tion factor 88 (MyD88) (2, 3); MyD88 adaptor-like (Mal) (4),
also called TIR domain-containing adaptor protein (TIRAP) (5,
6); TIR domain-containing adaptor-inducing IFN-? (TRIF)
(7–9), also called TIR domain-containing adaptor molecule 1
(TICAM-1) (10); and TRIF-related adaptor molecule (TRAM),
or TICAM-2 (11–13) (for review, see ref. 14). Exactly how the
adaptor molecules initiate signaling to these pathways once a
TLR is triggered by its ligand is still unclear. This event is
particularly complicated in the case of TLR4, in which all four
of the above adaptors are required for a complete LPS response.
LPS (endotoxin) is the major constituent of the outer membrane
of Gram-negative bacteria (15, 16). The recognition of LPS by host
phagocytes stimulates the release of inflammatory mediators and
cytokines from a variety of target cells. In conjunction with CD14
and its coreceptor, MD-2, TLR4 binds LPS and serves to elicit a
‘‘danger’’ signal, thus initiating the host immune response (for a
unique among TLRs because it utilizes all four TIR domain-
containing adaptor molecules: MyD88, Mal, TRIF, and TRAM
oll-like receptors (TLRs) recognize microbial products de-
rived from all of the major classes of pathogens and initiate
(18). In recent years, considerable progress has been made delin-
eating the requirement for each of these adaptor molecules in the
TLR4-activated immune response. The recruitment of MyD88 to
proximal TIR domains of activated TLRs allows for the interaction
and activation of the IRAK family members, IRAK1 and IRAK4
(19, 20), and the subsequent activation of TNF receptor-associated
factor 6 [TRAF-6 (21)], a RING finger domain-containing protein
complex (22, 23), which phosphorylates I?B?. This phosphoryla-
tion marks I?B for ubiquitination and degradation by the proteo-
some. NF-?B is then released, translocates to the nucleus, and
also activates MyD88-independent responses, which lead to the
activation of IFN regulatory factor (IRF) 3, the induction of IFN-?
and IFN-inducible genes, and the up-regulation of costimulatory
molecules (18, 24–26). Mal?TIRAP appears also to regulate in-
flammatory cytokine genes, suggesting that Mal may cooperate
with MyD88 to control these responses (4–6, 27).
Using an RNA interference approach or gene targeting, several
reports have suggested that TRAM is uniquely required in the
TLR4 signal transduction pathway and together with TRIF coor-
dinates the activation of IRF3 and the MyD88-independent re-
sponses outlined above (9, 11, 28, 29). The TRAM–TRIF module
also controls MyD88-dependent inflammatory responses, suggest-
ing that TRAM is a master regulator of both arms of the TLR4
signaling pathway. Our earlier studies with overexpression systems
suggested that TRAM functioned upstream of TRIF in TLR4
signaling (11). This conclusion was supported by the observation
as by the observation that TRIF dominant-negative constructs
eliminated the direct activation of the MyD88-independent path-
way by TRAM, but not vice versa (12). In the case of TLR3
signaling, where TRAM is not required, TRIF binds directly to the
TLR3 TIR domain (10).
To characterize further the unique role of TRAM in the initi-
ation of TLR4 signaling, we analyzed the subcellular localization of
TRAM and the consequences of TRAM localization for efficient
signal transduction. Our study shows that TRAM is localized in the
plasma membrane and Golgi apparatus by N-terminal myristoyl-
ation, where it colocalizes with TLR4. In fact, TRAM contains a
putative N-terminal myristoylation site, similar to that found in
mammalian Src kinases. Mutation of this predicted myristoylation
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
ribosomal entry site; IRF, IFN regulatory factor; Mal, MyD88 adaptor-like; MyD88, myeloid
differentiation factor 88; Pam3CSK4, tripalmitoyl-Cys-Ser-(Lys)4; RANTES, regulated on
activation, normal T expressed and secreted; TIR, Toll?IL-1 resistance; TIRAP, TIR adaptor
protein; TLR, Toll-like receptor; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-
containing adaptor-inducing IFN-?; YFP, yellow fluorescent protein.
¶K.A.F. and D.T.G. contributed equally to this work.
?To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
April 18, 2006 ?
vol. 103 ?
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site (TRAM-G2A) redistributes TRAM from the plasma mem-
brane to the cytosol. TRAM-G2A did not signal on overexpression
and was unable to reconstitute LPS responses in macrophages
deficient for wild-type TRAM. These results indicate that myris-
toylation and plasma membrane localization of TRAM are critical
for responses to LPS and also indicate the potential for a hitherto
unappreciated mechanism of regulation of LPS responses.
Subcellular Localization of TRAM. To understand further the role of
By using a C-terminal cyan fluorescent protein (CFP)-tagged
version of TRAM (TRAMCFP), we discovered that in resting cells,
TRAM resides at the plasma membrane and also in the Golgi
apparatus of human embryonic kidney (HEK)293 cells. Similar
localization patterns were observed in stable cell lines expressing
TLR4 tagged with yellow fluorescent protein (TLR4YFP). TLR4
was both surface-expressed as well as localized in the Golgi
apparatus, consistent with previous reports (30). In fact, TRAM
and TLR4 appeared to colocalize on the plasma membrane (Fig.
1A, Right, Overlay). There was no colocalization of TRAM with
TLR9 (Fig. 1A), which resides in the endoplasmic reticulum (31).
In addition to the plasma membrane, TRAM appears to localize in
the Golgi apparatus as seen in Fig. 1A (Bottom). Cell fractionation
localization of MyD88, which resides in the cytosolic fraction
TRAM Contains a Putative Myristoylation Site. This localization
pattern of TRAM in living cells indicates that TRAM is anchored
to the membrane by direct interaction with a membrane-associated
molecule (such as TLR4), by fatty acid modification, or by both. To
examine the possibility that TRAM was anchored by acylation, we
subjected the TRAM sequence to N-myristoyltransferase program
tor.html), which revealed that TRAM contained a putative myris-
Ser and Lys (Fig. 2A). Similar myristoylation sequences have been
identified previously in members of the Src kinase family.
we took a number of complementary approaches. We generated a
mutant form of TRAM in which the glycine at position 2 was
mutated to alanine and examined its localization by confocal
microscopy. As a comparison, we compared the localization of
MyrYFP, a synthetic construct containing an acylation sequence
derived from the mammalian Src kinase, Lyn (32). MyrYFPwas
clearly membrane-localized. In fact, MyrYFPand TRAMCFPcolo-
calized, as indicated by the yellow overlay of the confocal image
(Fig. 2B). Mutation of glycine at position 2 to alanine (TRAM-
G2A) resulted in the relocalization of TRAM from the cell
membrane to the cytosol (Fig. 2B). This change was confirmed by
subcellular fractionation, with TRAM-G2A occurring only in the
cytosolic fraction (data not shown). As a second approach, we
compared the cellular localization of MyD88, a universal TLR
adaptor that resides in the cytosol (Fig. 2B). In contrast to TRAM,
MyD88CFPdid not colocalize with MyrYFP. Next, we performed a
gain-of-function experiment and generated a mutant form of
MyD88, in which the first seven amino acids of MyD88 were
replaced with those from TRAM (T7-MyD88). This experiment
of MyD88. T7-MyD88 was also capable of localizing in the plasma
membrane (Fig. 2C). Together, these results suggest that the
putative N-terminal myristoylation site was sufficient for targeting
TRAM to the plasma membrane.
we monitored TRAM myristoylation in vivo. 293T cells were
Stable TLR4YFP- or TLR9YFP-expressing HEK293 cells were transiently trans-
fected with TRAMCFP. In addition, HEK293 cells were cotransfected with
TRAMCFPand ?-galactosyltransferaseYFP(GolgiYFP). Twenty-four hours after
transfection, cells were visualized by confocal microscopy. (B) Membrane
fractionation was carried out on HEK293 cells stably expressing TLR4YFPand
either TRAMCFPor MyD88CFP. The fractions were resolved on an SDS?10%
polyacrylamide gel before transfer to nitrocellulose and immunoblotting.
TRAM is associated with the cell membrane and Golgi apparatus. (A)
of the consensus sequence for protein N-terminal myristoylation with amino
acids 1–18 of TRAM. Blue characters indicate amino acids that match myris-
transfected with TRAMCFPor TRAM-G2ACFPand visualized by confocal micros-
copy. (C) MyrYFPstable HEK293 cells were transiently transfected with
MyD88CFPor T7-MyD88CFPand visualized by confocal microscopy 24 h later.
TRAM contains a putative myristoylation sequence. (A) Comparison
www.pnas.org?cgi?doi?10.1073?pnas.0510041103Rowe et al.
MyD88, or T7-MyD88. Cells were metabolically labeled with
[3H]myristate. Fyn, an adaptor molecule involved in T cell receptor
signal transduction, was used as a positive control for in vivo
myristoylation. Likewise, Fyn-G2A served as a negative control for
the incorporation of [3H]myristate. The indicated proteins were
of [3H]myristate by autoradiography (Fig. 3). TRAM, but not
TRAM-G2A, was clearly myristoylated in vivo. Furthermore, nei-
ther Mal nor MyD88 was labeled with [3H]myristate. In contrast,
T7-MyD88 efficiently incorporated [3H]myristate. These results
provide clear evidence that TRAM is indeed a myristoylated
protein. Even though TRAM and Mal have similar molecular
weights, consisting of 232 and 235 aa, respectively, these adaptors
resolve differently on SDS?PAGE (Fig. 3 Lower), possibly because
of different posttranslational modifications.
TRAM-G2A Does Not Signal on Overexpression. As reported previ-
ously, transient expression of TRAM induces both NF-?B- and
BB loop of TRAM at position 117 (TRAM-C117H), a mutation
equivalent to the C3H?HeJ mutation in the TLR4 TIR domain BB
loop, abolished the ability of TRAM to signal (11). To determine
whether TRAM-G2A was compromised in its ability to signal on
NF-?B- or IRF3-dependent reporter gene activation in HEK293
cells. Wild-type TRAM, but neither the C117H nor the G2A
mutant, could drive NF-?B, RANTES, or ISRE (interferon-
stimulated response element) reporters (Fig. 4 A–C). Moreover,
4C). Similar results were obtained when LPS-induced NF-?B
activation was examined in cells transfected with TRAM or the
TRAM mutants (Fig. 4D). As anticipated, TRAM-G2A had no
effect on lipopeptide-induced NF-?B activation in TLR2-
expressing HEK293 cells (Fig. 4E). We also examined the ability of
example, transient expression of MyD88 induced NF-?B reporter
gene expression, as was also true in the case of TRAM and TRIF,
although to a lesser extent. TRAM-G2A had no effect on either
MyD88- or TRIF-induced NF-?B activation, but it inhibited the
activation of NF-?B induced by overexpressing wild-type TRAM
(Fig. 4F). These results suggest that TRAM-G2A cannot signal
efficiently and in fact can act as a dominant-negative interfering
mutant. Together, these results suggest that the membrane local-
ization of TRAM is critical for its ability to initiate efficient
Myristoylation of TRAM Is Critical for LPS Responses in Macrophages.
To investigate whether membrane localization of TRAM is critical
for LPS responses in vivo, we reconstituted bone marrow-derived
macrophages from TRAM-deficient mice. To this end, we gener-
ated retroviruses expressing wild-type TRAM, TRAM-G2A, or
TRAM-C117H in tandem with an internal ribosomal entry site
(IRES)-encoded GFP. This construct was designed to allow the
expression of a bicistronic mRNA that would give rise to the
translation of TRAM at a 1:1 molar ratio with GFP. Transduced
bone marrow-derived macrophages from TRAM-deficient mice
were sorted by flow cytometry for GFP fluorescence. As seen in
Fig. 5A, the levels of GFP fluorescence, and hence TRAM recon-
stitution, were comparable in the cells transduced with the wild-
GFP-sorted cells with Western blotting, presumably because of the
low-level expression of TRAM from the construct in these cells
of the three proteins in transfected HEK293 cells, we found no
the levels of the wild type and TRAM mutants are similar.
IRES vector that encoded only for GFP were also sorted for
productive infection (data not shown). The CC-chemokine RAN-
TES is a downstream target gene of the MyD88-independent
pathway after LPS stimulation (11). In addition, IL-6 production in
(13). We therefore examined IL-6 and RANTES production after
LPS stimulation in both wild-type and TRAM-deficient macro-
phages that had been transduced with either the control vector-
expressing retrovirus or the TRAM-expressing retroviruses. As
seen in Fig. 5B, GFP-positive, wild-type, empty vector-expressing
macrophages produced IL-6 and RANTES after LPS stimulation,
whereas GFP-positive, TRAM-deficient, empty vector-expressing
macrophages failed to induce either IL-6 or RANTES under
identical experimental conditions. Remarkably, TRAM-deficient
macrophages transduced with a virus encoding the wild-type
TRAM restored responsiveness to LPS challenge completely. As
predicted, TRAM-C117H was unable to restore LPS responses,
consistent with our earlier in vitro studies. Importantly, TRAM-
G2A was also incapable of restoring LPS responses (Fig. 5B). Cells
transduced with the TRAM-G2A mutant were unable to produce
provide clear evidence that the myristoylation and subsequent
membrane localization of TRAM are critical for responses to
A diverse range of viral and cellular proteins are known to be
modified covalently by lipophilic moieties, including protein
kinases, guanine nucleotide-binding proteins, transmembrane
receptors, and viral structural proteins. The attachment of lipid
groups to these molecules influences protein–protein interac-
tions, membrane-binding affinity, and cellular signal transduc-
tion. Here, we implicate fatty acid modification as a critical event
in TLR4 signaling. We show that the TIR domain-containing
membrane and in the Golgi apparatus as a result of myristoyl-
fection, cells were incubated in medium containing 250 ?Ci of [3H]myristate per
well for 4 h. Cells were lysed and immunoprecipitated with anti-GFP polyclonal
raphy. Whole-cell lysates were immunoblotted with anti-GFP mAb.
TRAM is myristoylated in vivo. 293T cells were transiently transfected
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April 18, 2006 ?
vol. 103 ?
no. 16 ?
ation. Previous studies have demonstrated that LPS traffics
rapidly to and from the Golgi apparatus with TLR4, MD2, and
CD14; however, these events are distinct from the initiation of
signal transduction (30). Therefore, the localization of TRAM
with TLR4 in an intact Golgi network is also unlikely to be
required for signaling.
Because TRAM does not contain a signal peptide sequence,
its presence on the membrane could be the result of protein–
protein interactions, prenylation, or fatty acid modification.
TRAM does not contain a predicted prenyl modification site
because it does not contain a CAAX box at or near its C
terminus. There are two distinct types of fatty acid modification:
myristoyl and palmitoyl (33–35). In this study, we show that
TRAM contains an N-terminal myristoylation sequence. For
myristoylation to occur, the initiating methionine is usually
removed by methionine aminopeptidase during translation, and
requirement for glycine at the N terminus is absolute; no other
amino acid can substitute. Such proteins are labeled with my-
ristate on these N-terminal glycines in an irreversible, cotrans-
lational manner by N-myristoyltransferase. This type of covalent
is central to their function (33). A well characterized myristoy-
lated protein is the Src kinase pp60v-src from Rous sarcoma
virus. A myristoylation mutant of this Src kinase (G2A) does not
bind to the membrane and is incapable of mediating cellular
transformation (33). Myristoylation is also required for mem-
brane association and virion formation by Gag polyproteins of
mammalian C- and D-type retroviruses. Nonmyristoylated mu-
tants of murine leukemia C virus Pr65gag and HIV-1 Pr55gag
are predominantly cytosolic, and infectious viral particles are not
ing of N-myristoylated proteins, myristoylation alone is not suffi-
is required for efficient membrane binding. The second signal is
most often either palmitoylation or the presence of a polybasic
cluster of amino acids. The latter signal is the mechanism used by
the Src kinase pp60v-src for membrane anchoring (33, 35). A basic
amino acid cluster in Src kinase, downstream from the glycine at
position 2, forms electrostatic interactions with acidic phospholipid
headgroups in the membrane. It is unclear at present what is the
second signal that tethers TRAM to the membrane. TRAM has a
reporters as indicated. FLAG-tagged versions of TRAM, TRAM-G2A, and TRAM-C117H or empty vector were cotransfected as indicated. (C–E) TLR2- or
TLR4?MD2-expressing HEK293 cells were transfected as indicated (EV, empty vector) and 24 h later were stimulated with 2 ?g?ml LPS or 2 ?g?ml tripalmitoyl-
Cys-Ser-(Lys)4(Pam3CSK4) for 6 h. (F) HEK293 cells were transfected with the NF-?B-luciferase reporter and cotransfected with wild-type TRAM, TRIF, or MyD88
Twenty-four hours after transfection, cell lysates were generated, and luciferase activity was assayed. All of the data are representative of three independent
TRAM-G2A fails to activate the RANTES promoter and NF-?B. (A and B) HEK293 cells were transiently transfected with the RANTES and NF-?B-luciferase
www.pnas.org?cgi?doi?10.1073?pnas.0510041103Rowe et al.
serve as a polybasic cluster. A combination of both myristate and
basic motifs in TRAM would allow the hydrophobic and electro-
static forces to synergize and facilitate membrane binding (37).
TRAM also has a number of cysteine residues, however, which
could potentially become palmitoylated. A third possibility for
membrane tethering might involve association with membrane-
anchored molecules, which, in the case of TRAM, could be TLR4.
transfected into HEK293 cells, which ordinarily lack expression of
TLR4, TRAM localization in the cell surface and Golgi was
nevertheless observed (data not shown). Of course, other cell
surface TLRs are theoretically capable of serving this function, but
cells from TRAM-deficient animals appear to have normal re-
sponses to a variety of TLR ligands, including those for TLR2,
TLR5, TLR7, and TLR9 (data not shown), suggesting that TRAM
binds the cytoplasmic domain of TLR4 selectively.
Proper localization of signaling molecules to specific cellular
membranes is critical for their function. It is tempting to speculate
microdomains, such as lipid rafts or caveolae, enhances particular
protein–protein interactions important for subsequent signal trans-
duction. LPS has been reported to result in the redistribution of
redistribution. Similarly, in the case of TRAM, colocalization with
TLR4 in lipid rafts may be critical for signaling.
A key question, which remains to be answered, is why TRAM
must be membrane-localized to allow cells to signal through TLR4.
One possible explanation is that membrane-localized TRAM must
leave the membrane and dissociate from TLR4 to interact with
pathways. Indeed, preliminary experiments suggest that TRAM
leaves the membrane after LPS stimulation. In this scenario, the
dissociation of TRAM from the membrane could be a regulated
event (e.g., demyristoylation, depalmitoylation, or phosphoryla-
tion). N-myristoylation is usually a permanent modification; how-
ever, there is evidence in particular situations (e.g., brain synapto-
somes) that a demyristoylase activity modifies myristoylated
alanine-rich C-kinase substrate (MARCKS), promoting its release
form the membrane (39). Depalmitoylation is a more common
mechanism of protein translocation because the thioester linkage
between palmitate and the polypeptide is quite labile. Another
interactions between polybasic residues and phospholipid head-
groups could be destabilized by phosphorylation events adjacent to
polybasic clusters. Ligand-induced phosphorylation of TRAM
would elicit its dissociation from the membrane. Recent evidence
from O’Neill and colleagues suggests that TRAM is indeed phos-
often phosphorylated by PKC near their N termini, leading to their
dissociation from the membrane (40–43). Phosphorylation of
TRAM by PKC? is critical for LPS signaling (L.A.O., personal
communication), consistent with the possibility that TRAM phos-
phorylation could facilitate it to leave the membrane after LPS
In conclusion, the data described in this work show that TRAM
on it membrane localization as a prerequisite for LPS signaling.
Defining the functional significance of membrane-localized
TRAM in LPS signaling remains to be elucidated completely, but
the data strongly suggest that the acylation of TRAM is one means
by which cells regulate their responses to bacterial endotoxin.
Materials and Methods
Reagents. Unless otherwise stated, all reagents were purchased
from Sigma. Escherichia coli O111:B4 LPS was subjected to a
second phenol extraction to remove contaminating lipopeptides
(44). Lipopeptide Pam3CSK4 was purchased from EMC Micro-
collections (Tuebingen, Germany).
Plasmids. Most of the constructs described in this work have been
described elsewhere. These include: pCDNA3-TRAMCFP, pEF-
BOS-TRAMFLAG, pEF-BOS-TRAM-C117HFLAG, pEF-BOS-
TRIFFLAG(11); pCDNA3-MyD88CFP(31); MyrYFP(32); ?-galac-
tosyltransferaseYFP(GolgiYFP) (30); NF-?B-luciferase, RANTES-
luciferase, ISRE-luciferase, and Renilla-luciferase (11); and the
retroviral vector pMSCV2.2-ISRE-GFP (45). pCDNA3-TRAM-
G2A and pEF-BOS-TRAM-G2A were generated by using a site-
directed mutagenesis kit (Stratagene). The plasmid pCDNA3-Fyn
was subcloned using PCR cDNA obtained from E. Kurt-Jones
(University of Massachusetts, Worcester); pCDNA3-Fyn-G2A was
generated by site-directed mutagenesis. pMSCV2.2-IRES-GFP-
murine TRAM, murine TRAM-G2A, and murine TRAM-C117H
were subcloned from pEF-BOS vectors expressing murine con-
structs. pCDNA3-T7-MyD88CFPwas generated by PCR. The pack-
aging vector for the retroviruses was the proviral clone of Moloney
murine sarcoma virus ?Ecu from O. Witte (University of Califor-
nia, Los Angeles).
conditioned supernatant from L929 cells as a source of macrophage colony-stimulating factor. On day 2, cells were transduced with viral supernatant derived
from empty vector, WT TRAM, TRAM-G2A, or TRAM-C117H viral constructs. On day 10, cells were sorted for GFP and seeded in 96-well plates at 40,000 cells per
well. (A) Transduced and GFP-sorted cells were examined for GFP fluorescence by FACS analysis. (B) GFP-positive cells were stimulated with LPS as indicated, and
cell supernatants were assayed for IL-6 and RANTES by ELISA.
TRAM myristoylation is critical for optimal LPS responses. Bone marrow-derived macrophages from C57BL?6 or TRAM?/?mice were cultured in 20%
Rowe et al.PNAS ?
April 18, 2006 ?
vol. 103 ?
no. 16 ?
Cell Lines. HEK293 cells stably expressing TLR2YFP, TLR4YFP,
and TLR9YFPwere as described previously (30, 31).
Confocal Microscopy. HEK293 cells stably expressing TLR4YFPand
TLR9YFPor MyrYFPwere transfected with TRAMCFP, TRAM-
G2ACFP, MyD88CFP, or T7-MyD88CFPwhere indicated. In addi-
tion, HEK293 cells were cotransfected with TRAMCFPand
?-galactosyltransferaseYFP(GolgiYFP). Twenty-four hours after
TCS SP2 AOBS microscope.
Subcellular Fractionation. HEK293 cells stably expressing TLR4YFP
and TRAMCFP, TRAM-G2ACFP, or MyD88CFPwere seeded in
10-cm dishes at a density of 1 ? 106cells per dish. After 24 h, the
medium was removed, and the cells were washed in PBS and then
scraped into fractionation buffer (20 mM Tris?HCl, pH 7.5?10 mM
MgCl2?1 mM EDTA?250 ?M sucrose?200 ?M PMSF). The sam-
ples were subjected to 20 strokes of a Dounce homogenizer and
removed to a fresh tube, and the pellet (membrane fraction) was
resuspended in 50 ?l of SDS sample buffer [50 mM Tris?Cl, pH
6.8?10% (vol?vol) glycerol?2% (wt?vol) SDS?0.1% (wt?vol) bro-
mophenol blue?5% (vol?vol) 2-mercaptoethanol]. The fractions
were run on an SDS?10% polyacrylamide gel, transferred onto
nitrocellulose, and blotted with the appropriate antibody.
Luciferase Reporter Assay. Cells were seeded into 96-well plates at
a density of 40,000 cells per well and transfected 24 h later with 80
ng of the indicated luciferase reporter genes and the indicated
amounts of the TRAM constructs, MyD88 or TRIF, by using 0.8
?l of GeneJuice (EMD Biosciences, San Diego) per well. The
thymidine kinase Renilla-luciferase reporter was also cotransfected
(40 ng) so that the data could be normalized for transfection
efficiency. Cells were either left untreated or treated with 1 ?g?ml
LPS or 2 ?g?ml Pam3CSK4 as indicated for 6 h. Cell lysates were
Dual Luciferase Assay System (Promega). Data are expressed as
the mean relative stimulation ? SD.
Radiolabeling. 293T cells were transfected by using GeneJuice with
the CFP-tagged constructs as indicated. Eighteen hours after
transfection, cells were incubated in medium containing 250 ?Ci (1
Ci ? 37 GBq) of [3H]myristate (PerkinElmer) for 4 h. Cells were
then lysed in 0.5 ml of lysis buffer [20 mM Tris?HCl?2 mM
EDTA?137 mM NaCl?0.5% Triton X-100?10% (vol?vol) glycerol,
with protease inhibitors]. Polyclonal anti-GFP (Molecular Probes)
was incubated with the cell lysates in protein A–Sepharose for 2 h.
The immune complexes were precipitated and subjected to SDS?
4–15% PAGE. The gel was then dried and developed by using
Amplify (Amersham Pharmacia) according to the manufacturer’s
Reconstitution of Bone Marrow-Derived Macrophages.Theretroviral
vector pMSCV expressing TRAM, TRAM-G2A, TRAM-C117H,
and GFP from the IRES element was used to generate high-titer,
transient cotransfection of 293T cells (45). After 24–72 h, cell
supernatants were harvested, filtered, and used to transduce target
cells. Bone marrow-derived macrophages were cultured from
C57BL?6 mice or age- and sex-matched TRAM?/?mice for 8–10
days. Conditioned supernatant from L929 cells comprised 20% of
the total volume as a source of macrophage colony-stimulating
factor. Cells were transduced with virus encoding the indicated
versions of TRAM or empty vector on day 2 while cells were
actively dividing. On day 10, cells were sorted for GFP and seeded
into 96-well plates at a density of 35,000 cells per well. Cells were
stimulated with LPS overnight after being allowed to recover from
presence of IL-6 and RANTES by ELISA (R & D Systems).
We thank K. Halmen for helpful discussions and A. Cerny for animal
husbandry and care.
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