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: email@example.com.
© 2006 by The National Academy of Sciences of the USA
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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