Download Epstein-Barr virus (EBV)

Epstein-Barr virus (EBV) and
pathogenesis of Nasopharyngeal
carcinoma (NPC)
Epstein-Barr virus (EBV)
• A human herpesvirus , -subfamily of herpesviridae
1958- first description of Burkitt's lymphoma
1964- Epstein and Barr isolate continuous cell lines from
Burkitt lymphoma cells
1968- EBV demonstrated as the etiological agent of
infectious mononucleosis
1969- EBV shown to immortalize lymphocytes in culture
•172 kb viral genome
Epstein-Barr virus (EBV)
•Latent Infection
latency I : Epstein-Barr virus nuclear antigens (EBNA1)
latency II: EBNA1 and LMP1, LMP2
latency III: EBNA1, 2, 3, LP and LMP1, 2
•Lytic Activation
•Associated several malignancies
Burkitt's lymphoma, Hodgkin’s Disease, post-transplant
lymphoproliferative disorders, T cell lymphoma
Gastric cancer, Nasopharyngeal carcinoma (NPC)
Nasopharyngeal carcinoma (NPC)
•Occurs world wide, rare in western countries, high in
south-east Asia, northern Africa, Greenland, and Alaska
•Two main histological types of NPC by WHO classification:
(I) Squamous cell carcinoma --- desmoplastic, without lymphoid
(II) Non-keratinizing carcinoma --- associated with lymphoid
stroma
stroma
differentiated non-keratinizing/undifferentiated carcinoma
(Nicholls, 1996)
•Undifferentiated carcinoma associated with prominent lymphoid
stroma ---- the most common form of NPC (~80% of cases)
Association of EBV with NPC
•Initially suggested by serological studies
•Subsequently substantiated by the detection of viral genomes
and gene products in the NPC tumor cells
(Wolf et al., Nature, 1973; Klein et al., PNAS USA, 1974)
•Which types of NPC are associated with EBV?
The viral genomes is of monoclonal origin in undifferentiated NPC, so
virus infection must have occurred before clonal expansion of tumor cell
By contract, conflicting results have been reported about
association of EBV with squamous cell NPC
Squamous cell NPCs from areas where NPC is endemic may be mostly EBV-associated
whilst morphologically identical cases from areas where NPC occurs as a sporadic tumor
are commonly not EBV-related
•EBV genomes in epithelial cells of in-situ NPC lesions
The complement receptors (CR2/CD21)
are the EBV receptors on B cells
The regulation of B cell activation by complement receptor
Binding of complement proteins (C3d) to antigens directly or via attached antibodies leads to
coligation of antigen receptors (Ig) and complement receptors (CR2, CD21) on antigen-specific
B cells. This enhances the Ig-mediated signal and the subsequent B cell response.
The EBV viral envelope protein gp350/200, which shares a region
of sequence similarity with C3d, can interact with CR2/CD21
The complement receptors (CR2/CD21)
on epithelial cells
JOURNAL OF VIROLOGY, 73, 2115–2125 (1999)
CD21
RT-PCR
for EBNA1
For EBER1
Northern
blotting
1969- EBV shown to immortalize lymphocytes in culture
Latent Infection of EBV --latency I : Epstein-Barr virus nuclear antigens (EBNA1)
latency II: EBNA1 and LMP1, LMP2
latency III: EBNA1, 2, 3, LP and LMP1, 2
What gene product(s) of EBV is (are)
responsible for cell transformation?
Latent membrane protein (LMP1)
• latent membrane proteins: LMP1 and 2A, 2B
LMP1--- present in > 60% of all NPC and Hodgkin's lymphoma
--- can transform rodent fibroblasts, which grow as tumors
in nude mice Wang et al., (1985) Cell; Sugden, B. (1988) Oncogene
--- induce B lyphoma in transgenic mice
Raab-Traub, N. (1998) PNAS USA
BLMP1
----B95-8 strain
NLMP1
-- NPC strain, have 10 amino acid deletion
essential in tumorigenesis
Chen et al., (1992) Oncogene; Lee et al., (1996) Oncogene
LMP1 structure
Hammerschmidt W.
• 62 kDa polypeptide:
25 amino acid at N-terminus
6 transmembrane domain
(1999) EMBO J.
200 amino acid at C-terminus
• CTAR: C-terminal activation region
• C-terminal required for the signal transduction
LMP1 affects multiple signaling events
in cells
CTAR: C-terminal activation region
Hammerschmidt W.
(1999) EMBO J.
LMP1 acts like a permanently activated receptor
Morphological transformation of human keratinocytes
expressing the LMP1 gene of Epstein-Barr virus
Klein et al. (1990) Nature
RHEK-1: non-tumorigenic
keratinocyte cell line
C
LMP1
W.B: PKK-1
(cytokeratin 19, 18, 8)
C
LMP1
W.B: PKK-2
(cytokeratin 19, 17, 16, 7)
LMP1 C
W.B: AE1/AE3
(cytokeratin 40-66K)
LMP1 transfected RHEK-1 cells shows a down-regulation
of cytokeratin expression
Control cell
Transfected with LMP1
Change in morphology and growth in spindle-shaped bundles
LMP expression may be an important causal factor in the
development of NPC
Induction of bcl-2 expression by Epstein-Barr virus
latent membrane protein 1 protects infected B cell
from programmed cell death
Henderson et al. (1991) Cell
W.B with LMP1 mAb
EBV-negative BJAB lymphoma cell line
apoptosis assay
LMP1 mediates the protection from apoptosis by upregulating expression of bcl-2
The Epstein-Barr virus LMP induces A20 zinc finger
protein expression by activating nuclear factor-B
Laherty et al. (1992) J. Biol. Chem.
Involvement of two NF-kB binding elements in tumor
necrosis factor alpha-, CD40-, and Epstein-Barr
virus latent membrane protein 1-mediated induction
of the cellular inhibitor of apoptosis protein 2 gene
Hong et al. (2000) J. Biol. Chem.
A20 and cIAP2 are antiapototic proteins
Cellular proteins interacted with LMP1
The Epstein-Barr virus transforming protein
LMP-1 engages signaling proteins for the tumor
necrosis factor receptor family
Mosialos et al. Cell (1995)
The cytoplasmic C-terminus of Epstein-Barr virus (EBV) latent infection
membrane protein 1 (LMP1) is essential for B lymphocyte growth
transformation and is now shown to interact with a novel human protein
(LMP1-associated protein 1 [LAP1]). LAP1 is homologous to a murine protein,
tumor necrosis factor receptor-associated factor 2 (TRAF2), implicated in
growth signaling from the p80 TNFR. A second novel protein (EBI6), induced
by EBV infection, is the human homolog of a second murine TNFR-associated
protein (TRAF1). LMP1 expression causes LAP1 and EBI6 to localize to LMP1
clusters in lymphoblast plasma membranes, and LMP1 coimmunoprecipitates
with these proteins. LAP1 binds to the p80 TNFR, CD40, and the
lymphotoxin-beta receptor, while EBI6 associates with the p80 TNFR. The
interaction of LMP1 with these TNFR family-associated proteins is further
evidence for their role in signaling and links LMP1-mediated transformation
to signal transduction from the TNFR family.
(identified by yeast two-hybrid system)
TRAF2
(TNFR-associated factor 2) --An important signal transducer
for TNFR to activate
NF-kB pathway
The Epstein–Barr virus oncogene product latent
membrane protein 1 engages the tumor necrosis factor
receptor-associated death domain protein to mediate B
lymphocyte growth transformation and activate NF-kB
IZUMI AND KIEFF* PNAS USA (1997)
The Epstein–Barr virus latent membrane protein 1 (LMP1) is essential for the transformation
of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model
thatLMP1is a constitutively activated receptor that transduces signals for transformation
through its carboxylterminal cytoplasmic tail. One transformation effector site (TES1),
located within the membrane proximal 45 residues of the cytoplasmic tail, constitutively
engages tumor necrosis factor receptor-associated factors. Signals from TES1 are
sufficient to drive initial proliferation of infected resting B lymphocytes, but most
lymphoblastoid cells infected with a virus that does not express the 155 residues beyond
TES1 fail to grow as long-term cell lines. We now find that mutating two tyrosines to an
isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause
lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2.
A yeast two-hybrid screen identified TES2 interacting proteins, including the tumor necrosis
factor receptor-associated death domain protein (TRADD). TRADD was the only protein that
interacte with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associated
with wild-type LMP1 but not with isoleucinemutated LMP1 in mammalian cells, and TRADD
constitutively associated with LMP1 in EBV-transformed cells. In transfection assays,
TRADD and TES2 synergistically mediated high-level NF-kB activation. These results
indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell
outgrowth. High-level NF-kB activation also appears to be a critical component of long-term
outgrowth.
TRADD
(TNFR-associated death
domain protein) --An important signal transducer
for TNFR to activate
NF-kB and JNK pathway
The Epstein–Barr virus LMP1 amino acid sequence that
engages tumor necrosis factor receptor associated factors
is critical for primary B lymphocyte growth transformation
(NF-kB/TRAF)
IZUMI et al., (1997) PNAS USA
Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for transforming
primary B lymphocytes into lymphoblastoid cell lines. EBV recombinants with LMP1 genes
truncated after the proximal 45 codons of the LMP1 carboxyl terminus are adequate for
transformation. The proximal 45 residues include a domain that engages the tumor necrosis
factor receptor associated factors (TRAFs). We investigated the importance of the TRAF
binding domain by assaying the transforming ability of recombinant EBV genomes with a
deletion of LMP1 codons 185–211. This mutation eliminates TRAF association in yeast and in
lymphoblasts but does not affect LMP1 stability or localization. Specifically mutated
recombinant EBV genomes were generated by transfecting P3HR-1 cells with overlapping EBV
cosmids. Infection of primary B lymphocytes resulted in cell lines that were coinfected with an
LMP1D185–211 EBV recombinant and P3HR-1 EBV, which has a wild-type LMP1 gene but is
transformation defective due to another deletion. Despite the equimolar mixture of wild-type
and mutated LMP1 genes in virus preparations from five coinfected cell lines, only the wildtype LMP1 gene was found in 412 cell lines obtained after transformation of primary B
lymphocytes. No transformed cell line had only the LMP1D185–211 gene. An EBV recombinant
with a Flag-tagged LMP1 gene passaged in parallel segregated from the coinfecting P3HR-1.
These data indicate that the LMP1 TRAF binding domain is critical for primary B lymphocyte
growth transformation.
LMP1 activates transcription factor NF-B through a
pathway that includes the NF- B-inducing kinase and
the I B kinases IKK  and IKK 
Mosialos et al. PNAS USA (1998)
NIK

IKK/

NF-B
Dominant
negative mutant
NIK
IKK
IKK
Epstein-Barr virus latent membrane protein-1 triggers
AP-1 activity via the c-Jun N-terminal kinase cascade
Hammerschmidt et al. (1997) EMBO J
JNK1 is induced by LMP-1
AP-1 induction by LMP-1
Dominant-negative mutants of SEK1 block
LMP-1-induced activities of JNK1 and AP-1
LMP-1 induces AP-1-dependent transcription via the
SEK1  JNK1  c-Jun N-terminus cascade
deletion of CTAR2 completely abolished
LMP-1’s ability to induce AP-1
The LMP-1 C-terminal CTAR2 region is essential for
LMP-1's effects on AP-1
LMP1 structure and signal transduction
Eliopoulos and Young, Seminars Cancer Biol. (2001)
The oncogenic Epstein–Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has
structural features and functions reminiscent of a constitutively active TNF family
receptor. LMP1 aggregates at the plasma membrane and initiates the activation of signalling
pathways, such as NF-kB, the mitogen-activated protein kinases JNK and p38, the small
GTPase Cdc42 and the JAK/STAT cascade. The constitutive engagement of these signals
and the characteristic molecular interactions that regulate them provide the basis for the
molecular explanation of the transforming properties of this key EBV protein.
Rel/NF-kappaB/IkappaB proteins and cancer
Gilmore et al., Oncogene (1996)
The Epstein-Barr virus oncogene product, latent membrane
protein 1, induces the downregulation of E-cadherin gene
expression via activation of DNA methyltransferases
Y.S. Chang PNAS USA. (2002)
LMP1 Represses E-cadherin
Gene Expression
CTAR-2 is essential for LMP1-Mediated
Down-Regulation of the E-cadherin Gene
Two tumor supressor genes are hypermethylated
in NPC
High Frequency of Promoter Hypermethylation
of RASSF1A in Nasopharyngeal Carcinoma
Lo et al., Cancer Res (2001)
Hypermethylation of the p16 gene in
nasopharyngeal carcinoma
Lo et al., Cancer Res (1996)
Negative Regulation of Epstein-Barr Virus
Latent Membrane Protein 1-mediated functions
by Bone Morphogenetic Protein Receptoe IABinding Protein, BRAM1
Chung et al., J. Biol. Chem., October 18, 2002
yeast two-hybrid assay
LMP1 C-T
BRAM1
Bait: LMP1 cytoplasmic domain
cDNA library: BALB/c3T3 cells
Full-length BRAM1 (WT) and its deletion mutants
Identification of the interacting domains of
BRAM1 and LMP1
Complete C-terminal
CTAR2 and intervening
CTAR1
CTAR1 and intervening
C.T excluding YYD
The C-terminal 116-amino acid sequence of BRAM1
specifically interacts with CTAR2 of LMP1
BRAM1---Bone morphogenetic protein receptor
IA-binding protein
•TGF- superfamily:
Ligand family
TGF- 
BMP
Nodal
•Biological fuction:
Receptor family
TGF- Receptor
BMP Receptor
Regulate pattern formation (during development)
Influence cell behaviors: proliferation; cell death
differentiation
extracellular matrix production
Tissue repair
Immune regulation
Cellular localization of LMP1 and BRAM1
NPC-TW02 cells
 transfected with GFP-LMP1
and Flag-BRAM1
 anti-Flag
immunofluorescence staining
 observed with
luciferase microscope
conventional fluorescence
microscope
confocal microscope
in XY scanning
confocal microscope
in XZ scanning
BRAM1 and LMP1 are co-localized in cells
293 cells
LMP1
 transfected with B/N-LMP1
BRAM1
 in vitro kinase assay
BALB/c3T3 cells
 transfected with 4xB-Luc
B/N-LMP1, Flag-BRAM1
 luciferase values
 WB using anti-LMP1 (S12)
and anti-Flag (M2 )
BRAM1
?
BRAM1
Induction of inducible nitric oxide synthase by Epstein-Barr
virus B95-8-derived LMP1 in Balb/3T3 cells promotes stressinduced cell death and impairs LMP1-mediated transformation
Yu et al., Oncogene in press (2002)
Balb/3T3 cells
BLMP1(+++)
NLMP1 (+)
iNOS induction
iNOS inhibitor
NO production
More sensitive to stress (UV/HS)
200
150
100
50
0
0 4
8 12 16 20 24 28 32 36
Time (hr)
Cell Viability (%)
Nitrite (nmol/ml medium)
250
1.2
HS
1
0.8
0.6
Reduced anchorage-independent growth
0.4
D-11
E-9
0.2
Parental 3T3
3T3neo
0
01
22
Reduced tumorigenecity in nude mice (?)
43
Time (hr)
46
Effects of iNOS induction on the tumorigenecity of LMP1-expressing
Balb/3T3 cells in nude mice model
440
0
Neo
Neo+AG
E9
E9+AG
D11
D11+AG
D13
D13+AG
6000
6000
5000
5000
4000
4000
AG
--AG
數
列
1
30
30
+AG
+AG
數
列2
220
0
110
0
3000
3000
0
0
2000
2000
D11
D11+
AG
D13
D13+
AG
35
35
40
40
45
45
Days post
inoculation (day)
Time Post
CellcellInjection
50
LMP1
E9
-AG
+AG
D11
D13
Tumor volume (mm3)
iNOS
500
500
400
400
300
300
200
200
100
100
00
-
D11
+AG(200) +AG(400)
D11+AG200 D11+AG400
D13+AG
30
30
D13
25
25
D11+AG
20
20
D11
15
15
E9+AG
0
E9
0
Ctrl
E9
E9+A
G
Cells from tumor mass
1000
1000
D11
Tumor volume (mm )
3)
Tumor Volume (mm
3
Nitrite (nmol/ml)
7000
7000
Molecules That Interfere Interactions between
LMP1/TRADD (or TRAF2) May Represent Lead
Compounds for Potential Anti-EBV Drug Development
TRAF2
TRADD
Multiple signaling cascades (NF-B, AP-1)
Pleiotropic effects on cell functions
(A20, cIAPs, IL-6, IL-8, CD40, ICAM1, transformation)
ELISA for Measuring the Interactions between
EBV LMP1 and Its Downstream Signaling
Molecules: TRAF2 and TRADD
Recombinant TRAF2 or TRADD:
Recombinant LMP1 or peptides derived from LMP1 sequence :
Coating recombinant
TRAF2 or TRADD
Binding between LMP1
(or LMP1 peptide) and
TRAF2/TRADD
Detection by LMP1-specific
Ab or 2nd reagent
or
Molecules interfere
the interaction
between
LMP1/LMP1 peptide
and TRAF2/TRADD
Binding of LMP1specific Ab or 2nd
reagent
ELISA for Interactions between LMP1 and
ELISA for interaction
between LMP1 and TRAF2/TRADD
TRAF2/TRADD
LMP1B or LMP1C Ab
LMP1
TRAF2 or TRADD
0.14
0.18
0.17
0.12
0.16
0.1
A
O D405
405
O.D. 405
A405
0.15
0.14
0.13
0.08
0.06
0.12
0.04
0.11
0.02
0.1
0.09
0
0
1:729
1:243
1:81
1:27
1/729 1/243 1/81 1/27
TRAF2 concentration (g)
1:9
1/9
His6x-TRAF2 (g)
1:3
1/3
1
1
0
PBS
TRADD TRADD TRADD TRADD TRADD TRADD
0 1/TRADD
729
1/27
1/9
1/3
11:1
1:729 1/243
1:243 1/81
1:81
1:27
1:9
1:3
TRADD Conc.
His6x-TRADD
(g)
ELISA for Interactions between biotin-labeled
and TRADD
ELISA LMP1C16
for interactionpeptide
between biotin-labeled
LMP1C16
peptide and TRADD
TRADD & biotin-LMP1-C16 binding 020320
TRADD & biotin-LMP1-C16 binding 020320
00.2
.6
0 0.18
.6
-
0 0.16
.5
A405
0.12
00.14
.4
O.D.405
O.D.405
0 .30.1
0 0.08
.2
0.04
0
0.02
00.12
.3
0.1
0 .2
0.08
0.04
0
0
1:729
1:243
1:81
1:27
1:9
1:3
1
0.02
0
0
0
1:729
1:243
1:81
1:27
1:9
1:3
0 1/729 1/243 1/81 1/27 1/9 1/3
1
0
1:729
1:243
1:243
1:81
1:81
1:27
1:27
1:9
1:9
1:3
1:3
1
1 microgram
1
Biotin-LMP1C16 (g)
0 .6
+- o Biotin-LMP1C16
v a lb u m in /- b io tin LMP1 C1 6
++oBiotin-LMP1C16
v a lb u m in /+ b io tin LMP1 C1 6
0 .5
O. D. 405
A405
O.D.405
A405
Ovalbumin
Or BSA
0.3
1:729
Biotin-LMP1(C16)-peptide (g)
0.6
0.4
0
0 1/729 1/243 1/81 1/27 1/9 1/3
1
microgram
His6x-TRADD
His6x-TRADD (g)(g)
0.5
TRADD
00.06
.1
0 0.06
.1
TRADD
+ oTRADD
v a lb u m in /- b io tin LM P1 C 1 6
++o TRADD
v a lb u m in /+ b io tin LM P1 C 1 6
0.16
A405
0.14
0 .4
-
00.18
.5
+ o Biotin-LMP1C16
v a lb u m in /- b io tin LM P1 C1 6
++o vBiotin-LMP1C16
a lb u m in /+ b io tin LM P1 C1 6
0 .4
0 .3
0 .2
TRADD
0.2
0 .1
0.1
0
0
1:729
1:243
1:81
1:27
1:9
00 1/729
1/243
1/81
1/27
1/9
ovalbumin concentration (g)
1:3
1/3
Ovalbumin or BSA (g)
11
TR ADD + biotin-LM P1C16
+
+
TR ADD + LM P1C16 + biotinLM P1C16
+
+
+
LM P1C16 + biotin-LM P1C16
+
+
TRADD
LMP1C16
biotin-LMP1C16
Thank you
for your attention
LMP1
BRAM1
TNFR
?
BRAM1
Fig. 4.Whether BRAM1 is also involved in TNF signaling
293 cells
 transfected with Flag-BRAM1
 treatment with TNF-
 luciferase activity
BRAM1 acts as a negative regulator for NF-B activation
in response to TNF- treatment
LMP1
BRAM1
BRAM1
?
TNFR
?
BRAM1
BRAM1
Fig. 5A. Whether BRAM1 abrogate NF-κB activation
through preventing the phosphorylation and degradation
of IκBα
BALB/c3T3 cells
 transfected with B-LMP1
and/or BRAM1
 WB using anti-phospho-IB antibody (B-9)
and anti-IB-specific antibody (C-21)
BRAM1 prevented LMP1-induced IκBα phosphorylation
and degradation
Fig. 5B.C. The role of BRAM1 in the inhibition of
phosphorylation and degradation of IB
BALB/c3T3 cells
 transfected with BRAM1
 treated with TNF-
 WB using anti-IB antibody (C-21)
and anti-phospho-IB antibody (B-9)
BRAM1 significantly reduced the IκBα activation in
response to TNF-α treatment
?
BRAM1
BRAM1
Fig. 5D. BRAM1 may interfere with the phosphorylation
of IκBα by its kinase IKK
BALB/c 3T3 cells
 transfected with BRAM1
 treated with TNF-
 in vitro phosphorylation reactions
 immunocomplex kinase assay using
anti-IKK- antibody (M-110)
BRAM1 interferes LMP1 and TNF-α-induced NF-κB
activation by targeting the posttranslational modifications
of IκBα molecules
Fig. 6. To further examine the inhibitory role of BRAM1
293 cells
 transfected with BLMP1 and/or BRAM1
 treated with TNF- and cycloheximide
 stained with PI
 Flow cytometry
BRAM1 interferes with the anti-apoptotic effect of LMP1
DISCUSSION
• BRAM-1 plays a negative role in LMP1-mediated
signaling and promoting cell death induced by TNF-
• BRAM1 inhibits NF-B activation by interfering the
activation of the upstream NIK-IKK-IB complex
• BRAM1 can co-precipitate with a small amount of
TRADD, indicating that these three proteins can be
present within a complex
?
BRAM1
BRAM1
Epstein-Barr virus latent membrane protein-1 triggers
AP-1 activity via the c-Jun N-terminal kinase cascade
W. Hammerschmidt et al. (1997) EMBO Journal
JNK1 is induced by LMP-1
AP-1 induction by LMP-1
c-Jun induction by LMP-1
Epstein-Barr Virus-Encoded Latent Membrane Protein
1 Activates the JNK Pathway through Its Extreme C
Terminus via a Mechanism Involving TRADD and TRAF2
L.S. Young et al. (1999) J Virol
TRAF6 is a critical mediator of signal transduction by
the viral oncogene latent membrane protein 1
W. Hammerschmidt et al. (2001) EMBO J
 LMP1 affects multiple signaling events
in cells
• induces the anti-apoptotic protein, A-20
R.T. Nancy 1999
Virology
•inhibits epithelial cell differentiation
Young, L. S. (1990) Nature
LMP1 signal transduction differs substantially from TNF
receptor 1 signaling in the molecular functions of TRADD
and TRAF2
Hammerschmidt et al. (1999) EMBO Journal
1-194: N-terminal
195-312: death domain
296-299A: mutant
Whether BRAM1 interacts with BMPR-IA in vivo
293 cells
 Transfected with Flag-BRAM1
HA-BMPR-IA and Flag-BS69
 IP with anti-HA
 WB with anti-Flag
BRAM1 specifically interacts with BMPR-IA in mammalian cells
BRAM1, a BMP receptor-associated molecule involved in
BMP signaling
S. Hiroshi. et al. Genes to Cells 1998
 Result
• BRAM1, a BMPR-IA associated molecule, found to associate
specifically with BMPR-IA
• BRAM1 may facilitate the formation of a complex with TAB1
and the receptor
• BRAM1 may thus serve as an interacting protein in the BMP
signal pathway
Fig. 3A. Whether BRAM1 interferes NF-B activation
BALB/c3T3 cells
 transfected with 4xB-Luc
B/N-LMP1, Flag-BRAM1
 luciferase values
 WB using anti-LMP1 (S12)
and anti-Flag (M2 )
BRAM1 interferes with LMP1-mediated NF-B
activation in a dose-dependent manner
Fig. 3B. Whether BRAM1 interferes JNK pathway
293 cells
 transfected with B/N-LMP1
BRAM1
 in vitro kinase assay
BRAM1 was not involved in LMP1-mediated JNK signaling
in 293 cells
BRAM1, a BMP receptor-associated molecule involved in
BMP signaling
S. Hiroshi. et al. Genes to Cells 1998
BS69
1
377
562
BRAM1
MLLEPPSPVPWT
BS69
BRAM1
BS69
BRAM1
Northern blot with BRAM1 cDNA
BRAM1 transcripts were detected in various human tissues and
functions in the cytoplasm
Fig.1C. To further confirm the physical association
between LMP1 and BRAM1
BALB/c3T3 cell
 or BALB/c3T3 LMP1 cell lysates
 incubated with immobilized GST-BRAM1c
or GST control
 pull-down assay
 WB using anti-LMP1 antibody (S12)
LMP1 and BRAM1 are physically associated
Whether BMPR-IA, BRAM1 and TAB1 form a complex
COS7 cells
 Transfected with HA-BRAM1
GST-BMPR-IA, GST and
Myc-TAB1
 pull-down assay
 WB
TAB1: TAK1 (MAPKKK) activator protein
BMPR-IA, TAB1 and BRAM1 form a ternary complex
Fig.1D. Whether LMP1 and BRAM1 interact in vivo
293 cells
 transfected with Flag-BRAM1 or LMP1
 IP with S12 or anti-Flag M2
affinity gel
 WB with S12 or M2
 Cell lysates
 WB with S12 or M2
LMP1 and BRAM1 are associated in vivo