From the a Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, the f Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, the h Albert Einstein College of Medicine, New York, and i The Molecular Sciences Institute, Berkeley, California 94704
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ABSTRACT |
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Tvl-1 is a 269-amino acid ankyrin repeat protein
expressed primarily in thymus, lung, and testes that was identified by
screening a murine T-cell two-hybrid cDNA library for proteins that
associate with the serine-threonine kinase Raf-1. The interaction of
Tvl-1 with Raf-1 was confirmed by co-immunoprecipitation of the two proteins from COS-1 cells transiently transfected with Tvl-1 and Raf-1
expression constructs as well as by co-immunoprecipitation of the
endogenous proteins from CV-1 and NB2 cells. Tvl-1 interacts with Raf-1
via its carboxyl-terminal ankyrin repeat domain. The same domain also
mediates Tvl-1 homodimerization. Tvl-1 was detected by
immunofluorescence in both the cytoplasm and the nucleus suggesting that in addition to Raf-1 it may also interact with nuclear proteins. Activated Raf-1 phosphorylates Tvl-1 both in vitro and
in vivo. In baculovirus-infected Sf9 insect cells,
Tvl-1 potentiates the activation of Raf-1 by Src and Ras while in COS-1
cells it potentiates the activation of Raf-1 by EGF. These data suggest
that Tvl-1 is both a target as well as a regulator of Raf-1. The human
homologue of Tvl-1 maps to chromosome 19p12, upstream of
MEF2B with the two genes in a head to head arrangement.
Following stimulation of membrane receptors, the Raf-1 kinase is
activated and transduces signals to several signaling pathways (1-3).
The molecular mechanism of Raf-1 activation is not well understood but
appears to be under the control of a host of Raf-1-interacting macromolecules including both proteins and phospholipids (2, 4).
One of the mechanisms by which interacting macromolecules regulate the
activity of Raf-1 is phosphorylation. Mitogenic stimulation leads to an
increase in Raf-1 phosphorylation on serine, threonine, and in some
cases, tyrosine residues (5, 6). Studies using overexpression systems
have demonstrated that Raf-1 can be activated both by tyrosine
phosphorylation mediated by members of the Src kinase family (7, 8) and
by serine phosphorylation, mediated by protein kinase C (9). In
contrast, cAMP-dependent protein kinase A (10) and an
unidentified G-protein-coupled tyrosine phosphatase down-regulate the
activity of Raf-1 (11). The significance of these kinases/phosphatases
in regulating Raf-1 activation under physiological conditions is still
inconclusive. However, it was recently shown that the Raf-1 activating
phosphorylation at Tyr340, which is mediated by members of
the Src kinase family, is triggered by CD4 cross-linking in T cells
(12) and by Fc Phosphorylation may be the final outcome of the interaction of Raf-1
with other macromolecules. However, the range of the effects of these
interactions is quite diverse. Thus, it has been suggested that the
interaction of Raf-1 with 14-3-3 (14-16), Hsp90·p50Cdc37
(17-20), or Ras (21-23) may promote the proper folding of the kinase and may enhance its stability. Moreover, the interaction of Raf-1 with
Ras may be responsible for the translocation of Raf-1 to the plasma
membrane (24, 25) while the interaction of Raf-1 with Ras or 14-3-3 may
promote its oligomerization (26, 27).
The mechanisms by which these interacting macromolecules regulate the
activation of Raf-1 are overlapping and interdependent, as exemplified
by the interaction of Raf-1 with Ras. The GTP-charged form of Ras binds
directly to the amino-terminal conserved region 1 which includes the
Ras-binding domain and the cysteine-rich domain of Raf-1 (28-31). This
interaction causes translocation of Raf-1 from the cytosol to the
plasma membrane where Raf-1 activation takes place (24, 25). The nature
of the membrane events and the macromolecules involved in Raf-1
activation have not been well defined to date. One such mechanism,
however, may involve binding of Raf-1 to the oligomer forming adaptor
protein 14-3-3 (32). Binding to this protein may induce oligomerization
and activation of Raf-1 via auto- or trans-phosphorylation.
To study the role of Raf-1 in the transduction of downstream signals we
need not only to understand its regulation but also to know its
targets. Despite intensive efforts, the only known physiological
substrate of Raf-1 to date is MEK (33-35). A recent report has shown
that Raf-1 also phosphorylates the phosphatase Cdc25A, a cell cycle
regulator, and that phosphorylation enhances the Cdc25A phosphatase
activity in vitro (36). In addition, Raf-1 was found to
phosphorylate Bad, a pro-apoptotic Bcl-2 family member, and may inhibit
Bad-induced apoptosis (37). However, whether Cdc25A and Bad can
function as physiological downstream effectors of Raf-1 remains to be determined.
The purpose of the work presented here was to identify novel Raf-1
interacting proteins and to determine their role in Raf-1 regulation
and signaling. Screening a yeast two-hybrid cDNA library derived
from mouse thymus RNA, for such proteins led to the isolation of a
novel ankyrin repeat protein, Tvl-1 (from the Greek word touvlo which
means brick). Tvl-1 interacts with Raf-1 through its carboxyl-terminal
ankyrin repeat domain. Similar to 14-3-3, Tvl-1 also forms oligomers.
However, in contrast to 14-3-3, Tvl-1 is phosphorylated by Raf-1.
Co-expression of Raf-1, Src, and Ras with Tvl-1 in Sf9 cells
reproducibly enhanced Raf-1 activity by 3-4-fold. Furthermore,
overexpression of Tvl-1 in COS-1 cells enhanced the
EGF1-induced Raf-1
activation. These findings suggest that Tvl-1 may contribute to the
regulation of Raf-1 activation as well as to the transduction of Raf-1 signals.
Yeast Interaction Trap--
The open reading frame of human
Raf-1 (from D. Morrison) was inserted into the pNLex vector
so as to be expressed as a fusion with the DNA-binding domain of LexA,
a bacterial transcriptional repressor (38). A murine CD4+ T
cell cDNA library (from Stratagene) was subcloned into the expression vector pJG4-5, which utilizes the galactose-inducible GAL-1 promoter to express library clones as fusions to a
nuclear localization sequence, a portable transcriptional activation
domain (the "acid blob", B42), and a hemagglutinin epitope tag
(39). Bait and library plasmids were transformed into the yeast strain EGY48/pSH18-34, in which the upstream regulatory elements of the chromosomal LEU2 gene have been replaced by three copies of
the LexA operator and which carries the reporter plasmid SH18-34
containing the LacZ gene also under the control of
LexA operators. Library clones encoding proteins interacting
with Raf-1 were identified based on their ability to activate the LEU2
and LacZ reporters, thus enabling yeast cells to grow and form blue
colonies in leucine-free, 5-bromo-4-chloro-3-indoyl
To determine the specificity of the interaction between Tvl-1 and
Raf-1, we carried out yeast interaction mating assays as described (40,
41). Briefly, EGY48 (MAT RNA Isolation, Northern Blotting, and cDNA
Libraries--
Poly(A)+ RNA was isolated using the method
of Chomczynski and Sacci (42). Northern blots were hybridized with the
partial Tvl-1 cDNA probe cloned from the interaction
trap library. The same probe was used to screen a murine
CD4+ T cell cDNA library (Stratagene, CA) and a
cDNA library constructed by us from rat spleen mRNA. The rat
spleen cDNA library was constructed using the Stratagene cDNA
cloning kit according to the protocol provided by the manufacturer.
Mammalian Cell Lines and Transfections--
COS-1 and CV1 cells
were obtained from the American Type Tissue Collection (ATCC) and
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum and penicillin (50 units/ml), streptomycin (50 µg/ml), and kanamycin (100 µg/ml) (PSK). NIH 3T3 cells cultured in
Dulbecco's modified Eagle's medium supplemented with 10% calf serum
and PSK. NB2, a prolactin-dependent rat T cell lymphoma
line was provided by P. Gout (University of British Columbia). NB2
cells were grown in RPMI 1640 media supplemented with fetal bovine
serum (10%) and PSK. COS-1 cells were transiently transfected using
the DEAE-dextran/chloroquine method as described previously (43). NIH
3T3 cells were transfected using LipofectAMINE (Life Technologies,
Inc.) according to the manufacturer's protocol.
Expression Constructs--
A Tvl-1 expression construct was
generated by subcloning the full-length Tvl-1 cDNA
insert into the pCMV5 expression vector (44). FLAG epitope-tagged Tvl-1
(FLAG-Tvl-1) and hemagglutinin (HA) epitope-tagged Tvl-1 (HA-Tvl-1)
constructs were generated by inserting the Tvl-1 coding sequence
in-frame with the FLAG or the HA epitope tag into the pCMV-5 expression
vector. A series of truncated Tvl-1 expression constructs were
generated by cloning cDNA fragments of truncated Tvl-1, epitope
tagged at their amino terminus with FLAG, into the pcDNA3
expression vector (Invitrogen). CMV-Raf-1 expression constructs were
generated by inserting the wild type or constitutively active
Raf-1(Y340D) mutant cDNA (7) into the pCMV5 vector. To generate
plasmids expressing GST fusion proteins of Raf-1 and its truncated
mutants, the cDNA inserts of the full-length Raf-1, its
NH2-terminal domain (1-323 amino acids) and its
COOH-terminal catalytic domain (305-648 amino acids) were,
respectively, subcloned into the pEBG expression vector which encodes
glutathione S-tranaferase driven by EF1
Baculoviruses expressing Tvl-1, the constitutively active Raf-1 mutant
Y340D, and the kinase-negative Raf-1 mutant K375M were generated by
ligating the appropriate inserts into the transfer vector pVL1393
(PharMingen). The resulting constructs were transfected into Sf9
cells using the Baculogold Transfection Kit (PharMingen). Baculoviruses
expressing Raf-1, v-Src, and v-Ha-Ras were provided by Dr. J. Chernoff (Fox Chase Cancer Center).
Antibodies--
Rabbits were inoculated with
(His)6-tagged Tvl-1 protein purified from Escherichia
coli strain M15 (Qiagen) transformed with the construct
pQE30-Tvl-1. pQE30-Tvl-1 was constructed by inserting the coding
sequence of the full-length Tvl-1 cDNA into the bacterial expression vector pQE30 (Qiagen). E. coli transformed with
this construct were grown at 37 °C in the presence of ampicillin (50 µg/ml) and kanamycin (25 µg/ml) to OD600 = 0.7. Protein
expression was induced by growing cells in 0.5 mM
isopropyl-1-thio-
Rabbit polyclonal antibody against the carboxyl terminus of Raf-1 (C12)
was purchased from Santa Cruz Biotechnologies. An anti-Raf-1 monoclonal
antibody (R19120) was purchased from Transduction Labs. The M2
anti-FLAG monoclonal antibody was purchased from Kodak IBI. Anti-HA
mouse monoclonal antibody 12CA5 was purchased from Babco.
Immunoprecipitations and Western Blotting--
Cultured cells
were lysed in Nonidet P-40 lysis buffer (20 mM Hepes, pH
7.6, 137 mM NaCl, 0.5% Nonidet P-40, 2 mM
EGTA, 10% glycerol, 5 µg/ml leupetin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 5 mM sodium
fluoride, 1 mM sodium vanadate). Cell lysates were
clarified by centrifugation at 4 °C for 10 min at 12,000 × g and were precleared by a 30-min incubation with protein
A-agarose beads (Life Technologies, Inc.). Immunoprecipitations were
carried out by incubating these lysates with the appropriate antibodies for 3 h. The resulting antigen-antibody complexes were collected with protein A- or protein G-agarose beads. The immunoprecipitates were
washed three to four times with cold Nonidet P-40 lysis buffer prior to
their analysis by SDS-PAGE. Western blots of the immunoprecipitates or
of total cell lysates were carried out using Immobilon-P membranes (Millipore).
Immunofluorescence--
NIH 3T3 cells seeded on glass coverslips
were transfected with pCMV5-FLAG-Tvl-1 using LipofectAMINE (Life
Technologies, Inc.). Thirty-six hours after transfection, the cells
were washed twice with phosphate-buffered saline (PBS), fixed with
3.7% paraformaldehyde (pH 7.4), and permeabilized with 0.1% Triton
X-100 in PBS. To block nonspecific antibody binding, the cells were
incubated in 10% goat serum in PBS for 60 min at room temperature,
prior to being incubated for 60 min with the M2 mouse monoclonal
anti-FLAG antibody or the rabbit anti-Tvl-1 antiserum (1:400 dilution
in 5% goat serum). Following washing with PBS, the cells were
incubated for 30 min with fluorescein isothiocyanate-conjugated goat
anti-mouse or anti-rabbit antibodies (Sigma, dilution at 1:400) plus 1 µg/ml bisbenzimid (Hoechst 33258, Sigma). At the end, the coverslips were washed twice in PBS and mounted on glass slides with anti-fade medium (90% glycerol with 1 mg/ml paraphenylene diamine). Fluorescence was recorded by confocal microscopy.
Protein Kinase Assays in Sf9 Cells--
Raf-1 kinase
assays were performed using standard procedures (45). Briefly, 2 × 106 Sf9 cells in 60-mm Petri dishes were infected
with the desired combination of baculoviruses. After 48 h, cells
were washed twice in cold PBS and lysed in the Nonidet P-40 lysis
buffer for Raf/Tvl-1 co-immunoprecipitation or in RIPA buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol,
1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotin/leupeptin, 5 mM sodium fluoride, and 5 mM sodium vanadate). Insoluble material was removed by
centrifugation at 12,000 × g for 10 min at 4 °C.
Clarified lysates were incubated with rabbit anti-Raf-1 antibody (C12,
Santa Cruz Biotechnology) for 3 h at 4 °C, followed by
incubation with protein A-agarose beads for 1 h at 4 °C to
collect the antigen-antibody complexes. Immunoprecipitates were washed
twice with lysis buffers and twice with kinase buffer containing 30 mM Hepes (pH 7.5), 5 mM MgCl2, 5 mM MnCl2, and 1 mM dithiothreitol.
Kinase reactions were carried out by incubating the immune complexes
for 20 min at 25 °C in 40 µl of Raf-1 kinase buffer containing 10 µM unlabeled ATP, 10 µCi of [ Protein Kinase Assays in COS-1 Cells--
Raf-1 kinase assays in
COS-1 cells were performed as described previously (20, 26). Briefly,
COS-1 cells were transfected with a pEBG-Raf-1 expression construct
(20, 26) alone or in combination with the CMV-Tvl-1 expression
construct. Twenty-four hours later, cells were serum-starved for
20 h, and then treated with EGF (50 ng/ml, R & D) for 20 min.
Cells were lysed using the Triton X-100 lysis buffer (50 mM
Tris-Cl, pH 7.6, 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 2 mM sodium vanadate, 20 mM
Metabolic Labeling and Phosphopeptide
Analysis--
Two-dimensional phosphopeptide analysis was carried out
as described by Boyle et al. (47). Briefly, COS-1 cells were
transfected with the CMV-Tvl-1 expression construct alone or in
combination with the CMV-Raf-1 (Y340D) expression construct. At 24 h after transfection, the cells were cultured in serum-free medium.
Sixteen hours later, cells were cultured for 4 h in phosphate-free
Dulbecco's modified Eagle's medium containing 1 mCi/ml
ortho[32P]phosphate (NEN Life Science Products Inc.).
Thirty minutes prior to lysis, half of the cells transfected only with
the Tvl-1 expression construct were stimulated with 10% fetal calf
serum. 32P-Tvl-1 was immunoprecipitated from the
metabolically labeled cells using rabbit anti-Tvl-1 antiserum and
electroblotted onto nitrocellulose membranes. The Tvl-1 protein band
excised from the membrane was digested with trypsin. The resulting
phosphopeptides were separated using a Hunter thin layer peptide
mapping electrophoresis apparatus (CBS Scientific Company, Inc., Del
Mar, CA) and visualized by autoradiography.
Interaction Trap and Interaction Mating Assays- Identify a
Ubiquitously Expressed Ankyrin Repeat Protein, Tvl-1, That Specifically
Binds Raf-1--
To identify novel proteins that interact with Raf-1
we screened a CD4+ murine T cell cDNA library using the
interaction trap two-hybrid system in the yeast Saccharomyces
cerevisiae, as described by Gyuris et al. (39). Out of
50 isolated clones, 15 were sequenced and found to be derived from
a single gene, Tvl-1 (data not shown). The specificity of
interaction of the B42-Tvl-1 fusion with Raf-1 was confirmed by the
experiment in Fig. 1 which shows that
growth is galactose and therefore B42-Tvl-1-dependent and
that a full-length Raf-1 interacts with B42-Tvl-1 but not with the B42
domain alone. The same figure shows that Tvl-1 also interacts with the
amino-terminal domain of Raf-1 (amino acids 1-330) although with lower
efficiency.
To determine whether Tvl-1 interacts with other signaling proteins we
carried out interaction mating experiments (40, 41) between EGY48 MAT
Hybridization of a Tvl-1-specific cDNA probe to a
Northern blot of poly(A)+ RNA from normal adult mouse
tissues revealed that Tvl-1 mRNA is widely expressed
(Fig. 2A). Western blots of
lysates from normal adult mouse tissues probed with a Tvl-1-specific
antibody raised against bacterially expressed Tvl-1 protein (Fig.
2B, "Experimental Procedures") revealed that Tvl-1 is
expressed primarily in thymus, lung, and testes. The levels of
Tvl-1 mRNA and protein in different tissues correlate
only partially, suggesting that protein expression of Tvl-1 may be
regulated in part at the post-transcriptional level.
Sequencing of full-length cDNA clones obtained by screening a
murine CD4+ T cell cDNA library revealed that
Tvl-1 encodes a novel 269-amino acid protein containing four
ankyrin repeats (48, 49) at its carboxyl terminus (Fig. 2, C
and D). Some of the cDNA clones contain a small internal
deletion. These clones represent a differentially spliced mRNA
species that, when translated, is expected to give rise to a short
Tvl-1 protein (deletion marked by a box in Fig. 2C). Deletion of the indicated 10 amino acids generates a
potential 5th ankyrin repeat motif that is located upstream of the
first one (Fig. 2, C and D). Since both the long
and short forms of Tvl-1 interact with Raf-1 (data not shown), the
significance of the two forms is currently unknown. Sequencing a
full-length cDNA clone isolated from a rat spleen cDNA library
showed that the mouse and rat genes exhibit 95% identity at the amino
acid level. Screening existing data bases for Tvl-1-related
sequences identified two overlapping human cosmid clones (R2770 and
F14150) which map to human chromosome 19p12 and contain the entire
sequence of the human Tvl-1 homologue (accession numbers AC002126 and AC003110). The same clone contains a myocyte enhancer-binding factor
MEF2B (50, 51) upstream of Tvl-1 in a head to
head orientation with Tvl-1. Human and mouse Tvl-1 share
85% identity at the amino acid level (data not shown).
The Tvl-1 clones isolated from the two-hybrid cDNA
library encode protein fusions between the COOH-terminal two-thirds of Tvl-1 (amino acids 81-269) and the activation domain B42. Following cloning of the full-length Tvl-1 gene, we showed that while
the full-length protein also interacts with Raf-1, its
NH2-terminal portion (amino acids 1-96) does not (data not
shown). Therefore, the interaction between Raf-1 and Tvl-1 is mediated
by the carboxyl-terminal domain of Tvl-1.
Tvl-1 Co-immunoprecipitates with Raf-1--
To determine whether
Tvl-1 interacts with Raf-1 in mammalian cells, expression constructs of
Tvl-1 tagged at their amino terminus with a FLAG epitope tag
(FLAG-Tvl-1) and wild type Raf-1 or activated Raf-1 (Y340D) were
co-transfected into COS-1 cells in the combinations shown in Fig.
3A. Forty-eight hours later,
Tvl-1 or Raf-1 were immunoprecipitated from Nonidet P-40 lysates of the
transfected COS-1 cells, with anti-FLAG or anti-Raf antibodies,
respectively. Co-precipitating proteins were detected by probing
Western blots of the immunoprecipitates with anti-Raf-1 and anti-Tvl-1
antibodies. The results confirmed that Tvl-1 interacts with both the
wild type and activated forms of Raf-1 in mammalian cells (Fig.
3A). Similar experiments using NH2- or
COOH-terminal truncated Raf-1 expression constructs revealed that both
truncated Raf-1 proteins interact equally well with Tvl-1 (Fig.
3B). Therefore, Tvl-1 interacts with multiple sites in the
NH2- and COOH-terminal domains of Raf-1. Moreover, since
the kinase-dead Raf-1K375M also binds Tvl-1 (data not shown), the
interaction between the two proteins is independent of the Raf-1 kinase
activity.
To map the Tvl-1 domain that interacts with Raf-1, a series of Tvl-1
deletion mutants in-frame with an amino-terminal FLAG tag were
constructed and cloned in the pCMV5 expression vector. Co-transfection
of these constructs with Raf-1 in COS-1 cells (Fig. 3C)
confirmed the results of the two-hybrid experiments in yeast by showing
that it is indeed the carboxyl-terminal ankyrin repeat region of Tvl-1
that interacts with Raf-1.
The preceding experiments were carried out using lysates of transiently
transfected COS-1 cells that express high levels of both Tvl-1 and
Raf-1. To determine whether the two proteins interact also when
expressed at physiological levels, we immunoprecipitated Raf-1 from
Nonidet P-40 lysates of CV-1 and NB2 cells, two cell lines that express
both proteins. Immunoprecipitations were carried out using an
anti-Raf-1 antiserum raised against the Raf-1 peptide C12 (Santa Cruz)
or the monoclonal anti-Raf-1 antibody (R19120, Transduction Labs) (Fig.
4). Control immunoprecipitations of the NB2 cell lysates were also carried out using a control antibody against
the interleukin-9 receptor. The immunoprecipitates were subjected to
SDS-PAGE, and following Western blotting, they were probed with a
rabbit antiserum raised against the full-length Tvl-1 protein (Fig.
4A). In the NB2 cell experiment Western blots were probed
with the anti-Tvl-1 antibody raised against the Tvl-1 peptide (amino
acids 100-113) in the presence or absence of excess peptide (Fig.
4B). The results (Fig. 4, A and B)
showed that endogenous Tvl-1 co-immunoprecipitates with endogenously
expressed Raf-1 from lysates of both CV-1 and NB2 cells.
Tvl-1 Expressed in Transiently Transfected COS-1 Cells Forms
Homodimers--
Notch, an ankyrin repeat-containing protein has been
shown to oligomerize via homotypic intermolecular interactions between ankyrin repeats (52). To determine whether Tvl-1, also an ankyrin repeat protein, homodimerizes, expression constructs of HA-tagged and
FLAG-tagged Tvl-1 were co-transfected into COS-1 cells. Tvl-1 was
immunoprecipitated using the anti-FLAG antibody from Nonidet P-40
lysates of the transfected cells harvested 48 h later. Following SDS-PAGE and Western blotting, the resulting immunoprecipitates were
probed with the anti-HA antibody. The results (Fig.
5) revealed that Tvl-1 molecules indeed
homodimerize. Since HA-Tvl-1 and a FLAG-tagged Tvl-1 deletion lacking
the ankyrin repeat domain do not dimerize (Fig. 5, lane 3),
we conclude that homodimerization is likely to be mediated by the
ankyrin repeat region.
Subcellular Localization of Tvl-1--
To define the subcellular
localization of Tvl-1, we transfected NIH 3T3 cells with a
FLAG-tagged Tvl-1 construct, either transiently or stably, and we
stained the transfected cells with the anti-FLAG antibody M2. The
results (Fig. 6) revealed that Tvl-1 is
present in both the cytoplasm and the nucleus. Similar results were
obtained using affinity purified anti-Tvl-1 antibody instead of the
FLAG antibody (data not shown). The detection of Tvl-1 in the cytoplasm is compatible with its interaction with Raf-1, a cytoplasmic protein. However, since Tvl-1 is also present in the nucleus, it is likely that
it may interact with nuclear proteins and may contribute to the
regulation of nuclear functions.
Raf-1 Phosphorylates Tvl-1 in Vitro and in Vivo--
To determine
whether Tvl-1 serves as a Raf-1 substrate we first carried out in
vitro kinase assays using Raf-1 immunoprecipitated from
baculovirus-infected Sf9 cells co-expressing Raf-1 and Tvl-1. The kinase inactive mutant of Raf-1, K375M, and the activated mutant of
Raf-1, Y340D, were used as controls. Wild type Raf-1 was activated by
co-expression of v-Ha-Ras and v-Src (Fig.
7A). In a separate experiment,
bacterially expressed (His)6-Tvl-1 was added as the
exogenous substrate in Raf-1 in vitro kinase assays. The
results in Fig. 7, A and B, revealed that Tvl-1
co-immunoprecipitates with, and is phosphorylated only by catalytically
active Raf-1. By comparing the degree of phosphorylation of Tvl-1 and
MEK-1 by Raf-1 in vitro, we found that both proteins served
equally well as Raf-1 substrates (data not shown). Inspection of the
Tvl-1 sequence revealed a motif
SALSLASMGG starting at
amino acid 168, which is similar to the MEK-1 motif
(L/A)-S-(T/S)-G phosphorylated by
Raf-1 and represents a candidate phosphorylation site (53).
To determine whether Raf-1 induces phosphorylation of Tvl-1 in
mammalian cells, 32P-Tvl-1 was immunoprecipitated from
ortho[32P]phosphate-labeled COS-1 cells expressing Tvl-1
alone or in combination with constitutively active Raf-1 (Y340D). Tvl-1
immunoprecipitated from singly transfected 32P-labeled
cells stimulated with serum for 30 min was used as a control.
Immunoprecipitated Tvl-1 was electroblotted onto nitrocellulose membranes, digested with trypsin. The resulting products were separated
using a Hunter thin layer peptide mapping electrophoresis apparatus. As
shown in Fig. 7C, the tryptic phosphopeptide map of Tvl-1
from COS-1 cells stimulated with serum is very similar, if not
identical, to the one from serum-starved COS-1 cells co-expressing Tvl-1 and active Raf-1. These data indicate that Raf-1 indeed phosphorylates Tvl-1 in vivo and is the primary kinase to
phosphorylate Tvl-1 in the serum-stimulated cells.
Tvl-1 Potentiates the Activation of Raf-1--
To determine
whether Tvl-1 modulates the activity of the Raf-1 kinase we examined
the kinase activity of Raf-1 in Sf9 cells co-expressing Tvl-1
and Raf-1 in the presence or absence of v-Src plus v-Ha-Ras. To this
end, Sf9 cells were infected with baculoviruses expressing
Tvl-1, Raf-1, v-Src, and v-Ha-Ras in the combinations shown in Fig.
8A. Forty-eight hours later
the infected cells were lysed in a RIPA lysis buffer. An in
vitro kinase reaction carried out using Raf-1 immunoprecipitated
from these lysates, and histidine-tagged kinase-dead MEK-1 as the
exogenous substrate revealed that Tvl-1 potentiates the activation of
Raf-1 by Src and Ras. The potentiation of the activation of Raf-1 by
Tvl-1 is highly reproducible, with very similar results obtained in
three independent experiments. To determine whether Tvl-1 has a similar
effect on the activity of the Raf-1 kinase in mammalian cells, we
overexpressed Tvl-1 in COS-1 cells and examined the kinase activity of
Raf-1 following overnight serum starvation and EGF stimulation for 20 min. As shown in Fig. 8B, while overexpression of Tvl-1 had
no effect on the kinase activity of Raf-1 in unstimulated cells, it
significantly enhanced the activation of Raf-1 by EGF. Similar results
were obtained in three independent experiments.
In this report we presented the cloning and initial
characterization of a gene (Tvl-1) that encodes a novel
ankyrin repeat protein that interacts with Raf-1 and is phosphorylated
by the Raf-1 kinase. Through its interaction with Raf-1, Tvl-1 also
promotes the activation of the Raf-1 kinase by EGF or Src/Ras. The
interaction between Tvl-1 and Raf-1 is specific since Tvl-1 failed to
interact with other kinases such as cdc2, CDK2, ERK1, Akt2, Akt3, and
ILK-1 as well as a series of additional signaling molecules (data not shown). Moreover, Raf-1 failed to co-immunoprecipitate with other ankyrin repeats containing proteins including Cdk inhibitor p16 and
ILK-1. Finally, Tvl-1 was shown to interact with Raf-1 not only in
cells overexpressing the two proteins, but also in untransfected cells
expressing physiological levels of the endogenous proteins. The
phosphorylation of Tvl-1 by Raf-1 is also specific in that it is due to
Raf-1 and not a contaminating co-immunoprecipitated kinase. Thus, Tvl-1
is not phosphorylated by Raf-1 expressed in the absence of Src and/or
v-Ha-Ras, but is phosphorylated by the constitutively active Raf-1
mutant Y340D. Moreover, the kinase-dead mutant Raf-1K375M did not
phosphorylate Tvl-1 when immunoprecipitated from lysates of Sf9
cells co-expressing this mutant and v-Ha-Ras plus v-Src. Tvl-1 was
detected in both the cytosol and the nucleus of cells transiently
transfected with Tvl-1 expression constructs. These findings suggest
that the interaction of Tvl-1 with Raf-1 and its consequences are
physiologically possible.
Although studied extensively, the molecular mechanism(s) of Raf-1
activation by growth factors remain relatively poorly understood. The
process of activation is regulated by the interaction of Raf-1 with a
host of cellular proteins, one of which is the Tvl-1 protein described
in this report. Tvl-1, similar to other interacting proteins such as
14-3-3, does not activate Raf-1 by itself (32). Instead, it potentiates
the activation of Raf-1 by signals transduced by other signaling
molecules such as Src/Ras or induced by growth factors such as EGF.
Earlier studies had shown that Raf-1 can be activated by both
Ras-dependent and independent mechanisms (54-56). The
findings presented in this report suggest that Tvl-1, similar to
14-3-3, is involved in the activation of Raf-1 by signals that are
Ras-dependent.
Tvl-1 may potentiate the activation of Raf-1 by affecting its
conformation. This in turn may influence the association of Raf-1 with
other activating molecules. Alternatively, Tvl-1 may function as a
scaffold protein that contributes to the assembly of Raf-1-activating
multimolecular complexes, or may contribute to the association of Raf-1
with the plasma membrane. A scaffolding role for Tvl-1 is supported by
the fact that it contains multiple ankyrin repeat motifs that function
as domains of protein-protein interaction, and oligomerize
spontaneously. The potential involvement of Tvl-1 in promoting the
association of Raf-1 with the plasma membrane was suggested by findings
that other Raf-1-interacting proteins, such as Ras, may enhance Raf-1
activity by this mechanism (24, 25, 57). In the case of Tvl-1 this does
not seem to be the case, however, based on two lines of evidence.
First, a myristylated form of Tvl-1, which is membrane-associated,
failed to enhance the activation of Raf-1 (data not shown). Second,
following serum or EGF stimulation, we failed to convincingly detect
the membrane translocation of Tvl-1 by immunofluorescence staining (data not shown). We conclude that Tvl-1 either forms a bridge between
Raf-1 and other Raf-1 activating proteins or directly affects Raf-1
protein conformation facilitating its activation by other interacting proteins.
The only other known Raf-1 substrates to date are MEK-1, as well as
perhaps Cdc25A and Bad. Phosphorylation of MEK-1 activates the kinase
and contributes to the propagation of the signal from Raf-1 to the
mitogen-activated protein kinase (33-35, 58). Phosphorylation of
Cdc25A may contribute to the activation of this phosphatase (36).
Finally, phosphorylation of Bad may prevent its interaction with
anti-apoptotic Bcl-2 family members in the mitochondria, thus
inhibiting apoptosis (37). The outcome of Tvl-1 phosphorylation is
presently unknown. Given that Tvl-1 enhances Raf-1 activation, phosphorylation of Tvl-1 by Raf-1 may represent a feedback inhibitory signal that may alter the binding affinity between the two proteins and
promote their dissociation. Alternatively, phosphorylated Tvl-1 may
contribute to the transduction of Raf-1 signals. To date we know that
Tvl-1 does not activate the mitogen-activated protein kinase in NIH 3T3
cells.2 It remains to be
determined whether Tvl-1 affects the phosphorylation of Cdc25A and Bad
by Raf-1, or whether it transduces signals to other growth
factor-activated pathways.
A recent report (59) that appeared after the original submission of
this manuscript, identified Tvl-1 as a protein that contributes to the
assembly of the transcriptional complex regulating the expression of
the major histocompatibility complex class II genes. Tvl-1 (named
RFXANK) enhances the stability of binding of the transcription factors
RFX5 and RFXAP to the sequence in the major histocompatibility complex
class II promoter. These findings combined with the data presented here
suggest that indeed Tvl-1 may define a novel class of scaffold proteins
that promote the assembly of a variety of macromolecular complexes. In
addition, they raise the question whether Raf-1 regulates gene
expression by directly phosphorylating the transcriptional adaptor
Tvl-1.
Overall, the data presented in this report show that Tvl-1 interacts
with Raf-1 in mammalian cells even when it is expressed at natural
levels. Although the precise role of this interaction in mammalian
cells has not been fully explored, our data from overexpression systems
clearly show that Tvl-1 has the potential to both regulate Raf-1 and to
function as a Raf-1 downstream target.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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RI cross-linking in myeloid cells (13).
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-D-galactoside containing medium.
) haploid cells harboring the plasmid
expressing the acid blob-Tvl-1 chimera were mated with a panel of
haploid RFY206 (MAT a) cells harboring constructs encoding LexA fusion
proteins. Besides LexA-Raf-1, the LexA fusions included the
constitutively active Raf-1 mutant, Raf-1 Y340D, the amino-terminal 330 amino acids of Raf-1, and 27 other proteins that function in pathways
regulating cell proliferation. As in the original screen, the
interaction of Tvl-1 with these proteins was determined on the
activation of the LEU2 and LacZ reporter genes.
promoter (20,
32).
-D-galactopyranoside at 25 °C for
5 h. Bacteria were collected, resuspended in 10 mM Tris (pH 7.6), 0.5 M NaCl, 0.1% Nonidet P-40, 10%
glycerol, 4 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and lysed by sonication on ice.
His-tagged Tvl-1 was collected by incubating the clarified lysate with
Ni2+-NTA resin (Qiagen). The resin beads were washed with
lysis buffer containing 25 mM imidazole. The proteins were
eluted from the resin by incubation with 100 mM imidazole.
His-tagged Tvl-1 isolated by this procedure was shown to be at least
85% pure as determined by Coomassie Brilliant Blue staining of the
eluted proteins separated by SDS-polyacrylamide gel electrophoresis
(PAGE). Tvl-1 protein was excised from the gel and inoculated into
rabbits. A second rabbit polyclonal antibody was raised against a
Tvl-1-derived multiple antigen peptide (amino acids 100-113)
(synthesized by Research Genetics Inc., Huntsville, AL) according to
standard protocols.
-32P]ATP
(Amersham), and 0.2 µg of purified recombinant His-tagged Tvl-1
(His-Tvl-1) or His-tagged kinase inactive MEK (His-MEK
(K
)). His-MEK (K
) was induced and purified
as described by Gardner et al. (46). Kinase assays using
Raf-1 and co-immunoprecipitated Tvl-1 were carried out without added
exogenous Tvl-1. Kinase reactions were stopped by adding 3× SDS sample
buffer (120 mM Tris-HCl, pH 6.8, 100 mM
dithiothreitol, 3% SDS, 10% glycerol, 0.02% bromphenol blue). The
products of the kinase reactions were then resolved by SDS-PAGE,
transferred onto polyvinylidene difluoride membranes (Millipore), and
visualized by autoradiography.
-glycerophosphate, and protease inhibitors). GST-Raf was pulled down
by incubating the lysates with glutathione-Sepharose beads (Pharmacia)
at 4 °C for 3 h. The beads were washed twice with lysis buffer,
twice with lysis buffer containing 0.5 M LiCl, and twice
with kinase buffer (40 mM Tris-Cl, pH 7.5, 0.1 mM EDTA, 5 mM MgCl2, 2 mM dithiothreitol). The Raf kinase reaction was carried out
by incubating the pulled down Raf-1 in 60 µl of the kinase reaction
buffer containing 100 µM ATP, 10 µCi of
[
-32P]ATP, and 200 ng of recombinant kinase-dead
His-MEK1. Incubations were carried out at 25 °C for 30 min. The
products of the kinase reaction were analyzed by SDS-PAGE and autoradiography.
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Fig. 1.
Tvl-1 interacts with Raf-1 in the yeast
two-hybrid system. A, B42·Tvl-1 interacts with LexA
fusions of Raf-1 (1), Raf-1 Y340D (3) and, less
efficiently, with Raf-1(1-330) (4). However, the acid blob
B42 alone does not interact with LexA-Raf-1 (2). The
observed interactions occurred only in the presence of galactose. The
intensity of the interactions of Raf-1 with Tvl-1 and c-Ha-Ras in the
yeast two-hybrid system were comparable (data not shown).
haploid cells harboring the construct encoding the fusion protein
B42·Tvl-1 and a panel of 26 RFY206 MAT
haploid cells harboring
constructs encoding LexA fusion proteins. The results showed that Tvl-1
does not interact with cdc2 (cdk1), cdk2, c-Ha-Ras, c-Ha-Ras/C186A, Rb,
ftz, SSN6, CDI1, daughterless, goosecoid, CDK3, CDI7, CDI11, DMcdc2,
LAR, cyclin C, cyclin E, CLN3, hairy, MYC, MAX, Mxi-1, p53, thyroid
hormone receptor, bicoid, and a synthetic polyglutamine peptide (data
not shown). These results indicated that the interaction of Tvl-1 with
Raf-1 is specific.
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Fig. 2.
Tvl-1 is widely expressed and
encodes a novel ankyrin repeat protein. A, upper panel:
Northern blot analysis of 5 µg of poly(A)+ RNA isolated
from the indicated normal adult mouse tissues and probed with
32P-labeled Tvl-1 cDNA. Lower
panel: profile of the ethidium bromide gel of the Northern blot.
B, Western blot of total cell lysates from normal mouse
tissues probed with a polyclonal rabbit antiserum raised against
bacterially expressed His-tagged-Tvl-1 protein. While Tvl-1 RNA is
expressed at similar levels in all tissues, Tvl-1 protein is expressed
at higher levels in thymus, lung, and testis. The observed 33-kDa
protein band is specific for Tvl-1 because: (a) it
comigrates with the Tvl-1 protein encoded by a CMV-Tvl-1 expression
construct transfected into COS-1 cells and (b) detection of
this band was competed by preadsorption of the anti-Tvl-1 antiserum
with the recombinant Tvl-1 protein (data not shown). C,
amino acid sequence of the murine Tvl-1 protein deduced from the
nucleotide sequence of clone 19 isolated from a murine CD4+
T cell cDNA library (Stratagene). The underlined
sequence defines the ankyrin repeat region. The boxed
sequence starting at amino acid 112 is absent from some of the cDNA
clones. This is due to differential splicing (data not shown).
D, alignment of the four ankyrin repeats (ANK) in the
COOH-terminal portion of Tvl-1 with the consensus ankyrin repeat
sequence (48, 49). The Tvl-1 protein encoded by the differentially
spliced mRNA contains a 5th ankyrin repeat motif (conditional ANK
and the dashed line in C) that is generated
following deletion of the boxed 10-amino acid region
shown in C.
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Fig. 3.
Tvl-1 interacts with Raf-1.
A, COS-1 cells were transfected with FLAG-tagged Tvl-1, wild
type Raf-1 (wt) and constitutively active Raf-1
(Y340D) in the vector pCMV5. Cells were lysed 48 h
later, and FLAG-Tvl-1 was immunoprecipitated (IP) with the
anti-FLAG M2 monoclonal antibody (lanes 1-3). Raf-1 and
Flag-Tvl-1 were detected in the immunoprecipitates by Western blotting
using rabbit anti-raf antiserum (C12, upper part) and rabbit
anti-Tvl-1 antiserum (lower part). COS-1 cell lysates were
also immunoprecipitated with the anti-raf-1 C12 antibody which detects
very weakly the endogenous Raf-1 protein of COS-1 cells. Western blots
of these immunoprecipitates were probed with the same anti-raf-1 and
anti-Tvl-1 antibodies (lanes 4-6). B, both
NH2-terminal and COOH-terminal domains of Raf-1 interact
with Tvl-1. The EBG-based expression constructs of full-length
(lane 2), NH2-terminal (lane 4) and
COOH-terminal (lane 3) domains of Raf-1 were co-transfected
with the FLAG-tagged Tvl-1 expression construct into COS-1 cells.
GST-fusion proteins from transfected cell lysates were pulled down and
Western blotted with the anti-FLAG monoclonal antibody (top
panel). Total cell lysates were also immunoblotted with anti-FLAG
(middle panel) and anti-GST antibodies (bottom
panel). C, Mapping the Raf-1 interacting Tvl-1 domains.
Upper panel, schematic representation of FLAG-tagged
wild-type Tvl-1 (WT) and five Tvl-1 deletion mutants.
Lower panel, co-immunoprecipitation of Raf-1 and the
FLAG-tagged Tvl-1 deletion mutants (shown in the upper
panel) from COS-1 cells transfected with the indicated expression
constructs. Cell lysates were immunoprecipitated with anti-FLAG M2
monoclonal antibody, Western blotted either with the rabbit anti-Raf-1
C12 antibody (upper panel), or with the rabbit anti-FLAG
antibody (lower panel). Note that the interaction between
Raf-1 and the Tvl-1 1 mutant representing the
NH2-terminal domain of Tvl-1 was very weak.
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Fig. 4.
Interaction of endogenous Raf-1 and
Tvl-1. Panel A, Raf-1 was immunoprecipitated from
lysates of CV-1 cells using the anti-Raf peptide antiserum (C12) from
Santa Cruz (S.C., lane 2) or the anti-Raf-1 monoclonal
antibody from Transduction Labs (T.L., lane 3). A Western
blot of the resulting immunoprecipitates was probed with the anti-Tvl-1
antiserum. Co-immunoprecipitating protein band comigrates with Tvl-1 in
total cell lysates. Panel B, Raf-1 was immunoprecipitated
from NB2 Cell lystaes using anti-Raf C12 antibody (Raf-IP, lanes
2 and 3). Anti-interleukin 9 receptor antibody was used
a negative control (control-IP, lanes 4 and 5).
Both the immunoprecipitates (ppt) and the supernatants
(sup) of the immunoprecipitation reactions were Western
blotted with an anti-Tvl-1 peptide antiserum in the absence ( ) as
well as in the presence (+) of excess peptide. Note that the protein
band comigrating with Tvl-1 (Raf-IP, ppt; lane 3) from
anti-Raf-1 immunoprecipitates was absent from the control anti-IL-9
receptor immunoprecipitate (control-IP, ppt; lane 5).
Moreover, this Tvl-1 band was detected only in the absence of peptide
competition (compare lanes 3 and 6).
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Fig. 5.
Tvl-1 expressed in transiently transfected
COS-1 cells homodimerizes. COS-1 cells were transfected with the
indicated combinations of HA-tagged wild type Tvl-1 and FLAG-tagged
wild type or COOH-terminal truncated Tvl-1 (FLAG-Tvl-1 1, Fig.
3C) expression constructs. Anti-FLAG immunoprecipitates of
transfected cell lysates was Western blotted with the anti-HA
monoclonal antibody (upper panel). The lower two
panels show the Western blots of total lysates derived from the
same cells and probed with anti-HA or anti-FLAG monoclonal
antibodies.
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Fig. 6.
Subcellular localization of Tvl-1.
Immunofluorescence of NIH 3T3 cells transfected transiently with a
FLAG-Tvl-1 expression construct and co-stained with the anti-FLAG
monoclonal antibody (left panel) and the nucleus staining
dye Hoechst 33258 (right panel).
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Fig. 7.
Tvl-1 is phosphorylated by Raf-1 in
vitro and in vivo. A, upper
panel: in vitro kinase assays of Raf-1
immunoprecipitated form Sf9 cells infected with baculoviruses
directing the expression of wild type (wt) Raf-1,
kinase-dead (K375M) Raf-1 or constitutively active (Y340D) Raf-1,
v-Src, and v-Ras as well as Tvl-1 as indicated. Tvl-1
co-immunoprecipitating with Raf-1 was highly phosphorylated only in the
lysates derived from cells expressing both active Raf-1 and Tvl-1.
Lower panel, Raf-1 was expressed approximately equally in
all the baculovirus-infected cultures of Sf9 cells. B,
upper panel: in vitro kinase assays of Raf-1
immunoprecipitated from lysates of Sf9 cells, infected with the
indicated baculoviruses. Recombinant (His)6-Tvl-1 protein
purified from E. coli was used as the exogenous kinase
substrate. Lower panel, Western blot of the same Sf9
cell lysates probed with the anti-Raf-1 antibody. C, Raf-1
phosphorylates Tvl-1 in vivo. In the top panel,
COS-1 cells transfected with Tvl-1 alone (lanes 1 and
2) or in combination with a constitutively active
Raf-1-Y340D construct (lane 3) were serum-starved overnight
and labeled with ortho[32P]phosphate for 4 h. The
cells in lane 2 were stimulated with 10% fetal calf serum
for 30 min prior to harvesting. Cells were lysed, and the
32P-labeled Tvl-1 was immunoprecipiated, fractionated by
SDS-PAGE, electroblotted onto a nitrocellulose membrane, and visualized
by autoradiography. In the lower three panels, the
32P-labeled Tvl-1 bands were cut out from the membrane and
digested with trypsin. The resulting tryptic phosphopeptides were
separated electrophoretically on a thin-layer cellulose plate (pH 1.9),
followed by ascending chromatography in isobutyric acid buffer. The
phosphopeptides were visualized by autoradiography. Note that the
tryptic peptide map of Tvl-1 from serum-stimulated cells is similar to
that from the cells co-expressing active Raf-1(Y340D).
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Fig. 8.
Tvl-1 enhances the activation of Raf-1
induced by v-Ras/v-Src in Sf9 cells and by EGF in COS-1
cells. A, top panel: in vitro kinase reactions were
carried out using Raf-1 immunoprecipitated from lysates of Sf9
cells infected with the indicated baculovirus combinations. Bacterially
expressed kinase-dead His6-MEK1 was used as the exogenous
kinase substrate. Middle panel, Western blot of the total
infected cell lysates probed with anti-Raf, anti-Ras, and anti-Src
antibodies. Bottom panel, quantitation of the Raf-1 kinase
activity from the top panel by PhosphorImager. B,
COS-1 cells were transfected with pEBG (lanes 1 and
2) or pEBG-Raf-1 expression construct (lanes
3-6) alone or in combination with the pCMV-FLAG-Tvl-1 expression
construct (lanes 4 and 5). Twenty-four hours
after transfection, cells were serum-starved overnight, and stimulated
with EGF (50 ng/ml) for 20 min. Cells were lysed and GST-Raf-1 was
pulled down for kinase assays using recombinant kinase-dead
His6-MEK1 as the exogenous substrate (top
panel). Total cell lysates were Western blotted and probed with
anti-Raf-1 or anti-FLAG antibody (lower two panels). The
experiments in both A and B were repeated three
times with similar results.
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ACKNOWLEDGEMENTS |
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We thank N. Grammatikakis for providing pEBG-Raf-1 construct, J. Chernoff for providing the v-Src, Ras, and Raf-1 baculovirus constructs, and D. Morrison for providing the wild type and mutant Raf-1 constructs. We also thank all members of the Tsichlis lab for helpful discussions, and J. Estojak for technical help.
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FOOTNOTES |
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* This work was supported by in part United States Public Health Service Grant RO1-CA38147 (to P. N. T.), American Cancer Society Grant RPG-94-025-04-CCG (to E. G.), United States Public Health Service Grant CA06927, and by an appropriation from the Commonwealth of Pennsylvania to the Fox Chase Cancer Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF123704.
b Contributed equally to the results of this work.
c Supported by National Institutes of Health Postdoctoral Training Grant T32-CA09683.
d Fellow of the Leukemia Society of America, Inc. Present address: Mediterranean Agronomic Institute, Alsylion agrokepion, P. O. Box 85, 73100, Chania, Crete, Greece.
e Present address: Dana Farber Cancer Institute, Boston, MA.
g Special Fellow of the Leukemia Society of America, Inc.
j To whom correspondence should be addressed: Kimmel Cancer Center, Thomas Jefferson University, 233 10th St. Philadelphia, PA 19107. Tel.: 215-503-0689; Fax: 215-503-1607; E-mail: P_Tsichlis{at}lac.jci.tju.edu.
2 J-H. Lin and P. N. Tsichlis, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: EGF, epidermal growth factor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.
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