From INSERM U343 and Laboratoire d'Immunologie,
Hôpital de l'Archet I, Nice 06202, France and the
¶ Division of Cell Biology, La Jolla Institute for Allergy and
Immunology, San Diego, California 92121
Received for publication, September 17, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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Lymphocyte stimulation by immunoreceptors
is achieved through the activation of multiple signaling pathways
leading to cytokine gene transcription. Adapter proteins are critical
signaling components that can integrate multiple pathways by allowing
the assembly of multimolecular signaling complexes. We previously
showed that the cytoplasmic adapter 3BP2 (also known as SH3BP2)
promotes NFAT/AP-1 transcriptional activities in T cells through the
activation of Ras- and calcineurin-dependent pathways.
However, the molecular mechanisms by which 3BP2/SH3BP2 regulates cell
signaling and activation remain poorly documented. In this study, using
a combination of yeast two-hybrid analysis and biochemical approaches,
we present evidence for a physical interaction between 3BP2 and the
chaperone protein 14-3-3. This interaction was direct and
constitutively detected in yeast and in mammalian cells. Phorbol ester,
pervanadate, and forskolin/isobutylmethylxanthine stimulations
enhanced this interaction, as well as co-expression of constitutive
active mutants of serine/threonine kinases, including protein kinase C. We found that dephosphorylation of 3BP2 by alkaline phosphatase
disrupted its interaction with 14-3-3 and that 3BP2 was a substrate of
purified protein kinase C in vitro, suggesting that the
phosphorylation of 3BP2 by upstream kinases was required for 14-3-3 binding. Using deletion mutants of 3BP2, two 14-3-3 binding domains
were mapped to two proline-rich (residues 201-240 and 270-310)
domains of 3BP2. These domains were shown to contain two 14-3-3 consensus binding motifs. We identified residues
Ser225 and Ser277 of 3BP2 as being
essential for interaction with 14-3-3 family proteins, optimal 3BP2
serine phosphorylation, and then for 3BP2-dependent function. Indeed, a 3BP2 mutant protein incapable of binding 14-3-3 showed increased capacity to stimulate NFAT transcriptional activities, suggesting that 14-3-3 binding to 3BP2 negatively regulates 3BP2 adapter function in lymphocytes.
Antigen receptor engagement on lymphocytes induces the
activation of the protein-tyrosine kinases
(PTKs)1 of the Src and Syk
families. In turn, the activated PTKs promote the assembly of
intracellular protein complexes that transduce signals to the cytoplasm
and nucleus (1). In recent years, it has become apparent that adapter
molecules play critical roles in the coupling of receptor-proximal
events, such as protein-tyrosine kinases activation, to specific
signaling processes linked to cell growth and differentiation (2, 3).
Adapter proteins lack intrinsic catalytic activity, but they possess
motifs and domains capable of mediating protein-protein and
protein-lipid interactions (4). The assembly of large signaling
complexes, through the multiple interactions stabilized by adapters,
allows the activation of downstream effectors, including phospholipase C We previously showed that the cytoplasmic adapter protein 3BP2 (also
known as Abl SH3-binding protein 2 or SH3BP2) positively regulates T
cell signaling (9). 3BP2 has been originally described as an in
vitro binding partner of the SH3 domain of the PTK c-Abl (10).
3BP2 is composed of a N-terminal pleckstrin homology domain, a central
proline-rich (PR) region that interacts with c-Abl, and a C-terminal
SH2 domain. Human 3BP2 transcripts are ubiquitously expressed but can
be preferentially found in spleen, in peripheral blood leukocytes, and,
to a lesser extent, in thymus (9, 11). During a screen for Syk
kinase-interacting proteins in lymphocytes, we found a direct,
activation-induced interaction of 3BP2 with the SH2 domain of Syk and
Zap-70. In addition, the SH2 domain of 3BP2 was also found to associate
in vitro several tyrosine-phosphorylated proteins including
PLC The 14-3-3 protein family constitutes a highly conserved group family
of molecules present in high abundance in all eukaryotic cells (15,
16). They mediate signal transduction, cell cycle control, apoptosis,
stress response, and malignant transformation by binding to
phosphoserine-containing proteins such as c-Raf, Cdc25c, and BAD (17).
14-3-3 proteins are considered as scaffold molecules modulating
protein-protein interactions, protein subcellular localization, and
enzyme activities. Evidences of their involvement in lymphocyte
signaling have been provided. For example, it has been shown that the
binding of 14-3-3 In this report, we have identified a novel interaction between 14-3-3 proteins and the adapter 3BP2. By using a yeast two-hybrid system,
pull-down assays and Western ligand blot analysis, we show that 3BP2
specifically and directly associates in vitro and in
vivo with 14-3-3. Our studies also provide ample evidence that 3BP2 interacts with 14-3-3 via a serine
phosphorylation-dependent mechanism and that PKC is one of the
Ser/Thr kinases that phosphorylates 3BP2 to facilitate this
association. 3BP2 deletion analysis revealed the presence of two 14-3-3 binding sites located to the central proline-rich domain of 3BP2 and
containing two serine residues (Ser225 and
Ser277) critical for 14-3-3/3BP2 interaction. Finally, gene
reporter analysis showed that mutation of the 14-3-3 binding motif on
3BP2 increases its ability to activate NFAT in T and B lymphoid cells, suggesting that 14-3-3 interaction with 3BP2 negatively regulates its
adapter function in lymphocytes.
Antibodies and Reagents--
Culture media, oligonucleotides,
and enzymes were from Invitrogen. The chemicals were obtained from
Sigma. Antibodies specific for Cbl, Raf-1, 14-3-3, and GST were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Anti-Myc (9E10) and anti-hemagglutinin (HA; 12CA5) monoclonal
antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY) and
Roche Diagnostics, respectively. The GST 3BP2 SH2 domain fusion protein
was previously described (9). Polyclonal antibodies against 3BP2 were
prepared following rabbit immunization with the GST 3BP2 SH2 domain
fusion protein. Alkaline phosphatase was from Roche Diagnostics, and
purified catalytic subunit of rat brain PKC was obtained from
Calbiochem.
Plasmids--
The yeast expression plasmids pLex9, pACTII, and
the HA-tagged 3BP2 constructs in the expression vector pMT3 were
described before (9). 3BP2 mutants were subcloned into a modified
version of the pLex9 vector using an EcoRI fragment obtained
from the pMT3 HA-3BP2 constructs. All of the additional point and
deletion mutants were generated with the QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA). The mutations were verified
by DNA sequence analysis. In-frame insertion of the murine 3BP2
cDNA into the pCS3 6-Myc vector was performed by PCR using High
Fidelity Platinium Taq DNA polymerase (Invitrogen). The NFAT
luciferase reporter construct was from G. Crabtree (Stanford, CA).
Constitutively active PKA, Akt, and PKC Cell Culture and Transfection--
The cells were obtained from
the American Type Culture Collection (Manassas, VA). The human leukemic
Jurkat, U937 monocytic, and Burkitt's lymphoma Daudi cells were grown
in RPMI 1640 medium (Invitrogen), supplemented with 10% fetal calf
serum (Dutcher, Brumath, France), 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, nonessential
amino acids solution, and 100 units/ml of penicillin and streptomycin.
Cells grown in a logarithmic growth phase were transfected with the
indicated amounts of plasmids by electroporation using the Gene Pulser
apparatus (Bio-Rad). Simian COS-1 cells were cultured in Dulbecco's
modified Eagle's medium (Invitrogen) with 10% fetal calf serum.
1 × 107 cells were plated the day before
transfection. Transfections were then performed by the calcium
phosphate precipitation method.
Yeast Two-hybrid System--
Growth and transformation of the
yeast strain L40 were performed as described before (23). Briefly, a
human lymphocyte cDNA library encoded by the yeast two-hybrid
vector pACTII (Clontech) was transformed into the
yeast strain L40 that was previously transformed by the pLex9 3BP2 bait
vector. The transformed cells were plated on synthetic drop-out medium
lacking tryptophan, leucine, histidine, and uracil and grown for 4 days
at 30 °C. The colonies were then assayed for GST Pull-down Assays--
GST fusion proteins were expressed in
BL21 bacteria with 1 mM
isopropyl- Immunoprecipitation, Immunoblotting, and Western Ligand Blot
Analysis--
The cells were lysed at 1 × 108
cells/ml in ice-cold lysis buffer for 30 min on ice. Cleared lysates
were then incubated for 3 h at 4 °C with the indicated
antibodies and for 1 h with protein G-Sepharose beads (Sigma). The
pellets were then washed three times with ice-cold lysis buffer
containing 0.2% Nonidet P-40 and resuspended in SDS sample buffer.
Eluted immunoprecipitates or whole cell lysates were separated by
SDS-PAGE and analyzed by immunoblotting. SDS-PAGE-resolved samples were
transferred to nitrocellulose membranes and probed with primary and
horseradish peroxidase-conjugated secondary antibodies followed by
enhanced chemiluminescence detection. For Western ligand blot,
electrophoresed immunoprecipitates were transferred to membranes and
incubated with GST-14-3-3 In Vitro Phosphorylation and Dephosphorylation of
3BP2--
COS-1 cells were transiently transfected with 3BP2. After
cell lysate preparation, 3BP2 was immunoprecipitated using anti-3BP2 antibodies coupled to protein A-Sepharose beads for 3 h at
4 °C. The beads were then washed four times with ice-cold lysis
buffer containing 0.2% Nonidet P-40. For dephosphorylation of 3BP2,
the beads were washed twice with phosphatase buffer (50 mM
Tris-HCl, pH 8.5, 0.1 mM EDTA), resuspended in 50 µl of
phosphatase buffer, and incubated or not with alkaline phosphatase (20 units/ml; Roche Diagnostic). After 2 h at 30 °C, the beads were
spun down and eluted with SDS-PAGE buffer. Immunoprecipitates were then
subjected to Western ligand blotting with GST-14-3-3
For phosphorylation of 3BP2, the beads were washed twice in 20 mM HEPES (pH 7.5), resuspended in 30 µl of kinase
reaction mix (20 mM HEPES, pH 7.5, 3 mM
Fluorescent Staining and Confocal Microscopy--
The cellular
localization of 3BP2 and 14-3-3 proteins was performed as described
before (9). Briefly, COS-1 cells were transfected with the indicated
plasmids. After 48 h, the transfected cells were fixed with 3.7%
paraformaldehyde, permeabilized in phosphate-buffered saline containing
1% bovine serum albumin, 0.05% saponin, and processed for indirect
immunofluorescence microscopy. 3BP2 proteins were stained with anti-Myc
antibody, followed by incubation with a secondary fluorescein
isothiocyanate-labeled antibody to mouse immunoglobulins. 14-3-3 proteins were stained with anti-14-3-3 antibody, followed by incubation
with a secondary Texas Red-labeled antibody to rabbit immunoglobulins
(Jackson ImmunoResearch). The stained cells were analyzed with a Leica confocal laser scanning microscope.
Reporter Assays--
For luciferase assays, Jurkat TAg and Daudi
cells were transiently transfected with 5-15 µg of the NFAT reporter
plasmid together with the indicated expression plasmids. The luciferase
activity was assayed and normalized by luminometry as described before (23). The luciferase activity was determined in triplicate and expressed as fold increase relative to the basal activity seen in
unstimulated mock transfected cells.
Identification of 14-3-3 as 3BP2 Interacting Proteins--
In an
attempt to identify proteins able to bind to 3BP2, we employed the
yeast two-hybrid interaction analysis. 3BP2 was fused in frame to the
DNA-binding domain of LexA and used to screen a human lymphocyte
library. 2.5 × 106 transformants were screened. Of
the 48 clones selected for sequence analysis, three 14-3-3 Constitutive Interaction between 14-3-3 and 3BP2 in Resting
Cells--
Biochemical interaction between 3BP2 and 14-3-3 was next
tested by co-immunoprecipitation assay. COS-1 cells were transiently transfected with plasmids encoding Myc-tagged 3BP2, HA-tagged 14-3-3
To further localize the association of 3BP2 and 14-3-3, we used double
immunofluorescence staining and confocal microscopy and analyzed COS-1
cells co-transfected with Myc-tagged 3BP2 and HA-tagged 14-3-3. 3BP2
was mainly localized in the cytoplasm with a small fraction
constitutively associated with the membrane (Fig. 2D and
Ref. 9). 14-3-3 staining was found diffusely in the cytoplasm,
partially overlapping with 3BP2 staining (Fig. 2D, middle and right panels). This suggests that a
significant fraction of 3BP2 and 14-3-3 proteins co-localized in
COS-1 cells.
The Interaction between 14-3-3 and 3BP2 Is Direct and Increased by
Agonists of PKC and PKA Pathways--
Next, we used an in
vitro pull-down assay and a Western ligand blot technique to study
the effect of mitogenic stimulation on 3BP2/14-3-3 interaction. First,
COS-1 cells were transfected with Myc-tagged 3BP2, and stimulated with
PMA or pervanadate treatment. The cell lysates were incubated with
recombinant GST or GST-14-3-3
To determine whether the association between 3BP2 and 14-3-3
A similar in vitro pull-down assay using a GST-14-3-3 3BP2 Phosphorylation Increases 14-3-3 Binding--
To determine
whether phosphorylation on serine residues was involved in the
interaction between 3BP2 and 14-3-3, we immunoprecipitated 3BP2 from
resting COS-1 cells and treated these immune complexes with 20 units/ml
alkaline phosphatase. The samples were then analyzed for binding of
GST-14-3-3
PMA stimulation increases both 3BP2/14-3-3 interaction and PKC
activation. To determine whether PKC directly phosphorylates 3BP2, 3BP2
immunoprecipitates from transfected COS-1 cells were used in in
vitro kinase assay with a purified catalytic domain of rat brain
PKC. 3BP2 phosphorylation was not detectable in the absence of PKC.
When PKC was added, a band of phosphorylated 3BP2 was clearly visible
(Fig. 4B). Probing the membrane with anti-3BP2 antibody
showed that similar amounts of 3BP2 were immunoprecipitated in all of
the samples (bottom panel). These results support the notion
that PKC directly phosphorylates 3BP2 in vitro.
To further define the nature of the 3BP2/14-3-3 association, we
evaluated the ability of different Ser/Thr kinases to modulate it.
COS-1 cells were transfected with 3BP2-Myc expression vector and with
cDNAs encoding for constitutively active forms of PKA, Akt, and
PKC Mapping of the 14-3-3 Binding Domain on 3BP2--
Next, we used
the yeast-two hybrid system to map the minimum region of 3BP2 involved
in this interaction. Structurally, 3BP2 is composed of three regions: a
N-terminal pleckstrin homology domain, a central region containing
three proline-rich domains, and a C-terminal SH2 domain. Various
deletion mutants of 3BP2 fused to LexA protein were tested for binding
to Gal4-14-3-3 fusion proteins in yeast. As shown in Fig.
5A, the deletion of either the
pleckstrin homology domain (3BP2 130-553) or the SH2 domain (3BP2
1-455) did not alter the interaction with 14-3-3. In contrast, the
central proline-rich region of 3BP2 was essential for the 3BP2/14-3-3
interaction. In this region, we defined three PR domains based on the
presence of several putative SH3 domain binding sequences PXXP (4). The first (amino acids 201-240) and the second
(amino acids 270-310) proline-rich domains were particularly
important, as demonstrated by the decreased ability of the respective
deletion mutant to bind 14-3-3, whereas the third domain (amino acids
340-384) was not (Fig. 5A).
Next, COS-1 cells were transfected by different 3BP2-cDNA
constructs and activated or not with PMA 15 min at 37 °C. A Western ligand blot experiment was then done using a GST-14-3-3 Serine 277 in 3BP2 Is Critical for 14-3-3 Binding--
14-3-3
isoforms have been shown to bind conserved motifs in a manner dependent
on the phosphorylation of serine residues (24). We thus searched for
serine residues matching predicted consensus sequences within the first
and the second proline-rich domains of 3BP2. Two serine residues,
Ser225 and Ser277, were identified within such
consensus motifs in the first and the second proline-rich domain,
respectively (Fig. 6A). These two residues were substituted with alanine residues using site-directed mutagenesis, and 14-3-3/3BP2 interaction was analyzed using the two-hybrid system. 3BP2 S225A mutation decreased the interaction, whereas S277A mutation abolished it (Fig. 6B). Next, Jurkat
TAg cells were transfected with wild-type 3BP2, S225A, or S277A point mutants. Following cell treatment with PMA, the lysates were analyzed for GST-14-3-3 14-3-3 Negatively Regulates 3BP2-induced NFAT Activation in
Lymphoid Cells--
3BP2 is a positive regulator of NFAT activities in
T cells (9). The effect of 14-3-3 expression on 3BP2 activity was
investigated in transfection assays using a NFAT reporter gene. Jurkat
TAg cells were co-transfected with plasmids encoding NFAT reporter construct and wild-type 3BP2, 3BP2 S277A, 14-3-3, or a combination of
these plasmids. As previously described, 3BP2 overexpression potentiated NFAT activity, whereas 14-3-3 had the opposite effect (Fig.
7A). The expression of the
S277A mutant, which shows reduced binding to 14-3-3, led to a stronger
activation of NFAT relative to wild-type 3BP2. On the other hand,
14-3-3 overexpression completely prevented NFAT activation induced by
3BP2, whereas it only reduced by 50% the activity of the S277A mutant.
Immunoblot analysis with anti-3BP2 and anti-HA antibodies showed
similar levels of protein expression. To ascertain this finding in
another cell type, 3BP2 plasmids were transfected in Daudi B cells,
together with the NFAT reporter. As shown in Fig. 7B,
expression of 3BP2 S277A mutant in these cells led to a dramatic
increase of NFAT activation, compared with the effect of wild-type
3BP2.
PMA stimulation increased 14-3-3 binding to 3BP2 as demonstrated in
previous sections. We therefore questioned the effect of PMA on
3BP2-induced NFAT activation. Jurkat cells were electroporated with
plasmids encoding for NFAT reporter and wild-type 3BP2. One hour after
the transfection, the cells were washed and resuspended in culture
medium containing or not 50 ng/ml of PMA. The luciferase activities
were then determined 24 h after. Fig. 7C shows that PMA
stimulation causes a 55% decrease of 3BP2-induced NFAT activation, whereas it did not affect 3BP2 expression. This suggests that the PKC
phosphorylation might mediate 14-3-3 binding on 3BP2 and then inhibit
its positive effect on NFAT activity. Together, these results suggest
that 14-3-3 binding to 3BP2 following PMA stimulation negatively
regulates its positive adapter function in lymphoid cells.
In the present study, we found that the cytoplasmic adapter
protein 3BP2 (also known as SH3BP2) specifically and directly associates in vitro and in vivo with 14-3-3 proteins. This interaction, which is constitutively detected in yeast
and in mammalian cells, is enhanced by phorbol ester stimulation and
co-expression of different serine/threonine kinases, including PKC.
This interaction most likely requires the phosphorylation of 3BP2 by
upstream kinases because we found that: (i) treatment of 3BP2 by
alkaline phosphatase disrupts the 14-3-3/3BP2 interaction, (ii)
in vitro 3BP2 is a substrate of purified PKC, and (iii)
point mutation of two serine residues (Ser225 and
Ser277) disrupts 14-3-3/3BP2 interaction and decreased
PKC-dependent 3BP2 phosphorylation. Functionally, a 3BP2
mutant protein incapable of binding 14-3-3 was a more potent activator
of NFAT transcriptional activities than the wild-type protein,
suggesting that 14-3-3 binding to 3BP2 negatively regulates 3BP2
adapter function in lymphocytes.
14-3-3 constitutes a highly conserved and ubiquitously expressed family
of proteins. They function as dimeric scaffold proteins involved in the
formation of complexes with serine phosphorylated proteins (15, 24). A
consensus sequence that mediated phosphorylation-dependent association to 14-3-3 was defined as RSXpSXP
(16). However, other 14-3-3 binding sequences containing phosphorylated
serine have been identified (25-27). Interestingly, 3BP2 does not
contain the exact consensus sequence RSXpSXP.
However, by using deletion mutants of 3BP2, we mapped the
14-3-3-binding site on 3BP2 to a central proline-rich region (residues
201-310) that contains two serine-based putative 14-3-3-binding sites
lying in the sequences RAHS225FTS and
RRMS277DP. The first sequence matches an unconventional
14-3-3 binding sequence
RX1-2pS225X2-3S,
which was initially found in Cbl (25), whereas the second sequence
RXXpS277XP represents an almost
perfect match of the consensus 14-3-3 binding sequence found in several
14-3-3 binding proteins including Cdc25c, PKC Our studies do not identify the Ser/Thr kinases that directly
phosphorylate 3BP2 to increase its association with 14-3-3 proteins. However, they provide ample evidence that 3BP2 interacts with 14-3-3 via a serine phosphorylation-dependent mechanism and that PKC is one of the Ser/Thr kinases phosphorylating 3BP2 to facilitate this association. The two 14-3-3 binding motifs identified by our
experiments contain a basic arginine residue in position 3BP2 was initially cloned as an Abl SH3-binding protein of unknown
function (10). We next identified 3BP2 as a Syk kinase-interacting protein that plays a positive adapter function on NFAT and AP-1 transcriptional activation through the binding of its SH2 domain to the
T cell signaling proteins (9). More recently, a positive regulatory
function of 3BP2 downstream surface receptors coupled to cytoplasmic
PTKs was also reported in NK (12) and mast cells (13). In addition, our
studies have shown that 3BP2 is tyrosine-phosphorylated by several
PTKs, including Syk and Fyn (data not shown). Thus, 3BP2 adapter
functions appear to be linked to PTK-dependent signaling pathways through the capacity of its SH2 domain to interact with tyrosine-phosphorylated proteins and by being itself a PTK substrate. Our findings that 3BP2 also represents a target for serine/threonine kinases such as PKC and binds several 14-3-3 isoforms (namely 14-3-3
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ABSTRACT
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DISCUSSION
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(PLC
), guanine nucleotide exchange factors of the Vav
family, and phosphatidylinositol 3-kinase, which couple extracellular signals to small GTPases signaling, cytoskeletal reorganization, gene
expression, and, ultimately, to lymphocyte activation and functions (1,
5). For example, by SH2 domain-containing leukocyte
phosphoprotein of 76 kDa (SLP-76) and by linker for activation of T
cells (LAT), two adapter molecules expressed in T cells, are required
for proper activation of PLC
, intracellular calcium fluxes, and Ras
stimulation during T cell activation (6, 7). In an analogous manner,
the B cell linker protein (BLNK) is required for tyrosine
phosphorylation of PLC
and activation of Jun N-terminal kinase in
activated B lymphocytes (8).
1, c-Cbl, and LAT, proteins that have all been implicated in T
cell signaling pathways (9). We also showed that 3BP2 was a potent
inducer of both basal and TCR-mediated activities of NFAT and AP-1, two
transcription factors involved in interleukin 2 gene transcription.
Furthermore, 3BP2-induced gene activation in T cell required the
activation of a cyclosporin A-sensitive signaling pathway and of a
Ras-dependent signaling pathway (9). More recently, 3BP2
was shown to also have a positive regulatory role during the
development of NK cell-mediated cytotoxicity through the binding of
several signaling molecules like PLC
1 and Vav1 (12) and to regulate
Fc
R1-mediated degranulation in basophilic cells (13). Finally,
mutations in the 3bp2/sh3bp2 gene have been
linked to a rare human disease of childhood characterized by
proliferative lesions within the mandible and maxilla, suggesting that
3BP2 could be implicated in osteoclasts differentiation (14). However,
the exact role of 3BP2 in hematopoietic/lymphoid cell signaling remains
ill-defined.
to PKC
prevents its translocation to the
membrane, thereby decreasing its ability to activate the interleukin 2 promoter (18). In addition, the interaction of 14-3-3 with the
catalytic subunit of the phosphatidylinositol 3-kinase and the Cbl
protooncogene has been involved in the regulation of the
Ras-dependent pathway leading to NFAT activation
(19-21).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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expression vectors were
provided by M. Montmigny, B. Hemmings, and A. Altman, respectively.
Plasmids encoding 14-3-3
in frame with Gal4 activation domain, GST,
and HA were previously described (22).
-galactosidase
activity using a
-galactosidase filter assay (23). After a second
round of screening performed with a LexA-lamin plasmid as a negative
control, library plasmid cDNAs were rescued, sequenced, and
identified using the BLAST algorithm.
-D-thiogalactopyranoside for 3 h at
37 °C. The proteins were affinity purified using
glutathione-Sepharose beads. Purified proteins were resolved by
SDS-PAGE and analyzed by Coomassie staining. For GST precipitations,
the cells were lysed at 1 × 108 cells/ml in ice-cold
lysis buffer (1% Nonidet P-40 in 150 mM NaCl, 50 mM HEPES, pH 7.4, 5 mM NaF, 5 mM
sodium pyrophosphate, 1 mM sodium orthovanadate, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl
fluoride) for 30 min on ice. The lysates were clarified by
centrifugation at 15,000 × g for 15 min at 4 °C and
incubated with 10 µg of the indicated GST fusion protein and
glutathione-Sepharose beads (Sigma) for 3 h at 4 °C. After extensive washing in lysis buffer, the bound proteins were detected by
immunoblotting as indicated below.
(10 µg/ml) for 16 h at 4 °C.
After extensive washings, the membranes were successively incubated
with anti-GST and horseradish peroxidase-conjugated antibodies followed
by enhanced chemiluminescence detection.
as described above.
-mercaptoethanol, 10 mM MgCl2, 0.1 µg/ml
bovine serum albumin, 20 µCi of [
-32P]ATP, and 0.1 unit/ml of purified catalytic subunit of PKC (Calbiochem, Germany) and
incubated at 30 °C for 1 h. The reactions were stopped by the
addition of 5× hot sample buffer and run on SDS-PAGE. The gel was then
transferred to nitrocellulose membrane and exposed to film. After
autoradiography, the membrane was saturated and subjected to immunoblot
analysis using anti-HA to quantitate the levels of 3BP2 proteins in the
kinase reaction.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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clones,
one 14-3-3
clone, and one 14-3-3
clone were identified by
comparison with the nucleotide data base at the National Library of
Medicine using the BLAST algorithm (Fig.
1). For further experiments, we elected
to use the 14-3-3
isoform, which is expressed abundantly in lymphoid
cells.
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Fig. 1.
Identification of 14-3-3 as 3BP2 interacting
proteins by yeast two-hybrid system. A, a yeast two-hybrid
screen of a human lymphocyte library using LexA-3BP2 as bait was
performed. Shown are the accession numbers of the different 14-3-3 cDNAs isolated during this screen and the number of independent
clones for each cDNA. B, yeast were co-transformed with
plasmids encoding LexA-3BP2, and either Gal4 activation domain alone,
Gal4 activation domain (GAD)-14-3-3 ,
, or
, and
interactions were assayed using a
-galactosidase filter assay.
Similar results were obtained by assessing yeast growth on
histidine-deficient medium. A LexA-lamin expression vector was used as
a negative control.
, or empty vectors. Lysates from unstimulated cells were incubated with anti-HA antibody, resolved by SDS-PAGE, and
immunoblotted with anti-Myc antibody. As shown in Fig.
2A, 3BP2 co-immunoprecipitated with 14-3-3, indicating that there is a constitutive complex in resting
COS-1 cells between 3BP2 and 14-3-3. This result was confirmed by an
in vitro pull-down assay using GST-14-3-3
(Fig.
2B). Lysates of COS-1 cells transfected with Myc-tagged 3BP2
were incubated with recombinant GST or GST-14-3-3
fusion protein.
3BP2 bound to immobilized GST-14-3-3
fusion proteins but not to GST
protein when lysate proteins were eluted from immobilized GST proteins and immunoblotted with anti-Myc antibody. To further demonstrate that
3BP2 interacts with 14-3-3, we immunoprecipitated endogenous 3BP2 (or
Raf-1 as control) from U937 cells (Fig. 2C). Western blot
with anti-14-3-3 antibody showed that 3BP2 co-immunoprecipitated with a
significant fraction of endogenous 14-3-3 proteins in resting monocytic
U937 cells, reaching 0.5-1% of total 14-3-3
, as judged by Western
blot analysis of one-tenth of total cell lysate. Binding of 14-3-3
to Raf-1 is also showed for comparison (Fig. 2C).
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Fig. 2.
Constitutive interaction between 14-3-3 and
3BP2 in resting cells. A, 3BP2 co-immunoprecipitates
with 14-3-3 in resting cells. COS-1 cells were transiently transfected
with expression plasmids encoding Myc-tagged 3BP2, HA-tagged 14-3-3 ,
or both, as indicated. The lysates (0.5 × 107 cell
equivalents) were immunoprecipitated with 5 µg of anti-HA antibody
and protein G-Sepharose beads. The washed beads were analyzed by
Western blotting with anti-Myc antibody. Fractions of the same lysates
(0.5 × 106 cell equivalents) were directly
subjected to Western blot analysis with anti-Myc antibody as
transfection controls. The membranes were then stripped and reprobed
with anti-HA antibody to control 14-3-3 protein expression.
B, COS-1 cells were transiently transfected with expression
plasmid encoding Myc-tagged 3BP2. The lysates were precipitated with 10 µg of GST or GST-14-3-3
and recovered with glutathione-Sepharose
beads. The washed beads were subjected to SDS-7.5% PAGE, transferred
onto nitrocellulose membrane, immunoblotted with anti-Myc, and
visualized with ECL. C, lysates from U937 cells were
precipitated with anti-3BP2 or anti-Raf1 antibodies and protein
A-Sepharose beads. The washed beads were subjected to SDS-7.5% PAGE,
transferred onto nitrocellulose membrane, immunoblotted with
anti-14-3-3, anti-3BP2 or anti-Raf1 antibodies, and visualized with
ECL. D, 3BP2 co-localizes with 14-3-3
in resting cells.
COS-1 cells were transiently transfected with expression plasmids
encoding Myc-tagged 3BP2 and HA-tagged 14-3-3. After fixation and
permeabilization, the expressed proteins were detected by staining with
anti-Myc (green) and anti-14-3-3 (red)
antibodies. The localization of 3BP2 and 14-3-3 was examined by
immunofluorescence staining and confocal microscopy analysis. The
right panel is a superposition of the two stainings, in
which COS-1 stained regions appear in yellow. The
scale bar represents 10 µm. IP,
immunoprecipitation.
fusion protein, and bound proteins,
which were recovered with glutathione-Sepharose beads, were subjected
to SDS-PAGE and immunoblotting with anti-Myc antibody. Raf-1, which
associates directly with 14-3-3 proteins, was used here as a positive
control. More 3BP2 was associated with GST-14-3-3
in lysates from
PMA or pervanadate-stimulated cells than in unstimulated samples (Fig.
3A).
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Fig. 3.
The interaction between
14-3-3 and 3BP2 is direct and increased by
agonists of PKC and PKA pathways. A, COS-1 cells were
transfected with 3BP2-Myc expression vector. After 48 h, they were
either left unstimulated or stimulated for 15 min at 37 °C with PMA
(100 ng/ml) or pervanadate (10 µM). The lysates (0.5 × 107 cell equivalents) were precipitated with 10 µg of
GST or GST-14-3-3
and recovered with glutathione-Sepharose beads.
The washed beads were subjected to SDS-7.5% PAGE, transferred onto
nitrocellulose membrane, immunoblotted with antibodies against Myc
epitope or Raf, and visualized with ECL (left panel).
Fractions of the same lysates (0.5 × 106 cell
equivalents) were directly subjected to Western blot analyses with
anti-Myc or anti-Raf antibodies (right panel). B,
COS-1 cells transfected with 3BP2-Myc vector were stimulated or not
with PMA for 15 min at 37 °C. The lysates were immunoprecipitated
with anti-Myc antibody and protein G-Sepharose. Immunoprecipitates were
resolved by SDS-7.5% PAGE and transferred onto nitrocellulose
membrane. The membranes were incubated with GST-14-3-3
, and binding
was detected with anti-GST antibody and ECL. The membrane was then
stripped and reprobed with anti-Myc antibody. C, COS-1 cells
were transfected with 3BP2-Myc expression vector and with cDNA
encoding constitutively active Ras. The lysates were analyzed with
pull-down assay using GST-14-3-3
and anti-Myc immunoblotting like
above. D, COS-1 cells transfected with HA-3BP2 vector were
either stimulated or unstimulated with forskolin (10 µM)
and isobutylmethylxanthine (100 µM) for 15 min at
37 °C. The lysates were immunoprecipitated with anti-HA antibody and
subjected to Western ligand blot with GST-14-3-3
. IP,
immunoprecipitation; PV, pervanadate.
is
direct, we used the Western ligand blot technique. COS-1 cells were
transfected with Myc-tagged 3BP2, either unstimulated or stimulated
with PMA, and the lysates were immunoprecipitated with anti-3BP2
antibody. Electrophoresed precipitated 3BP2 was probed with a
recombinant GST-14-3-3
fusion protein, and binding was detected with
anti-GST monoclonal antibody (Fig. 3B). As expected, GST-14-3-3
bound immunoprecipitated 3BP2 from both unstimulated and
activated cells. Immunoblotting of the same membrane with anti-3BP2
antibody showed that a similar amount of 3BP2 proteins was present in
immunoprecipitates from resting and PMA-stimulated cells.
was
performed from unstimulated COS-1 cells transfected with plasmids encoding for 3BP2-Myc without or with a constitutively active form of
Ras. As shown in Fig. 3C, activation of Ras mitogenic pathway led to increases 3BP2/14-3-3 interaction. Next, COS-1 cells
were transfected with HA-tagged 3BP2 construct and either unstimulated
or stimulated with PKA agonists, forskolin, and isobutylmethylxanthine. The lysates were immunoprecipitated with anti-HA antibody, and Western
ligand blot was performed as described above. Fig. 3D shows
that cell stimulation with forskolin and isobutylmethylxanthine increased the basal binding of 14-3-3
to 3BP2. Altogether, our results indicate that the constitutive and direct association between
14-3-3 and 3BP2 is reinforced when cells are stimulated by agonists of
PKC and PKA pathways.
by Western ligand blotting. Treatment of anti-HA
precipitates with alkaline phosphatase totally abrogated 3BP2/14-3-3
interaction (Fig. 4A).
Stripping and reprobing the membrane with anti-HA antibody showed that
similar amounts of 3BP2 were present in all samples. These results
suggest that phosphorylation of 3BP2 was required for its association
with 14-3-3.
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Fig. 4.
3BP2 phosphorylation increases 14-3-3 binding. A, dephosphorylation of 3BP2 disrupts its
interaction with 14-3-3. COS-1 cells were transfected with HA-3BP2
expressing vector. After 48 h, the cells were left unstimulated or
stimulated with PMA (100 ng/ml) for 15 min at 37 °C. The lysates
were immunoprecipitated with anti-HA antibody and protein G-Sepharose
beads. HA immunoprecipitates from unstimulated or PMA-stimulated cells
were incubated without or with alkaline phosphatase for 2 h at
30 °C. The samples were then separated by SDS-7.5% PAGE,
electrotransferred onto nitrocellulose membrane, and analyzed by
probing with GST-14-3-3 followed by detection with anti-GST
antibody. The membrane was then stripped and reprobed with anti-HA
antibody. B, 3BP2 is phosphorylated by PKC in
vitro. HA-tagged 3BP2 was expressed in COS-1 cells and
immunoprecipitated with anti-HA and protein G-Sepharose beads. The
immunoprecipitates were incubated without or with a purified catalytic
subunit of rat PKC (0.1 unit/ml) and with 20 µCi of
[
-32P]ATP at 30 °C for 1 h. The samples were
run on SDS-PAGE (7.5% gel), and the gel was then dried and exposed to
film. After autoradiography, the membrane was subjected to immunoblot
analysis using anti-3BP2. C, COS-1 cells were transfected
with 3BP2-Myc expression vector and with cDNAs encoding
constitutively active PKA, Akt, and PKC
. The lysates were subjected
to pull-down assay using GST-14-3-3
, and nitrocellulose membranes
were immunoblotted with anti-Myc or anti-HA antibodies as indicated and
visualized with ECL. IP, immunoprecipitation.
. The lysates were incubated with GST-14-3-3
fusion protein or
GST alone as control, and binding was detected with anti-Myc antibody
(Fig. 4C). Surprisingly, all of the Ser/Thr kinases tested
increased the interaction between 14-3-3 and 3BP2, suggesting that 3BP2
could be a substrate for several Ser/Thr kinases, including PKC.
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Fig. 5.
Mapping of the 14-3-3 binding domain on
3BP2. A, yeast two-hybrid interactions. The indicated
3BP2 mutants were fused to the DNA-binding domain of LexA, whereas
14-3-3 ,
, and
cDNAs were fused to Gal4 activation domain.
Yeast were co-transformed with the indicated plasmid combinations, and
interactions were assayed using a
-galactosidase filter assay.
,
no interaction; ++, strong interaction. Similar results were obtained
by assessing yeast growth on histidine-deficient medium. A LexA-lamin
expression vector was used as a negative control. B, COS-1
cells were transfected with 10 µg of empty pMT3 expression vector or
the indicated 3BP2-cDNA constructs. The cells were unstimulated or
stimulated with PMA (100 ng/ml) for 15 min at 37 °C. HA
immunoprecipitates were resolved by SDS-7.5% PAGE and transferred onto
nitrocellulose membrane. The membranes were incubated with
GST-14-3-3
, and binding was detected with an anti-GST antibody and
ECL. The membrane was then stripped and reprobed with anti-HA antibody.
IP, immunoprecipitation.
fusion protein. Immunoblotting with anti-HA antibody was used to normalize for
expression of each 3BP2 construct. In resting and PMA-stimulated cells,
the interaction was lost when the first or second PR domain of 3BP2
were deleted. The third PR domain, which does not contain any putative
14-3-3 binding site, was not required for this interaction (Fig.
5B). These results show that two PR domains in 3BP2 are critical for 14-3-3 binding.
binding by Western ligand blotting. 14-3-3 binding was perturbed by the S225A mutation and completely abolished by the
S277A mutation (Fig. 6C). Immunoblotting with anti-3BP2
antibody was used to normalize for expression of each 3BP2 construct.
Together, these results show that 3BP2 serine residues
Ser225 and Ser277 are required for basal and
stimulation-dependent association between 3BP2 and 14-3-3. To test whether serine residues Ser225 and
Ser277 are important for PKC-dependent 3BP2
phosphorylation, COS-1 cells were transfected with plasmids encoding
HA-tagged 3BP2 or S225A or S277A mutants, and an in vitro
kinase assay was performed as described above. c-Cbl was used here as
substrate to control PKC activity under our experimental conditions.
Fig. 6D shows a reduced level of phosphorylation of 3BP2
when serine 225 or 277 was mutated to alanine. Apparently, the effect
of mutation of serine 277 to alanine had a greater impact on 3BP2
phosphorylation by PKC, with an ~50% decrease, an effect consistent
with the decreased binding of 14-3-3 shown above. Together, this
suggests that serines 225 and 277 are required for optimal 3BP2
phosphorylation by PKC.
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Fig. 6.
Serine 277 in 3BP2 is critical for 14-3-3 binding. A, identification of 14-3-3 consensus binding
motifs in 3BP2. Shown is the alignment of PR1, PR2, and PR3 amino acid
sequences of murine and human 3BP2. Two conserved binding sites for
14-3-3 on 3BP2 are boxed, and the putative phosphorylated
serine residues indicated. B, yeast two-hybrid interactions.
The indicated serine-to-alanine 3BP2 mutants were fused to the
DNA-binding domain of LexA, whereas 14-3-3 ,
, and
cDNAs
were fused to Gal4 activation domain (GAD). Yeast were
co-transformed with the indicated plasmid combinations, and the
interactions were assayed using a
-galactosidase filter assay.
,
no interaction; ++, strong interaction. Similar results were obtained
by assessing yeast growth on histidine-deficient medium. A LexA-lamin
expression vector was used as a negative control. C, Jurkat
TAg cells were transfected with 10 µg of empty pMT3 expression vector
or the indicated 3BP2-cDNA constructs. The cells were unstimulated
or stimulated with PMA (100 ng/ml) for 15 min at 37 °C. 3BP2
immunoprecipitates were resolved by SDS-7.5% PAGE and transferred onto
nitrocellulose membrane. The membranes were incubated with
GST-14-3-3
, and binding was detected with anti-GST antibody and ECL.
The membrane was then stripped and reprobed with anti-3BP2 antibody.
D, HA-tagged 3BP2 constructs or HA-tagged c-Cbl were
expressed in COS-1 cells and immunoprecipitated with anti-HA and
protein G-Sepharose beads. The immunoprecipitates were incubated with a
purified catalytic subunit of rat PKC (0.1 unit/ml) and with 20 µCi
of [
-32P]ATP at 30 °C for 1 h. The samples
were run on SDS-PAGE (7.5% gel); the gel was then dried and exposed to
film. After autoradiography, the membrane was subjected to immunoblot
analysis using anti-HA antibody. IP,
immunoprecipitation.
View larger version (22K):
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Fig. 7.
14-3-3 negatively regulates 3BP2-induced NFAT
activation in lymphoid cells. A, Jurkat TAg cells were
transfected with a combination of HA-3BP2, HA-3BP2 S277A, and
HA-14-3-3 expression vectors as indicated, together with
NFAT-luciferase reporter. After 24 h, the cells were harvested and
assayed for luciferase activity. Expression levels of 3BP2 and 14-3-3 were detected by immunoblotting with anti-3BP2 or anti-HA antibodies,
respectively. B, NFAT activity was assayed in Daudi B cells
transfected with an empty pMT3 vector or a pMT3 vector encoding HA-3BP2
or HA-3BP2 S277A. Expression levels of 3BP2 proteins were detected by
immunoblotting with anti-3BP2 antibody. C, Jurkat TAg cells
were transfected with an empty pMT3 vector or a pMT3 vector encoding
HA-3BP2, together with NFAT-luciferase reporter construct. One hour
after transfection, the cells were left unstimulated or stimulated with
PMA (100 ng/ml). Luciferase activity was determined 24 h after
transfection. The inset at the top of
C represents an anti-3BP2 immunoblot showing the expression
of the transfected 3BP2 protein.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, BAD, and Raf-1 (16).
This second motif differs from the consensus only by the absence of a
serine residue in position
2 relative to the putative phosphorylated
serine residue. Nevertheless, Western ligand blot and yeast two-hybrid
analysis of 3BP2 point mutations showed that a mutant of 3BP2 in which serine 277 was replaced by a nonphosphorylatable alanine (S277A) failed
totally to bind 14-3-3, when 3BP2 Ser225 mutant only
displayed a decreased ability to bind 14-3-3. Several 14-3-3 binding
proteins such as Raf-1, c-Cbl, and BAD contain two 14-3-3 binding
sequences separated by polypeptides fragments of various length, and
tandem binding to adjacent 14-3-3 sites has been shown to facilitate
the formation of high affinity dimeric complexes (24). Recently, it has
been postulated that a first site called "gatekeeper" is
indispensable for a stable 14-3-3 interaction, whereas a second site
enhances the interaction but has too weak an affinity to bind 14-3-3 alone (24). Consistent with this model, our studies suggest that 3BP2
Ser277 residue might constitute the gatekeeper residue,
whereas Ser225 might represent the "enhancer" residue,
to facilitate 14-3-3 dimer fixation on 3BP2. Additional experiments are
clearly needed to ascertain the mechanism of 14-3-3/3BP2 interaction.
For example, it would be interesting to perform phosphoamino acid
analysis on serine-mutated 3BP2 proteins.
3 relative
to the putative phosphorylated serine residue. Such a sequence could
represent a consensus phosphorylation motif for various Ser/Thr
kinases, including PKA, PKC, or calmodulin-dependent protein kinase II (28). Indeed, co-expression of 3BP2 with constitutive active PKA and PKC in COS-1 cells enhanced 3BP2/14-3-3 interaction. Our
observation that co-expression of active Akt (PKB) also led to a
stronger interaction between 14-3-3 and 3BP2 is the likely result of an
indirect phosphorylation event because 3BP2 does not contain a
consensus phosphorylation sequence as previously defined (29).
Moreover, the basal interaction between 3BP2 and 14-3-3 was reinforced
following cell stimulation with PMA, a known PKC activator, and
treatment of wild-type 3BP2 with alkaline phosphatase significantly
reduced the amount of 3BP2 that binds to 14-3-3. In addition, 3BP2 was
phosphorylated by purified rat brain PKC in vitro, and a
reduced phosphorylation of 3BP2 by PKC was observed when serine 277 was
mutated to alanine, indicating that this residue is a direct target for
PKC at least in vitro. This effect is also consistent with a
marked decreased binding of 14-3-3 to the 3BP2 S277A mutant. Finally,
stimulation of Jurkat T cells with PKA agonists (forskolin and
isobutylmethylxanthine) also increased the association between 14-3-3 and 3BP2, suggesting that 3BP2 can be directly or indirectly
phosphorylated by different Ser/Thr kinases to induce its interaction
with 14-3-3.
,
, and
isoforms) suggest that 3BP2 could integrate PTK- and PKC-dependent signaling pathways in various cell
types, including lymphocytes. It is interesting to note that in T cells a similar function was attributed to c-Cbl (25), and PLC
(30), two
signaling proteins found to interact with 3BP2 SH2 domain (9). However,
the role of 3BP2 serine phosphorylation is presently unclear. Our
studies show that, relative to wild-type 3BP2, the S277A mutant, which
is incapable of binding 14-3-3, was a more potent activator of NFAT in
both T and B cells. Also, under conditions promoting the association
between 14-3-3 and 3BP2, PMA stimulation decreased 3BP2-induced NFAT
activation in Jurkat T cells. Together, these results suggest that
14-3-3 may negatively regulates 3BP2 adapter function on the signaling
pathways leading to NFAT transcriptional activities. In our model,
activation of Ser/Thr kinases like PKC (or PKA) following receptor
stimulation would lead to serine phosphorylation of 3BP2. This would
allow the direct binding of 14-3-3 dimers on 3BP2 (Fig.
8). Subsequent events leading to
inhibition of 3BP2 function in NFAT pathway by 14-3-3 remain elusive.
14-3-3 binding could modify 3BP2 subcellular localization in a manner
similar to the regulation of PKC
(18) or the Ras effector RIN1 (31). Alternatively, the binding of 14-3-3 to 3BP2 could prevent the formation of 3BP2-associated protein complexes, including proteins like
Syk family PTKs (9), Vav1 (Ref. 12 and our unpublished observations),
PLC
(9, 12, 13), and LAT (9, 13), which are required for signal
transduction.
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Fig. 8.
A model for 3BP2 and 14-3-3 proteins
interaction. Phosphorylation of 3BP2 by Ser/Thr kinases, like PKC,
permits recruitment of 14-3-3 dimers, which in turn leads to
down-modulation of 3BP2-dependent gene activation.
Growing evidence supporting the importance of adapter proteins has been
presented in numerous signal transduction cascades (3, 4). Together
with our previous finding that 3BP2 regulate a Ras- and a
calcineurin-dependent pathways, respectively, required for
AP-1 and NFAT activation in T cells (9), the results reported in the
present study indicate that 3BP2 may also play an important role as an
integrator of multiple signaling pathways, opening the way to future
investigations aimed at deciphering the function of 3BP2 in immune cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Amnon Altman for reagents and continuous support, Anne Doye for confocal microscopy, and Séverine Le Bras and Céline Chavet for discussion.
![]() |
FOOTNOTES |
---|
* This work was supported by INSERM, the Fondation pour la Recherche Médicale, the Association pour la Recherche sur le Cancer, and the Ministère de l'Enseignement Supérieur et de la Recherche.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.
§ Recipient of a doctoral fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche.
To whom correspondence should be addressed: INSERM U343, IFR
50, Hôpital de l'Archet I, 06202 Nice Cedex 3, France. Tel.: 33-4-92-15-77-00; Fax: 33-4-92-15-77-09; E-mail:
deckert@unice.fr.
Published, JBC Papers in Press, December 24, 2002, DOI 10.1074/jbc.M209509200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PTK, protein-tyrosine kinase;
PLC, phospholipase C
;
SH, src
homology;
PR, proline-rich;
PMA, phorbol 12-myristate 13-acetate;
GST, glutathione S-transferase;
HA, hemagglutinine;
PKC, protein
kinase C;
PKA, cyclic AMP-dependent protein
kinase.
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