From the Instituto de
Biotecnología/Universidad Nacional Autónoma de
México, Apartado Postal 510-3, Cuernavaca, MOR 62250, Mexico, the § Dana Farber Cancer Institute, Boston,
Massachusetts 02115, and the ¶ La Jolla Institute for Allergy and
Immunology, La Jolla, California 92121
Received for publication, September 18, 2000
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ABSTRACT |
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CD43, one of the most abundant glycoproteins on
the T cell surface, has been implicated in selection and maturation of
thymocytes and migration, adhesion, and activation of mature T cells.
The adapter molecule Cbl has been shown to be a negative regulator of
Ras. Furthermore, it may also regulate intracellular signaling through
the formation of several multi-molecular complexes. Here we
investigated the role of Cbl in the CD43-mediated signaling pathway in
human T cells. Unlike T cell receptor signaling, the interaction
of the adapter protein Cbl with Vav and phosphatidylinositol 3-kinase,
resulting from CD43-specific signals, is independent of Cbl tyrosine
phosphorylation, suggesting an alternative mechanism of interaction.
CD43 signals induced a Cbl serine phosphorylation-dependent interaction with the T cell stimulation through the
TcR1 and/or co-receptor
molecules leads to the activation of several protein-tyrosine
kinases (1), thus inducing a cascade of tyrosine phosphorylation
events. Phosphorylation of tyrosine residues provides docking sites for adapter molecules that recognize phosphorylated residues through Src
homology 2 (SH2) domains or phosphotyrosine-binding (PTB) domains (2).
Adapter molecules have emerged as critical components of cellular
signaling (3, 4). The formation of tyrosine phosphorylation-dependent multi-protein complexes involving
adapter and effector proteins has been shown to be essential for signal transduction. Disruption of these complexes by mutating specific phosphotyrosine residues of either the adapter or the effector proteins
leads to the inhibition of cellular responses (5-7). On the contrary,
hyperphosphorylation of adapter molecules such as Shc resulted in
enhanced cellular response (8).
Cbl, the oncogene product of the Casitas B-lineage lymphoma has been
implicated in several signal transduction pathways. Cbl is transiently
phosphorylated on tyrosine residues after activation of several
receptors and co-receptors by their cognate ligands, including: TcR, B
cell receptor, Fc The T cell surface glycoprotein CD43, is a transmembrane glycoprotein.
Two isoforms of CD43 (115 and 130 kDa) are generated by
post-translational modifications of the glycosylation pattern. The
115-kDa form of CD43 is expressed on all T cells, whereas the 130-kDa
is more abundant on resting CD8+ cells and is up-regulated
on both CD4+ and CD8+ lymphocytes upon
activation (32-34). CD43 functions are not well defined. CD43 has been
shown to play a positive role in cellular adhesion mediated by
integrins (35, 36) and in T cell homing (37). On the contrary, based on
the negative charge of the sialic acid residues of CD43, it has been
suggested that it participates in anti-adhesive processes (38, 39).
Also during thymic development, CD43 may play a role in the positive
selection of immature cortical thymocytes, through its interaction with
galectin-1 on thymic epithelial cells (40-42). A role for CD43 in
negative selection has also been proposed, probably through mechanisms
involving CD43-ICAM interactions (43). Co-ligation of CD43 with the TcR enhances T cell proliferation above levels observed when cross-linking the TcR alone, in normal as well as in CD28 Despite the fact that CD43 participates in different immunological
processes, the molecular event involved in CD43 signaling are still
poorly understood. We have recently shown that cross-linking CD43 on
the cell surface of human T lymphocytes with the anti-CD43 mAb L10
leads to a CD43-Fyn kinase interaction and to Fyn phosphorylation on
tyrosine residues. This interaction is mediated by the Fyn SH3 domain
and presumably a proline-rich sequence located in the cytoplasmic
domain of CD43 (48). Other groups have shown that CD43 can also
interact with Lck (49), although no functional data were provided
regarding this association. We have also shown that CD43-specific
activation of human T lymphocytes induces tyrosine phosphorylation of
the adapter protein Shc and of the GEF Vav, leading to the formation of
a macromolecular complex that comprises Shc, Grb2, and Vav. CD43
ligation results also in the formation of Vav-SLP-76 complexes and the
activation of the MAPK pathway (50). Recent data suggest that the
interleukin-2 gene expression mediated by CD43 signaling is
Ca2+- and PKC-dependent and that it
involves activation of the transcription factors AP-1, NFAT, and
NFkB.2
In the present report we show that cross-linking CD43 on human
peripheral T lymphocytes induced the association of Cbl with the GEF
Vav, the lipid kinase PI3K, and the Reagents--
L10, an IgG1 mAb that recognizes CD43, was either
purified from ascites on protein A-Sepharose columns or used as ascites (1:500). Rabbit anti-mouse IgG antiserum was generated by repeated immunizations with purified mouse IgG, and anti-mouse IgG
immunoglobulins were affinity purified. 3D6, an IgG1 mAb that
recognizes the VP7 protein from human rotavirus (51), was used as a
negative control in all experiments and was the kind gift from Dr. Luis
Padilla-Noriega (Instituto de Investigaciones Biomedicas/Universidad
Nacional Autónoma de México). The anti-Vav,
anti-14-3-3, and anti-Cbl antibodies were from Santa Cruz Biotechnology
(Santa Cruz, CA). The anti-phosphotyrosine 4G10 mAb has been described
elsewhere (52). The anti-GST antibody was generated by rabbit
immunization with purified GST protein. Protein A-Sepharose was from
Zymed Laboratories Inc. (San Francisco, CA) and
Ficoll-Hypaque was from Sigma. The plasmid containing the AP-1 element
linked to the thymidine kinase promoter and the luciferase
reporter gene was the kind gift of Dr. M. Rincon from the University of Vermont.
Cell Culture--
Jurkat cells were cultured in RPMI 1640 (Hyclone, Logan, UT) supplemented with 5% fetal calf serum (Hyclone)
and 5% bovine iron supplemented calf serum (Hyclone), 2 mM
L-glutamine (Sigma), 50 units/ml penicillin, 50 µg/ml
streptomycin, and 50 µM T Cell Activation and Binding of Cellular Protein to GST-14-3-3
Fusion Proteins--
Purified T cells (2 × 107) or
Jurkat cells (2 × 107) were incubated in 0.5 ml of
cold RPMI for 15 min at 4 °C with the following antibodies: L10
(1:500 dilution of ascites or 1 µg/ml of purified IgG), 3D6, or OKT3
at 1 µg/ml. Cross-linking was achieved by further incubating the
cells with rabbit anti-mouse IgG (1 µg/ml) for 15 min at 4 °C,
following which cells were activated by incubation at 37 °C for the
indicated times. Alternatively, when indicated, cells were stimulated
with 4 µg/ml of L10 anti-CD43 or 4 µg/ml of OKT3 anti-CD3 mAbs, and
cross-linking was achieved using 10 µg/ml of rabbit anti-mouse IgG at
37 °C for different time periods. After activation, cells were lysed
in 100 µl of lysis buffer (25 mM Hepes, pH 7.7, 150 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, O.5% Triton X-100, 0.5 mM
dithiothreitol, 20 mM Immunoblotting--
Proteins were transferred to Immobilon-P
membranes (Millipore, Medford, MA). Membranes were blocked with 5%
nonfat milk in Tris-buffer saline (TBS; 10 mM Tris, pH 7.5, 150 mM NaCl), followed by incubation with the indicated
antibody diluted in TBS with 0.05% Tween 20 (TBS-T; Bio-Rad). After
three washes with TBS-T, the membranes were incubated with the
appropriate second antibody coupled to horseradish peroxidase (Biomeda
Corp, Foster City, CA), and proteins were visualized by ECL (Amersham
Pharmacia Biotech), following the manufacturer's instructions.
Immunoprecipitation--
Protein A precleared lysates from
activated or nonactivated T cells (2 × 107 cellular
equivalents) were immunoprecipitated with the indicated antibody (1 µg/ml) for 2 h at 4 °C. Immune complexes were harvested with
protein A-Sepharose for 1 h on ice and washed once with cold TNE-T
(150 mM NaCl, 50 mM Tris, pH 7.5, 5 mM EDTA, 1% Triton X-100 (w/v)), twice with TNE (150 mM NaCl, 50 mM Tris, pH 7.5, 5 mM EDTA), and once with H2O. When indicated, Cbl precipitates
were treated for 30 min at 37 °C with 5 units of alkaline
phosphatase or 25 units of protein-tyrosine phosphatase (PTPase from
Yersinia) in 50 µl of the buffer suggested by the manufacturer (Roche
Molecular Biochemicals). To inhibit alkaline phosphatase, 10 mM Lipid Kinase Assay--
PI3K assay was performed basically as
described previously (53), with the following modifications. Cbl
precipitates were washed twice with 100 mM Tris, pH 6.8, containing 500 mM LiCl and two final washes with 10 mM Tris, pH 7.5, containing 100 mM NaCl and 1 mM EDTA. 10 µg of phosphatidylinositol were added to each
immunoprecipitate and equilibrated for 5 min at 30 °C and then 40 µl of kinase buffer were added (100 mM Hepes, pH 7.5, 5 mM MgCl2, 10 µCi of
[ Transfection and Luciferase Assays--
Transient transfection
of Jurkat cells was performed by electroporation as described
previously (54). Briefly, 107 cells were washed twice with
phosphate-buffered saline, resuspended in 400 µl of RPMI and mixed
with an equal volume of RPMI containing 10 µg of DNA and 40 µg of
DEAE-dextran prior to electroporation at 270 V, 950 µF in a
Bio-Rad gene pulser. After electroporation, cells were allowed to stand
for 10 min at room temperature. Cells transfected with the same plasmid
were pooled and cultured for 48 h in complete RPMI. Finally, cells
were stimulated for the indicated time under different experimental
conditions. Luciferase activity was determined as described previously
(55).
Cbl Associates with Vav and the p85 Subunit of the PI3K upon
CD43-specific T Cell Activation--
We have previously shown that
cross-linking CD43 on the cell surface of human peripheral T cells
induces the formation of a macromolecular complex containing Grb2 and
tyrosine-phosphorylated Shc and Vav. Because we identified Vav as the
p95 tyrosine phosphorylated protein, we found that Vav precipitates
contained the adapter molecule Cbl. Fig.
1A shows that in resting human
peripheral T cells, Cbl and Vav can associate. Cbl precipitates from
cells treated with an isotype control mAb (3D6) contained Vav
(lanes 6 and 8, lower panel). This
association was clearly enhanced by CD43 cross-linking with the L10 mAb
and activation at 37 °C for 1 or 5 min (lanes 2 and
4). Similarly, Vav immunoprecipitates from 3D6-treated cells
contained basal levels of associated Cbl (lanes 5 and
7, upper panel), and the amount of Cbl found in
Vav immunoprecipitates increased with time when cells were stimulated with the anti-CD43 mAb L10 (lanes 1 and 3,
upper panel). Equivalent amounts of Cbl (lanes 2,
4, 6, and 8, upper panel)
or Vav (lanes 1, 3, 5, and
7, upper panel) were precipitated from T cells
lysates, independently of the treatment.
Because we had previously shown that CD43 ligation induced Vav tyrosine
phosphorylation (50), we asked whether the Vav molecules associated
with Cbl were tyrosine phosphorylated. Fig. 1B shows that
Cbl immune complexes isolated from isotype control antibody-treated cells contained low levels of tyrosine phosphorylated Vav (lanes 3 and 4). On the other hand, following CD43 engagement,
the amount of tyrosine phosphorylated Vav associated with Cbl increased
(lanes 1 and 2). Identification of Vav in Cbl
precipitates was performed using anti-Vav polyclonal antibodies (data
not shown). Thus, these data show that Vav molecules associated with
Cbl in resting or activated T lymphocytes are tyrosine phosphorylated
and that CD43-mediated signals induce the association of tyrosine
phosphorylated Vav to Cbl.
Cbl has also been shown to interact with the p85 subunit of the PI3K,
upon T and B cell activation through the TcR or the BcR (16, 56). A
similar interaction has been described to be induced in macrophages by
colony-stimulating factor stimulation (14, 57). We tested whether
CD43-specific activation of human T lymphocytes induced Cbl-p85
interactions. As shown in Fig.
2A, p85 immunoprecipitates
from L10-treated cells contained Cbl. This association was
time-dependent, with maximum levels of association observed
at 1 and 5 min after activation (lanes 1 and 2)
and decreasing after 10 min (lane 3). Only very low levels
of Cbl were found in p85 precipitates from control antibody-treated
cells (lane 4). To further characterize this interaction, we
tested whether the PI3K associated with Cbl upon CD43 cross-linking was
active. Cbl immunoprecipitates from CD43-stimulated or control-treated peripheral human T cells were subjected to a PI3K assay. Fig. 2B shows that the PI3K associated with Cbl in response to
CD43-mediated signals was active in a time-dependent
manner, with maximum activation levels reached after 1 min of
activation and decreasing thereafter (Fig. 2B, lanes
1-3). Basal levels of the PI3K activity associated with Cbl
remained constant in control isotype mAb-treated lymphocytes (Fig.
2B, lanes 4-6). The CD43-induced PI3K activity
associated with Cbl was approximately four times lower than that
resulting from TPA stimulation of the same cells (data not shown).
These data clearly indicate that T cell activation through the CD43 molecule induces Cbl interaction with activated PI3K, suggesting a role
for both Cbl and PI3K in the CD43 signal pathway.
Cbl-Vav Interaction Induced by CD43 Occurs Independently of Cbl
Tyrosine Phosphorylation--
Association of Cbl with Vav and p85 in
response to TcR signaling is mediated by Vav and p85 SH2 domains and
tyrosine phosphorylation of Cbl on residues Tyr700
(17) and Tyr731 (58), respectively. Therefore, we
investigated whether signaling through CD43 in normal T cells induced
Cbl tyrosine phosphorylation. Cell lysates from T lymphocytes activated
with the L10 mAb, OKT3 mAb, or the control mAb 3D6 were
immunoprecipitated with anti-Cbl, and the presence of
tyrosine-phosphorylated Cbl was assessed by immunoblotting with the
anti-phosphotyrosine antibody 4G10. As shown in Fig.
3A, only basal levels of
tyrosine phosphorylated Cbl could be detected after CD43 cross-linking
for different time periods (lanes 2-5, upper
panel). As expected, T cell stimulation through the TcR clearly
induced Cbl tyrosine phosphorylation (lanes 6-9,
upper panel). When cells were treated with the control
antibody (data not shown) or left unstimulated, only basal levels of
tyrosine phosphorylated Cbl were observed (lane 1).
Reprobing the same membranes with anti-Cbl antibodies showed that
similar amounts of Cbl were precipitated in all cases (Fig.
3A, lower panel). Similarly, the Cbl molecules
that co-precipitated with Vav or p85 following CD43 ligation were not
tyrosine phosphorylated (data not shown).
To determine whether phosphorylation on residues other than tyrosines
was involved in the CD43-induced Cbl-Vav interaction, we
immunoprecipitated Cbl from CD43- or TcR-activated T lymphocytes and
treated these immune complexes with alkaline phosphatase or with a
tyrosine specific phosphatase (PTPase) from Yersinia. As shown in Fig.
3B, activation of human peripheral T cells with the L10 mAb
for 5 min had no effect on Cbl tyrosine phosphorylation as compared
with unstimulated T cells (lanes 1 and 2,
top panel). However, CD43 signals induced Cbl-Vav
interaction (lanes 1 and 2, middle
panel). Activation through the CD3 complex induced Cbl tyrosine
phosphorylation and Cbl-Vav interaction (compare lanes 1 and
7, top and middle panels,
respectively). Treatment of Cbl precipitates with alkaline phosphatase
totally dephosphorylated Cbl precipitated from L10-stimulated (compare
lanes 2 and 3, top panel) or
OKT3-stimulated cells (compare lanes 7 and 8,
top panel), whereas the addition of the phosphatase
inhibitor CD43 Signaling Leads to Cbl and Raf Serine Phosphorylation and to
Their Interaction with the 14-3-3 Molecule--
Data presented above
and the multiplicity of different domains present in Cbl suggest that
domains other than the PTB domain or post-translational modifications
of Cbl rather than phosphorylation of Tyr700, could
participate in the Cbl-Vav interactions induced by CD43. Recently, it
was reported that T cell treatment with TPA resulted in the association
of Cbl with the isoform
Raf is a direct effector of Ras and is the first kinase activated in
the MAPK pathway; furthermore, it is constitutively associated with a
dimer of 14-3-3. Even though the role for this association has been
controversial, it was recently demonstrated that 14-3-3 interacts as a
dimer with Raf phosphoserines 259 and 621. Upon cell activation, new
sites on Raf become phosphorylated, probably by PKC, inducing the
association of 14-3-3 to these new sites, thus stabilizing the active
state of Raf, in the absence of GTP-bound Ras (59). As expected, we
found that the association of Raf to the GST-14-3-3 fusion protein was
constitutive (Fig. 4B, lane 8). Moreover, a shift
in Raf mobility was observed in Jurkat cells in response to CD43-
(lane 2), TcR- (lane 4), or TPA-mediated signals
(lane 6), as compared with control-treated cells (lane 8). This shift is indicative of Raf activation (60).
On the contrary, Cbl-14-3-3 association was clearly
activation-dependent (Fig. 4B, lanes
2, 4, and 6) because no association was
observed in control-treated cells (lane 8). Neither Cbl nor Raf were found in GST precipitates (lanes 1, 3,
5, and 7). These data suggested that
CD43-mediated PKC activation induces serine phosphorylation of both Cbl
and Raf. To test this possibility we pretreated Jurkat cells with
RO-31-8220 or staurosporine (two PKC inhibitors) prior to cell
activation. RO-31-8220 pretreatment diminished CD43-mediated Cbl-14-3-3
and active Raf-14-3-3 interactions (Fig. 4C, compare
lanes 1 and 2, upper panel) and had no
effect on the basal Raf-14-3-3 association (Fig. 4C,
lanes 3 and 4, upper panel). Blotting
the same membranes with anti-14-3-3 antibodies showed that CD43
signaling may also induce 14-3-3 dimerization (Fig. 4C,
compare lanes 1 and 3, lower panel),
because endogenous 30-kDa 14-3-3 molecules were detected in the
precipitates. 14-3-3 dimerization was also PKC-dependent
because RO-31-8220 pretreatment decreased this interaction (compare
lanes 1 and 2, lower panel). Similar
results were obtained with staurosporine (data not shown). These data
clearly show that CD43 signaling leads to PKC activation and serine
phosphorylation of Cbl and Raf.
To further prove that CD43 signals lead to Cbl serine phosphorylation
and 14-3-3 association, Jurkat cells were transfected with plasmids
containing the human IgG1-Fc fragment fused in frame with the wild type
or mutated Cbl-serine-rich region required for the interaction with
14-3-3 (27), and their ability to interact with GST-14-3-3 in
vitro, after CD43 engagement, was monitored. Fig.
5 shows that GST-14-3-3 was able to
precipitate the wild type Cbl fragment (amino acids 615-644 (S4)),
whereas mutation of the serine residues 619, 623, 639, and 642 for
alanine residues (A4) prevented this interaction (compare lanes
1 and 2 with lanes 3 and 4,
upper panel). Consistent with the data presented above, the
14-3-3-Cbl interaction was enhanced following CD43 ligation with the
L10 mAb (lanes 1 and 2, upper panel).
Blotting the same membrane with anti-GST antibodies shows that
equivalent amounts of GST-14-3-3 fusion protein were used for
precipitation (lanes 1-4, lower panel). Thus,
these results demonstrate that in human T cells, CD43 signaling leads
to Cbl serine phosphorylation and that through this mechanism, CD43
induces the interaction between Cbl and 14-3-3.
Cbl Overexpression Negatively Regulates CD43 Signaling
Pathway--
Recently it became clear that in T cells, Cbl may
function as a negative regulator of the MAPK-dependent TcR
signal transduction pathway (29, 30). It is not yet clear, however,
whether tyrosine or serine phosphorylation of Cbl play a role in
regulating Cbl negative effect on T cell activation. Contrary to TcR
signaling, we have shown here that T cell activation through CD43
engagement has no effect on Cbl tyrosine phosphorylation levels.
Moreover, CD43 signaling induced the phosphorylation of Cbl on serine
residues and its interaction with 14-3-3. Because we had previously
shown that CD43 cross-linking induced MAPK pathway activation (50), we
tested the effect of Cbl overexpression on the
CD43-dependent activation of the MAPK pathway in Jurkat
cells. Fig. 6A (upper panel) shows that CD43 (lane 2) and TcR (lane
3) cross-linking, as well as TPA treatment (lane 4) of
cells transfected with the empty vector (lanes 1-4),
resulted in a different pattern of ERK activation as determined by
blotting with anti-p-ERK antibodies, when compared with untreated cells
(lane 1). Independent of the stimulus (lanes
6-8), overexpression of Cbl drastically diminished ERK
activation. The same membrane was blotted with anti-ERK antibody to
show that equivalent amounts of protein were loaded in each lane (Fig.
6A, lower panel). To determine whether Cbl had
also a negative effect downstream of the MAPK pathway, at the
transcriptional level, Jurkat cells were co-transfected with a plasmid
containing four AP-1-binding sites upstream the thymidine kinase
promoter linked to the luciferase reporter gene and either an empty
vector or the vector encoding for Cbl. As shown in Fig. 6B,
in cells transfected with the empty vector and the reporter plasmid,
CD43 as well as CD3 ligation induced AP-1 trans-activational
activity (approximately 2-fold over background levels), whereas TPA
treatment resulted in a stronger effect (approximately 4-fold). Cbl
overexpression prevented the induction of luciferase activity mediated
by the different stimuli. Thus, these results suggest that
independently of the fact that CD43 signaling induces Cbl-serine
phosphorylation and interaction with 14-3-3, Cbl overexpression has a
negative role on the CD43-dependent MAPK and AP-1
activation.
PKC Activation through CD43 Signaling Is Necessary to Activate the
MAPK Pathway in Human T Lymphocytes--
Activation of the Ras-MAPK
pathway can be induced by positive signals (reviewed in Ref. 61) or by
eliminating the default negative effect of Cbl (30). The strong
induction observed after TcR engagement results probably from
activating both mechanisms. We asked whether the phosphorylation of Cbl
on serine residues by PKC activation after CD43 engagement could
prevent the negative effect of Cbl on MAPK activation. The rationale
for these experiments was that CD43 signaling requires PKC activation
to cancel the inhibitory effect of Cbl, thus inducing MAPK activation.
Human peripheral T cells were pretreated with the PKC inhibitor
RO-31-8220 or with the PLC Following receptor engagement, the formation of tyrosine
phosphorylation-dependent multimeric complexes involving
adapter and effector proteins has been shown to be essential for signal transduction. Upon cell activation through different stimuli, the
adapter protein Cbl becomes phosphorylated on tyrosine residues, favoring its interaction with SH2-containing signaling molecules. In
primary murine T cells as well as in Jurkat T cells, TcR activation induces Cbl tyrosine phosphorylation on tyrosine residues 700 and 731, resulting in the interaction with the SH2 domains of Vav and PI3K,
respectively (17, 58). Here we show that in human normal peripheral T
cells, CD43 engagement induced also the interaction of Cbl with Vav and
PI3K. Unlike with TcR signaling, these interactions were independent of
Cbl tyrosine phosphorylation because only basal tyrosine
phosphorylation levels were detected when cross-linking CD43 for
different periods of time. Nonetheless, under the same experimental
conditions, the association with Vav and PI3K was enhanced after 5 min
of activation. Furthermore, even though PTPase treatment of Cbl
precipitates from CD43- or CD3-stimulated T cells resulted in complete
Cbl tyrosine dephosphorylation, the interaction with Vav was not
affected. Removal of phosphate groups from serine, threonine, and
tyrosine residues with alkaline phosphatase resulted in complete Cbl
dephosphorylation and in partial blockade of the CD43-induced Cbl-Vav
interaction. Interestingly, this treatment had only a minor effect on
the formation of the Cbl-Vav complex mediated through the TcR.
Transient expression of a mutated form of the Cbl molecule
(Y700A) in Jurkat cells did not prevent the formation of this
complex,3 further suggesting
an alternative mechanism through which Cbl and Vav interact following
CD43 engagement. The highly conserved amino-terminal region of Cbl
(Cbl-N) binds to phosphorylated tyrosine residues and has a cell
transforming activity. Point mutations in Cbl that disrupt its
recognition of phosphotyrosines also interfere with its negative
regulatory function and, in the case of v-Cbl, with its oncogenic
potential (62, 63). Cbl-N is composed of three interacting domains: a
four-helix bundle (4H), an EF-hand calcium-binding domain, and a
divergent SH2 domain. Mutations in the 4H, EF-hand, and SH2 domains
confirm that the three domains together form an integrated
phosphoprotein-recognition module (64). The possibility that the Cbl
PTB domain could bind tyrosine phosphorylated Vav was eliminated,
because neither the Wt PTB nor the G306E mutant PTB domain were able to
bind Vav after CD43 engagement (data not shown). This was particularly
important, because we had previously shown that CD43 signaling leads to
Vav tyrosine phosphorylation (50). Altogether these data suggest that
the interaction between Cbl and Vav induced by CD43 signaling is
independent of Cbl tyrosine phosphorylation and that phosphorylation on
residues other than tyrosine may be involved in this interaction. Therefore, CD43 and the TcR induce Cbl-Vav interactions by two independent mechanisms.
The dephosphorylation experiments bring evidence that phosphorylation
of Tyr700 of Cbl is not the only mechanism by which Cbl and
Vav interact in response to TcR engagement, suggesting that
interactions between these two molecules are also mediated by domains
that do not recognize phosphoaminoacids, like SH3-proline-rich regions.
Using a yeast two-hybrid system, Vav-Cbl-b interactions have been shown
to be mediated by the SH3 domain of Vav and the proline-rich region of
Cbl-b (65). Furthermore, the proline-rich region of Cbl has been
reported to interact with the amino-terminal SH3 domain of Crk-L,
leading to lamellopodia and membrane ruffle formation, indicating that
Cbl participates in cytoskeleton rearrangements essential for cell
adhesion, spreading and migration (31, 66, 67). Vav was also shown to
be localized to the lamellopodia and to regulate
Rac-dependent lamellopodia formation after
In the present report we show that CD43 signaling induced Cbl-PI3K
interaction and the activation of Cbl-associated PI3K. Engagement of
CD43 on different hematopoietic cells induces homotypic cell adhesion,
a process described as partially dependent on integrin activation
(71-74). In human peripheral T cells, this process is prevented by
wortmannin and Ly294002, two specific inhibitors for
PI3K.4
CD43-dependent signals have been shown to have a regulatory
role on integrin-mediated T cell adhesion to endothelial cells and extra-cellular matrix components (36, 75). Cbl-associated PI3K activity
induced after CD28 engagement is required for integrin T cell activation through CD43 ligation with the L10 mAb also promoted
the interaction of the We described previously the activation of the MAPK pathway resulting
from CD43 ligation (50) that is consistent with a shift in SDS-PAGE
mobility of 14-3-3-associated Raf described by others (60).
Furthermore, activation of Raf depends on 14-3-3 dimerization (59), a
phenomenon we found in response to CD43 cross-linking. The fact that RO
31-8220 and staurosporine prevented the shift in mobility
characteristic of active Raf suggests that, in response to CD43
engagement, PKC is phosphorylating 14-3-3 and inducing its
dimerization. Different 14-3-3 isoforms have been shown to be
phosphorylated by PKC on Ser64 (79, 80), by a
sphingosine-dependent kinase on Ser58 (81), and
by casein kinase I on Thr233 (82). Therefore,
phosphorylation of 14-3-3 by different protein kinases may control its
own dimerization, regulating the formation of specific complexes and
thus signal transduction.
Cumulative genetic and biochemical evidences point at a negative role
for the Cbl family members in T cell signal transduction. Overexpression of c-Cbl in Jurkat cells inhibits both the MAPK activation pathway and AP-1 transcriptional activity (29). c-Cbl null
mice show enhanced T cell signaling after TcR engagement, and ZAP70
kinase as well as MAPK activation levels were higher in those mice as
compared with wild type mice (30, 83). Consistent with these data,
overexpression of c-Cbl in Jurkat cells prevented induction of the
CD43- and TcR-dependent MAPK pathway as well as of
AP-1-mediated transcriptional activity, suggesting that when
overexpressed, Cbl may be acting as a negative modulator of CD43 signaling.
During the last year several groups showed that, to negatively modulate
cellular signaling, Cbl requires its PTB domain to bind tyrosine kinase
receptors (epidermal growth factor and platelet-derived growth factor
(63, 84)) and tyrosine kinases (ZAP70 and SYK (64, 85, 86)) as well as
the RING finger to bind components of the ubiquitination machinery and
target them for degradation by the proteasome degradation or endocytic
degradation pathways (87-89). It was recently shown that Cbl
overexpression in the Ramos B-lymphoma cell line resulted in a decrease
in Syk protein levels and that the Cbl RING finger domain was essential
to induce Syk down-modulation (90). One of the earliest events
following CD43 engagement in T cells is activation of the ZAP70
kinase5 and that of the
Ras-dependent MAPK pathway (50). The blockade of
CD43-dependent signals we observe upon c-Cbl overexpression may result from reduced ZAP70 protein levels or from the generation of
Cbl-CrkL-C3G inhibitory complexes. These in turn may activate the Rap1
GTPase, a molecule shown to have negative effects on T cells signaling
(91), probably by counteracting the Ras-dependent activation (92). c-Cbl overexpression may also negatively modulate the
Vav GEF activity. Although there is a direct correlation between Vav
tyrosine phosphorylation levels and its GEF activity (93, 94), whether
Vav associated with Cbl, either through its SH2 domain or the
Cbl-tyrosine phosphorylation-independent mechanism described here, has
any GEF activity remains to be determined.
From the overexpression experiments it is clear that Cbl has a negative
effect on CD43 signaling. However, it is not possible to assess the
role of serine-phosphorylated Cbl in T cell activation resulting from
CD43 engagement under these experimental conditions. Nonetheless, the
fact that PKC and PLC Serine phosphorylation of Cbl has been shown to prevent phosphorylation
of tyrosine residues and the interaction of this molecule with
SH2-containing signaling proteins like PI3K and CrkL (95, 96),
suggesting a cross-talk between serine/threonine and tyrosine phosphorylation signaling targeted to Cbl. Differential phosphorylation patterns of Cbl may regulate its functions, probably by modifying its
capacity to interact with different signaling molecules, thus modulating cellular signaling. Phosphorylation of Cbl serine residues as a result of CD43-specific signals may play a role in modulating Cbl
negative effect on T cell signaling. Ongoing work is in progress to
elucidate the biological role of Cbl serine phosphorylation and
Cbl-14-3-3 interaction induced by CD43 engagement in T cell activation.
-isoform of 14-3-3. protein. Protein
kinase C-mediated Cbl serine phosphorylation was required for this
interaction, because the PKC inhibitor RO-31-8220 prevented it, as well
as 14-3-3 dimerization. Moreover, mutation of Cbl serine residues 619, 623, 639, and 642 abolished the interaction between Cbl and 14-3-3. Overexpression of Cbl in Jurkat cells inhibited the
CD43-dependent activation of the mitogen-activated protein
kinase (MAPK) pathway and AP-1 transcriptional activity, confirming
nevertheless a negative role for Cbl in T cell signaling. However,
under normal conditions, PKC activation resulting from CD43 engagement
was required to activate the MAPK pathway, suggesting that
phosphorylation of Cbl on serine residues by PKC and its association
with 14-3-3 molecules may play a role in preventing the Cbl inhibitory
effect on the Ras-MAPK pathway. These data suggest that by inducing its phosphorylation on serine residues, CD43-mediated signals may regulate
the molecular associations and functions of the Cbl adapter protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor, Fc
RI, colony-stimulating factor-1
receptor, interleukin-3 receptor, CD38, CD28, interferon
receptor, epidermal growth factor receptor, platelet-derived growth factor receptor, nerve growth factor receptor, and integrin receptors, among others (reviewed in Ref. 9). Cbl tyrosine phosphorylation is directly mediated by ZAP-70 and Syk kinases, although it is not yet clear whether it may also be a direct target for
the Src family kinases, Fyn and Lck (10, 11). Cbl tyrosine phosphorylation induces its association with several signaling molecules including tyrosine kinases such as ZAP-70 and Fyn, (12, 13);
the lipid kinase PI3K (14-16); the guanine exchange factor (GEF) Vav
(17); and the adapter molecule Crk (18-21). Through its proline-rich
regions, Cbl may interact with the SH3 domain of kinases from the Src
family Fyn (22) or Lyn (23) and with the adapter molecules Grb2 (24,
25) and Nck (26). Cbl phosphorylation on serine residues favors its
association with proteins of the 14-3-3 family (27). This complex array
of interactions suggests a central role for Cbl in cellular signaling.
Recent data point to a negative role for Cbl in cellular signaling.
Genetic evidences from the nematode Caenorhabditis elegans
suggested that the Cbl homologue Sli-1 is a negative regulator of the
Ras-dependent epidermal growth factor signal transduction
pathway (28). In Jurkat cells, overexpression of Cbl inhibited the
Ras-dependent activation of the MAPK pathway and AP-1
transcriptional activity (29). Recent data obtained from the
c-Cbl
/
mouse clearly showed that the absence of Cbl
leads to enhanced T cell signaling upon CD3 ligation, involving ZAP-70
kinase activity (30). However, a positive role for Cbl in
integrin-mediated cell adhesion has also been described (31). Thus, Cbl
seems to be a complex adapter molecule that, depending on the signal, may function both as positive and negative regulator.
/
mice (44).
Furthermore, it was recently shown that CD43 may modulate TcR signaling
and immune responses (45). CD43 ligation with different monoclonal
antibodies (mAbs) was reported to induce the generation of
diacylglycerol and inositol phosphates, Ca2+ mobilization,
and PKC activation (46). These effects are mediated by the highly
conserved cytoplasmic domain of CD43 because deletion of the entire
cytoplasmic domain abolished the co-receptor functions of CD43 when
expressed in an antigen-specific murine T cell hybridoma (47).
-soform of the 14-3-3 family of
proteins; interestingly, these interactions did not require
Cbl-tyrosine phosphorylation. The induction of 14-3-3 dimerization, the
14-3-3-Cbl interactions, and MAPK activation mediated by CD43 ligation
were found to be PKC-dependent. Furthermore, we show that
the CD43-induced 14-3-3-Cbl interactions required serine
phosphorylation of Cbl. Nevertheless, overexpression of Cbl in Jurkat
cells inhibited the CD43-dependent ERK kinase and AP-1
transcriptional activity, suggesting a negative role for Cbl in
CD43-dependent early signaling events. Altogether, these data clearly show that CD43 signaling induces Cbl-serine
phosphorylation rather than Cbl-tyrosine phosphorylation and that Cbl
serine phosphorylation may contribute to regulate the molecular
associations and functions of this adapter molecule.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol. Peripheral
blood T cells were isolated from healthy adult donors by Ficoll-Hypaque
gradient centrifugation. The buffy coat was washed three times with
phosphate-buffered saline and resuspended in supplemented RPMI.
Adherent cells were removed by plating the cells onto 100-mm Petri
dishes (4 × 107 cells/plate) for at least 2 h at
37 °C in a 5% CO2 atmosphere. Nonadherent cells were
collected and loaded on a nylon column pre-equilibrated with
supplemented RPMI, incubated for 45 min at 37 °C, and eluted with
supplemented RPMI. The resultant purified cells were predominantly
OKT3+ (>80%) and L10+ (>95%), as determined
by fluorescence-activated cell sorter analysis.
-glycerophosphate, 1 mM Na3VO4, 5 mM NaF, 4 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin) for 30 min at 4 °C. Lysates were spun at
14,000 × g for 15 min at 4 °C, and supernatant
volume was adjusted to 300 µl with fresh lysis buffer and incubated
with approximately 10 µg of fusion protein noncovalently coupled to glutathione-Sepharose beads for 2 h at 4 °C. Beads were washed four times with cold lysis buffer, and bound proteins were resolved by
SDS-PAGE and subjected to immunoblotting.
-glycerophosphate was added to the reaction mix, and
100 µM of Na3VO4 was used to inhibit PTPase. Immunoprecipitated proteins were subjected to SDS-PAGE
and immunoblotted as described above.
-32P]ATP, and 10 µM ATP). After
incubating at 30 °C for 10 min, reactions were stopped by adding 400 µl of 1 N HCl and 400 µl of chloroform/methanol (1:1),
the aqueous phase was discarded, and lipids were extracted with 1 N HCl/methanol (1:1). Phospholipids were separated by thin layer chromatography using 1-propanol/2 N acetic acid
(65:35) as solvent and detected by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CD43 cross-linking induces Vav-Cbl
interactions in human T cells. A, 2 × 107 T cells were stimulated at 37 °C for different
periods of time with anti-CD43 mAb L10 (lanes 1-4) or the
isotype control mAb 3D6 (lanes 5 and 6) or left
untreated (lanes 7 and 8). Precleared cell
lysates were immunoprecipitated with anti-Vav (lanes 1,
3, 5, and 7) or anti-Cbl polyclonal
Abs (lanes 2, 4, 6, and 8).
Precipitated proteins were separated by SDS-PAGE and blotted with the
indicated antibodies. B, Cbl was precipitated from
precleared T cell lysates of anti-CD43 mAb-stimulated cells
(lanes 1 and 2) or isotype control mAb-treated
cells (lanes 3 and 4); precipitated proteins were
separated by SDS-PAGE and blotted with anti-phosphotyrosine 4G10 mAb.
The position of Vav is indicated. IP,
immunoprecipitation.
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Fig. 2.
Active p85 associates to Cbl upon T
cell activation through CD43. A, 2 × 107 human peripheral T cells were stimulated at 37 °C
for the indicated time periods with anti-CD43 mAb L10 (lanes
1-3) or with the isotype control mAb 3D6 (lane 4). p85
was precipitated from the precleared cell lysates. Proteins were
separated by SDS-PAGE and blotted with anti-Cbl antibodies.
B, human peripheral T cells were stimulated with anti-CD43
mAb L10 (lanes 1-3) or isotype control mAb 3D6 (lanes
4-6) for different periods of time. Cbl was precipitated from
precleared cell lysates and subjected to PI3K assay. The position of
phosphatidylinositol phosphate (PIP) is indicated.
IP, immunoprecipitation.
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Fig. 3.
Activation of human peripheral T cells
through CD43 cross-linking does not induce Cbl tyrosine
phosphorylation. A, 2 × 107 T cells
were unstimulated (lane 1) or stimulated at 37 °C for the
indicated time periods with the anti-CD43 mAb L10 (lanes
2-5) or OKT3 (lanes 6-9). Cbl was precipitated from
precleared cell lysates, and proteins were separated by SDS-PAGE and
blotted with anti-phosphotyrosine 4G10 mAb (upper panel) or
with anti-Cbl polyclonal Ab (lower panel). B, Cbl
was precipitated from precleared T cell lysates (2 × 107) of unstimulated (lane 1) or stimulated
cells for 5 min with anti-CD43 mAb L10 (lanes 2-6) or with
anti-CD3 mAb OKT3 (lanes 7-11). Cbl precipitates were left
untreated (lanes 2 and 7) or treated with
alkaline phosphatase in the absence (lanes 3 and
8) or the presence of -glycerophosphate (lanes
4 and 9) or treated with PTPase in the absence
(lanes 5 and 10) or presence of NaVO4
(lanes 6 and 11). Proteins were separated by
SDS-PAGE and blotted with anti-phosphotyrosine 4G10 mAb (top
panel), anti-Vav polyclonal Ab (middle panel), or
anti-Cbl polyclonal Ab (bottom panel). IP,
immunoprecipitation.
-glycerophosphate prevented this effect (lanes
4 and 9, top panel). Cbl dephosphorylation resulted in a partial blockade of the CD43-induced Cbl-Vav interaction that was overcome by the addition of phosphatase inhibitors (compare lanes 3 and 4, middle panel). Alkaline
phosphatase treatment had a minor effect on TcR-induced Cbl-Vav
interactions as compared with the precipitates containing phosphatases
inhibitors (lanes 8 and 9, middle
panel). PTPase treatment of Cbl precipitates from CD43- or
TcR-stimulated cells resulted also in complete Cbl dephosphorylation (lanes 5 and 10, top panel), whereas
dephosphorylation was prevented by the addition of the PTPase inhibitor
NaVO4 (lanes 6 and 11, top
panel). Equivalent levels of Cbl-Vav interactions were found in
Cbl precipitates from L10-stimulated T cells after PTPase treatment in
the absence or presence of NaVO4 (lanes 5 and
6, middle panel); the same was true for Cbl
precipitated from OKT3 stimulated cells (lanes 10 and
11). Blotting the same membrane with anti-Cbl antibodies shows that equivalent amounts of Cbl were precipitated (Fig.
3B, bottom panel). These results suggest that the
CD43-mediated Cbl-Vav interactions are independent of Cbl-tyrosine
phosphorylation. The Cbl-PTB domain does not bind
tyrosine-phosphorylated Vav after CD43 engagement (data not shown),
ruling out the possibility that this Cbl-Vav interaction could be
mediated by Cbl-PTB domain and Vav tyrosine residues. Altogether, these
data suggest that Cbl-Vav interactions may be partially mediated by
phosphorylation on serine or threonine residues.
of the 14-3-3 family of proteins and that
this association was dependent on the PKC-mediated Cbl-serine
phosphorylation (27). Because previous reports suggest a role for PKC
in the CD43 signaling pathway (46), we tested whether activation of T
cells through CD43 could induce Cbl-14-3-3 interactions, as indicative
of Cbl serine phosphorylation in response to CD43 activation. As shown
in Fig. 4A (upper
panel), cross-linking CD43 in human peripheral T cells with the
L10 mAb induced the association of Cbl to a GST-
-14-3-3 fusion
protein in a time-dependent manner. Maximum association was
found after 10 min of activation (lanes 1-5). TcR or TPA
stimulation also induced 14-3-3-Cbl association (lanes 6 and
7); no association was found in control cells (lane 8), even though equivalent amounts of fusion protein were used in
all cases, as determined by anti-GST immunoblotting (Fig.
4A, lower panel).
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Fig. 4.
CD43 cross-linking induces Cbl-14-3-3
interaction in a PKC-dependent manner. A,
2 × 107 human peripheral T cells were stimulated at
37 °C for the indicated periods of time with anti-CD43 mAb L10
(lanes 1-5), anti-CD3 mAb OKT3 (lane 6), 50 ng/ml TPA (lane 7), or isotype control mAb 3D6 (lane
8). Cells lysates were incubated with Sepharose 4B-GST-14-3-3
fusion protein for 2 h at 4 °C. Bound proteins were separated
by SDS-PAGE and blotted with anti-Cbl or anti-GST polyclonal Abs.
B, 2 × 107 Jurkat were stimulated as
described above for 5 min with anti-CD43 mAb L10 (lanes 1 and 2), anti-CD3 mAb OKT3 (lanes 3 and
4), or isotype control mAb 3D6 (lanes 7 and
8) or for 15 min with 50 ng/ml TPA (lane 5 and
6). Total cell extracts were incubated with Sepharose-4B-GST
(lanes 1, 3, 5, and 7) or
with Sepharose 4B-GST-14-3-3 fusion protein (lanes 2,
4, 6, and 8) for 2 h at 4 °C.
Bound proteins were separated by SDS-PAGE and blotted with anti-Cbl or
anti-Raf polyclonal Abs. C, 2 × 107 Jurkat
cells were incubated for 30 min in the absence or in the presence of 10 µM RO-31-8220 prior to activation with anti-CD43 mAb L10
(lanes 1 and 2) or isotype control mAb 3D6
(lanes 3 and 4) for 5 min. Total cell extracts
were incubated with Sepharose 4B-GST-14-3-3 fusion protein for 2 h
at 4 °C. Bound proteins were separated by SDS-PAGE and blotted with
anti-Cbl or anti-Raf (upper panel) or anti-14-3-3
(lower panel) polyclonal Abs.
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Fig. 5.
Cbl serine residues S619, S623, S639 and S642
are required for the CD43-induced Cbl-14-3-3 interaction. Jurkat
cells were electroporated with 10 µg of the vector S4 encoding for
the wild type c-Cbl 14-3-3 binding site, fused to the human IgG Fc
(lanes 1 and 2) or with 10 µg of the vector A4
encoding for the mutated c-Cbl 14-3-3 binding site (lanes 3 and 4). After 36 h, cells were treated for 10 min with
isotype control mAb 3D6 (lanes 1 and 3) or
anti-CD43 mAb L10 (lanes 2 and 4). Total cell
extracts were incubated with Sepharose 4B-GST-14-3-3 fusion protein for
2 h at 4 °C. Bound proteins were separated by SDS-PAGE and
blotted with anti-hFc or anti-GST polyclonal Abs.
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Fig. 6.
Cbl plays a negative role on the CD43
signaling pathway. A, Jurkat cells were electroporated
with 10 µg of empty vector (lanes 1-4) or with 10 µg of
the vector coding for Cbl (lanes 5-8). After 36 h,
cells were left unstimulated (lanes 1 and 5) or
stimulated with anti-CD43 mAb L10 (lanes 2 and
6), anti-CD3 mAb OKT3 (lanes 3 and 7),
or 50 ng/ml TPA (lanes 4 and 8) for 15 min. Cell
lysates (10 µg) were separated by SDS-PAGE and blotted with anti-pERK
monoclonal Ab (upper panel) or anti-ERK polyclonal Ab
(lower panel). B, Jurkat cells were
electroporated with 5 µg of empty vector and 5 µg of the reporter
plasmid AP-1-PRL-Luc or with 5 µg of the vector coding for Cbl and 5 µg of the reporter plasmid AP-1-PRL-Luc. After 12 h of
stimulation as described above, total cell extracts were prepared, and
luciferase activity was measured.
inhibitor U73122 before stimulation
through CD43 or TPA and activation of the MAPK pathway was monitored by immunobloting with anti-active ERK antibodies. Fig.
7 (upper panel) shows that
CD43 engagement or TPA treatment resulted in ERK activation when
compared with isotype control antibody-treated cells (lanes 1, 2, and 5). The PKC inhibitors RO-31-8220
(lanes 3 and 6) and Gö6976 (data not shown)
partially prevented the CD43-dependent ERK activation and
totally blocked ERK activation induced by TPA. Stimulation of PKC
activity in response to CD43 ligation depends on the activation of
PLC
(46). As expected, inhibition of PLC
with the specific
inhibitor U73122 blocked MAPK activation in response to CD43 signaling
and had no effect on TPA-stimulated cells (compare lanes 4 and 7). Equivalent amounts of ERK were present in all lanes
as determined by blotting the same membrane with anti-ERK antibodies
(lanes 1-7, lower panel). These results suggest
that CD43-dependent MAPK activation requires the activation of PKC, which depends on previously activated PLC
. Together these results suggest that the PKC-dependent Cbl phosphorylation
on serine residues and its subsequent association with 14-3-3, resulting of CD43 engagement on the surface of normal human T
lymphocytes, probably blocks the default negative effect that Cbl has
on MAPK activity, resulting in activation of the MAPK pathway.
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Fig. 7.
CD43-dependent MAPK pathway
induction requires PKC activation. 2 × 107 human
peripheral T cells were incubated for 30 min in the absence or presence
of 10 µM RO-31-8220 or 2 µM U73122, prior
to activation with isotype control mAb 3D6 (lane 1),
anti-CD43 mAb L10 (lanes 2-4), or with 50 ng/ml TPA
(lanes 5-7) for 10 min. Total cell extracts were separated
by SDS-PAGE and blotted with anti-phosphorylated ERK (upper
panel) or anti-ERK (lower panel) polyclonal Abs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IIb
3-integrin activation (68). Although
at the moment we do not know whether the Vav molecules associated to
Cbl in response to CD43 ligation are active, it is possible that the
Cbl-Vav complexes we find participate in the CD43-dependent
cytoskeleton reorganization (69, 70).
1
activation (31). Altogether, these data suggest that multimolecular complexes containing Cbl-Vav and/or PI3K may play a role in the cytoskeleton remodeling required for cell adhesion and migration induced by the interaction of CD43 on the surface of a T cell with a
putative receptor on the surface of endothelial or dendritic cells (37,
76-78).
isoform of the 14-3-3 family of proteins
with the Cbl and Raf molecules. Phosphorylation of c-Cbl serine
residues Ser619, Ser623, Ser639,
and Ser642 through a PKC-dependent mechanism
was found to be responsible for this association (27). Mutation of
those residues prevented this interaction in response to CD43
cross-linking. Consistent with this, 14-3-3-Cbl as well as
14-3-3-active Raf complex formation resulting from CD43 ligation, were
diminished by pretreatment of the cells with the PKC inhibitors RO
31-8220 or staurosporine, suggesting that a CD43-dependent
PKC activation (46), leading to Cbl and Raf phosphorylation, was
responsible for these interactions.
inhibitors prevented the activation of the
MAPK pathway and the association between Cbl and 14-3-3 after CD43
engagement, strongly suggests that the CD43-induced MAPK activation
results from a blockade of the negative effect of Cbl on the MAPK
pathway by promoting the interaction of serine phosphorylated Cbl and
14-3-3 molecules. Activation of the Ras-MAPK pathway may result of two
events: (i) activation of SOS GEF activity by tyrosine phosphorylation
and translocation to the membrane where it binds and activates Ras and
(ii) blocking the negative regulation mediated by Cbl. In human
peripheral T cells, TcR signaling induces a faster and stronger
activation of MAPK activity when compared with CD43
engagement.6 The
TcR-dependent signals could activate both processes
concomitantly. On the contrary, because we have not been able to detect
SOS or SOS-1 tyrosine phosphorylation in response to CD43 cross-linking in human T lymphocytes,6 MAPK activation following CD43
engagement may result from blocking the Cbl negative effects rather
than activating SOS GEF activity.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Mercedes Rincón for the AP-1-Luc reporter plasmid, Dr. Eduardo Huerta for leukocyte concentrates, and Dr. Leonor Pérez and Angélica Santana for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by Grant IN217498 from Dirección General de Apoyo al Personal Académico/Universidad Nacional Autónoma de México and Grant 25307-M from Consejo Nacional de Ciencia y Tecnologia, México.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.
Present address: Kyoto University Faculty of Medicine.
Sakyo-ku, Kyoto 606-8315, Japan.
** To whom correspondence should be addressed: Instituto de Biotecnología/UNAM, APDO Postal 510-3, Cuernavaca, MOR 62250, Mexico. Tel.: 52-73-291606; Fax: 52-73-172388; E-mail: yvonne@ibt.unam.mx.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M008494200
2 Santana, M. A., Pedraza-Alva, G., Oliveras-Zavaleta, N., Madrid-Marina, V., Horejsi, V., Burakoff, S. J., and Rosenstein, Y. (2000) J. Biol. Chem. 275, 31460-31468.
3 G. Pedraza-Alva, unpublished data.
4 E. Layseca, unpublished data.
5 M. Cruz-Muñoz, manuscript in preparation.
6 H. Gordillo, G. Pedraza-Alva, and Y. Rosenstein, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: TcR, T cell receptor; SH2, Src homology domain 2; Ab, antibody; mAb, monoclonal antibody; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PTB, phosphotyrosine-binding domain; PI3K, phosphatidylinositol 3-kinase; GEF, guanine exchange factor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PTPase, protein-tyrosine phosphatase; TPA, 12-O-tetradecanoylphorbol-13-acetate; PLC, phospholipase C; PKC, protein kinase C.
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REFERENCES |
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