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Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval QC, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 activation, and
extracellular signal-regulated kinase 1/2 activation. Importantly,
these cellular events are not affected in the signaling cascade induced
by engagement of the CD3/TCR complex. However, both CD3- and
CD2-induced NF-AT activation and IL-2 secretion are impaired in
p62dok-overexpressing cells. In addition, we
show that CD2 but not CD3 stimulation induces
p62dok and Ras GTPase-activating protein
recruitment to the plasma membrane. These results suggest that
p62dok plays a negative role at multiple steps
in the CD2 signaling pathway. We propose that
p62dok may represent an important negative
regulator in the modulation of the response mediated by the
TCR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In T cells, p62dok and
p56dok are expressed (10), whereas
dok-3 is absent in thymus and in most T cell lines examined
(9). Little information is available on the biological
function of these proteins in T cells. Recent evidence suggests that
they play a specific role in signal transduction pathways initiated by
costimulatory receptors. The phosphorylation of
p62dok has been reported to occur following CD2
(10) or CD28 stimulation (11), whereas CD3
stimulation does not induce p62dok
phosphorylation. Phosphorylation of the other Dok member,
p56dok, seems to be regulated in the same way
since we have shown that it is phosphorylated upon CD2 stimulation and
not upon CD3 stimulation (10). Several lines of evidence
support the conclusion that Src family kinases phosphorylate Dok
proteins. Cell adhesion-dependent tyrosine phosphorylation of Dok is
mediated by Src tyrosine kinases (12). We have shown that
Lck is required for CD2-mediated phosphorylation of Dok proteins
(10). In transient transfection assays, Dok proteins are
good substrates for Src family kinases (9).
Phosphorylation of p56dok by Lyn generates
binding sites for RasGAP and Nck (7). Depending on the
transduction pathway examined, Dok proteins can act as a positive or
negative regulator. In B cells, phosphorylation of
p62dok is involved in the FcRIIB-mediated
inhibition of B cell receptor signaling (13, 14) most
likely by negatively regulating the activity of Ras (15),
thereby inhibiting the Ras-dependent activation of extracellular
signal-regulated kinase 1/2 (Erk1/2) (13, 14). By
contrast, transient overexpression of p62dok in
293 cells (8) and in Chinese hamster ovary cells
expressing insulin receptors (12) has been reported to
have no effect on v-abl-dependent and insulin-dependent
mitogen-activated protein kinase activation, respectively.
Moreover, overexpression of p62dok
enhances cell migration in response to insulin.
p56dok has been reported to be a negative
regulator of the Ras activation pathway. Transient overexpression of
p56dok diminishes the IL-2-induced activation of
mitogen-activated protein kinase and AP-1 (5). The lowered
p56dok expression in hr/hr T cells
induces an increase of T cell proliferation in response to cytokine and
TCR stimulation (5).
In this study, to gain insight into the mechanisms involved in Dok
function in T cells, we examined the role of
p62dok in regulating T cell signaling. We
studied the effect of p62dok overexpression on
CD2- and CD3-mediated signal transduction events in Jurkat cells. We
demonstrate that overexpression of p62dok
specifically inhibits CD2-mediated phospholipase C1 (PLC1)
phosphorylation, Erk1/2 activation, and Ca2+
mobilization, and that these events remain unaffected when initiated
via the CD3/TCR complex.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat cells, clone 77-6, were grown in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, penicillin, and streptomycin. Puromycin at 1 µg/ml was added to the medium when required.
mAbs used included: anti-CD3
UCHT1 (IgG2a; kindly provided by A.
Alcover, Institut Pasteur, Paris, France); anti-CD2
(anti-T11-2 and T11-3, kindly provided by E. Reinherz, Harvard
Medical School, Boston, MA); anti-RasGAP (B4F8; Santa Cruz
Biotechnology, Santa Cruz, CA); anti-phosphotyrosine (4G10; Upstate
Biotechnology, Lake Placid, NY); and anti-PLC1 (a mixture of
mAbs; Upstate Biotechnology). Polyclonal Abs used included:
anti-p62dok PTB directed against
p62dok PTB domain (produced by immunizing
rabbits with a GST fusion protein bearing residues 152–259);
anti-p62dok directed against aa residues
425–439 of p62dok (kindly provided by B.
Stillman, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY);
phospho-specific anti-Erk1/2 (Promega, Madison, WI); and total
anti-Erk1/2 (New England Biolabs, Beverly, MA).
Plasmids and transfections
The plasmid pSR
-dok was generated by cloning an
EcoRI fragment corresponding to the human
p62dok cDNA (kindly provided by B. Stillman,
Cold Spring Harbor, NY) into the plasmid pSR puromycin. The
hemagglutinin (HA)-p62dok construct
(pSR-HA-dok) was generated as follows. The first methionine residue
of p62dok was deleted by PCR and cloned into the
plasmid MT073 (kindly provided by M. Thome, Institut de Biochimie,
Epalinges, Swizerland) in frame with the sequence encoding the HA
epitope. The HA-p62dok was then subcloned into
the plasmid pSR puromycin. J.77-6 cell line was transfected with 20
µg of pSR-HA-dok or pSR-dok by electroporation using a Gene
Pulser (Bio-Rad, Hercules, CA) set at 250 mV and 960 µF.
Drug-resistant cells were cloned by limited dilution in
puromycin-containing medium. Expression levels of
p62dok were evaluated by immunoblotting of cell
extracts with anti-p62dok. Expression levels
of CD2 and CD3 were evaluated by flow cytometric analysis with an EPICS
XL (Coulter Electronics, Hialeah, FL). Clones expressing similar levels
of CD3 and CD2 compared with the parental Jurkat cells were kept for
further studies. To quantify the amounts of
p62dok expressed in the transfectants, we
performed serial dilution of p62dok
immunoprecipitates. After Western blotting with
p62dok Abs, the ECL signal was quantified using
Kodak Image Station 440cf to acquire the image and the 1D Image
Analysis software.
Measurement of intracellular Ca2+
Cells were washed twice with HBSS and incubated at
107 cells/ml with 3 µM Indo-1 (Molecular
Probes, Eugene, OR) and 0.4 mg/ml Pluronic acid F-127 (Molecular
Probes) for 25 min at room temperature. Cells were washed in HBSS and
resuspended at 106 cells/ml, and
Ca2+ mobilization studies were conducted on an
EPICS ELITE ESP cell sorter (Coulter Electronics). Cells were
stimulated with anti-CD3 mAbs (UCHT1, 1/1000 dilution of
ascites) or the anti-CD2 mAb pair T11-2 + T11-3 (1/1000 dilution of
ascites). Successful loading with Indo-1 was confirmed by subsequently
treating the cells with 1 µM ionomycin. Violet/blue ratio signals
were analyzed using the MultiTime software (Phoenix Flow Systems, San
Diego, CA).
Immunoprecipitations and immunoblotting
Cells were washed twice in RPMI 1640 and resuspended at 5
x 107 cells/ml in RPMI 1640. Cells were left
unstimulated or stimulated with anti-CD3 (UCHT1 at 1/500
dilution of ascites) or anti-CD2 (a combination of T11-2 and T11-3
at 1/1000 dilution of ascites) for the indicated times. Cells were
harvested and solubilized for 30 min at 4°C in 1% Nonidet P-40
containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM
MgCl2, and 1 mM EGTA in the presence of
inhibitors of proteases and phosphatases (10 µg/ml leupeptin and
aprotinin, 1 mM Pefabloc-sc, 50 mM NaF, 10 mM
Na4P2O7,
and 1 mM NaVO4). Immunoprecipitations and
immunoblotting were performed as described previously
(10).
Subcellular fractionation
Cells were washed twice in RPMI 1640 and resuspended at 5
x 107 cells/ml in RPMI 1640. Cells were left
unstimulated or stimulated with anti-CD3 (UCHT1 at 1/500
dilution of ascites) or anti-CD2 (a combination of T11-2 and T11-3
at 1/1000 dilution of ascites) for the indicated times. To determine
p62dok and RasGAP cellular
redistribution, cells were fractionated into cytosolic and
membrane fractions, as described (16). Both fractions were
immunoprecipitated with either anti-p62dok
or anti-RasGAP Abs.
Luciferase assays
Jurkat cells (106 cells) were transfected
with 2.5 µg NF-AT-firefly luciferase (kindly provided by O. Acuto,
Institut Pasteur) and 0.5 µg thymidine kinase (TK)-Renilla
luciferase constructs (Promega, Madison, WI) using Fugene transfection
assays (Roche Diagnostics, Laval, Quebec, Canada). For transient
overexpression of p62dok, 1 µg of pSR-dok
or empty vector was used in combination with 1.5 µg NF-AT-firefly
luciferase and 0.5 µg TK-Renilla luciferase constructs.
After 24 h, 3 x 105 cells were
stimulated with plate-bound anti-CD3 or soluble anti-CD2 Abs
for 6 h in a 24-well plate. Maximal stimulation was obtained by a
combination of PMA (10 ng/ml) and iononycin (1 µM). Cells were then
lysed and assayed for luciferase activity using the dual luciferase
reporter assay system (Promega, Madison, WI) and a luminometer
(Berthold, LUMAT LB 9507). The NF-AT-firefly luciferase values were
normalized based on the constitutive Renilla luciferase
activity.
IL-2 assays
A total of 105 Jurkat cells was stimulated
by plate-bound anti-CD3 mAb (50 µl of UCHT1 at 1/100 dilution
of ascites in PBS), soluble anti-CD2 (a combination of T11-2 and
T11-3 at 1/1000 dilution of ascites), or PMA (10 ng/ml) and ionomycin
(1 µM) for 20 h in 96-well plates. Fifty microliters of
supernatant were assayed for IL-2 production using the IL-2-dependent
cell line CTLL-2, as described previously (17).
3H incorporation was assessed on a liquid
scintillation counter (MicroBeta Trilux).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the importance of p62dok in T
cell function, we stably transfected Jurkat cells with an expression
vector containing an unmodified or an HA-tagged version of
p62dok. Drug-resistant clones overexpressing
p62dok were identified by immunoblotting of
total cell lysates with anti-p62dok Abs
(data not shown). Clones were also tested for expression of CD2 and CD3
cell surface molecules, and those expressing levels comparable with
that of the Jurkat parental cell line were kept for further study (data
not shown). Three representative clones (clones 2, 74, and HA-1), shown
in Fig. 1
, that express at least 2.5
times more p62dok than the parental Jurkat cells
were subsequently characterized for their signaling capacity.
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A). Phosphorylation patterns remained
unaffected in CD3-stimulated cells in clones overexpressing
p62dok, indicating that
p62dok overexpression did not noticeably
prevent the activation of CD3-mediated tyrosine phosphorylation
pathways. CD2 stimulation led to the phosphorylation of a similar set
of proteins in all the clones, with the exception of a 62-kDa protein,
which is hyperphosphorylated in clones overexpressing
p62dok. We have previously shown that CD2
stimulation of Jurkat cells induces tyrosine phosphorylation of
p62dok (10). To ascertain that
the 62-kDa band corresponded to p62dok,
p62dok immunoprecipitations were performed
from the different Jurkat clones that were left unstimulated, CD2
stimulated, or CD3 stimulated, and were analyzed by
anti-phosphotyrosine immunoblotting (Fig. 1B). As
expected, CD2 stimulation of the parental Jurkat cell line and clones
that overexpress p62dok led to an increased
phosphorylation of p62dok. In contrast, CD3
stimulation did not induce p62dok
phosphorylation. It should be noted that there is a higher basal level
of p62dok phosphorylation in unstimulated
clones overexpressing p62dok compared with
the parental Jurkat cells. The increase in constitutively
phosphorylated p62dok correlates with the
levels of p62dok expressed in these clones.
Therefore, overexpression of p62dok does not
seem to affect the constitutive phosphorylation of
p62dok. Effect of p62dok overexpression on Ras signaling
To assess the role of p62dok in the Ras
signaling pathway, we examined whether
p62dok overexpression interferes with the
activation of Erk1/2 following CD2 or CD3 engagement (Fig. 2A). To monitor the activation
of Erk1/2, we used Abs specific for the phosphorylated form of Erk1/2.
As shown in Fig. 2, phospho-Erk1/2 induction after CD3 stimulation was
similar in all clones, indicating that CD3-induced Erk1/2 activation
was not inhibited by p62dok overexpression.
In contrast, there was a decrease in CD2-mediated Erk1/2 activation in
p62dok-overexpressing clones. The intensity
of this inhibitory effect correlated with the levels of
p62dok overexpression, as evidenced by a
greater decrease in CD2-induced Erk1/2 phosphorylation in clone 74 and
HA-1 when compared with clone 2. Moreover, the inhibition of
CD2-induced Erk1/2 phosphorylation could be rescued by PMA (Fig. 2B). These results show that
p62dok overexpression selectively inhibits
CD2- but not CD3-mediated activation of Erk1/2.
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We evaluated the effect of p62dok
overexpression on Ca2+ mobilization after CD2 and
CD3 stimulation. In Fig. 3, we show that
Ca2+ mobilization after CD3 stimulation was
unaltered in all cell lines, regardless of
p62dok expression levels. In contrast, the
CD2-induced Ca2+ response was abolished or
greatly diminished in clones overexpressing
p62dok. Inositol 3,4,5-triphosphate-mediated
mobilization of Ca2+ requires the activation of
PLC1. Since tyrosine phosphorylation of PLC1 contributes to the
activation of the enzymatic activity of PLC1, we next investigated
whether PLC1 tyrosine phosphorylation is affected by
p62dok overexpression after CD2 or CD3
stimulation (Fig. 4). After CD3
stimulation, PLC1 was highly phosphorylated in all cell lines
tested. However, there was a slight decrease in the phosphorylation
levels of PLC1 after CD2 stimulation when we compared clones 2 and
74 with the control J.77-6 cells. More importantly, PLC1
phosphorylation was abolished after CD2 stimulation of the HA-1 cell
line. The decreased or absence of phosphorylation of PLC1 in
p62dok-overexpressing cells might be, at
least in part, responsible for the diminished
Ca2+ influx following CD2 stimulation.
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The activation of signaling pathways initiated by engagement of
the CD3/TCR or the CD2 receptor leads to IL-2 production in Jurkat
cells. We examined whether p62dok
overexpression would influence IL-2 production. Jurkat clones were left
unstimulated or stimulated by anti-CD3, anti-CD2 Abs, or by PMA
and ionomycin. After 24 h, the culture supernatants were assayed
for IL-2 production (Fig. 5A).
Activation of the different clones by PMA and ionomycin resulted in
equivalent levels of IL-2 production, indicating that
p62dok overexpression did not alter the
capacity of the cells to produce IL-2. CD2 stimulation of
p62dok-overexpressing clones led to
significantly reduced levels of IL-2 production compared with the
parental cell line. This result was expected given that IL-2 gene
expression is dependent on Ca2+ mobilization and
Erk1/2 activation. The inhibition of CD2-induced IL-2 secretion seems
to correlate with the amount of p62dok
present in the cells. Surprisingly, PMA restored CD2-induced IL-2
secretion (Fig. 5A). In the clones 74 and HA-1, CD3-induced
IL-2 secretion was decreased, whereas CD3-induced IL-2 production in
the clone 2 was comparable with that of the parental cell
line.
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B). Cells were left unstimulated, or stimulated by
anti-CD3 or anti-CD2 Abs or PMA and ionomycin for 6 h. The
activation of NF-AT was evaluated by a luciferase activity assay.
p62dok overexpression inhibited CD2-induced
activation of NF-AT (Fig. 5B). Moreover, the intensity of
the inhibition correlates with the levels of
p62dok expressed in the cells. We observed a
5- and 15-fold decrease in NF-AT activation for clones 2 and clones 74
and HA-1, respectively. These results were confirmed by performing
transient transfection assays with plasmids containing
p62dok cDNA and the NF-AT luciferase
reporter construct. Transient overexpression of wild-type
p62dok or HA-tagged
p62dok inhibited (2- to 3-fold) CD2-induced
NF-AT activation (Fig. 5C).
As shown in Fig. 5D, PMA increases NF-AT activity upon CD2
stimulation in both wild-type Jurkat cells and cells overexpressing
p62dok. However, overexpression of
p62dok considerably inhibited NF-AT
activation in response to CD2 and PMA when compared with the parental
cell line. The NF-AT activity in clone HA-1 following treatment with
anti-CD2 and PMA is similar to the CD3-induced NF-AT activity in
wild-type cells. This result might, at least partially, explain why
CD2-induced IL-2 secretion is restored in presence of PMA in clones
overexpressing p62dok. NF-AT activation
requires the cooperative binding between NF-AT and AP-1 to the IL-2
promoter NF-AT site. NF-AT nuclear translocation is mediated by the
Ca2+-regulated phosphatase calcineurin, whereas
the synthesis and activation of Fos and Jun, components of the AP-1
family of transcription complexes, are mediated by the protein kinase C
(PKC)/Ras pathway. Therefore, the inability of PMA treatment to rescue
CD2-induced NF-AT activity is most likely due to the absence of
Ca2+ mobilization in these clones. As expected,
in cells overexpressing p62dok, restoration
of Ca2+ flux by treatment with ionomycin is not
sufficient to rescue the CD2-induced NF-AT activation, which is partly
dependent on Erk1/2 activation. In contrast, treatment with PMA and
ionomycin restored CD2-mediated activation of NF-AT in these cells
(Fig. 5E). This indicates that
p62dok overexpression does not lead to
nonspecific CD2-mediated inhibition of NF-AT activity.
We were surprised to find that p62dok
overexpression interfered with CD3-induced NF-AT activation given that
p62dok overexpression does not seem to
affect Ca2+ response and Erk1/2 activation (Fig. 5B). The CD3-induced activation of NF-AT was only slightly
reduced in clone 2, whereas there was a marked decrease in CD3-mediated
NF-AT activation in clones 74 and HA-1 (2-, 6-, and 28-fold decrease in
NF-AT activation for clones 2, 74, and HA-1, respectively). Therefore,
as shown for CD2 stimulation, the
p62dok-mediated inhibition of CD3-induced
NF-AT activation is dependent on the level of Dok overexpression. The
inhibition of CD3-induced NF-AT activation can be bypassed by treatment
with PMA and calcium ionophore used in combination, but not alone. We
have shown that the CD3-mediated Ca2+ response is
not affected in clones overexpressing p62dok
(Fig. 3). These data clearly suggest that there is a difference between
anti-CD3 Abs and Ca2+ ionophore with respect
to Ca2+ mobilization. Moreover, CD3-mediated
activation of Erk1/2 activation seems normal in clones overexpressing
p62dok (Fig. 2). Therefore, in these clones,
PMA treatment most likely compensates for a defective component that is
required in the NF-AT activation pathway and is downstream or
independent of Erk1/2 activation. In addition, in wild-type Jurkat
cells, CD3 stimulation or CD2 stimulation further increased NF-AT
activity induced by the combination of PMA and ionomycin. Therefore,
compared with antireceptor stimuli, the pharmacological stimuli PMA and
ionomycin may utilize an additional Ras-independent pathway, which
leads to NF-AT activation. These results underline the complexity of
the regulation of NF-AT activation. Additional experiments will be
required to elucidate the mechanisms by which
p62dok mediates inhibition of CD3-induced
activation of NF-AT.
Membrane localization and interaction of p62dok with RasGAP following CD2 stimulation
p62dok binding to the Src homology 2
(SH2) domain of RasGAP requires p62dok
phosphorylation, and RasGAP is known to be an important negative
regulator of Ras. Therefore, Dok-mediated inhibition of Ras signaling
might occur through its interaction with RasGAP. To test this
hypothesis, we compared the amount of RasGAP associated with
p62dok in clones ovexpressing
p62dok and in the parental cell line.
Lysates from cells that were left unstimulated or stimulated with
anti-CD2 mAbs for 3 and 10 min were immunoprecipitated with
anti-p62dok Abs. CD2 stimulation
resulted in an increase in the amount of RasGAP associated with Dok in
cells overexpressing Dok and in the parental cell line (Fig. 6). Importantly, the amount of RasGAP
associated with p62dok correlates with the
amount of phosphorylated p62dok in the cells
(Fig. 6).
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). In all the cell lines, the majority of
p62dok was found in the soluble fraction
(Fig. 7A–C). The amount of membrane-bound
p62dok in clones overexpressing
p62dok was significantly higher than in the
parental cell line. However, the percentage of total
p62dok present in the membrane fraction in
unstimulated cells did not change significantly with varying levels of
p62dok. There was a slight increase in the
total amount of p62dok in the particulate
fraction after CD2 stimulation, whereas CD3 stimulation did not alter
the localization of p62dok. The relative
proportion of phosphorylated p62dok at each
stimulation condition and cytofraction was calculated by normalizing
the phosphotyrosine signal to the amount of precipitated protein, and
the ratio of these two signals is presented in Fig. 7. These data
clearly show that in all the clones, the percentage of phosphorylated
p62dok was higher in the membrane fraction
than in the cytosolic fraction at each stimulation condition. These
results indicate that the phosphorylation of
p62dok is mainly occurring at the membrane
or that once phosphorylated, p62dok is
preferentially recruited to the membrane.
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D). As shown for
p62dok, CD2 stimulation induced the
translocation of RasGAP to the plasma membrane, whereas CD3 stimulation
had no effect on RasGAP subcellular localization. Altogether, these
results suggest that recruitment of RasGAP to the membrane following
CD2 stimulation is mediated by tyrosine-phosphorylated Dok present at
the membrane. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 phosphorylation, Erk1/2 activation, and
Ca2+ mobilization, whereas these events remain
unaffected when initiated via the CD3/TCR complex.
Our finding that overexpression of Dok affects PLC1 phosphorylation
upon CD2 stimulation indicates that p62dok
functions early in the signal transduction cascade initiated via CD2.
Activation of PLC1 requires several targeting signals for its
recruitment to the plasma membrane, where its substrate,
phosphatidylinositol 4,5-biphosphate, is found. SH2-mediated
interaction of PLC1 with linker for activation of T cells
(18) and binding of its PH domain to the phosphoinositide
3-kinase products, phosphatidylinositol 3,4,5-triphosphate molecules in
the membrane, represent two important steps for PLC1 activation
(19, 20). In addition, tyrosine phosphorylation of PLC1
is required for complete stimulation of its enzymatic activity and is
most likely mediated by the tyrosine kinase Itk (21, 22).
The Gads/SLP-76 complex recruited by linker for activation of T
cells also plays an important role for PLC1 activation (23, 24). p62dok overexpression may
interfere with the formation of any of these signaling complexes.
Additional experiments are required to define the molecular basis of
p62dok involvement in PLC1
activation.
Importantly, we found that Dok-mediated inhibition of PLC1
phosphorylation, Erk1/2 activation, and Ca2+
mobilization correlates with phosphorylation of specific tyrosine
residues that are phosphorylated upon CD2 cross-linking. Indeed,
although the amount of tyrosine-phosphorylated
p62dok is higher in unstimulated or
CD3-stimulated clones 74 and HA-1 than in CD2-stimulated clone 2, there
is no inhibition of CD3-mediated PLC-1 phosphorylation, Erk1/2
activation, and Ca2+ mobilization in these
clones. Therefore, if phosphotyrosine residues are involved in
CD2-mediated inhibition of PLC1, Erk1/2, and/or
Ca2+ mobilization, these tyrosine residues are
specifically phosphorylated upon CD2 stimulation and are different from
those that are constitutively phosphorylated.
Since PLC1 is a critical regulator of
Ca2+ mobilization, defect in PLC1
activation is a likely cause of the impaired Ca2+
response in clones overexpressing p62dok.
However, we cannot exclude the possibility that overexpression of Dok
may directly interfere with Ca2+ mobilization
downstream of inositol 3,4,5-triphosphate production.
The inhibition of Erk1/2 activation following CD2 stimulation in clones
overexpressing p62dok may also be secondary
to defective induction of PLC1 activation and diacylglycerol (DAG)
production. DAG, a product of PLC1, activates PKC, which in turn
stimulates Erk1/2 via the Ras/Raf pathways (25).
Consistent with a role of PKC-mediated Erk1/2 activation, PMA rescued
CD2-induced IL-2 secretion and Erk1/2 activation in clones
overexpressing Dok (Figs. 5A and 2A). Since the
contribution of PKC activation in the CD2 regulation of Erk1/2 activity
has not been studied, it is not possible to discriminate whether
p62dok acts in CD2 signaling upstream of PKC
or acts on a CD2-induced Erk1/2-activating pathway independent of
PKC.
The recently described Ras-guanine nucleotide exchange factor, RasGRP, has been shown to be involved in CD3-induced Ras activation (26, 27). DAG binding to RasGRP can recruit RasGRP to the membrane and thereby promote Ras signaling. Although the importance of RasGRP in the regulation of CD2-induced Ras activation is unknown, impaired production of DAG in clones overexpressing p62dok might be in part responsible for the decrease in Ras-mediated Erk1/2 activation.
Ras function is negatively regulated by RasGAP, which stimulates the GTPase activity of Ras (28). In addition, RasGAP binds to tyrosine-phosphorylated p62dok via an SH2-mediated interaction (2, 3). Therefore, RasGAP was a good candidate for mediating the negative effect conducted by p62dok in the Ras/Erk1/2 signaling pathway. We have shown that there is a specific recruitment of RasGAP to the plasma membrane following CD2 stimulation and not CD3 stimulation. Moreover, the relocalization of RasGAP is mediated by phosphorylated p62dok. Although it was recently reported using an in vitro assay that p62dok binding to RasGAP diminished its catalytic activity (29), recruitment of RasGAP in the proximity of Ras is likely to have a global negative effect on Ras function. In addition, binding of p62dok to RasGAP might induce a conformational change that exposes domains of RasGAP (such as SH3) to binding partners. This would allow RasGAP to undergo additional protein interactions that might be important in Dok-mediated functions.
Surprisingly, we find that IL-2 secretion and NF-AT activation induced by CD3 cross-linking are inhibited by p62dok overexpression. The level of inhibition correlates with the amount of p62dok overexpression. The inhibition of CD3-induced IL-2 expression might be related to the increased amount of tyrosine-phosphorylated p62dok present in clones overexpressing p62dok. Alternatively, but not exclusively, other structural elements of p62dok might be responsible for Dok-mediated effects on CD3-induced IL-2 secretion. Activation of Erk1/2 is required, but not sufficient for NF-AT induction (30). Since we have shown that CD3-induced Erk1/2 activation is not affected by overexpression of p62dok, p62dok may inhibit the Rac-regulated pathway involved in the regulation of NF-AT induction (31). In unstimulated cells, the amount of membrane-bound p62dok in clones overexpressing p62dok is significantly higher than in the parental cell line. Our current data do not allow us to distinguish whether CD3-mediated inhibition of NF-AT activation takes place in the cytosol or/and in the membrane. In any case, it is important to point out that CD2 stimulation, and not CD3 stimulation, specifically increases tyrosine phosphorylation and induces membrane translocation of p62dok. Therefore, the involvement of p62dok in the signaling cascade initiated by CD2 is likely to be different from that initiated by CD3.
Our data support a model in which phosphorylation of
p62dok following CD2 engagement enables its
translocation from the cytosolic to the membrane compartment.
Activation of phosphoinositide 3-kinase upon CD2 stimulation (32, 33) is likely to be involved in the PH domain-dependent
recruitment of p62dok to the membrane
(12). Recently identified CD2-binding proteins
(34, 35, 36) might also regulate CD2-mediated
p62dok membrane localization. Once at the
membrane, p62dok is in the proximity of
various protein tyrosine kinases, such as Lck, thereby allowing
phosphorylation of tyrosine sites that act as SH2 acceptor sites for
downstream signaling molecules such as RasGAP.
p62dok-mediated RasGAP recruitment to the
proximity of Ras leads to the accumulation of RasGDP and consequently
to the down-regulation of CD2-mediated Erk1/2 activation. Although this
model takes into account the p62dok-mediated
Ras inhibition, other molecules are likely to be recruited by
p62dok and involved in Dok-mediated
inhibition of PLC1. To identify such molecules, we have tested the
co-association of p62dok with inhibitory
molecules such as Csk, SH2-containing inositol 5'-phosphatase-1, or
-2, but were not able to detect any significant interactions
(data not shown).
Maximal phosphorylation of p62dok occurred
10 min following CD2 stimulation and remained high for 30 min (data not
shown). This slow time course supports a model in which
p62dok could be considered a hinge molecule
that comes into play to down-regulate CD2-mediated activation signals.
Although originally described as a receptor delivering stimulatory
signals to the cell upon ligand binding (37), CD2 has also
been described as a molecule capable of transducing inhibitory signals
(38, 39, 40). Since p62dok is
clearly involved in the negative regulation of B cell signaling
mediated by FcRIIB (13, 14),
p62dok might be considered as a key molecule
involved in the modulation of the response mediated by the TCR and the
B cell receptor.
In conclusion, p62dok is a multifunctional adaptor protein that most likely plays a negative role at multiple steps in the CD2 signaling pathway. Better insight into the molecules recruited by p62dok will help to identify the molecular mechanisms involved in Dok-mediated inhibition of T cell signaling.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Pascale Duplay, Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies, Laval QC, H7V 1B7, Canada.
3 Abbreviations used in this paper: PH, pleckstrin homology; DAG, diacylglycerol; Erk, extracellular signal-regulated kinase; HA, hemagglutinin; PKC, protein kinase C; PLC, phospholipase C; PTB, phosphotyrosine binding domain; RasGAP, RasGTPase-activating protein; SH, Src homology; TK, thymidine kinase.
Received for publication August 30, 2000. Accepted for publication January 30, 2001.
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