|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 275, Issue 19, 14590-14597, May 12, 2000
From the Institut National de la Recherche Scientifique, Institut
Armand-Frappier, Université du Québec, Laval,
Québec H7V 1B7, Canada
The Lck tyrosine kinase is involved in signaling
by T cell surface receptors such as TCR/CD3, CD2, and CD28. As other
downstream protein-tyrosine kinases are activated upon stimulation of
these receptors, it is difficult to assign which
tyrosine-phosphorylated proteins represent bona fide Lck
substrates and which are phosphorylated by other tyrosine kinases. We
have developed a system in which Lck can be activated independently of
TCR/CD3. We have shown that activation of an epidermal growth factor
receptor/Lck chimera leads to the specific phosphorylation of Ras
GTPase-activating protein (RasGAP) and two RasGAP-associated proteins,
p56dok and p62dok.
Activation of the chimeric protein correlates with an increase in
cellular Ca2+ in the absence of ZAP-70 and phospholipase
C Lck is a member of the Src family
PTKs1 that participates in
signal transduction pathways initiated by T cell surface receptors such
as TCR/CD3, CD2, CD4, CD8, and CD28. Following cross-linking of these
receptors, an increase in its kinase activity has been reported (1-3).
Moreover, the absolute requirement of Lck in these pathways has been
demonstrated through the use of mutant cell lines that lack Lck
expression (4-8). Two regulatory tyrosine residues, Tyr-505 and
Tyr-394, control Lck activity. Dephosphorylation of tyrosine 505, likely by the tyrosine phosphatase CD45, is thought to allow the
disruption of an inhibitory intramolecular interaction between the
phosphorylated Tyr-505 and the SH2 domain of Lck (9-11). Moreover, the
catalytic and functional activities of Lck are dependent on the
phosphorylation of tyrosine 394 (12). Structural domains including the
unique SH2 and SH3 domains contribute to regulate Lck kinase activity
and specificity (13-20). In addition, Lck function depends on the
ability of the SH3 and SH2 domains to interact with cellular molecules
that may represent specific substrates or regulatory proteins
(21-25).
Although the structural basis for Lck activation is well known, the
molecular mechanisms that take place in vivo in Lck
activation and function are not well understood. CD4 cross-linking has
been shown to enhance Lck activity (3). This might be a consequence of
oligomerization of CD4/Lck, a process that takes place upon binding of
CD4 to MHC/TCR/Ag (reviewed in Ref. 26). One of the consequences of CD4
activation of Lck is the translocation of Lck to the Nonidet P-40
insoluble cytoskeleton fraction (27). In addition, cross-linking of
defined epitopes of the CD4 molecule can lead to the induction of
Ca2+ flux, phosphorylation of Shc, and activation of the
nuclear factor of activated T cells transcription factor (28). CD4
cross-linking has also been reported to inhibit CD3-mediated signaling
by sequestering the CD4-associated Lck from TCR/CD3 (29). Lck can also
be activated independently of the CD4 or CD8 coreceptor. Indeed,
Lck activity has been reported to increase shortly after CD3 or CD2
cross-linking (2). Stimulation through these receptors results in the
phosphorylation of a partially overlapping set of membrane and
cytosolic proteins (30, 31). These proteins represent potential
in vivo substrates of Lck and several of them such as p95Vav
(32), SHP-1 (33), RasGAP (34), CD5 (35, 36), and the To identify potential Lck substrates, we designed a system where Lck
activation can be achieved independently of TCR/CD3 or the CD2
receptor. We introduced into Jurkat cells a chimeric protein that
contains the Lck sequence fused to the extracellular and transmembrane
domains of EGFR. We have studied the effect of EGF-mediated activation
of the EGFR/LckF505 chimera on signal transduction events. We show that
phosphorylation of RasGAP and two RasGAP-associated proteins,
p62dok and p56dok, are
specifically induced after Lck activation. Moreover, their phosphorylation correlates with an increase in intracellular
Ca2+. Importantly, CD2 stimulation induces the
phosphorylation of RasGAP, p62dok, and
p56dok, whereas CD3 stimulation does not. We
propose that phosphorylation of these proteins by Lck plays an
important role in CD2-mediated signaling.
Cell Lines and Antibodies--
The J-CD45-64 Jurkat cell line
is a derivative of the CD45
mAbs used included anti-CD3 Transfection of the EGFR/Lck Chimeras--
The EGFR/Lck
constructs have been described previously (41). Transfections of the
J-CD45-64 cell line were performed with a Gene Pulser (Bio-Rad) set at
250 mV and 960 microfarads. Drug-resistant cells were cloned by limited
dilution in puromycin-containing medium as described (41). Expression
of the transfected EGFR/Lck chimera and CD3 cell surface expression
were screened by flow cytometric analysis with an EPICS XL (Coulter
Electronics, Hialeah, FL).
Immunoprecipitations and Immunoblotting--
Cells were washed
twice in RPMI 1640 and resuspended at 5 × 107
cells/ml in RPMI 1640. Cells were left unstimulated or stimulated with
anti-CD3 (UCHT1 at 1:500 dilution of ascites), anti-CD2 (a combination
of T11-2 and T11-3 at 1/500 dilution of ascites), or EGF (100 ng/ml)
for the time indicated. 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). Cells were lysed in buffer containing 0.25%
n-dodecyl- Measurement of Intracellular Ca2+--
Cells were
washed twice with Hanks' balanced salt solution (Life Technologies,
Inc.) and incubated at 107 cells/ml with 3 µM
indo-1 (Molecular Probes) and 0.4 mg/ml Pluronic acid F-127 (Molecular
Probes) for 25 min at room temperature. Cells were washed in Hanks'
balanced salt solution and resuspended at 5 × 106
cells/ml, and Ca2+ mobilization was conducted on an EPICS
ELITE ESP cell sorter (Coulter Electronics, Hialeah, FL). Cells were
stimulated with the mAb UCHT1 (1:1000 dilution of ascites) or EGF (100 ng/ml) as indicated. Successful loading with Indo-1 was confirmed by subsequently treating the cells with ionomycin. Violet/blue ratio signals were analyzed using the MultiTime sofware (Phoenix, Flow Systems Inc., San Diego, CA).
SH2 Binding Assay--
MBP and MBP-SH2-Sepharose beads were
prepared as described (22). Postnuclear lysates were precleared for
1 h with MBP-Sepharose beads and incubated for 2 h with
MBP-SH2 beads. The complexes were washed in the same conditions as the
immune complexes and eluted by boiling in the presence of SDS sample
buffer or by incubating twice for 10 min at 4 °C in the presence of
50 mM phenyl phosphate (a structural analog of
phosphotyrosine) in 1% detergent lysis buffer under constant agitation.
Oligomerization of Lck F505 Induces the Specific Phosphorylation of
RasGAP and p62dok--
To identify novel Lck substrates,
we have developed T cell lines in which Lck activation is induced
independently of TCR/CD3. In this system, the entire Lck sequence
except the 28 first amino acid residues is fused to the extracellular
and transmembrane domains of the EGFR. Three chimeric PTKs containing
either a wild-type (WT), a constitutively active (Phe-505), or a kinase
inactive (Ala-273) version of Lck were generated (41). Multiple clones were established following transfection of a Jurkat cell line, J-CD45-64, with the different EGFR/Lck constructs, and levels of
expression of EGFR/Lck were analyzed. Data of representative clones,
which express comparable levels of TCR/CD3 at their cell surface, are
shown. In unstimulated cells, constitutive tyrosine phosphorylation of
the EGFR/LckWT chimera is much higher than that of the EGFR/LckF505
chimera (Fig. 1). This is likely because of the phosphorylation of Tyr-505, this site being absent in the EGFR/LckF505. The accessibility of this site to the CD45 tyrosine phosphatase might be limited in the EGFR/Lck chimera. Consequently, the
high phosphorylation of Tyr-505 is likely to prevent EGF-induced activation of the WT chimera (see below). The addition of EGF induces
the tyrosine phosphorylation of the EGFR/LckF505 chimeric protein (Fig.
1). The increase of EGFR/LckF505 tyrosine phosphorylation is likely to
occur by autophosphorylation of Tyr-394, which is the predominant site
of increased tyrosine phosphorylation after CD4 cross-linking. In
contrast, the addition of EGF has no effect on the phosphorylation of
the EGFR/LckWT and EGFR/LckA273 chimeras (Fig. 1). This result suggests
that EGF-induced phosphorylation of the EGFR/Lck chimera requires a
kinase domain that is constitutively activated.
We next investigated whether the increased tyrosine phosphorylation of
the EGFR/LckF505 chimera correlates with an increase of the activity of
the EGFR/Lck kinase by analyzing the effect of LckF505 oligomerization
on tyrosine phosphorylation of cellular proteins (Fig.
2). The pattern of phosphorylation
induced following CD3 cross-linking was similar in all the cell lines
expressing the EGFR/Lck and in the parental cell line. This result
indicates that expression of the EGFR/Lck chimera does not interfere
with the CD3-mediated signaling pathway. No change in the pattern of phosphorylated proteins is detected following EGF stimulation in the
cells expressing the EGFR/LckWT or the EGFR/LckA273 chimera. In
contrast, in the EGFR/LckF505 cell line, EGF induces the specific tyrosine phosphorylation of 4 proteins of Mr
56,000, 62,000, 80,000, and 120,000.
Previous reports suggested that RasGAP and RasGAP-associated proteins
represent potential substrates of the Lck tyrosine kinase (43). To
investigate whether the p56, p62, and p120 phosphoproteins correspond
to RasGAP and to the two recently cloned RasGAP-associated proteins
(44-47), p62dok and
p56dok, we performed immunoprecipitation
experiments. Anti-RasGAP and anti-p62dok
immunoprecipitates were obtained from EGFR/LckF505 Jurkat cells that
were unstimulated, EGF-stimulated, or CD3-stimulated and were analyzed
by antiphosphotyrosine immunoblots (Fig.
3A). In unstimulated cells,
the RasGAP protein is not phosphorylated, whereas a very low level of
phosphorylation of RasGAP-associated p62 protein is detected. EGF
stimulation induces an increase in tyrosine phosphorylation of RasGAP
and RasGAP-associated p62 protein. In contrast, CD3 stimulation has no
effect on the phosphorylation status of RasGAP and RasGAP-associated
p62 protein. The RasGAP-associated protein of Mr
190,000, likely to correspond to p190RhoGAP (48), is
constitutively phosphorylated with no further increase in tyrosine
phosphorylation following EGF or CD3 stimulation. To investigate
whether the p62 RasGAP-associated protein corresponds to
p62dok, we performed
p62dok immunoblots on RasGAP immunoprecipitates
(Fig. 3A). The amount of tyrosine-phosphorylated p62
RasGAP-associated protein correlates with the amount of
p62dok present in RasGAP immunoprecipitates. In
addition, phosphorylation of p62dok following
activation of the EGFR/Lck chimera was verified by antiphosphotyrosine
immunoblotting of p62dok immunoprecipitates.
Activation of the EGFR/Lck chimera leads to the phosphorylation of
another member of the Dok family, p56dok, as
shown in Fig. 3B.
Because the pattern of proteins phosphorylated by the EGFR/LckF505
chimera appeared different from that of proteins phosphorylated following CD3 cross-linking (see Fig. 2), we assessed the
phosphorylation status of specific proteins implicated as effector
proteins downstream of CD3-mediated Lck activation. Whereas CD3
stimulation induces an important increase in ZAP-70 and PLC Phosphorylation of RasGAP, p56dok, and
p62dok Correlates with an Increase in the Concentration of
Intracellular Ca2+--
To evaluate the effect of the
phosphorylation of RasGAP and RasGAP-associated proteins on
intracellular Ca2+ mobilization, we compared the
Ca2+ response of the various cell lines after EGF and CD3
stimulation. Oligomerization of LckF505 by EGF induces a rapid and
sustained rise in the concentration of intracellular Ca2+
(Fig. 5). In contrast, no
Ca2+ increase can be detected after oligomerization of the
EGFR/LckWT or EGFR/LckA273 chimeras (Fig. 5). Expression of EGFR/LckWT,
EGFR/LckA273, or EGFR/LckF505 does not alter the CD3-mediated
Ca2+ response because CD3 cross-linking resulted in the
characteristic increase in intracellular Ca2+. Because
PLC CD2-mediated Lck Activation Induces RasGAP, p56dok, and
p62dok Phosphorylation--
The CD2-mediated activation
pathway leads to the phosphorylation of RasGAP and a RasGAP-associated
p62 protein (4, 49). Therefore, we investigated whether there is an
increase in p62dok and
p56dok phosphorylation following CD2 stimulation
of Jurkat cells. As reported previously (49), tyrosine phosphorylation
of RasGAP is induced upon CD2 cross-linking with a mAb pair,
anti-T11-2 and anti-T11-3 (Fig.
6A). Similarly, CD2
stimulation induced an increase in the phosphorylation of
p56dok and p62dok as
detected by antiphosphotyrosine immunoblotting of
p56dok and p62dok
immunoprecipitates (Fig. 6A). Phosphorylation of the Dok
proteins allows their subsequent binding to RasGAP as detected by an
increase in the amounts of p56 and p62-associated proteins in the
RasGAP immunoprecipitates (Fig. 6A). Because CD2-mediated
activation occurs in the absence of ZAP-70 phosphorylation (Fig.
6B), these results suggest that activation of EGFR/LckF505
induces signal transduction pathways overlapping with those induced by
CD2 stimulation.
To determine whether Dok phosphorylation required Lck, CD2 stimulation
was performed in JCaM1.6 cells (Fig. 6C). These cells lack
functional Lck and express at their cell surface a similar level of CD2
to that of the wild-type Jurkat cells (data not shown). No
tyrosine-phosphorylated p56dok and
p62dok were detectable following CD2 stimulation
in p56dok, p62dok, and
RasGAP immunoprecipitates. Reconstitution of JCaM1.6 cells with
wild-type Lck restores the CD2-induced phosphorylation of p56dok and p62dok (Fig.
6D). This result demonstrates that CD2-mediated
phosphorylation of Dok proteins requires Lck.
Association of Lck with p56dok and p62dok
via an SH2-mediated Interaction--
To test if an interaction between
Lck and p62dok occurs in vivo,
immunoprecipitations of EGFR/Lck were performed with anti-EGFR antibodies and analyzed by antiphosphotyrosine and
anti-p62dok immunoblottings (Fig.
7). Immunoprecipitation of the
EGFR/LckF505 chimera in EGF-stimulated cells compared with unstimulated
cells revealed two additional phosphorylated proteins of
Mr 62,000 and 80,000. The 62-kDa phosphoproteins
may correspond to p62dok. Immunoblots with
anti-p62dok antibodies confirmed that a fraction
of phosphorylated p62dok was present in EGFR/Lck
immunoprecipitates (Fig. 7). The antibodies against
p56dok that we used in this study react weakly
in Western blotting with human p56dok.
Therefore, we were not able to conclusively identify
p56dok in the EGFR/Lck immunoprecipitate.
To gain better insight into the mechanisms by which Lck is involved in
the phosphorylation of RasGAP and p62dok, we
examined whether the SH2 domain of Lck is able to bind to these
proteins. Tyrosine-phosphorylated proteins that bind to the Lck SH2
domain were identified using in vitro binding assays with
MBP·LckSH2 fusion protein and antiphosphotyrosine immunoblotting (Fig. 8A). The MBP·SH2
fusion protein bound proteins of 62- and 56-kDa when incubated with a
lysate of EGF-induced LckF505 cells. The 56- and 62-kDa proteins
correspond to the p56dok and
p62dok proteins, respectively, as shown by
immunoprecipitations with anti-p56dok and
anti-p62dok antibodies after elution of the
SH2-bound proteins (Fig. 8A). Moreover, the interaction of
the Lck SH2 domain with p56dok and
p62dok can be reconstituted in vitro
in SDS-denatured cell lysates (Fig. 8B) indicating that it
is a direct interaction. Although CD3 stimulation of the same cells
induced the phosphorylation of several proteins able to bind to Lck SH2
domain, there is no binding of p56dok and
p62dok. This result is consistent with the
absence of phosphorylation of p56dok and
p62dok after CD3 stimulation (see Fig. 3). As
shown in Fig. 8C, an association of
p56dok and p62dok was
detected with Lck·SH2 fusion protein using lysates isolated from
CD2-activated Jurkat cells, whereas this association is barely detectable in resting or CD3-activated cells. Interestingly, the pattern of proteins able to bind to the Lck SH2 domain after CD2 cross-linking is very similar to that after EGFR/Lck activation. Altogether these results suggest that upon CD2 stimulation,
phosphorylated p56dok and
p62dok associate with Lck via a SH2-mediated
interaction.
In this study, we identified three potential Lck substrates. We
showed that oligomerization of an activated version of a EGFR/Lck chimera enhances the specific phosphorylation of four major cellular proteins including RasGAP, two RasGAP-associated proteins,
p62dok and p56dok, and an
unidentified 80-kDa protein. Lck-dependent phosphorylation of two of these proteins has been previously reported (34, 43). RasGAP
and an associated protein p62, that likely corresponds to
p62dok, are tyrosine-phosphorylated in the LSTRA
thymoma cell line in which Lck is overexpressed and in
LckF505-transformed fibroblasts (43). Moreover, RasGAP can serve as an
in vitro substrate for Lck (34). Once phosphorylated, two of
these proteins, namely p56dok and
p62dok, are recognized by the SH2 domain of Lck
in vitro. Given that cytosolic PTKs preferentially
phosphorylate sites recognized by their own SH2 (50), this result
supports the idea that p56dok and
p62dok represent Lck substrates. Activation of
Lck leads to subsequent activation of downstream PTKs such as ZAP-70
and Itk (51, 52). None of these kinases are activated following EGF
treatment (see Fig. 4 and data not shown). Although we cannot rule out
that yet unidentified tyrosine kinases are activated, taken together
these results favor a direct phosphorylation of these proteins by the EGFR/Lck chimera.
We believe that the proteins phosphorylated following EGFR/Lck
activation represent relevant Lck substrates involved in CD2- but not
CD3-mediated signaling. Two points argue against the hypothesis that
substrates of the EGFR/Lck chimera do not correspond to those phosphorylated by Lck in physiological conditions. (a)
Activation of the chimera induces the phosphorylation of many of the
same proteins induced by CD2 stimulation. This includes RasGAP,
p62dok, and p56dok. In
contrast, none of these proteins are phosphorylated after CD3
stimulation. (b) Activation of the CD2-signaling pathway and activation of the EGFR/Lck chimera are not coupled with ZAP-70 tyrosine kinase.
Since both CD3 and CD2 stimulation lead to Lck activation (2), why does
the phosphorylation of Dok proteins occur only in the CD2 pathway and
not in CD3 pathway? The difference in Lck substrate specificity
observed after CD2 and CD3 activation might result from different
relocalization of Lck induced by these two stimuli. In CD2-stimulated T
cells, Lck has been shown to be internalized in the endosomal fraction
(39). This Lck redistribution is not observed upon CD3 stimulation.
Cell surface expression of EGFR/LckF505 is modulated upon the addition
of EGF (data not shown). It is tempting to speculate that the chimera
will be localized in the same cellular compartment as Lck after CD2 stimulation.
What are the effects of the tyrosine phosphorylation of RasGAP and
RasGAP-associated proteins on T cell signaling? We showed in this study
that there is a correlation between phosphorylation of these proteins
and Ca2+ influx from the extracellular medium. Consistent
with this finding, previous reports suggested that Lck activation by
itself is sufficient for Ca2+ mobilization. Introduction of
LckF505 in Jurkat can substitute for the Ca2+ signal
required for interleukin-2 promoter activation (53). CD4/Lck
cross-linking with some anti-CD4 mAbs results in an increase in the
concentration of intracellular Ca2+ (28). It will be
interesting to examine tyrosine phosphorylation of RasGAP and
RasGAP-associated proteins in these conditions. However, using several
CD4 antibodies directed against different epitopes, we were unable to
detect tyrosine phosphorylation of RasGAP and RasGAP-associated
proteins as well as Ca2+ mobilization following CD4
cross-linking in Jurkat cells. The reason for this discrepancy is
unknown but might reflect different conditions used to cross-link the
CD4 molecule. The mechanism by which Lck mediates Ca2+
influx is unknown, but our results suggest that the Ca2+
response can occur with very little activation of PLC We provide evidence that Lck physically associates in vivo
with phosphorylated p56dok and
p62dok. This association was detectable only in
conditions where sufficient amounts of phosphorylated Dok were present,
i.e. after activation of the EGFR/Lck chimera. We have been
unable to detect the association between Lck and
p62dok in lysates from CD2-stimulated Jurkat
cells, likely because the level of p62dok
associated with Lck (i.e. phosphorylated
p62dok) is too low to be detected by Western
blotting. The data presented here support the conclusion that this
association is mainly mediated by a specific binding of the SH2 domain
of Lck to Dok. We did not detect significant amounts of
p62dok bound to Lck in its unphosphorylated
form. Upon CD2 stimulation or EGF treatment, the phosphorylated Dok
proteins are capable of binding directly to the SH2 of Lck in
vitro. This interaction is remarkably specific, because these two
Dok proteins represent the two major phosphorylated bands present in
the SH2-binding assay. Phosphorylated RasGAP is barely detectable in
these conditions (data not shown), which supports the hypothesis that
the binding of Dok proteins to the Lck SH2 domain is direct and not
mediated by RasGap. At least two tyrosine binding motifs,
YXX(I/L/V), within p62dok
and p56dok sequences could act as acceptor sites
for the Lck SH2 domain.
Very little is known about p56dok and
p62dok function and the role of phosphorylation
in the modulation of these functions. Based on the presence of several
signaling domains (pleckstrin homology, phosphotyrosine-binding domain,
tyrosine residue, and proline-rich regions), it has been proposed that
they act as docking proteins that link receptor-coupled PTKs to signal
transduction pathways (44-46). Tyrosine phosphorylation of
p62dok and p56dok occurs
upon stimulation of cells with a variety of stimuli or in cells
transformed by oncogenic tyrosine kinases such as v-Src and BCR-Abl
(44, 45, 56-60). In T cells, phosphorylation of p62dok has been reported upon CD2 (this study
and Ref. 49) and CD28 stimulations (61, 62).
p56dok has been proposed to be a negative
regulator of cytokine-induced proliferation in T cells (47), and we
demonstrated here that phosphorylation of p56dok
is specifically induced upon CD2 stimulation. The interaction of the
Lck SH2 domain with Dok is likely to represent an important step in the
course of CD2-mediated T cell activation. Phosphorylation of Dok
proteins by Lck might provide a mechanism by which SH2-containing proteins can be recruited and co-localized with their substrates. A
SH2-mediated interaction has been reported between members of the Dok
family and Nck, RasGAP, and Csk (44-46, 56, 58, 60, 63). Interaction
of Dok with Csk might provide a mechanism by which Dok participates in
the down-modulation of the CD2 response by localizing Csk near Lck.
Recently, a role for Nck in regulating the T cell cytoskeleton has been
proposed (64). Moreover, induction of tyrosine phosphorylation of
p62dok by cell adhesion to extracellular matrix
proteins has been shown to be mediated by Src family kinases (65). It
is therefore conceivable that a trimolecular complex Lck/Dok/Nck is
involved in the rearrangement of the cytoskeleton initiated by CD2
cross-linking (reviewed in Ref. 66). The RasGAP-associated p190 protein
acts as a GAP for members of the Rho GTPase family (48, 67). Therefore,
through its binding to RasGAP this protein might also regulate actin microfilaments.
In conclusion, our data provide the first evidence that in T cells Lck
phosphorylates and associates with members of the Dok family,
p56dok and p62dok. The
Dok family appears to have a selective function in accessory receptor
signal transduction mechanisms as they are not a substrate for
TCR-regulated PTKs. Better insight into the molecules they recruit will
help to elucidate the function of these molecules in T cell signaling
and delineate the respective roles of p56dok and
p62dok in T cell signaling.
We thank A. Alcover, W. Paul, B. Stillman, D. Straus, and E. Reinherz for kindly providing reagents; M. Desrosier and
J. Roger for technical assistance with this work; and A. Descoteaux, K. Gehring, and A. Veillette for critical reading of this manuscript.
*
This work was supported by Grant MT-14811 from the Medical
Research Council of Canada.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
PTK, protein-tyrosine kinase;
TCR, T cell receptor;
CD, cluster of
differentiation;
SH, Src homology;
EGF, epidermal growth factor;
EGFR, epidermal growth factor receptor;
mAb, monoclonal antibody;
MBP, maltose-binding protein;
WT, wild type;
PLC, phospholipase C;
RasGAP, Ras GTPase-activating protein.
Evidence That Lck-mediated Phosphorylation of
p56dok and p62dok May
Play a Role in CD2 Signaling*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 phosphorylation. Furthermore, we have found that
p62dok co-immunoprecipitates with the activated
epidermal growth factor receptor/LckF505 and that phosphorylated Dok
proteins bind to the Src homology 2 domain of Lck in vitro.
In addition, we have shown that activation via the CD2 but not the
TCR/CD3 receptor leads to the phosphorylation of
p56dok and p62dok.
Using JCaM1.6 cells, we have demonstrated that Lck is required for
CD2-mediated phosphorylation of Dok proteins. We propose that phosphorylation and Src homology 2-mediated association of
p56dok and p62dok with
Lck play a selective function in accessory receptor signal transduction mechanisms.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain (37)
serve as in vitro substrates for Lck. However, it is not
clear whether Lck is the kinase directly phosphorylating these proteins
in vivo. One of the most likely physiological substrates of
Lck in TCR/CD3-mediated activation is the chain (reviewed in Ref.
38). In contrast, or ZAP-70 phosphorylation is barely detectable
following CD2 stimulation (4). Moreover, CD2-mediated Lck increased
activity can be detected in CD3
cells (2). This suggests
that Lck activation can occur independently of TCR/CD3. Lck
redistribution to the endosomal fraction has been reported upon CD2 but
not CD3 stimulation (39). T cells expressing high levels of surface CD2
can signal via CD2 in absence of TCR/CD3 expression (40). Altogether,
these results suggest that important differences exist in the
Lck-mediated signal transduction pathways following CD3 or CD2
cross-linking.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
clone 6.6, which expresses
the p180 CD45 isoform, and Jurkat cells clone 77.6 have been described
previously (41). JCaM1.6 cells were purchased from the American Type
Culture Collection (Rockville, MD). JCaM1.6 cells transfected with Lck
(JCaM1/Lck, 42) were kindly provided by D. Straus (University of
Chicago, Chicago, IL). Jurkat cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM
L-glutamine, penicillin, and streptomycin. Selective media
included 2 mg/ml G418, 500 µg/ml hygromycin, and 1 µg/ml puromycin
when required.
UCHT1 (IgG2a; kindly provided by A. Alcover, Institut Pasteur, France), anti-CD2 (anti-T11-2 and T11-3,
kindly provided by E. Reinherz, Harvard Medical School, Boston, MA),
anti-EGFR (579, American Type Culture Collection, Rockville, MD and
LA-22, Upstate Biotechnology, Lake Placid, NY), anti-p62dok (M-276, Santa Cruz Biotechnology,
Santa Cruz, CA), anti-RasGAP (B4F8, Santa Cruz Biotechnology, Santa
Cruz, CA), antiphosphotyrosine (4G10, Upstate Biotechnology, Lake
Placid, NY), and anti-PLC1 (a mixture of mAbs, Upstate
Biotechnology, Lake Placid, NY). Rabbit polyclonal antibodies used
included anti-p62dok directed against a amino
acid residues 425-439 of p62dok (kindly
provided by B. Stillman, Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY), anti-p56dok directed against amino
acids residues 271-284 of murine p56dok (kindly
provided by W. Paul, National Institutes of Health, Bethesda, MD),
anti-Lck (kindly provided by A. Veillette, McGill University, Montréal, Canada), and anti-ZAP-70, which has been described (22).
-D-maltoside (Anatrace, Maumee, OH)
instead of 1% Nonidet P-40 for co-immunoprecipitation experiments of
the EGFR/Lck chimera. Immunoprecipitations and immunoblotting were performed as described previously (22). For detection of biotinylated antibodies, blots were probed with a 1/3000 dilution of
streptavidin-biotinylated horseradish peroxidase complex (Amersham
Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
Fig. 1.
EGFR/LckF505 chimera phosphorylation
increases after EGF stimulation. Jurkat cells (J-CD45-64) were
left unstimulated ( 
) or stimulated with anti-CD3 mAb or EGF at 100 ng/ml for 1 min. Lysates of cells expressing the indicated chimeras
were immunoprecipitated with anti-EGFR mAbs (579.2) and analyzed by
immunoblotting with either antiphosphotyrosine mAb or anti-EGFR mAb
(LA22).
View larger version (34K):
[in a new window]
Fig. 2.
Activation of an EGFR/Lck F505 chimera leads
to the specific phosphorylation of four proteins. Total cell
lysates from Jurkat parental cell line (J-CD45-64) and from cells
expressing the EGFR/LckF505, EGFR/LckWT, or EGFRLckA273 chimera were
analyzed by phosphotyrosine immunoblotting. Cells were left
unstimulated ( ) or stimulated with anti-CD3 mAb or EGF at 100 ng/ml
for 1 min. Arrows indicate the position of the four major
tyrosine-phosphorylated proteins (p55, p62, p80, and p120) following
EGFR/LckF505 chimera activation. Left, positions of
molecular mass markers are shown in kilodaltons.
View larger version (38K):
[in a new window]
Fig. 3.
p56dok, p62dok, and RasGAP
are tyrosine-phosphorylated after EGFR/LckF505 chimera
activation. A, Jurkat cells (J-CD45-64) expressing
EGFR/LckF505 were left unstimulated ( ) or stimulated with CD3 mAb or
with EGF at 100 ng/ml for 1 min. Total cell lysates were
immunoprecipitated with anti-p62dok or
anti-RasGAP antibodies as indicated. Whole cell lysates are also shown.
Immunoprecipitates were immunoblotted with antiphosphotyrosine mAb and
anti-p62dok antibodies. The antibody against
p62dok used in this experiment is not able to
immunoprecipitate the totality of Dok present in the cell extract. This
explains the lower intensity of tyrosine-phosphorylated
p62dok present in the
p62dok immunoprecipitate (IP) than in
the RasGAP immunoprecipitate. Arrows indicate the positions
of the three RasGAP-associated proteins, p56, p62, and p190.
Left, positions of molecular mass markers are shown in
kilodaltons. B, Jurkat cells (J-CD45-64) expressing
EGFR/LckF505 were left unstimulated () or stimulated with EGF at 100 ng/ml for 1 min. Total cell lysates were immunoprecipitated with
anti-p56dok and analyzed by immunoblotting with
biotinylated antiphosphotyrosine mAbs. The p62 band indicated by an
asterisk might correspond to a highly phosphorylated form of
p56dok or to an unidentified protein recognized
by the anti-p56dok antiserum or
co-immunoprecipitated with p56dok. It is
unlikely that it corresponds to p62dok because
it is not detected by anti-p62dok immunoblotting
(data not shown). Left, positions of molecular mass markers
are shown in kilodaltons. WB, Western blot.
1
phosphorylation, the increase in phosphorylation of ZAP-70 and PLC1
is barely detectable in EGF-stimulated cells (Fig.
4). Taken together, these results showed
that LckF505 oligomerization induces the specific phosphorylation of
RasGAP, p56dok, and
p62dok proteins. Presumably, the phosphorylation
of p56dok and p62dok
allows its association with RasGAP.
View larger version (26K):
[in a new window]
Fig. 4.
Oligomerization of the EGFR/Lck chimera does
not induce phosphorylation of ZAP-70 and
PLC 1. Jurkat cells (J-CD45-64)
expressing EGFR/LckF505 were left unstimulated (), CD3-stimulated, or
EGF-stimulated for 1 or 10 min as indicated. Total cell lysates were
immunoprecipitated with anti-ZAP-70 and anti-PLC1 antibodies and
immunoblotted with antiphosphotyrosine. The membranes were stripped and
reprobed with the corresponding antibodies to ensure that equivalent
amounts of protein were present in the immunoprecipitates
(IP) as indicated. Right, positions of molecular
mass markers are shown in kilodaltons. WB, Western
blot.
1 phosphorylation is barely detectable after EGF stimulation,
this result indicates that the EGF-mediated increase in intracellular
Ca2+ may not be dependent of PLC1 activation and
therefore may activate a signaling pathway distinct from that of the
TCR. To analyze the effect of EGF on intracellular Ca2+
release and extracellular Ca2+ influx, we performed similar
experiments in the presence of EGTA to chelate extracellular
Ca2+. Because EGTA prevents Ca2+ rise, these
results suggest that Lck regulates Ca2+ influx by mediating
the phosphorylation of a protein involved directly or indirectly in the
regulation of Ca2+ channel opening.
View larger version (18K):
[in a new window]
Fig. 5.
Oligomerization of LckF505 leads to
Ca2+ mobilization. A, Jurkat cells
expressing the indicated Lck chimeras were loaded with indo-1, and
calcium mobilization was assessed following CD3 or EGF stimulation of
the cells at the time indicated by an arrow. B,
Jurkat cells expressing the LckF505 chimera were stimulated with either
CD3 or EGF at the time indicated by an arrow. Calcium
mobilization was evaluated in the presence or in the absence of
EGTA.
View larger version (32K):
[in a new window]
Fig. 6.
Phosphorylation of p56dok
and p62dok is increased following CD2
stimulation and requires Lck. A, Jurkat cells (J77-6)
were left unstimulated ( ) or stimulated with an anti-CD2 mAbs pair
for 1 min. Total cell lysates were immunoprecipitated with
anti-p56dok, anti-p62dok,
and anti-RasGAP antibodies as indicated. Immunoprecipitates were
immunoblotted with biotinylated antiphosphotyrosine mAbs. B,
Jurkat cells were left unstimulated () or stimulated with CD3 mAb or
an anti-CD2 mAb pair (CD2) for 1 min. Total cell lysates were
immunoprecipitated with anti-ZAP-70 antibodies and immunoblotted with
antiphosphotyrosine mAbs. The membrane was stripped and reprobed with
anti-ZAP-70 antibodies to ensure that equivalent amounts of protein
were present in the immunoprecipitates (IP). C,
JCaM1.6 cells were left unstimulated () or stimulated with an
anti-CD2 mAb pair for 3 min. Total cell lysates were immunoprecipitated
with anti-p56dok,
anti-p62dok, and anti-RasGAP antibodies as
indicated. Immunoprecipitates were immunoblotted with biotinylated
antiphosphotyrosine mAbs. The arrow indicates a nonspecific
band, present in all immunoprecipitates, recognized by the biotinylated
antiphosphotyrosine mAbs. Left, positions of molecular mass
markers are shown in kilodaltons. D, JCaM1/Lck cells were
left unstimulated () or stimulated with an anti-CD2 mAbs pair for 3 min. Total cell lysates were immunoprecipitated with
anti-p56dok and anti-RasGAP antibodies as
indicated. Immunoprecipitates were immunoblotted with biotinylated
antiphosphotyrosine mAbs or anti-p62dok
antibodies as indicated. WB, Western blot.
View larger version (24K):
[in a new window]
Fig. 7.
p62dok associates in vivo
with EGFR/LckF505. Jurkat cells (J-CD45-64) expressing
EGFR/LckF505 were left unstimulated ( ), CD3-stimulated, or
EGF-stimulated for 1 min. Total cell lysates were immunoprecipitated
with anti-EGFR mAbs and immunoblotted with antiphosphotyrosine mAbs or
anti-Lck antibodies as indicated. The bands indicated by an
asterisk likely correspond to degradation products of the
EGFR/Lck chimera, because they are detected by anti-Lck antibodies. The
relative positions of the p80 and p62 Lck-associated proteins are
indicated. The same blot was stripped and probed with
anti-p62dok antibodies. The band indicated by an
asterisk is nonspecific, because it is present in anti-EGFR
immunoprecipitation using the parental cell that does not express the
EGFR chimera. Left, positions of molecular mass markers are
shown in kilodaltons. IP, immunoprecipitate; WB,
Western blot.
View larger version (35K):
[in a new window]
Fig. 8.
Tyrosine-phosphorylated p56dok and
p62dok bind to Lck SH2 domain. A, Jurkat cells
(J-CD45-64) expressing EGFR/LckF505 were left unstimulated ( ),
CD3-stimulated, or EGF-stimulated for 1 min. Proteins bound to the
MBP·LckSH2 fusion protein were eluted with 50 mM phenyl
phosphate and subjected to immunoprecipitations with
anti-p56dok and
anti-p62dok antibodies as indicated. Proteins
were revealed by antiphosphotyrosine immunoblotting. Left,
positions of molecular mass markers are shown in kilodaltons.
B, Jurkat cells (J-CD45-64) expressing EGFR/LckF505 were
left unstimulated (), CD3-stimulated, or EGF-stimulated for 1 min.
Lysates were boiled in the presence of 1% SDS, diluted to 0.1% SDS in
lysis buffer, and incubated with MBP-SH2 beads as for the nondenatured
lysates. Proteins were revealed by antiphosphotyrosine immunoblotting.
Left, positions of molecular mass markers are shown in
kilodaltons. C, Jurkat cells (J77-6) were left unstimulated
() or stimulated with an anti-CD2 mAbs pair for 1 min. Lysates were
precipitated with MBP-SH2 beads. SH2-binding proteins were analyzed by
antiphosphotyrosine immunoblotting. The same blot was stripped and
revealed by anti-p62dok immunoblotting.
Left, positions of molecular mass markers are shown in
kilodaltons. WB, Western blot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1. An inositol 1,4,5-trisphosphate-independent Ca2+ regulatory pathway has
been evidenced in T cells. Introduction of a constitutively active
v-Src PTK induced a substantial increase of Ca2+ in
unstimulated cells in the absence of PLC1 activation and inositol
1,4,5-trisphosphate production (54). Moreover, a CD4-associated Ca2+ influx occurs independently of the
phosphatidylinositol-PLC pathway in Jurkat cells (55). Our results
suggest that once activated, Lck mediates the phosphorylation of a
protein involved in the regulation of Ca2+ channel opening.
Phosphorylated RasGAP and RasGAP-associated proteins might play a role
in that process. It is important to note that this Ca2+
increase is not followed by a substantial extracellular
signal-regulated kinase 1/2 and nuclear factor of activated T cells
activation (data not shown). These results suggest that by itself
phosphorylation of RasGAP and Dok proteins is not sufficient to elicit
a full response.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Institut National de
la Recherche Scientifique, Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies, Laval Québec, H7V
1B7, Canada. Tel.: 450-687-5010; Fax: 450-686-5501; E-mail:
pascale_duplay@ inrs-iaf.uquebec.ca.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
August, A.,
and Dupont, B.
(1994)
Biochem. Biophys. Res. Commun.
199,
1466-1473[CrossRef][Medline]
[Order article via Infotrieve]
2.
Danielian, S.,
Alcover, A.,
Polissard, L.,
Stefanescu, M.,
Acuto, O.,
Fischer, S.,
and Fagard, R.
(1992)
Eur. J. Immunol.
22,
2915-2921[Medline]
[Order article via Infotrieve]
3.
Luo, K.,
and Sefton, B. M.
(1990)
Mol. Cell. Biol.
10,
5305-5313 4.
Hubert, P.,
Lang, V.,
Debre, P.,
and Bismuth, G.
(1996)
J. Immunol.
157,
4322-4332[Abstract]
5.
Sunder-Plassmann, R.,
and Reinherz, E. L.
(1998)
J. Biol. Chem.
273,
24249-24257 6.
Straus, D. B.,
and Weiss, A.
(1992)
Cell
70,
585-593[CrossRef][Medline]
[Order article via Infotrieve]
7.
Gibson, S.,
August, A.,
Branch, D.,
Dupont, B.,
and Mills, G. B.
(1996)
J. Biol. Chem.
271,
7079-7083 8.
Karnitz, L.,
Sutor, S. L.,
Torigoe, T.,
Reed, J. C.,
Bell, M. P.,
McKean, D. J.,
Leibson, P. J.,
and Abraham, R. T.
(1992)
Mol. Cell. Biol.
12,
4521-4530 9.
Gervais, F. G.,
Chow, L. M.,
Lee, J. M.,
Branton, P. E.,
and Veillette, A.
(1993)
Mol. Cell. Biol.
13,
7112-7121 10.
Sieh, M.,
Bolen, J. B.,
and Weiss, A.
(1993)
EMBO J.
12,
315-321[Medline]
[Order article via Infotrieve]
11.
Veillette, A.,
Caron, L.,
Fournel, M.,
and Pawson, T.
(1992)
Oncogene
7,
971-980[Medline]
[Order article via Infotrieve]
12.
Veillette, A.,
and Fournel, M.
(1990)
Oncogene
5,
1455-1462[Medline]
[Order article via Infotrieve]
13.
Luo, K.,
and Sefton, B. M.
(1992)
Mol. Cell. Biol.
12,
4724-4732 14.
Caron, L.,
Abraham, N.,
Pawson, T.,
and Veillette, A.
(1992)
Mol. Cell. Biol.
12,
2720-2729 15.
Carrera, A. C.,
Paradis, H.,
Borlado, L. R.,
Roberts, T. M.,
and Martinez-A, C.
(1995)
J. Biol. Chem.
270,
3385-3391 16.
Straus, D. B.,
Chan, A. C.,
Patai, B.,
and Weiss, A.
(1996)
J. Biol. Chem.
271,
9976-9981 17.
Reynolds, P. J.,
Hurley, T. R.,
and Sefton, B. M.
(1992)
Oncogene
7,
1949-1955[Medline]
[Order article via Infotrieve]
18.
Thome, M.,
Duplay, P.,
Guttinger, M.,
and Acuto, O.
(1995)
J. Exp. Med.
181,
1997-2006 19.
Xu, H.,
and Littman, D. R.
(1993)
Cell
74,
633-643[CrossRef][Medline]
[Order article via Infotrieve]
20.
Lewis, L. A.,
Chung, C. D.,
Chen, J.,
Parnes, J. R.,
Moran, M.,
Patel, V. P.,
and Miceli, M. C.
(1997)
J. Immunol.
159,
2292-2300 21.
Peri, K. G.,
Gervais, F. G.,
Weil, R.,
Davidson, D.,
Gish, G. D.,
and Veillette, A.
(1993)
Oncogene
8,
2765-2772[Medline]
[Order article via Infotrieve]
22.
Duplay, P.,
Thome, M.,
Herve, F.,
and Acuto, O.
(1994)
J. Exp. Med.
179,
1163-1172 23.
Fukazawa, T.,
Reedquist, K. A.,
Trub, T.,
Soltoff, S.,
Panchamoorthy, G.,
Druker, B.,
Cantley, L.,
Shoelson, S. E.,
and Band, H.
(1995)
J. Biol. Chem.
270,
19141-19150 24.
Prasad, K. V.,
Kapeller, R.,
Janssen, O.,
Repke, H.,
Duke, C. J.,
Cantley, L. C.,
and Rudd, C. E.
(1993)
Mol. Cell. Biol.
13,
7708-7717 25.
Takemoto, Y.,
Furuta, M.,
Li, X. K.,
Strong, S. W.,
and Hashimoto, Y.
(1995)
EMBO J.
14,
3403-3414[Medline]
[Order article via Infotrieve]
26.
Li, S.,
Satoh, T.,
Korngold, R.,
and Huang, Z.
(1998)
Immunol. Today
19,
455-462[CrossRef][Medline]
[Order article via Infotrieve]
27.
Veillette, A.,
Bookman, M. A.,
Horak, E. M.,
Samelson, L. E.,
and Bolen, J. B.
(1989)
Nature
338,
257-259[CrossRef][Medline]
[Order article via Infotrieve]
28.
Baldari, C. T.,
Milia, E.,
Di, S. M.,
Baldoni, F.,
Valitutti, S.,
and Telford, J. L.
(1995)
Eur. J. Immunol.
25,
1843-1850[Medline]
[Order article via Infotrieve]
29.
Haughn, L.,
Gratton, S.,
Caron, L.,
Sekaly, R. P.,
Veillette, A.,
and Julius, M.
(1992)
Nature
358,
328-331[CrossRef][Medline]
[Order article via Infotrieve]
30.
Ley, S. C.,
Davies, A. A.,
Druker, B.,
and Crumpton, M. J.
(1991)
Eur. J. Immunol.
21,
2203-2209[Medline]
[Order article via Infotrieve]
31.
Jin, Y. J.,
Kaplan, D. R.,
White, M.,
Spagnoli, G. C.,
Roberts, T. M.,
and Reinherz, E. L.
(1990)
J. Immunol.
144,
647-652[Abstract]
32.
Crespo, P.,
Schuebel, K. E.,
Ostrom, A. A.,
Gutkind, J. S.,
and Bustelo, X. R.
(1997)
Nature
385,
169-172[CrossRef][Medline]
[Order article via Infotrieve]
33.
Lorenz, U.,
Ravichandran, K. S.,
Pei, D.,
Walsh, C. T.,
Burakoff, S. J.,
and Neel, B. G.
(1994)
Gene (Amst.)
138,
219-222[CrossRef][Medline]
[Order article via Infotrieve]
34.
Amrein, K. E.,
Flint, N.,
Panholzer, B.,
and Burn, P.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
3343-3346 35.
Burgess, K. E.,
Yamamoto, M.,
Prasad, K. V. S.,
and Rudd, C. E.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
9311-9315 36.
Raab, M.,
Yamamoto, M.,
and Rudd, C. E.
(1994)
Biochemistry
33,
5070-5076[CrossRef][Medline]
[Order article via Infotrieve]
37.
Watts, J. D.,
Wilson, G. M.,
Ettenhadieh, E.,
Clark-Lewis, I.,
Kubanek, C. A.,
Astell, C. R.,
Marth, J. D.,
and Aebersold, R.
(1992)
J. Biol. Chem.
267,
901-907 38.
Weiss, A.,
and Littman, D. R.
(1994)
Cell
76,
263-274[CrossRef][Medline]
[Order article via Infotrieve]
39.
Marie-Cardine, A.,
Fischer, S.,
Gorvel, J. P.,
and Maridonneau-Parini, I.
(1996)
J. Biol. Chem.
271,
20734-20739 40.
Ohno, H.,
Ushiyama, C.,
Taniguchi, M.,
Germain, R. N.,
and Saito, T.
(1991)
J. Immunol.
146,
3742-3746[Abstract]
41.
Duplay, P.,
Alcover, A.,
Fargeas, C.,
Sekaly, R.,
and Branton, P. E.
(1996)
J. Biol. Chem.
271,
17896-17902 42.
Denny, M. F.,
Kaufman, H. C.,
Chan, A. C.,
and Straus, D. B.
(1999)
J. Biol. Chem.
274,
5146-5152 43.
Ellis, C.,
Liu, X. Q.,
Anderson, D.,
Abraham, N.,
Veillette, A.,
and Pawson, T.
(1991)
Oncogene
6,
895-901[Medline]
[Order article via Infotrieve]
44.
Carpino, N.,
Wisniewski, D.,
Strife, A.,
Marshak, D.,
Kobayashi, R.,
Stillman, B.,
and Clarkson, B.
(1997)
Cell
88,
197-204[CrossRef][Medline]
[Order article via Infotrieve]
45.
Yamanashi, Y.,
and Baltimore, D.
(1997)
Cell
88,
205-211[CrossRef][Medline]
[Order article via Infotrieve]
46.
Di Cristofano, A.,
Carpino, N.,
Dunant, N.,
Friedland, G.,
Kobayashi, R.,
Strife, A.,
Wisniewski, D.,
Clarkson, B.,
Pandolfi, P. P.,
and Resh, M. D.
(1998)
J. Biol. Chem.
273,
4827-4830 47.
Nelms, K.,
Snow, A. L.,
Hu, L. J.,
and Paul, W. E.
(1998)
Immunity
9,
13-24[CrossRef][Medline]
[Order article via Infotrieve]
48.
Settleman, J.,
Narasimhan, V.,
Foster, L. C.,
and Weinberg, R. A.
(1992)
Cell
69,
539-549[CrossRef][Medline]
[Order article via Infotrieve]
49.
Hubert, P.,
Debre, P.,
Boumsell, L.,
and Bismuth, G.
(1993)
J. Exp. Med.
178,
1587-1596 50.
Songyang, Z.,
Carraway, K. L., III,
Eck, M. J.,
Harrison, S. C.,
Feldman, R. A.,
Mohammadi, M.,
Schlessinger, J.,
Hubbard, S. R.,
Smith, D. P.,
Eng, C.,
Lorenzo, M. J.,
Ponder, B. A. J.,
Mayer, B. J.,
and Cantley, L. C.
(1995)
Nature
373,
536-539[CrossRef][Medline]
[Order article via Infotrieve]
51.
Howe, L. R.,
and Weiss, A.
(1995)
Trends Biochem. Sci.
20,
59-64[CrossRef][Medline]
[Order article via Infotrieve]
52.
Heyeck, S. D.,
Wilcox, H. M.,
Bunnell, S. C.,
and Berg, L. J.
(1997)
J. Biol. Chem.
272,
25401-25408 53.
Baldari, C. T.,
Heguy, A.,
and Telford, J. L.
(1993)
J. Biol. Chem.
268,
8406-8409 54.
Niklinska, B. B.,
Yamada, H.,
O'Shea, J. J.,
June, C. H.,
and Ashwell, J. D.
(1992)
J. Biol. Chem.
267,
7154-7159 55.
Lafont, V.,
Fischer, T.,
Zumbihl, R.,
Faure, S.,
Hivroz, C.,
Rouot, B.,
and Favero, J.
(1997)
Eur. J. Immunol.
27,
2261-2268[Medline]
[Order article via Infotrieve]
56.
Jones, N.,
and Dumont, D. J.
(1998)
Oncogene
17,
1097-1108[CrossRef][Medline]
[Order article via Infotrieve]
57.
Holland, S. J.,
Gale, N. W.,
Gish, G. D.,
Roth, R. A.,
Songyang, Z.,
Cantley, L. C.,
Henkemeyer, M.,
Yancopoulos, G. D.,
and Pawson, T.
(1997)
EMBO J.
16,
3877-3888[CrossRef][Medline]
[Order article via Infotrieve]
58.
Neet, K.,
and Hunter, T.
(1995)
Mol. Cell. Biol.
15,
4908-4920[Abstract]
59.
Gold, M. R.,
Crowley, M. T.,
Martin, G. A.,
McCormick, F.,
and DeFranco, A. L.
(1993)
J. Immunol.
150,
377-386[Abstract]
60.
Vuica, M.,
Desiderio, S.,
and Schneck, J. P.
(1997)
J. Exp. Med.
186,
259-267 61.
Nunes, J. A.,
Truneh, A.,
Olive, D.,
and Cantrell, D. A.
(1996)
J. Biol. Chem.
271,
1591-1598 62.
Yang, W. C.,
Ghiotto, M.,
Barbarat, B.,
and Olive, D.
(1999)
J. Biol. Chem.
274,
607-617 63.
Catipovic, B.,
Schneck, J. P.,
Brummet, M. E.,
Marsh, D. G.,
and Rafnar, T.
(1996)
J. Biol. Chem.
271,
9698-9703 64.
Bubeck, W. J.,
Pappu, R.,
Bu, J. Y.,
Mayer, B.,
Chernoff, J.,
Straus, D.,
and Chan, A. C.
(1998)
Immunity
9,
607-616[CrossRef][Medline]
[Order article via Infotrieve]
65.
Noguchi, T.,
Matozaki, T.,
Inagaki, K.,
Tsuda, M.,
Fukunaga, K.,
Kitamura, Y.,
Kitamura, T.,
Shii, K.,
Yamanashi, Y.,
and Kasuga, M.
(1999)
EMBO J.
18,
1748-1760[CrossRef][Medline]
[Order article via Infotrieve]
66.
Penninger, J. M.,
and Crabtree, G. R.
(1999)
Cell
96,
9-12[CrossRef][Medline]
[Order article via Infotrieve]
67.
Settleman, J.,
Albright, C. F.,
Foster, L. C.,
and Weinberg, R. A.
(1992)
Nature
359,
153-154[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. N. Zaman, M. E. Resek, and S. M. Robbins Dual acylation and lipid raft association of Src-family protein tyrosine kinases are required for SDF-1/CXCL12-mediated chemotaxis in the Jurkat human T cell lymphoma cell line J. Leukoc. Biol., October 1, 2008; 84(4): 1082 - 1091. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yasuda, K. Bundo, A. Hino, K. Honda, A. Inoue, M. Shirakata, M. Osawa, T. Tamura, H. Nariuchi, H. Oda, et al. Dok-1 and Dok-2 are negative regulators of T cell receptor signaling Int. Immunol., April 1, 2007; 19(4): 487 - 495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kashiwada, G. Cattoretti, L. McKeag, T. Rouse, B. M. Showalter, U. Al-Alem, M. Niki, P. P. Pandolfi, E. H. Field, and P. B. Rothman Downstream of Tyrosine Kinases-1 and Src Homology 2-Containing Inositol 5'-Phosphatase Are Required for Regulation of CD4+CD25+ T Cell Development J. Immunol., April 1, 2006; 176(7): 3958 - 3965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Boulay, J.-G. Nemorin, and P. Duplay Phosphotyrosine Binding-Mediated Oligomerization of Downstream of Tyrosine Kinase (Dok)-1 and Dok-2 Is Involved in CD2-Induced Dok Phosphorylation J. Immunol., October 1, 2005; 175(7): 4483 - 4489. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Okabe, S. Fukuda, Y.-J. Kim, M. Niki, L. M. Pelus, K. Ohyashiki, P. P. Pandolfi, and H. E. Broxmeyer Stromal cell-derived factor-1{alpha}/CXCL12-induced chemotaxis of T cells involves activation of the RasGAP-associated docking protein p62Dok-1 Blood, January 15, 2005; 105(2): 474 - 480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ito, H. Okazawa, K. Maruyama, K. Tomizawa, S.-i. Motegi, H. Ohnishi, H. Kuwano, A. Kosugi, and T. Matozaki Interaction of SAP-1, a Transmembrane-type Protein-tyrosine Phosphatase, with the Tyrosine Kinase Lck: ROLES IN REGULATION OF T CELL FUNCTION J. Biol. Chem., September 12, 2003; 278(37): 34854 - 34863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Dupuis, M. de Jesus Ibarra-Sanchez, M. L. Tremblay, and P. Duplay Gr-1+ Myeloid Cells Lacking T Cell Protein Tyrosine Phosphatase Inhibit Lymphocyte Proliferation by an IFN-{gamma}- and Nitric Oxide-Dependent Mechanism J. Immunol., July 15, 2003; 171(2): 726 - 732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Murakami, Y. Yamamura, Y. Shimono, K. Kawai, K. Kurokawa, and M. Takahashi Role of Dok1 in Cell Signaling Mediated by RET Tyrosine Kinase J. Biol. Chem., August 30, 2002; 277(36): 32781 - 32790. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Gugasyan, C. Quilici, S. T.T. I, D. Grail, A. M. Verhagen, A. Roberts, T. Kitamura, A. R. Dunn, and P. Lock Dok-related protein negatively regulates T cell development via its RasGTPase-activating protein and Nck docking sites J. Cell Biol., July 8, 2002; 158(1): 115 - 125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Liang, D. Wisniewski, A. Strife, Shivakrupa, B. Clarkson, and M. D. Resh Phosphatidylinositol 3-Kinase and Src Family Kinases Are Required for Phosphorylation and Membrane Recruitment of Dok-1 in c-Kit Signaling J. Biol. Chem., April 12, 2002; 277(16): 13732 - 13738. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Martelli, J. Boomer, M. Bu, and B. E. Bierer T Cell Regulation of p62dok (Dok1) Association with Crk-L J. Biol. Chem., November 30, 2001; 276(49): 45654 - 45661. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zhao, A. A.P. Schmitz, Y. Qin, A. Di Cristofano, P. P. Pandolfi, and L. Van Aelst Phosphoinositide 3-Kinase-dependent Membrane Recruitment of p62dok Is Essential for Its Negative Effect on Mitogen-activated Protein (MAP) Kinase Activation J. Exp. Med., July 30, 2001; 194(3): 265 - 274. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-G. Nemorin, P. Laporte, G. Berube, and P. Duplay p62dok Negatively Regulates CD2 Signaling in Jurkat Cells J. Immunol., April 1, 2001; 166(7): 4408 - 4415. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |