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ABSTRACT |
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The Tec protein-tyrosine kinase family includes
Btk, Itk/Tsk/Emt, Tec, Rlk/Txk, and Bmx which are involved in signals
mediated by various cytokines or antigen receptors. Itk is expressed
primarily in T cells and activated by TCR/CD3, CD28, and CD2. However,
the defect in T cell signaling in itk-deficient mice is
very modest. Thus, we looked for other Tec family kinases that could be
expressed in lymphoid cells and involved in T cell signal transduction. Here, we demonstrate that Tec, expressed in T cells, is activated following TCR/CD3 or CD28 ligation and interacts with CD28 receptor in
an activation-dependent manner. This interaction involves
the Tec SH3 domain and the proline-rich motifs in CD28. We also show that Tec can phosphorylate p62dok, one CD28-specific substrate,
whereas Itk cannot. Overexpression of Tec but not Itk can enhance the
interleukin-2 promoter activity mediated by TCR/CD3 or CD28 stimulation
and introduction of a kinase-dead Tec but not Itk can suppress
interleukin-2 expression, indicating that Tec is directly involved in T
cell activation. Altogether, these data demonstrate that Tec kinase is
an integral component of T cell signaling and that the two Tec family
kinases, Tec and Itk, have distinct roles in T cell activation.
The Tec family is a recently emerging subgroup of non-receptor
PTKs.1 The Tec family is
currently the second largest PTK subfamily encompassing five members:
Tec, Btk, Itk/Tsk/Emt, Rlk/Txk, and Bmx (1). However, little is known
about this family. They are all characterized by a pleckstrin homology
domain (PH)1 (except Rlk/Txk), a Tec homology domain (TH)
containing a region homologous to the GAP1 family of Ras GTPases at the
N terminus and one or two proline-rich motifs at its C terminus (2), an Src homology (SH) 3 domain, an SH2 domain, and an SH1/kinase domain. They are devoid of N-terminal myristoylation sites and C-terminal tyrosine residues corresponding to Tyr527 of c-Src involved
in its regulation. PH domains bind to phosphorylated inositol lipids
and are thought to anchor proteins possessing such domains to membrane
(3-6). Itk SH3 domains bind to cellular ligands such as Sam-68,
Wiskott-Aldrich syndrome protein, and human RNP-K (7, 8). In the same
way, Tec TH domains interact with SH3 domains of Grb-2, Src family PTKs
(Fyn, Lyn, and Hck) (9, 10), Vav (11), or c-Kit (12). In the
unactivated state, the interaction of Itk SH3 domain with the
proline-rich motif (KPLPPTP) present in its TH domain would result in
an intramolecular binding that could prevent the interaction of these
domains with other cellular interaction motifs and probably inhibit
their enzymatic activity. Regulation of Tec family depends on at least
two events, phosphorylation by Src family kinases (9, 13) and
recruitment to phospholipids produced by phosphatidylinositol 3-kinase
on membrane (4, 14).
Many members of Tec family are abundantly expressed in hematopoietic
tissues. Btk is expressed in B cells and myeloid cell lineages (15).
Itk/Tsk/Emt is primarily expressed in T cells (16-18), natural killer
cells, and mast cells (19, 20). Itk is activated by various T cell
surface receptors such as TCR/CD3, CD28, and CD2 (21, 22).
Interestingly, mice lacking Itk have both decreased numbers of mature
thymocytes and reduced proliferative responses to allogeneic major
histocompatibility complex stimulation or TCR cross-linking (23). These
signaling defects of the antigen receptors can be overcome by
stimulation with phorbol esters (PMA) and calcium ionophores
(ionomycin), indicating that this protein kinase functions at an early
stage in the signaling pathways. Tec is expressed in most hematopoietic
cells (24) and B cells (25). It is involved in the intracellular
signaling system of numerous cytokines such as IL-3, IL-6, stem cell
factor, granulocyte colony stimulating factor, erythropoietin, and
thrombopoietin (11, 12, 26-29). So far, Tec functions are hardly
understood, and its substrates still remain to be identified. However,
it was recently suggested that Tec can phosphorylate Jak-2 and the transient expression of Tec in BAF3 cells resulted in the marked elevation of the promoter activity of the c-fos
proto-oncogene (30, 31).
The full activation of T cells requires both interaction of the TCR
with antigen bound to major histocompatibility complex molecules and
costimulatory signals. The major costimulatory molecule is the adhesion
molecule CD28 that interacts with its ligands B7.1/CD80 and B7.2/CD86
on antigen presenting cells. These two signals induce T cell
proliferation, cytokine production, and regulation of T cell apoptosis
and survival. The signaling pathways for TCR/CD3 stimulation and CD28
costimulation still remain poorly understood. TCR/CD3 or CD28 signal
transduction induces various biochemical events inclusive of calcium
mobilization, tyrosine phosphorylation of downstream substrates, PTK
activation, activation of PI 3-kinase, and activation of
p21ras. TCR/CD3 and CD28 must recruit PTKs to phosphorylate
their substrates upon activation since they lack intrinsic kinase
activities. For example, it has been shown that Lck, Fyn, ZAP-70, and
Itk can be recruited and activated in TCR/CD3 pathway and that Lck,
Fyn, and Itk can be recruited and activated in CD28 pathway. Although similar PTKs can be recruited, TCR/CD3 and CD28 induce phosphorylation of their specific substrates such as SLP-76 (p76) and LAT (p36/38) for
TCR/CD3 and p62dok and the catalytic subunit of
phosphatidylinositol 3-kinase (32-35) for CD28 besides common
substrates such as phospholipase C Here, we demonstrate that Tec kinase is also expressed in T cells and
activated on TCR/CD3 or CD28 stimulation. We also show that Tec
inducibly binds to CD28 via SH3 domain proline-rich motif interaction
and Tec can phosphorylate in vivo p62dok protein, a
Ras GAP-associated adaptor that can be phosphorylated in CD28 but not
TCR/CD3 pathway. Furthermore, Tec but not Itk can induce IL-2 promoter
activity. In addition, kinase-dead Tec can suppress IL-2 activation in
TCR/CD3 and CD28 pathways. These data suggest that two Tec family
kinases, namely Itk and Tec, are involved in T cell signal transduction
and that Tec is a likely candidate for regulating T cell activation.
Cell Lines--
Murine T hybridoma parental cells DC27.1,
clone DWT6.11+ Plasmids--
Expression plasmids pSR Antibodies--
Anti-human CD28 monoclonal antibodies (mAbs)
248, CD28.2, and CD28.6 and anti-human CD3 mAb 289 have already been
described (40). The anti-Itk, anti-Tec, anti-p62dok rabbit
polyclonal antisera were raised against murine Itk, Tec, and
p62dok proteins using synthetic peptides corresponding to amino
acid residues 605-625 (DRPPFSQLLSQLAEIAEAGL), amino acid residues
162-179 (EIKKRRPPPPIPPEEENT), and amino acid residues 424-437
(PQGLILPESGTTRGS), respectively. Anti-Itk mAb Tuk-N1 has been
previously described (41). Anti-FLAG and 4G10 mAbs were purchased from
Eastman Kodak Co. and Upstate Biotechnology Inc., respectively.
Anti-rat CD2 mAb (OX34) was a kind gift from Dr. D. A. Cantrell
(London, UK) and 145 2C11 mAb is a hamster IgG specific for murine CD3
Cell Stimulation, Immunoprecipitation, and Immunoblot--
Ten
million cells per ml medium were stimulated at 37 °C for 2 min with
10 µg of CD28.2 mAb, then cross-linked with 30 µg of GAM antiserum
for 5 min at 37 °C. For nonstimulated controls, 10 × 106 of cells per ml of medium were left at 37 °C for 2 min and then cross-linked with 30 µg of GAM antiserum for 5 min at
37 °C. The cells were lysed with lysis buffer (1% Triton X-100, 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM
orthovanadate) on ice for 15 min and centrifuged at 4 °C for 15 min
at 13,000 rpm. Postnuclear supernatants were precipitated with
antibody. The precipitates were washed and subjected to
SDS-polyacrylamide gel electrophoresis, then electro-transferred to
polyvinylidene difluoride membrane (Millipore), and the membrane was
probed with antibody. Alternatively, 10 × 106 cells
per ml of medium were stimulated at 4 °C for 15 min with 20 µg of
CD28 mAb bound to beads and prepared according to the above described
protocol. The cells were lysed with lysis buffer, and CD28 molecules
were precipitated using a magnet. For nonstimulated control, 10 × 106 cells per ml of medium were left unstimulated at
4 °C for 15 min, lysed, and centrifuged at 4 °C for 15 min at
13,000 rpm. Postnuclear lysates were precipitated also with CD28 mAb
bound to beads. The precipitates were processed as described above.
GST Fusion Protein Production and Pulldown
Precipitation--
The bacterial hosts Escherichia coli
DH5 Transient Transfection--
COS-7 (6 × 106)
cells were transiently transfected by the DEAE-dextran method as
described (42).
In Vitro Kinase Assay--
The immune complex containing Tec was
precipitated from T murine hybridoma cells expressing endogenous Tec
with anti-Tec polyclonal antiserum and then washed three times with
lysis buffer and once with kinase buffer (20 mM Tris-HCl,
pH 7.5, 50 mM NaCl, 10 mM MgCl2, 2 mM MnCl2, 1 mM
Na3VO4). The immune complex was then incubated in 30 µl of kinase buffer containing 10 µCi of
[ Luciferase Assay--
Jurkat cells (10 × 106)
were electroporated at 960 microfarads and 250 V using a Bio-Rad Gene
Pulser with 15 µg of pIL-2-Luc plasmid, 5 µg of p Tec Expression in Myeloid and Lymphoid Cells Including T
Cells--
Four isoforms of Tec protein-tyrosine kinase type I to type
IV (527, 608, 602, and 630 aa long) have been described, resulting from
alternative splicing (26). They have estimated molecular masses of 62, 70, 68, and 72 kDa, respectively. The largest type IV which has an
estimated molecular mass of 72 kDa is also known as TecA. For clarity
we will hereafter refer to TecA/Tec type IV as Tec. By using specific
anti-Tec antiserum, Tec with an apparent molecular mass of 72 kDa was
found to be predominantly expressed in all cell lines examined here
with the exception of fibroblast cell line (Fig.
1A). These cells include a
monocytic cell line (THP1), B cell lines (Nalm6, BAF3, and U266), a
myeloid cell line (KG1a), T cell lines (WT and JH6.2), and resting T
cells. Moreover, another Tec isoform with 70 kDa can be found in
myeloid cell line KG1a, pro-B cell line BAF3, and T hybridoma cell line
WT (Fig. 1A, lanes 2, 5, and 7). The other band
at 79 kDa as described previously by Kitanaka et al. (25) is
probably nonspecific since it does not correspond to any predicted
molecular weight of Tec protein, is expressed in the fibroblast cell
line, and is not detected in COS-7 transfected with Tec plasmid (data
not shown). Fig. 1B illustrates the anti-Tec antiserum
specificity since anti-Tec antiserum can only recognize Tec but not Itk
or Btk protein expressed in COS-7 cells. Hence, Tec is expressed in
lymphoid cell lines, primary resting T cells and myeloid cell line.
Tec Is Activated Following TCR/CD3 or CD28 Ligation--
Itk, a
member of Tec family kinases, has been shown to be expressed in T cells
and be activated following TCR/CD3 or CD28 ligation. Because the defect
in T cell signaling in itk-deficient mice is quite modest
and Tec kinase is expressed in T cells, we wanted to know whether
another Tec family kinase member, Tec kinase, could be involved in T
cell signaling. To evaluate Tec activation, in vitro
autokinase assay indicating the Tec kinase activity (Fig. 2A, upper panel) and Western
blotting to quantify the Tec kinase amount in each lane (Fig. 2A,
lower panel) were carried out. Relative activity is shown at the
bottom of Fig. 2A. Ligation of CD3 or CD28 on the
CD28-transfected hybridoma line (WT) elicited a 2.3- or 2-fold increase
in Tec autokinase activity, respectively (Fig. 2A, lanes 2 and 3), in contrast to non-stimulated condition (Fig. 2A, lane 4). As in autokinase assay, tyrosine
phosphorylation in different stimulations was also evaluated by
immunoblot with anti-phosphotyrosine antibody (Fig. 2B).
Relative activity is shown at the bottom of Fig.
2B. Ligation of CD3 or CD28 on the CD28-transfected
hybridoma line (WT) also induced a 3- or 3.2-fold increase in tyrosine
phosphorylation level. The increase in relative activity of Tec
following ligation was relatively modest but reproducible because Tec
family kinases have a much lower autokinase activity than Src family
kinases, and there is no known substrate available for evaluating their
transphosphorylation activity. For example, the relative activity of
Itk is about 2-3-fold following CD28 or CD3 ligation (43, 44). Hence,
these results suggest that Tec can be involved in TCR/CD3 or CD28
pathway in T cells.
Tec Can Bind to CD28 via SH3 Domain Proline-rich Domain Interaction
Following CD28 Ligation--
Tec expression in T cells and activation
following CD28 ligation incited us to determine how Tec is involved in
CD28 signaling. Indeed, another member of the Tec family Itk/Emt/Tsk is
expressed in T cells and inducibly binds to CD28 (21). This interaction implicates Itk SH3 domain and the first proline-rich motif of CD28
(45). As depicted in Fig. 3, endogenous
Tec inducibly bound to CD28 following cross-linking (Fig. 2, lane
3), although a weak association was found in unstimulated cells
(Fig. 2, lane 2). Hence, Tec can bind to CD28 in
vivo in an activation-dependent manner. In our
hybridoma T cells, Itk associated with CD28 receptor following its
cross-linking (data not shown), confirming previous works (45).
Interestingly, the ratio of Itk and Tec that became recruited to CD28
receptor in comparison to the total kinase amount detected in total
cell lysates was similar (data not shown). This may mean that both Tec
and Itk play similarly important roles in CD28 pathway.
Tec possesses various domains that are likely candidates for
protein-protein and protein-lipid interaction, PH, TH, SH3, and SH2
domains. So far, the only region of Tec involved in its functional interaction with defined proteins is its TH domain that binds to
protein kinase, adaptor molecules, and to the receptor tyrosine kinase
c-Kit (9-12). To elucidate which region of Tec kinase is involved in
CD28 binding, recombinant proteins corresponding to these different
regions were constructed as GST fusion proteins, expressed and used to
precipitate CD28 from the murine T cell hybridoma transfected with
huCD28 cDNA. As depicted in Fig. 4, GSTTECSH3 can precipitate CD28 from nonstimulated (Fig. 4, lane 5) or stimulated cells by anti-CD28 antibody (Fig. 4, lane
6) to a similar degree. In contrast, the GSTTECSH2 domain (Fig. 4, lanes 7 and 8) did not bind CD28 even after CD28
cross-linking which was shown to induce the phosphorylation of its
intra-cytoplasmic domain and permit its association with p85 and Grb-2
SH2 domains. The GSTTEC5N containing the PH and TH domains was poorly
effective in precipitating CD28 (Fig. 4, lanes 3 and
4). Thus, Tec SH3 domain seems to be the only domain
responsible for CD28 interaction.
So far, some regions have been identified within CD28 intra-cytoplasmic
domain involved in the binding of adaptor molecules and tyrosine
kinases. The 173Tyr-Met-Asn-Met motif is involved in both
the binding of p85 and Grb-2 SH2 domains (46, 47). Two putative
proline-rich motifs, N-terminal 178Pro-Arg-Arg-Pro-Gly-Pro
and C-terminal 190 Pro-Tyr-Ala-Pro-Pro-Arg, called PR1 and
PR2, respectively (Fig. 5A),
are also likely candidates for SH3 domain binding. PR1 was recently
identified as the major binding site for Itk SH3 domain, whereas the
binding partner of the latter is unknown. We next investigated whether
the proline-rich motifs of CD28 are involved in Tec SH3 domain
interaction. We mutated prolines 181 and 183 in PR1 and prolines 193 and 194 in PR2 within CD28 (Fig. 5A). By using transfection
of mutant plasmids in murine T cell hybridoma, stable transfectants
with similar surface expression of CD28 molecules were selected and
analyzed by flow cytometry (Fig. 5B). We used as a control
the recombinant GST SH3 domain of Itk to precipitate different CD28
molecules. As already reported, Itk SH3 domain binds to wild-type CD28
in an activation-dependent manner (45). These two PR motifs
are functionally essential for Itk SH3 binding since CD28 mutated or
deleted within PR1 and PR2 abolished almost or completely this binding,
respectively. CD28 mutant Y173F+Y200F was still precipitated by Itk SH3
with enhanced binding following CD28 ligation (Fig. 5C).
This also confirms correct expression of CD28 molecules in these stable
clones. As indicated in Fig. 5D, Tec SH3 domain binds to
wild-type CD28 molecules, but in contrast to Itk no enhanced binding
was detected upon receptor ligation. The two PR motifs are functionally
essential for Tec SH3 binding since deletion mutant without PR motifs
abrogated completely this binding. Similarly to Itk SH3, mutations on
tyrosines 173 and 200 did not impede Tec SH3 precipitation. This
further demonstrates that its binding is independent of p85 and Grb-2
since these two adaptor molecules are recruited upon CD28 ligation (46,
47). In agreement with results of in vitro binding, the
in vivo binding between endogenous Tec and PR motif-mutated
CD28 molecules was impaired following CD28 activation in comparison to
that between endogenous Tec and wild-type CD28 molecule (Fig.
5E). Hence, we demonstrate that PR1 and PR2 within CD28 are
functionally important for Itk and Tec SH3 domains although they are
not typical proline-rich motifs (48, 49). Altogether, our data indicate
that Tec uses its SH3 domain to bind to PR motifs of CD28.
p62dok but Not CD28 Is an in Vivo Substrate for
Tec--
TCR activation results in tyrosine phosphorylation of
substrates such as Cbl (p120), SLP76 (p76), and p36/38 (LAT) (50-52). CD28 activation also leads to tyrosine phosphorylation of substrates such as p95vav, an adaptor molecule p62, Cbl, the catalytic
subunit of PI 3-kinase (p110), and CD28 itself (32-35, 46). To
understand the role of Tec kinase in TCR and CD28 pathways, we tried to
find its substrate(s). Molecule(s) of 62 kDa associated to Ras GAP was
demonstrated to be phosphorylated upon CD28 but not CD3 stimulation
(35). One molecule associated to Ras GAP has been recently cloned and
called p62dok (53, 54). To understand further whether or not
p62dok is actually a substrate for CD28 but not CD3 signaling,
CD28-transfected murine T cell hybridoma WT was transfected with
p62dok cDNA tagged with the hemagglutinin (HA) epitope, and
then stable transfected clones (WTHA62dok) with high expression
of p62dok were selected by Western blotting. As shown in Fig.
6A, upper panel, lane 3, CD28
ligation induced the tyrosine phosphorylation of p62dok. In
contrast, CD3 stimulation did not induce detectable tyrosine phosphorylation of p62dok (Fig. 6A, lower panel, lane
2). Fig. 6B showed the phosphorylation pattern of
clones WTHA62dok or WT stimulated with GAM, CD28 plus GAM, or
CD3 plus GAM. The proteins p120, p95, p76, and p36/38 were
phosphorylated on CD3 cross-linking, whereas p95 and p62 were
phosphorylated on CD28 cross-linking. These experiments confirm that
p62dok is a specific substrate of CD28 but not CD3 stimulation.
We next investigated whether Tec can phosphorylate p62dok. This
protein p62dok is a substrate for various kinases of the Src
family (Src and Lck) but also Abl (55-57). HAp62dok plasmid
was cotransfected with Src, Fyn, Lck, Tec, or Itk expression vector in
heterologous COS-7 cells. The relative phosphorylation level of
p62dok by PTKs was indicated in arbitrary units (AU). As shown
in Fig. 7A, Src family kinases
Src, Fyn, and Lck led to the tyrosine phosphorylation of p62dok
(lanes 3-5 in upper panels).
Interestingly, Tec induced robust tyrosine phosphorylation of
p62dok (Fig. 7A, upper panel, lane 6).
Under the same experimental conditions, Itk was unable to elicit a
significant p62dok tyrosine phosphorylation (Fig. 7A,
upper panel, lane 7) although similar quantities of Tec and Itk
proteins were detected (Fig. 7A, lanes 6 and 7 in
lower panels). Fig. 7B shows
phosphorylation level of Itk and Tec in heterologous COS-7 cells. Tec
could be more activated than Itk in these cells. Hence, one of the CD28 tyrosine-phosphorylated substrates, namely p62dok, can be
phosphorylated by Tec.
So far, CD28 has been shown to be phosphorylated on tyrosine residues
by Src family kinases such as Lck and Fyn and by Itk but not ZAP-70
(58, 59). Tec recruitment to CD28 receptor may be important for CD28
tyrosine phosphorylation. COS-7 cells were cotransfected with CD28
together with Lck or Tec. For the latter, we used Tec and its CD2
membrane-targeted versions either wild-type (rCD2flagTec) or
kinase-dead mutant (rCD2flagTeckd) where the lysine of the ATP-binding
site was replaced by glutamic acid. The tyrosine phosphorylation of
CD28 was analyzed by immunoblot using anti-phosphotyrosine mAb. CD28
was phosphorylated when coexpressed together with Lck tyrosine kinase
(Fig. 8, upper panel, lane 3). This confirms previous observations (58). Conversely, we were unable to
demonstrate a significant phosphorylation of CD28 using either
coexpression of Tec or its membrane targeted forms (Fig. 8, upper
panel, lanes 4-6). These experiments suggest that CD28 is
unlikely to be a major substrate for Tec in this experimental system.
To sum up, these data show that p62dok but not CD28 is a
substrate for Tec kinase.
Tec, but Not Itk, Is Involved in the IL-2 Promoter Activity in T
Cells--
To address the question of the role of Tec in T cell
activation, we analyzed its role in IL-2 transcription. We compared the ability of wild-type Itk and Tec to induce the IL-2 promoter activity upon transient transfection of Jurkat cells with plasmid vectors expressing the kinases and pIL-2 coupled to the luciferase gene (pIL-2-Luc). We used, as control kinase, kinase-dead mutants of Itk and
Tec. These mutants have both been generated by substitution of glutamic
acid for a critical lysine in the ATP-binding site at amino acid
positions 396 and 397 for Itk and Tec, respectively. As already
reported, activation of IL-2 promoter could be induced by
pharmacological drugs such as PMA which mimics protein kinase C
activation, PMA plus CD3 stimulation, and PMA plus CD28 costimulation in Jurkat cells. As shown in Fig.
9A, stimulation by PMA plus anti-CD3 antibody or PMA plus anti-CD28 antibody elicited IL-2 promoter-driven luciferase expression. The latter is stronger than the
former for IL-2 promoter (mock). Overexpression of wild-type Tec
(Tecwt), but not kinase-dead mutants of Tec and Itk or wild-type Itk
(Teckd, Itkkd, or Itkwt), can further increase significantly IL-2
promoter activity upon PMA, PMA plus anti-CD3 mAb, and PMA plus
anti-CD28 mAb stimulations. Moreover, overexpression of kinase-dead mutant of Tec (Teckd) can inhibit IL-2 promoter activity in CD3 or CD28
plus PMA stimulation, whereas kinase-dead mutant of Itk (Itkkd) was
devoid of any significant effect. This inhibition by overexpressed
kinase-inactive Tec suggests that Tec is located downstream of TCR/CD3
and CD28 receptors. More intriguingly, we measured endogenous IL-2
production in media collected in the above experiments by using
enzyme-linked immunosorbent assay. In accordance, we got similar
profiles (data not shown). That is to say, our isolated IL-2 promoter
can completely reflect endogenous IL-2 promoter, and Tec has the
similar effect on minimal and endogenous full-length IL-2 promoters.
Expression levels of Itk, Tec, or their mutants are indicated in Fig.
9B. Cytomegalovirus promoter-driven expression of these
expression vectors containing cDNA of Itk, Tec, or their mutants
was poor in Jurkat cells, whereas PMA can strongly induce their
expression. Apparently, PMA stimulation had no significant effect on
Tec kinase activity in COS cells (data not shown). Although a similar
quantity of wild-type Tec was overexpressed, CD3 or CD28 stimulation in
synergy with PMA increased IL-2 promoter activity to a greater extent
than PMA alone (Tecwt). These results suggest that CD3- or
CD28-mediated activation of Tec results in increase of IL-2 promoter in
these transient transfections.
Altogether, these experiments demonstrate that Tec can specifically
link signals from receptors TCR/CD3 and CD28 to IL-2 promoter (Fig.
10) and that the two Tec family members
Itk and Tec differ dramatically in their ability to modulate IL-2
promoter following TCR/CD3 or CD28 stimulation.
We have demonstrated that Tec protein-tyrosine kinase participates
in TCR/CD3 and CD28 pathways. CD3 or CD28 ligation induces Tec
activation. Moreover, CD28 engagement can lead to the recruitment of
Tec via its binding to proline-rich motifs (PR1 and PR2) found in the
CD28 intra-cytoplasmic domain and phosphorylation of downstream substrates such as p62dok, a substrate for Tec. Finally, Tec
activation results in the activation of the IL-2 promoter regulated by
TCR/CD3 and CD28 receptors.
PTKs are important for T cell functions (32, 34). PTK inhibitors
abolish TCR/CD3- and CD28-mediated protein phosphorylation and IL-2
production, a hallmark of activated T cells. The Src family kinases Lck
and Fyn are recruited and/or activated in TCR/CD3 and CD28 pathways
(60, 61). Besides, they have been shown to phosphorylate components
such as CD3 or CD28 in these two pathways (58). Tec family member
Itk/Emt/Tsk has been shown to be implicated in TCR/CD3 or CD28 pathway
and can phosphorylate CD28 in vitro. Our data show that Tec
kinase is expressed in T cells (Fig. 1) and activated in TCR as well as
CD28 pathways (Fig. 2).
The activation of Tec is still elusive. TCR/CD3 or CD28 ligation
induces Tec activation in T cells in addition to the activation of Src
family kinases including Lck and/or Fyn (60, 62-64). One mechanism by
which TCR/CD3 or CD28 can modulate Tec activity could be the activation
of Lck and Fyn. Studies performed with Btk, Itk, and Tec have
demonstrated that they are substrates of Src-related kinases that can
act as positive regulators (10, 13, 43, 65-67). For instance,
coexpression of Src family kinases induced the increased
phosphorylation and activity of Btk at two sites Tyr551 and
Tyr223. This latter located in the SH3 domain is conserved
among the different members of the Tec family and could be involved in
enzyme regulation (68). An alternative possibility is the modulation of
Tec by PI 3-kinase via targeting of its PH domain to membrane. PI
3-kinase has been shown to be activated following TCR/CD3 or CD28
ligation (69). This membrane targeting could render Tec accessible to
its partners such as regulators or substrates (4, 6, 70).
Tec kinase can interact with CD28 in an
activation-dependent manner in vivo. Tec SH3
domain seems to be responsible for direct binding to CD28, although we
cannot exclude the indirect binding between the other regions than SH3
domain of Tec and CD28 (Fig. 4). The CD28 regions involved in Tec SH3
binding are the two proline-rich motifs (PR1 and PR2) (Fig. 5). This
binding does not rely on inducible recruitment of p85 or Grb-2 to CD28
since mutants devoid of binding to p85 and Grb-2 retain their
interaction with Tec. Endogenous Tec increases its binding to CD28
following CD28 ligation although there is basal binding of Tec to CD28
in unstimulated condition (Fig. 3). However, Tec SH3 domain has similar
affinity for unstimulated or stimulated CD28 (Fig. 5B).
Explanation may be that endogenous Tec may be more accessible for CD28
following ligation since activated Tec becomes "opened" via
conformational change or induced indirect binding between other adaptor
molecules, CD28 and Tec. Another interesting question is why two PR
motifs of CD28 are essential for the binding of Tec containing one SH3
domain. This can be explained by cooperative binding between two Tec
kinases to CD28, the dimerization of Tec, or only an artifact since GST
proteins form a dimer. The protein sequence homology between Itk and
Tec is about 50%. Three sites (Trp208, Ser223,
and Ser224) located in murine Itk SH3-binding pocket have
been suggested to be involved in binding to its own PR motif in TH
domain (7). With the exception of Ser224, the first two
sites are conserved between Itk and Tec. This may explain why Itk SH3
domain is partially different from Tec SH3 domain.
Substrates for Tec kinases are almost unknown. Tec has been shown to
phosphorylate Jak kinase in hematopoietic cells and vice versa (31).
Following TCR and CD28 ligations, PTKs are responsible for the
phosphorylation of their specific substrates since TCR/CD3 and CD28
have no intrinsic kinase activity. p62dok containing some
functional domains (PH, PTB, and tyrosine residues) is a downstream
substrate of kinases such as Lck, Src, or Abl. Stimulation of CD2 and
CD28 but not CD3 can induce tyrosine phosphorylation of the
p62ras GAP-associated protein (35, 71). We first indicated that p62dok is an in vivo substrate for Tec kinase but
not Itk in COS-7 cells. This is probably because different Tec family
members have different substrates or because distinct regulatory steps
are involved in the regulation of Tec and Itk. For instance, Itk is
less activated than Tec in COS-7 cells (Fig. 7B). One
interesting question is why p62dok is phosphorylated only in
CD28 pathway but not in TCR/CD3 pathway, although TCR/CD3 and CD28
pathways appear capable of activating similar kinases (Lck, Fyn, Itk,
and Tec). A possible explanation is that p62dok is merely
accessible to kinases including Tec upon CD28 activation. Which one(s)
is responsible for p62dok phosphorylation remains to be studied
since kinases including Tec, Itk, Lck, and Fyn are activated and/or
recruited in CD28 pathway. Studies on Tec-mediated phosphorylation of
p62dok could be important for determining the role of Tec in
CD28 pathway. CD28 has been shown to be an in vitro
substrate for Itk. However, neither Tec nor its dominant positive
membrane-targeted forms of Tec kinase can phosphorylate CD28, whereas
Lck can. These results suggest that CD28 is a regulator but not a
potential substrate for Tec. On the contrary, CD28 could be a regulator
and substrate of Itk (59).
IL-2 production in T cells is induced following activation of the
TCR/CD3 together with costimulatory signals that are required for full
activation. IL-2 production can be mimicked by anti-TCR (or anti-CD3)
mAb and anti-CD28 mAb and be potentiated by pharmacological agents such
as PMA in Jurkat cells. As shown in Fig. 9A, Tec
overexpression but not Itk leads to the induction of IL-2 promoter
activity in combination with PMA alone as well as PMA plus CD3 or CD28
triggering. This effect is dependent on Tec kinase activity since
mutations that prevent ATP binding abrogated all Tec functions.
Expectedly, overexpression of kinase-inactive Tec with CD3 or CD28
stimulation plus PMA leads to inhibition of IL-2 promoter, suggesting
that Tec is a linker between receptors TCR/CD3 and CD28 and IL-2
promoter in a kinase-dependent manner. The mechanisms by
which Tec but not Itk can regulate IL-2 promoter in T cell signaling
need to be investigated, and this study is under way. IL-2 promoter can be induced via various transcriptional factors such as AP-1, NFAT, and
NFkB activated by TCR/CD3 or CD28 signals. One candidate regulated by
Tec may be the AP-1 site in IL-2 promoter since Ohya et al. (30) recently suggested a role for Tec in the regulation of c-fos transcription via ternary complex factors which is
also involved in the activation of Jun-B promoter in hematopoietic cells. Another candidate may also be the NFAT site in IL-2 promoter since Btk regulates Ca2+ entry which could induce
NFATp dephosphorylation and nuclear translocation via calcineurin
activation (72).
Taken together, we first indicated that Tec kinase is involved in T
cell signaling and that Tec can interact with CD28 (Fig. 10). More
interestingly, Tec can phosphorylate its substrate p62dok
specifically implicated in CD28 pathway and regulate IL-2 promoter whereas Itk cannot. These results suggest that although two Tec family
kinases Tec and Itk are involved in TCR and CD28 pathways, they have
different functions.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and p95vav. PTKs are
critical for T cell signaling since inhibition of PTKs abrogated the
substrate phosphorylation and IL-2 production. Nevertheless, in the
itk-deficient mice, CD3-mediated proliferative response was
severely compromised, whereas CD28-mediated proliferation was
significantly enhanced when compared with cells from control animals
(36). These data suggest that Itk negatively regulates the amplitude of
CD28 costimulation. Intriguingly, instead of being involved in the
amplification of IL-2 production which is the hallmark of CD28
costimulation, Itk could down-modulate these events. These observations
prompted us to look for other Tec family kinases that could be
expressed in lymphoid cells and involved in the amplification of IL-2 production.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
expressing wild-type huCD28 cDNA, mutant
DYF173+200 where Tyr has been substituted by Phe at positions 173 and
200, and DEL 21 where 21 amino acids in the C terminus of CD28 have
been truncated and contained no functional proline-rich motifs were
previously described (37). Here we renamed clones DWT6.11+- and
DYF173+200 for WT and Y173F+Y200F. Clones P181A+P183A, P193A, and P194A
were derived from DC27.1 cells that were stably transfected with the
plasmids PSR
CD28P181A+P183A, PSR
CD28P193A, and PSR
CD28P194A.
Clone WTHAp62dok was obtained from WT stably transfected with
the plasmid pMSCV-HADok. All T murine hybridoma cells were grown in
Dulbecco's modified Eagle's medium supplemented with 7% fetal calf
serum, penicillin, streptomycin,
-mercaptoethanol, sodium pyruvate,
and glutamate. COS-7 cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum, penicillin,
streptomycin, and glutamate. Besides human Jurkat T leukemia cells
JH6.2 (38), murine fibroblast cell line 3T6, murine
IL-3-dependent pro-B cell line BAF3, human monocytic cell
line THP1, human myeloid cell line KG1a, human pre-B cell line Nalm6,
and human myeloma cell line U266 were grown in RPMI 1640 medium
supplemented with 10% fetal calf serum, penicillin, streptomycin, and
glutamate except BAF3 medium which was further supplemented with
IL-3.
CD28, p48Src,
pF10, and BtkHA containing human wild-type CD28, Src, Fyn, and Btk
tagged with HA epitope cDNAs were gifts from Drs. R. Sweet, S. Courtneige, C. Bebbington, and R. Guinamard. Expression plasmids
pCDNALck, pCDNAItkflag, and pCDNAHAp62dok
possessing Lck, Itk tagged in 3' end, and p62dok tagged with HA
epitope were constructed by subcloning of pSMLck gifted from Dr. M. Marsh, p52.2.1, a gift from Dr. S. Desiderio, and pMSCV-HADok gifted
from Dr. Y. Yamanashi to pCDNA3 vector, respectively. Plasmid
pCDNAflagTec containing a wild-type full-length murine Tec cDNA
tagged with the FLAG epitope in 5' end was described previously. This
enzyme corresponds to the full-length enzyme, type IV or TecA (24).
Plasmid pCDNAflagTeckd was the kinase-dead version of
pCDNAflagTec with a point mutation at the ATP-binding site of Tec
corresponding to amino acid 397 (Lys to Glu). pCDNAItkkdflag was
the kinase-dead version of pCDNAItkflag with a point mutation at
the ATP-binding site of Itk corresponding to amino acid 396 (Lys to
Glu). Plasmid prCD2flagTec was constructed by in frame subcloning Tec
EcoRI fragment of pCDNAflagTec downstream of rat CD2
corresponding to amino acids 1-219 in pCDNArCD2BE vector. Plasmid
prCD2flagTeckd was a kinase-dead version of prCD2Tec with a point
mutation at the ATP-binding site of Tec corresponding to amino acid 397 (Lys to Glu). Plasmids PSR
CD28P181A+P183A, PSR
CD28P193A, and
PSR
CD28P194A in which human CD28 was point-mutated from Pro to Ala
at amino acid residues 181 and 183, 193, and 194, respectively, were
generated using the Transformer Site-directed Mutagenesis kit
(CLONTECH) according to the manufacturer's
instructions. The GST fusion plasmids pGSTTECSH1, pGSTTECSH2, and
pGSTTECSH3 were constructed by respectively subcloning the polymerase
chain reaction product of SH1, SH2, and SH3 regions of Tec into pGEX4T3 vector (Pharmacia Biotech Inc., Uppsala, Sweden). pGSTTEC5N containing PH and TH region of Tec has been described previously (12). These
constructs are further depicted in Fig. 3. pGSTITKSH3 containing Itk
SH3 domain was a gift from Dr. R. Guinamard. The promoter assay
plasmids pIL-2-Luc composed of IL-2 promoter fused with firefly
luciferase reporter gene and p
-actin-RLuc composed of
-actin
promoter fused with Renilla luciferase reporter gene were gifts from Drs. E. Verdin (39) and R. Castellano, respectively.
chain (ATCC). Anti-Lck, anti-Src, and anti-phosphotyrosine antisera were bought from Santa Cruz Biotechnology, and goat anti-mouse IgG
(GAM) antiserum was from Jackson ImmunoResearch. CD28-bound beads was
obtained by incubation of CD28.2 mAb with avidin-conjugated magnetic
beads (Immunotech) bound to biotinylated GAM (Jackson ImmunoResearch)
according to the manufacturer's instructions (Immunotech, France).
or BL21, transformed with GST fusion plasmids, were cultured
according to the manufacturer's instructions (Pharmacia Biotech Inc.,
Uppsala, Sweden). GST fusion proteins were purified according to the
manufacturer's instructions (Pharmacia Biotech Inc., Uppsala, Sweden).
For pulldown precipitation, 5 × 106 hybridoma cells
were stimulated, lysed, and centrifuged at 4 °C for 15 min at 13,000 rpm. Postnuclear lysates were incubated with 10 µg of GST recombinant
protein coupled with Sepharose at 4 °C overnight, and the
precipitates were treated as described above.
-32P]ATP at room temperature for 1 h. The immune
complex was subjected to gel electrophoresis, and gels were dried and
phosphorylated proteins were analyzed by autoradiography.
-actin-RLuc,
and 15 µg of the other plasmids expressing Tec and Itk or their
mutants. The cells were incubated for 2 h and then left
unstimulated or stimulated for 6 h with PMA (Sigma, 50 ng/ml), PMA
plus 248 (1/400 dilution), or PMA plus 289 (10 µg/ml). Following the
centrifugation, the cells were washed and lysed. Proteins were
quantified by Bradford reagent (Bio-Rad). Ten µg of cell lysates were
subjected to dual luciferase reporter assay (Promega) according to the
manufacturer's instructions. The efficiency of transfection was
corrected by the activity of firefly luciferase normalized by that of
Renilla luciferase in the lysates.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Expression pattern of Tec protein in
hematopoietic cells. A, 50 µg of total lysates from
different cells were analyzed by Western blotting with polyclonal
rabbit antiserum anti-Tec. Total lysates were prepared from different
cell lines 3T6 (lane 1), KG1a (lane 2), THP1
(lane 3), Nalm6 (lane 4), BAF3 (lane
5), U266 (lane 6), WT (lane 7), Jurkat clone
JH6.2 (lane 8), and human primary resting T cells
(lane 9). Data are representative of three independent
experiments. B, the anti-Tec antiserum was specific for Tec
rather than Itk or Btk proteins. COS-7 cells transfected with
pCDNA3 vector (lanes 1 and 2),
pCDNAflagTec (lanes 3 and 4),
pCDNAItkflag (lanes 5 and 6), or pSR BtkHA
(lanes 7 and 8) were lysed. Total lysates
(odd lanes), or immunoprecipitates with anti-epitope mAbs
(even lanes) were subjected to electrophoresis. Then
membrane was blotted with anti-Tec antiserum alone (upper
panel) or, following stripping, reblotted with anti-Itk antiserum
(middle panel) or anti-Btk antiserum (lower
panel).
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Fig. 2.
Tec is activated following CD3 and CD28
cross-linking. A, hybridoma cells WT were left
unstimulated (lanes 1 and 4) or stimulated with
anti-CD3 mAb cross-linking (lane 2) and with anti-CD28 mAb
cross-linking (lane 3) and then lysed. Total cell lysates
were precipitated by normal rabbit serum (lane 1) or
anti-Tec antibody (lanes 2-4). Immunoprecipitates were
divided into two parts, one for in vitro kinase assay
(upper panel) and the other for Western blotting
(lower panel). All values detected from upper and
lower panels were determined by densitometric analysis using
the BioImage system (Millipore); relative activities (RA)
were obtained by normalization of in vitro kinase activities
of Tec to the protein amounts. B, hybridoma cells WT were
left stimulated with anti-CD3 mAb cross-linking (lane 1),
with anti-CD28 mAb cross-linking (lane 2), or unstimulated
(lane 3). Following lysis, total cell lysates were
precipitated by anti-Tec antibody. Immunoprecipitates were subjected to
gel electrophoresis and then blotted with 4G10 (upper
panel). Following stripping, the membrane was blotted with
anti-Tec antiserum (lower panel). All values detected from
upper and lower panels were determined by
densitometric analysis using the BioImage system (Millipore), relative
activities were obtained by normalization of Tec tyrosine
phosphorylation to the protein amounts. Data are representative of two
independent experiments.
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Fig. 3.
CD28 receptor can bind Tec kinase in T cells
in vivo. Hybridoma cells WT were left unstimulated ( ) or
stimulated with anti-CD28 mAb and goat anti-mouse antiserum (+), before
lysis in lysis buffer and precipitation. IP,
immunoprecipitation. Total cell lysate (TL, lane
1) and precipitates from unstimulated cells (lane 2) or
from stimulated cells (lane 3) were subjected to gel
electrophoresis and immunoblotted with anti-Tec antiserum (upper
panel), or following stripping membrane was reblotted with
anti-CD28 antibody (lower panel). Immunoglobulin heavy chain
(Ig H) was indicated. Data are representative of two
independent experiments.
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Fig. 4.
SH3 domain of Tec is the only domain
responsible for the interaction of Tec with CD28 receptor in
vitro. GST fusion proteins containing different domains of
Tec were used to precipitate wild-type human CD28 receptor from
unstimulated cells ( , odd lanes) or cells stimulated by
CD28 antibody cross-linking (+, even lanes). Following gel
electrophoresis, proteins were transferred to membranes and blotted
with anti-CD28 antibody shown in upper panel. CD28X
represented CD28 cross-linking. GST fusion proteins containing
different domains of Tec are indicated at bottom of this
figure, the numbers correspond to the amino acid residues as
follows: GST 5N aa 1-178, SH3 aa 179-238, SH2 aa 239-344, and SH1 aa
345-630. Data are representative of two independent experiments.
TL, total cell lysate.
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Fig. 5.
Two proline-rich domains of CD28 receptor are
important for Tec binding. A, protein sequences of CD28
molecules mutated in intra-cytoplasmic domain. These mutations are
underlined. Stable clones with similar surface expression of
CD28 molecules were selected and analyzed for interaction.
B, flow cytometric analysis of T hybridoma cells. Parental
murine T hybridoma cells DC27.1 without human CD28 transfection or
different human CD28 stable clones were stained by anti-human CD28 mAb
CD28.2. C, interaction between GSTITKSH3 and various CD28
molecules expressed in T hybridoma cells. Following stimulation with
CD28 cross-linking (28×) or not (NS), lysates
obtained from stable transfected murine T cell hybridoma clones
expressing wild-type CD28 and its mutants were incubated with GSTITKSH3
protein coupled to Sepharose beads, washed three times with lysis
buffer, and processed as described under "Experimental Procedures."
The membrane was probed with anti-CD28 antibody. Following stripping,
the same membrane was reprobed with anti-Sam 68 antibody. AU displayed
on the y axis correspond to the ratio of the signal obtained
by immunoblotting with anti-CD28 antibody to that obtained with
anti-Sam 68 antibody. Data are representative of three independent
experiments. D, interaction between GSTTECSH3 and various
CD28 molecules expressed in T hybridoma cells. Similarly to
C, GSTTECSH3 protein was used instead of GSTITKSH3 protein.
E, interaction between endogenous Tec and various CD28
molecules expressed in T hybridoma clones. Different hybridoma clones
were treated as in Fig. 3. CD28 immunoprecipitates from total lysates
obtained from CD28-stimulated (+) or unstimulated ( ) samples were
subjected to gel electrophoresis and blotted with anti-Tec antiserum
(upper panel). Following stripping, the membrane was
reblotted with anti-CD28 mAb (lower panel).
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Fig. 6.
p62dok is phosphorylated
following CD28 cross-linking but not CD3 cross-linking.
A, murine T cell hybridoma transfected with expression
vector containing HAp62dok cDNA and puromycin resistance
gene and selected for puromycin resistance, WTHADok (lanes
1-3) or the parental T cell hydridoma WT (lanes 4-6)
were stimulated with GAM (lanes 1 and 4),
anti-CD3 mAb plus GAM (lanes 2 and 5), or
anti-CD28 mAb plus GAM (lanes 3 and 6). Lysates
were incubated with anti-HA mAbs, and immunoprecipitates were divided
in two halves, one processed and blotted with rabbit
anti-phosphotyrosine antiserum (upper panel) and the other
blotted with rabbit anti-p62dok antiserum (lower
panel). B, 50 µg of total lysates from these cells
were subjected to gel electrophoresis as in A, probed with
4G10 mAb. p120, p95, p76, p62, and p36/38 were indicated. Data are
representative of three independent experiments.
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Fig. 7.
p62dok is a common substrate for
Src, Fyn, Lck, and Tec rather than Itk in COS-7 cells.
A, COS-7 cells transiently transfected with pCDNA3 empty
vector (lane 1), pCDNAHAp62dok (lane
2), pCDNAHAp62dok plus p48Src (lane 3),
pCDNAHAp62dok plus pF10 (lane 4),
pCDNAHAp62dok plus pCDNALck (lane 5),
pCDNAHAp62dok plus pCDNAflagTec (lane 6),
and pCDNAHA62dok plus pCDNAItkflag (lane 7)
were harvested 2 days after transfection by trypsin treatment.
Trypsinized cells were lysed, and anti-HA antibody immunoprecipitates
were subjected to gel electrophoresis and then transferred to
membranes. The membrane was probed with rabbit anti-phosphotyrosine
(anti-phosphotyr) antiserum (upper panel), and
following stripping, it was reprobed with rabbit anti-p62dok
antiserum (middle panel). Data obtained from
upper and bottom panels by scanning using the
BioImage system were converted into relative phosphorylation levels in
AU by normalization of p62dok tyrosine phosphorylation to the
p62dok protein amounts and are indicated at the
bottom of the upper panel. Total lysates from the
same cells subjected to gel electrophoresis were then blotted with
anti-Src, anti-Fyn, anti-Lck, and anti-FLAG antisera. B,
tyrosine phosphorylation of Tec and Itk in Cos-7 cells. PCDNA3
(mock, lane 1), pCDNAItkflag (Itk, lane 2),
or pCDNAflagTec (lane 3) was transfected into COS-7
cells and treated as in A. Anti-FLAG immunoprecipitates
(IP) were transferred to membrane, followed by
immunoblotting with 4G10 (upper panel) or anti-FLAG
(upper panel). Data are representative of three independent
experiments.
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Fig. 8.
CD28 can be phosphorylated by Lck but not Tec
in vivo. COS-7 cells transiently transfected with
different expression vectors, pCDNA3 empty vector (lane
1), pSR CD28 (lane 2), pSR
CD28 plus pCDNALck
(lane 3), pSR
CD28 plus pCDNAflagTec (lane
4), pSR
CD28 plus prCD2flagTec (lane 5), and
pSR
CD28 plus prCD2flagTeckd (lane 6), were harvested with
trypsin treatment 2 days after transfection. Trypsinized cells were
lysed, and total cell lysates were divided in two parts. One was
precipitated with anti-CD28.2 mAb. Immunoprecipitates were subjected to
gel electrophoresis before transfer to membranes and immunoblotted with
anti-phosphotyrosine antibody 4G10 (upper panel). Following
stripping, the membrane was reprobed with anti-CD28 antibody CD28.6
(middle panel). The rest respectively precipitated with
anti-FLAG mAb (lower panel, lanes 1, 2, and 4),
anti-Lck (lower panel, lane 3), or anti-rat CD2 mAb OX34
(lower panel, lanes 5-6) was processed as described under
"Experimental Procedures." The membrane was probed with anti-Lck
and anti-Tec antibodies. Expression of Tec or Lck was depicted as
indicated in the lower panel. Data are representative of two
independent experiments.
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Fig. 9.
Tec but not Itk is implicated in IL-2
promoter activity following CD3 or CD28 ligation besides PMA
stimulation in Jurkat cells. A, 10 × 106
Jurkat cells were electroporated with pCDNA3 (mock),
pCDNAItkflag (Itkwt), pCDNAItkkdflag (Itkkd), pCDNAflagTec
(Tecwt), or pCDNAflagTeckd (Teckd) with pIL-2-Luc and
p -actin-RLuc. Following electroporation, cells were divided into
four parts, one left unstimulated (NS), the other three
stimulated with PMA, PMA plus 289, an anti-CD3 mAb
(PMA+CD3), and PMA plus 248, an anti-CD28 mAb
(PMA+CD28), respectively, for 6 h. After
centrifugation, cell pellets were lysed. Postnuclear supernatants were
analyzed according to the protocol described under "Experimental
Procedures." IL-2 promoter activity was indicated in AU representing
relative luminescence units of firefly luciferase normalized by that of
Renilla luciferase. B, Western blot with
anti-FLAG mAb indicates the expression level of transfected Itk, Tec,
and their mutants. Data are representative of two independent
experiments.
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Fig. 10.
Proposed model for Tec function in TCR/CD3
and CD28 pathway. Following TCR/CD3 or CD28 ligation, Tec kinase
can be activated. Src protein kinases such as Lck and/or Fyn activated
by these receptors could modulate Tec activation. Then Tec kinase can
phosphorylate its substrates such as CD28 pathway-specific
p62dok protein and lead to IL-2 expression. In addition, Tec
can associate with CD28 receptor following CD28 receptor ligation
through SH3 and proline-rich motif interaction. Arrowhead in
black shows activation or phosphorylation and
arrowhead in gray shows association.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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ACKNOWLEDGEMENTS |
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We thank Drs. D. A. Cantrell, R. Guinamard, M. Lopez, S. Baghdoyan, C. Bebbington, S. Courtneige, M. Marsh, S. Desiderio, Y. Yamanashi, R. Sweet, E. Verdun, J. Ihle, K. Sugamura, and R. Castellano for kindly providing reagents and helpful discussion; Drs. J. Nunes, Y. Collette, David Rawlings, Claude Mawas, and C. Lipcey for their critical review of this manuscript and their colleagues for their excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by INSERM, Ligue des Bouches du Rhone Contre le Cancer, and Association pour la Recherche Contre la Cancer.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.
Supported by France-Taiwan fellowship.
§ To whom correspondence should be addressed. Tel.: 33 491 75 84 15; Fax: 33 491 26 03 64; E-mail: olive{at}marseille.inserm.fr.
The abbreviations used are: PTK, protein-tyrosine kinase; PH, pleckstrin homology; TH, Tec homology; IL, interleukin; HA, hemagglutinin; mAb, monoclonal antibody; PMA, phorbol 12-myristate 13-acetate; TCR, T cell receptor; GAM, goat anti-mouse; PR, proline-rich; WT, wild type; AU, arbitrary units; aa, amino acid(s); GST, glutathione S-transferase; PI, phosphatidylinositol.
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