From the Department of Cell Signaling, DNAX Research
Institute of Molecular and Cellular Biology, Palo Alto, California
94304 and the ¶ Department of Molecular Genetics, Kansai Medical
University, 10-15 Fumizono-cho, Moriguchi 570, Japan
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ABSTRACT |
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Bruton's tyrosine kinase (Btk) is mutated in
X-linked agammaglobulinemia patients and plays an essential role in B
cell receptor signal transduction. Btk is a member of the Tec family of
nonreceptor protein-tyrosine kinases that includes Bmx, Itk, Tec, and
Txk. Cell lines deficient for Btk are impaired in phospholipase C- Bruton's tyrosine kinase
(Btk)1 is a member of the Tec
family of nonreceptor protein-tyrosine kinases (PTKs) that includes Bmx, Itk, Tec, and Txk (reviewed in Ref. 1). Tec family PTKs are
characterized by an NH2-terminal pleckstrin homology (PH) domain followed by a Tec homology domain (containing a
Zn2+-binding Btk motif and a proline-rich region), a
Src-homology 3 (SH3) and SH2 domain, and a COOH-terminal kinase domain
(1, 2) (Fig. 1). Tec PTKs share 50-60% amino acid sequence identity and are predominately expressed by cells of the immune system (1). Bmx
and Txk are somewhat atypical family members: Bmx shares little
sequence homology with other Tecs within the proline-rich and SH3
domains; Txk is particularly atypical at the NH2 terminus, lacking the PH and Tec homology domains (Fig.
1).
2 (PLC
2)-dependent signaling. Itk and Tec have recently
been shown to reconstitute PLC
2-dependent signaling in
Btk-deficient human cells, but it is not known whether the atypical Tec
family members, Bmx and Txk, can reconstitute function. Here we
reconstitute Btk-deficient DT40 B cells with Bmx and Txk to compare
their function with other Tec kinases. We show that in common with Itk
and Tec, Bmx reconstituted PLC
2-dependent responses
including calcium mobilization, extracellular signal-regulated
kinase (ERK) mitogen-activated protein kinase (MAPK) activation, and
apoptosis. Txk also restored PLC
2/calcium signaling but, unlike
other Tec kinases, functioned in a phosphatidylinositol 3-kinase-independent manner and failed to reconstitute
apoptosis. These results are consistent with a common role for Tec
kinases as amplifiers of PLC
2-dependent signal
transduction, but suggest that the pleckstrin homology domain of Tec
kinases, absent in Txk, is essential for apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Diagrammatic representation of the Tec family
of nonreceptor PTKs.
Btk, the prototypical Tec family PTK, was first identified in B cells but is also expressed in myelomonocytic and mast cells (1). An essential role for Btk in signal transduction through the B cell antigen receptor (BCR) was revealed when mutations in Btk were shown to be responsible for a human disease termed X-linked agammaglobulinemia and for X-linked immunodeficiency in the xid mouse (reviewed in Ref. 3). Agammaglobulinemia patients suffer an intrinsic defect in pre-B to B cell development, which results in a lack of mature B cells (4). xid and btk-null mice share a similar, though less severe, phenotype (5, 6). Btk was subsequently shown to be activated in B cells by a two-step mechanism involving Lyn and PI 3-kinase. After BCR cross-linking, Lyn activates Btk by transphosphorylation (7-9), which leads to Btk autophosphorylation (10, 11). The PI 3-kinase-dependent step involves Btk membrane-targeting by PH domain engagement with phosphatidylinositol 3,4,5-trisphosphate (PIP3) (12, 13). Btk activity can be down-regulated by the SH2-containing inositol phosphatase (SHIP), which dephosphorylates PIP3 (14, 15).
An effector function for Btk was identified in experiments that used
the DT40 chicken B cell system for gene knockouts. Btk-deficient DT40
cells exhibited a reduction in the level of phospholipase C (PLC)-2
phosphorylation upon BCR cross-linking and a consequent failure to
mobilize calcium and generate inositol 3,4,5-trisphosphate (16). In
addition, the downstream apoptotic response of DT40 cells after BCR
cross-linking was impaired in Btk-deficient cells (17). Analyses of
Syk-deficient DT40 cells has shown this PTK also to be crucial in
phospholipase-dependent signaling (18). A model has been
proposed in which the concerted actions of Btk and Syk in
phosphorylating PLC
2 are essential for BCR signal transduction
(reviewed in Refs. 3, 19-21).
The considerable structural homology exhibited among Tec family members
suggests that, similar to Src family kinases (22), these proteins may
have overlapping functions. A recent report has shown that Itk and Tec
can reconstitute Btk responses including BCR-dependent
PLC2 phosphorylation and calcium mobilization (23). This study
examined proximal signaling events and did not analyze the capacity of
Tec kinases to restore Btk-induced downstream responses including ERK
MAPK activation and apoptosis (23). Moreover, the roles of the atypical
Tec family PTKs, Bmx and Txk, have not been examined. Here we
reconstitute Btk-deficient DT40 cells with Bmx and Txk, in addition to
Itk and Tec. We confirm the role of Tec kinases in
phospholipase-dependent signaling (23) and extend our study
to show reconstitution of downstream ERK MAPK activation and apoptotic
responses. We find that Bmx can restore Btk signaling, whereas Txk,
although capable of restoring PLC
2 signaling, is unable to
reconstitute apoptosis and functions independently of PI 3-kinase.
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EXPERIMENTAL PROCEDURES |
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Cells, Antibodies, and Biochemical Analyses
Cells--
DT40 cells were cultured in RPMI 1640 supplemented
with 10% fetal calf serum, 1% chicken serum (Sigma), penicillin,
streptomycin, glutamine, and 50 µM mercaptoethanol. DT40
cell lines rendered deficient for btk and plc2
by homologous recombination were described previously (16, 24).
Antibodies--
Anti-chicken µ mAb M4 (18), anti-HA mAb 12CA5
(25), anti-phosphotyrosine mAb 5H1 (25), and antisera generated against Btk (25) were as described. Bmx, Itk, Tec, and Txk antisera were
produced by immunization of rabbits with glutathione
S-transferase fusion proteins (Amersham Pharmacia Biotech)
containing regions of each kinase spanning from the NH2
terminus to the SH3 domain. Antisera against PLC2, ERK1, and ERK2
were from Santa Cruz Biotechnology (Santa Cruz, CA).
Biochemical Analyses-- ERK MAPK assays were as described (26). The PI 3-kinase inhibitors wortmannin and LY 294002 were from Calbiochem. Immunoprecipitations, immune complex protein kinase assays, and immunoblotting were as described previously (27). In vitro translation of HA-tagged Tec family PTKs was performed using the TNTTM-coupled reticulocyte lysate system (Promega, Madison, WI). YOP protein-tyrosine phosphatase was from New England Biolabs (Beverly, MA).
Cloning and Transfection of Tec Family Kinases
Human Bmx (28), mouse Itk (29), mouse Tec (type IV) (30), and human Txk (31) were cloned by a polymerase chain reaction-based strategy using published sequences. The polymerase chain reaction utilized cDNA libraries (prepared by T. McClanahan, DNAX) from lung (Bmx) and from T cells (Itk, Tec, and Txk). The sequence was confirmed by DNA sequencing. Cloning of mouse Btk and production of HA-tagged versions of the Tec family PTKs were as described (25). For transfection into DT40 cells, Tec family PTKs were subcloned into the pApuroII vector, and cells were transfected by electroporation (18).
Calcium Flux Analyses
DT40 cells were labeled with 1 µM indo-1AM (Molecular Probes, Eugene, OR) for 30 min at room temperature. After labeling, cells were washed and resuspended in RPMI 1640 supplemented with 1% fetal calf serum and 20 mM Hepes buffer. Measurement of calcium flux was performed using a FACSVantage (Becton Dickinson, Mountain View, CA).
Apoptosis Assays
DT40 cells (1 × 105/ml) were incubated with M4
mAb ascites at 2 µg/ml. Flow cytometry analyses of apoptotic cells
was carried out after 24 h using the TUNEL in situ cell
death detection kit (Roche Molecular Biochemicals). Flow cytometry was
performed using a FACSCalibur (Becton Dickinson) and analyzed using
CellQuest software (Becton Dickinson).
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RESULTS |
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Reconstitution of Btk Function Is Dependent on Expression
Levels--
Human Btk was previously shown to restore BCR-induced
PLC2 phosphorylation, calcium flux, and apoptosis in Btk-deficient DT40 cells (16, 17). Here we use mouse Btk to reconstitute Btk-deficient DT40 cells and show that the level of functional reconstitution is dependent on expression levels (Fig.
2). Btk-deficient DT40 cells were
transfected with mouse Btk, and three stable clones were selected that
expressed different levels of Btk, as measured by Western blotting of
whole cell lysates followed by PhosphorImager quantitation (facilitated
by the use of 125I-conjugated second Abs) (Fig.
2A). A direct comparison of endogenous chicken Btk in WT
DT40 cells and transfected levels of mouse Btk was not possible because
of poor cross-reactivity of the mouse Btk antisera with chicken Btk.
Similar cell surface expression levels of the BCR on each clone was
confirmed by FACS analysis using the M4 anti-chicken µ mAb (data not
shown).
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The three DT40 cell lines that expressed different levels of mouse Btk were compared with WT and Btk-deficient cells for their capacity to flux calcium and undergo apoptosis after BCR cross-linking (Figs. 2, B-C). The initial peak calcium flux and its sustained elevation was relatively poorly reconstituted in clone 2A2, which had the lowest level of Btk. In contrast, clones 1B4 and 1B2, which had greater Btk expression levels, showed reconstitution comparable with WT cells (Fig. 2B). BCR-induced apoptosis was similarly dependent on Btk expression levels. The level of apoptosis increased approximately in proportion to Btk expression (Fig. 2C). These results are consistent with data from Btk-transgenic mice that show Btk to be a limiting component of BCR signaling (32). Taken together, this suggests a role for Btk as a signal amplifier.
Expression of Tec Family PTKs in Btk-deficient DT40 Cells and
Quantitation of Their Relative Expression Levels--
Btk-deficient
DT40 cells were transfected with Bmx, Itk, Tec, or Txk, and stable
clones were selected. Expression of each PTK was demonstrated by
Western blotting of whole cell lysates with specific antisera (Fig.
3A). Expression of endogenous
Bmx, Itk, Tec, or Txk was not detectable in the parental Btk-deficient cells (Fig. 3A). Similar cell surface expression levels of
the BCR on each clone was confirmed by FACS analysis (data not
shown).
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Because reconstitution of Btk function is dependent on expression levels (Fig. 2), the relative levels of each Tec family PTK were quantitated using HA-tagged versions as a common reference (Fig. 3, B-D). Tagged kinases were not used in reconstitution experiments because tagging was found to disrupt Btk function (data not shown). To allow relative quantitation, HA-tagged Tec family PTKs were in vitro translated. Translated products were protein-tyrosine phosphatase-treated with YOP to remove potential tyrosine phosphorylation from the HA tag that could affect the epitope. Western blotting was then performed with anti-HA-tag mAb 12CA5 (Fig. 3B). Relative expression levels were quantitated by phosphorimaging analysis. In Fig. 3C, a second set of Western blot analyses were performed, this time with specific Tec family antisera to compare the relative amounts of the quantitated HA-tagged versions from Fig. 3B with whole cell lysates of the transfected clones expressing each Tec family PTK. Quantitation of the data from Fig. 3C with reference to those from Fig. 3B allowed calculation of the relative efficacies of each specific antiserum followed by the relative expression levels of each PTK (Fig. 3D). Btk, Itk, Tec, and Txk were found to be expressed at similar expression levels in the clones tested (within a 4-fold range), but Bmx was expressed relatively strongly by DT40 cells (Fig. 3D). The high level of Bmx expression was a feature of all clones analyzed. In contrast, Txk was generally less well expressed, and clone 3H11 had the greatest level of expression among 15 clones analyzed (data not shown). The data shown in the following sections were generated using the clones shown in Fig. 3 but were representative of at least two clones analyzed in each assay.
Activation of Tec Kinases after Antigen Receptor Cross-linking-- Btk is transiently activated after BCR engagement, as measured by anti-phosphotyrosine blotting and in vitro kinase activity toward an exogenous substrate (33). Btk activation is a multistep process involving transphosphorylation of Btk by Lyn and subsequent Btk autophosphorylation (7, 8, 10, 11). Experiments using DT40 cells have generated data that is consistent with this mechanism (9).
Here we show that Bmx, in addition to Btk, Itk, and Tec, were rapidly
but transiently activated in DT40 cells after BCR cross-linking, as
measured by anti-phosphotyrosine blotting (Fig.
4A). Interestingly, some
differences in the kinetics of activation of each kinase were apparent
after phosphorimaging quantitation of the data (Fig. 4B). In
particular, Btk and Tec phosphorylation was more sustained than that
for Bmx and Itk. The pattern of whole cell lysate protein phosphorylation in these experiments was not affected by the presence of Btk, Bmx, Itk, or Tec (data not shown). This agrees with earlier data showing that Btk has no detectable affect on whole cell protein tyrosine phosphorylation, for which Lyn and Syk are largely responsible (16). Taken together, these results suggest that Tec kinases may be
specific for a relatively narrow range of substrates. Txk, which lacks
the PH domain common to the other Tec PTKs, was not inducibly
phosphorylated through the BCR and had a relatively high basal level of
phosphorylation on tyrosine (Fig. 4, A-B and data not
shown). Whole cell protein tyrosine phosphorylation showed a similar
pattern to WT DT40 cells (data not shown). These data suggest that Bmx
can be activated in a similar manner to Btk, Itk, and Tec but that Txk
may be regulated differently.
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PI 3-kinase activity plays a key role in Btk activation by generating PIP3, the membrane ligand for the Btk PH domain (12-15). Recently, antigen receptor-induced Itk and Tec activation were shown to be PI 3-kinase-dependent (15). To test whether Bmx and Txk are similarly dependent on PI 3-kinase, we analyzed their BCR-induced phosphorylation in the presence of PI 3-kinase inhibitors. In Fig. 4C, cells were stimulated through the BCR for a time period designed to give optimal phosphorylation of each Tec family member in the presence or absence of the PI 3-kinase inhibitor LY294002. Activation of individual kinases was measured by anti-phosphotyrosine blotting, and the data was quantified by phosphorimaging analyses (Fig. 4D). The BCR-induced tyrosine phosphorylation of Bmx, Btk, Itk, and Tec was reduced in the presence of LY294002, suggesting that the activation of Bmx (in common with Btk, Itk, and Tec) is partially dependent on PI 3-kinase activity. Differences in the extent of inhibition among Tec family members may reflect differences in PH domain affinities for PIP3. The tyrosine phosphorylation of Txk was not reduced in the presence of LY294002. The PI 3-kinase inhibitor wortmannin yielded similar results (data not shown). These data are consistent with a key role for PIP3-PH domain interactions in Tec family activation, but not for Txk, which lacks the PH domain and may be activated by a PI 3-kinase-independent mechanism.
Reconstitution of PLC2-dependent Signaling by Bmx
and Txk--
Studies using Btk-deficient DT40 cells have identified
PLC
2 as an important substrate for Btk (16). Moreover,
reconstitution of Btk-deficient human cells has shown that Itk and Tec
can replace Btk function in regulating PLC
2 phosphorylation (23).
Here we demonstrate that Bmx and, surprisingly, Txk, can restore
phospholipase-dependent signal transduction in
Btk-deficient cells.
In Fig. 5A, PLC2 tyrosine
phosphorylation was measured over a time course after BCR
cross-linking. In WT DT40 cells, PLC
2 was maximally phosphorylated
after 1 min and retained tyrosine phosphorylation above resting levels
for up to 1 h after stimulation. This pattern of inducible
phosphorylation was markedly reduced in the absence of Btk. Expression
of all Tec family PTKs, including Txk, restored PLC
2 phosphorylation
to at least WT levels.
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We previously demonstrated that BCR-induced Btk, Bmx, Itk, and Tec, but
not Txk, phosphorylation was dependent on PI 3-kinase (Figs. 4,
C-D). Here we have extended these observations by measuring the effect of PI 3-kinase inhibitors on PLC2 phosphorylation, which
is probably a more accurate measure of Tec PTK activity (Figs. 5,
B-C). Cells were stimulated in the presence or absence of
wortmannin with anti-BCR mAb for 60 min. This late time point was
chosen to reflect Tec PTK activity in sustaining PLC
2
phosphorylation. Earlier time points reflect a relatively large
component of Syk activity in phosphorylating PLC
2 (e.g.
compare WT versus Btk
in Fig. 5A).
We found that PLC
2 phosphorylation was partially inhibited by
wortmannin in cells expressing Btk, Bmx, Itk, and Tec but not Txk.
Similar results were obtained with the PI 3-kinase inhibitor LY294002
(data not shown). An actual enhancement of PLC
2 phosphorylation in
the presence of wortmannin was observed in Txk-expressing cells in
Figs. 5, B-C, but this was not seen consistently. These
data suggest that Txk is unique among Tec PTKs in not requiring PI
3-kinase activity for its normal function.
In addition to defective PLC2 phosphorylation, Btk-deficient cells
are impaired in BCR-induced calcium mobilization (16, 23). This
function of Btk in calcium signaling could be restored by Itk and Tec
(23). Here we have tested whether Bmx and Txk can reconstitute calcium
flux. In Fig. 6, DT40 intracellular
calcium flux was measured over an 8-min period, with the addition of
anti-BCR mAb after 1 min. WT DT40 cells showed an immediate rapid
increase in intracellular calcium concentration after BCR engagement,
which was sustained above basal levels over the course of the
experiment. The initial peak of calcium was reduced in Btk-deficient
cells, and no sustained elevation of calcium was observed. Consistent with the PLC
2 data (Fig. 5A), all Tec family PTKs
restored both the initial peak and later sustained phase of the calcium
flux to WT levels (Fig. 6). No BCR-induced calcium flux was observed in
PLC
2-deficient DT40 cells, as previously shown (24). Taken together,
these data demonstrate that all Tec family PTKs can replace Btk in B
cells and regulate PLC
2-dependent calcium
mobilization.
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A Role for Tec Kinases in Antigen Receptor-induced ERK MAPK
Activation--
Activation of the ERK MAPK pathway after BCR
cross-linking was recently shown to be impaired in Btk-deficient DT40
cells (34). Moreover, full ERK activation appears to be dependent on
protein kinase C, which is itself activated downstream of PLC2 (35). Here we have tested Tec PTKs for their capacity to reconstitute the ERK
MAPK signaling pathway in Btk-deficient DT40 cells (Fig. 7).
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Cells were stimulated with anti-BCR mAb, and cells were lysed over a
2-h time course. ERK1 and ERK2 MAPK activity was measured by immune
complex kinase assay using myelin basic protein as an exogenous
substrate (Fig. 7). WT DT40 cells exhibited sustained ERK MAPK
activation, whereas in the absence of Btk, ERK activation was transient
and delayed. The kinetics of ERK activation in Btk- and
PLC2-deficient cells were strikingly similar (Fig. 7). These data
are in agreement with recent studies (34, 35) and suggest that the
rapid and sustained activation of ERK is dependent on Btk and
PLC
2.
Bmx, Itk, Tec, and Txk were different in their capacities to fully restore ERK MAPK activation. Btk- and Bmx-expressing DT40 cells showed sustained BCR-induced ERK activation. The other Tec family expressing cells exhibited a more transient ERK activation. Nevertheless, ERK activity was sustained above basal levels throughout the 2-h time course in each case (Fig. 7). The data suggest that Tec kinases, including Txk, can play an important role in the rapid and sustained activation of ERK MAPK in antigen receptor signaling.
The Role of Tec Kinases in Antigen Receptor-induced
Apoptosis--
DT40 cells are programmed to die by apoptosis after BCR
engagement (17). This apoptotic response was impaired in Btk-deficient cells but was restored by re-expression of Btk (17). We therefore examined the capacity of other Tec family kinases to restore
BCR-induced apoptosis in DT40 cells (Fig.
8). Cells were treated with anti-BCR mAb,
and the extent of apoptosis was measured after 24 h by the TUNEL
method. TUNEL provides a quantitative measure of the DNA fragmentation,
which is a hallmark of apoptosis. In Btk-deficient cells, apoptosis was
barely detectable after BCR cross-linking (Fig. 8). In contrast, a
relatively high proportion of WT DT40 cells were apoptotic. Apoptosis
was efficiently restored in Btk-deficient cells by expression of Btk,
Bmx, and Itk. Reconstitution by Tec was relatively poor, but apoptosis
at levels above those of Btk-deficient cells was seen consistently in
several Tec-expressing clones (Fig. 8 and data not shown). In contrast,
Txk failed entirely to reconstitute BCR-induced apoptosis (Fig. 8).
Interestingly, PH domain mutants of Btk also failed to restore
BCR-induced apoptosis (data not shown), implying a role for the PH
domain in connecting Tec kinases to apoptosis induction. The effect of
antigen receptor engagement can be mimicked by phorbol 12-myristate
13-acetate and ionomycin, which can activate protein kinase C and
induce calcium influx, respectively. In Fig. 8, we show that all of the
DT40 cell lines tested apoptose to a similar extent when treated with
phorbol 12-myristate 13-acetate and ionomycin. These data suggest that Txk cannot restore the apoptotic pathway in DT40 cells despite effective reconstitution of PLC2/calcium/ERK MAPK signaling.
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DISCUSSION |
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Agammaglobulinemia patients, the xid mouse, and the Btk
knockout mouse show that Btk plays a nonredundant role in B cell
function and development (reviewed in Ref. 3). Analyses of
Btk-deficient B cell lines have revealed a role for Btk in BCR-induced
PLC2 phosphorylation and sustained calcium mobilization (16, 23). Reconstitution of human agammaglobulinemia transformed B cell lines
with Itk and Tec have shown these PTKs to be analogous in function to
Btk in PLC
2 signaling (23). Here we have focused on the atypical Tec
family PTKs, Bmx and Txk, whose roles are unclear. Bmx fully restored
signaling in Btk-deficient chicken DT40 cells, in common with Btk, Itk,
and Tec. Txk could also reconstitute PLC
2 signaling despite a
different mechanism of activation and a failure to restore downstream
apoptosis, suggesting an essential role for the PH domain and/or
membrane association in the apoptosis response.
Txk is predominantly expressed in T cells and mast cells (36, 37). The
mechanism of activation and functions of Txk are unknown. Txk shares
50-60% sequence identity with other Tec kinases but is atypical in
that it lacks a PH domain (Fig. 1). Our data suggest that Txk, in
common with other Tec kinases, can function in antigen receptor signal
transduction and that PLC2 is a downstream target. In Btk-deficient
cells, Txk effectively restored BCR-induced calcium mobilization and
partially restored ERK MAPK activation, which are both downstream of
PLC
2 activation in DT40 cells (35). We found that expression levels
of Txk and other Tec kinases correlated with the extent of functional
reconstitution (Fig. 2 and data not shown). This has been noted
previously (15, 23, 32) and implies a role for Tec kinases as
modulators or amplifiers of phospholipase dependent signaling.
Functional differences were observed between Txk and the other Tec
PTKs. First, no induction of Txk tyrosine phosphorylation, which is a
good measure of activation for other Tec family PTKs (25, 38-40), was
detected after BCR cross-linking (Fig. 4, A-B) despite the
apparent induction of Txk activity as measured by PLC2
phosphorylation (Fig. 5A). Second, Txk phosphorylation (Fig. 4C), PLC
2 phosphorylation (Fig. 5B), and
calcium mobilization (data not shown) were not dependent on PI 3-kinase
activity in Txk-expressing cells. Third, Txk could not reconstitute
apoptosis (Fig. 8), the functional response of DT40 cells to BCR signaling.
Interestingly, Txk can be palmitoylated and may be constitutively
membrane-associated (47). This would
potentially position Txk in close proximity to activating Src family
PTKs (47). This provides an explanation
for its relatively high basal phosphorylation state, which could mask
subtle changes in tyrosine phosphorylation that may occur upon Txk
activation. The PI 3-kinase-independent activity of Txk is not
surprising given the absence of a PH domain, and this has important
implications for T cell receptor signaling. PLC1 activation and
sustained calcium mobilization in T cells are likely to be modulated by
two Tec family PTKs, Itk and Txk. Itk function requires the activity of
PI 3-kinase, which is recruited by the costimulatory molecule CD28
(38). In contrast, Txk function does not require PI 3-kinase and may
function irrespective of CD28 costimulation. Such a model is consistent
with reports that Txk expression levels in T cells are regulated, being
down-regulated upon T cell activation (37) and preferentially expressed
in Th1 relative to Th2 T cell clones (36), perhaps suggesting a role in
the differential regulation of cytokine expression.
Txk failed to restore BCR-induced apoptosis in Btk-deficient DT40 cells
despite reconstituting PLC2 signaling and at least partially
reconstituting ERK MAPK activation. ERK MAPK plays a key role in
transducing signals from the cytoplasm to the nucleus (41). ERK
reconstitution by Txk and other Tec kinases was not surprising because
sustained ERK activation in DT40 cells requires a Btk/PLC
2/protein
kinase C signaling pathway (34, 35). This is consistent with our
kinetic analyses of ERK activation that show strikingly similar
profiles between Btk- and PLC
2-deficient cells (Fig. 7). In contrast
to ERK activation, the mechanism of BCR-induced DT40 cell apoptosis is
not known, but correlative data suggests that c-Jun
NH2-terminal kinase 1 (JNK1) activation may be required
(34). It is possible that Txk cannot activate this signaling pathway,
whereas other Tec family kinases can, perhaps by virtue of interactions
mediated by the PH domain. For example, the PH domain of Btk has been
shown to bind not only to PIP3 (12) but also to the
subunits of
heterotrimeric G proteins (42, 43) and to BAP-135 (44), a protein of
unknown function.
In addition to BCR-induced apoptosis, Btk was reported to play a
PLC2-independent role in radiation-induced apoptosis of DT40 cells
(17). We have been unable to repeat these data. Instead we find no
difference between WT and Btk-deficient DT40 cells in their apoptotic
responses to ionizing radiation (data not shown).
In common with Txk, Bmx is an atypical Tec family PTK but for different reasons. Bmx has the same domain organization as Btk, Itk, and Tec (Fig. 1), but the proline-rich Tec homology and SH3 domains are poorly conserved (39, 45). These domains are proposed to interact intramolecularly and function in negative regulation of Tec PTKs (46). It is possible that these domains in Bmx have co-evolved divergently from other Tec PTKs yet perform the same negative regulatory function. Our data is consistent with this hypothesis, because Bmx is activated in a PI 3-kinase-dependent manner in common with Btk, Itk, and Tec and fully reconstitutes signaling in Btk-deficient B cells.
We have shown that Bmx is able to regulate PLC2 activity in antigen
receptor signaling, but this is unlikely to be an in vivo
function because Bmx is not found in lymphocytes and is predominantly expressed in epithelial, endothelial, and granulomonocytic cells (39,
45). Our data suggest that Bmx may be activated in these cells by
stimuli that induce both Src family and PI 3-kinase activity. Bmx may
function in concert with receptor tyrosine kinases to regulate PLC
activity, calcium mobilization, and ERK activity. This idea is
supported by the observation that in prostate cancer cells, Bmx is
activated by interleukin 6 (IL-6) in a PI
3-kinase-dependent manner and functions in neuroendocrine
differentiation (39).
Reconstitution of Btk-deficient human cells demonstrated a role for Itk
and Tec in PLC2 activation and sustained calcium signaling (23). We
have performed a similar study in Btk-deficient DT40 chicken B cells.
We have confirmed the findings of Fluckiger et al. (23) and
have extended our study to show that the atypical Tec family PTKs, Bmx
and Txk, can also function in phospholipase-dependent signaling. We further show that Tec family PTKs can play a role in
sustained ERK MAPK activation. Interestingly, Txk is unique among Tec
PTKs as it functions independently of PI 3-kinase and fails to restore
apoptosis, the biological response of DT40 cells to antigen receptor
signaling. These data indicate that Tec PTKs provide at least two
different signals, namely to the PLC
2/ERK MAPK pathway that is not
sufficient for apoptosis and to a PH domain-dependent
pathway that is necessary for apoptosis.
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ACKNOWLEDGEMENTS |
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We thank Victoria Heath, Ronald Herbst, Alyssa Morimoto, Dave Parry, and Sarah Pogue for critically reviewing the manuscript and Jeng-Horng Her, Steve Miller, Alice Mui, and Pamela Schwartzberg for their helpful advice and comments. We are grateful to Debbie Liggett for oligonucleotide synthesis, to Eleni Callas and Jim Cupp for help with calcium flux analyses, and to the labs of Dan Gorman and Terri McClanahan for sequencing and cDNA libraries, respectively.
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FOOTNOTES |
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* DNAX Research Institute is supported by Schering-Plough.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.
§ To whom all correspondence should be addressed. Tel.: 650-858-7503; Fax: 650-496-1200; E-mail: tomlinson{at}dnax.org.
Supported by a grant from the Ministry of Education, Science,
Sports, and Culture of Japan.
** Current address: Hoechst Marion Roussel, Inc., Bridgewater, NJ 08807.
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ABBREVIATIONS |
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The abbreviations used are:
Btk, Bruton's
tyrosine kinase;
PTK, protein-tyrosine kinase;
PH, Pleckstrin homology;
SH3, Src-homology 3;
BCR, B cell antigen receptor;
PI 3-kinase, phosphatidylinositol 3-kinase;
PIP3, phosphatidylinositol
3,4,5-trisphosphate;
PLC, phospholipase C-2;
ERK, extracellular
signal-regulated kinase;
MAPK, mitogen-activated protein kinase;
mAb, monoclonal antibody;
HA, hemagglutinin;
TUNEL, terminal
deoxynucleotidyltransferase-mediated dUTP nick end-labeling;
WT, wild
type;
FACS, fluorescence-activated cell sorter.
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