By
From the * Department of Pathology, University of Massachusetts Medical Center, Worcester,
Massachusetts 01655; the Department of Molecular and Cellular Biology, Harvard University,
Cambridge, Massachusetts 02138; and the § Program of Immunology, Division of Medical Sciences,
Harvard University, Boston, Massachusetts 02115
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Itk, a Tec family tyrosine kinase, plays an important but as yet undefined role in T cell receptor
(TCR) signaling. Here we show that T cells from Itk-deficient mice have a TCR-proximal signaling defect, resulting in defective interleukin 2 secretion. Upon TCR stimulation, Itk/
T cells release normal amounts of calcium from intracellular stores, but fail to open plasma
membrane calcium channels. Since thapsigargin-induced store depletion triggers normal calcium entry in Itk
/
T cells, an impaired biochemical link between store depletion and channel opening is unlikely to be responsible for this defect. Biochemical studies indicate that
TCR-induced inositol 1,4,5 tris-phosphate (IP3) generation and phospholipase C
1 tyrosine
phosphorylation are substantially reduced in Itk
/
T cells. In contrast, TCR-
and ZAP-70
are phosphorylated normally, suggesting that Itk functions downstream of, or in parallel to, ZAP-70 to facilitate TCR-induced IP3 production. These findings support a model in which
quantitative differences in cytosolic IP3 trigger distinct responses, and in which only high concentrations of IP3 trigger the influx of extracellular calcium.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tcell activation is a complex process involving a number of independent signaling pathways. Of these, the best understood is the signaling downstream of the TCR (for review see reference 1). Biochemical as well as genetic studies have identified many of the important signaling molecules activated by TCR stimulation. For instance, the tyrosine kinases ZAP-70 and Lck are known to play a critical role during T cell activation. Evidence also supports a role for the Ras/Map kinase cascade, as well as the phospholipid modifying enzyme, phosphatidyl inositol-3 kinase (PI3K). To date, the precise role of each of these signaling branches, as well as the mechanism(s) that integrate these diverse signals, has not been completely elucidated. However, it has long been known that pharmacological agents such as phorbol esters plus calcium ionophores can bypass T cell surface receptors and directly activate resting T cells (2). This phenomenon clearly pinpoints the critical role of protein kinase C and calcium signaling in T cell activation.
The Tec family tyrosine kinases have been implicated in
antigen receptor signaling in a variety of hematopoietic cell
types. Itk, expressed in T cells and natural killer cells (3, 4), is tyrosine phosphorylated in response to cross-linking of
the TCR (5, 6), CD28 (7), or CD2 (8). A closely related family member, Btk, is expressed in B cells and mast cells;
immunodeficiency diseases in humans (XLA; references 9)
and mice (xid; references 12, 13) result from mutations in
the Btk gene, and are characterized by defects in B cell receptor (BCR) signaling (14). Furthermore, in Btk-deficient chicken DT40 B cells, the antigen receptor-induced tyrosine phosphorylation of the phospholipase C (PLC)1
isoform PLC-2 is substantially reduced, resulting in the
loss both of BCR-coupled phosphatidylinositol hydrolysis
and of calcium mobilization (18). Our studies suggested
that Itk might play an analogous role in TCR signaling in
T cells.
To test this possibility, we generated a line of Itk-deficient mice. Previous studies have shown that Itk is important for normal T cell development in the thymus and for
normal T cell activation (19). However, the precise role of
Itk in TCR signal transduction has not yet been described.
Our studies have extended these previous observations by
demonstrating biochemical defects in Itk/
T cells. We
show that IL-2 secretion is dramatically reduced after TCR
stimulation of Itk
/
T cells, and that this defect results, at
least in part, from impaired calcium signaling. We also
show that the failure to generate a sustained calcium flux in
Itk
/
T cells is correlated with a dramatic reduction in
TCR-induced inositol 1,4,5 tris-phosphate (IP3) generation and with reduced tyrosine phosphorylation of PLC-
1. In contrast, TCR-induced tyrosine phosphorylation of
ZAP-70 and the TCR-
chain are unaffected. These data
indicate that Itk is downstream of or parallel to ZAP-70, and, furthermore, that Itk is an important component of the
signaling pathway leading to the sustained elevation of cytoplasmic free calcium necessary for the activation of transcription factors such as c-rel and the nuclear factor of activated T cells (NFAT).
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of Itk-deficient Mice.
A murine strain 129 genomic clone encoding the Itk kinase domain (exons 9, 10, 11, 12, and 13 [see Fig. 1]) was isolated. A 2.6-kb HindIII fragment extending from the middle of exon 10 to beyond exon 12 was replaced by the 1.8-kb pGK-neo cassette; these deleted sequences encode a portion of the Itk kinase domain (amino acids 476-543). The targeting vector also contained 4.8 kb and 4.2 kb of flanking sequences at the 5' and 3' ends, respectively, and a 2.0-kb thymidine kinase cassette (pGK-tk). The construct was linearized and transfected into J1 (gift of Dr. A. Sharpe, Harvard Medical School, Boston, MA) and CCE (gift of Dr. E. Robertson, Harvard University, Cambridge, MA) embryonic stem (ES) cell lines. 360 clones resistant to Geneticin (Sigma Chemical Co., St. Louis, MO) and FIAU [1-(2'-deoxy-2'-fluro-
|
Cell Purification.
For CD4+ T cell purification, lymph node (inguinal, axillary, brachial, mandibular, and mesenteric) cells from multiple wild-type or Itk-deficient mice were isolated and were purified on columns (Biotex Laboratories, Alberta, Canada) according to the manufacturer's protocol. The resulting cells were 90-97% pure (Itk+/+ or Itk+/Antibodies and Flow Cytometry Analysis.
Before antibody staining 106 lymph node cells were plated in wells precoated with 5 µg/ ml goat anti-hamster IgG(H+L) followed by 5 µg/ml hamster anti-mouse CD3TCR Stimulation and IL-2 Assays.
For T cell stimulations, 5 × 104 purified CD4+ T cells were plated in wells precoated with goat anti-hamster antibody (5 µg/ml) followed by hamster anti- mouse CD3Calcium Flux.
5 × 106/ml purified CD4+ cells were incubated with 3 µg/ml fluo-3, 5 µg/ml Fura-Red, 0.5% pluronic acid (Molecular Probes, Eugene, OR), and 2% FCS in RPMI for 30-50 min. Dye-loaded cells were washed twice with RPMI and left at room temperature for 30 min in the dark before use. 1.5 × 106 (in 300 µl) dye-loaded cells were placed in 1 ml of serum-free RPMI and warmed to 37°C. Stimulated cells were analyzed on a FACScan®. The mean fluorescence ratio (FL-1:FL-3) was calculated using the FACSAssistant® program.Immunoprecipitation and Western Blotting.
Purified CD4+ T cells were incubated in serum-free RPMI for 2 h at 37°C before stimulation. Cells were then washed twice, resuspended in 120 µl of RPMI containing 25 µg/ml biotinylated anti-CD3Analysis of IP3 Release.
8 × 106 purified CD4+ T cells were starved in serum-free RPMI for 1 h at 37°C before stimulation. Cells were stimulated as for immunoprecipitation studies, but at 3.2 × 107/ml; signaling was terminated by adding 50 µl ice-cold 100% Trichloroacetic acid. IP3 levels were assessed using the IP3 Radioreceptor Assay Kit (NEN Life Science Products, Boston, MA) following the manufacturer's protocol. Controls included medium alone or mock-stimulated (no primary antibody) cells. ![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
To determine the function of Itk in T cell signaling, we
generated Itk-deficient mice by gene targeting. Exons of
the Itk gene encoding critical regions of the kinase domain
were deleted, and replaced with the neoR cassette (Fig. 1 a).
Mice homozygous for the mutated Itk allele (Itk/
; see
Fig. 1 b) fail to express any Itk protein in lymph nodes, spleen, or thymus (Fig. 1 c). Consistent with a previous report on Itk-deficient mice (19), T cell development is abnormal in the absence of the Itk protein. Specifically, the
size and general histology of the thymus are normal, but
CD4+ T cell maturation is diminished in Itk
/
mice. The
number of CD4+ T cells in peripheral lymphoid organs is
reduced to ~50% of that found in wild-type mice; in contrast, the number of CD8+ T cells is unchanged (data not
shown).
To investigate the role of Itk in TCR signaling, we examined the responses of Itk/
CD4+ T cells to TCR
stimulation. First, Itk
/
lymph node cells were stimulated
with plate-bound anti-CD3 antibody, and cells were assessed 18-20 h later for changes in surface marker expression. CD25 and CD69 were upregulated and CD62L (L-selectin) was downregulated after TCR stimulation on
both mutant and wild-type cells (Fig. 2 a). The only difference we consistently observed was a somewhat higher degree of CD25 upregulation on Itk+/+ cells compared with
Itk
/
cells; this difference is most likely due to the IL-2-
induced increase in IL-2 receptor
chain (CD25) expression, which is lacking in Itk
/
T cells (see below). These
data indicate that the TCR-dependent signaling pathways
leading to changes in activation marker and adhesion molecule expression are intact in Itk
/
T cells. Second, we examined IL-2 secretion in response to TCR stimulation.
When purified CD4+ Itk
/
T cells were stimulated with
anti-CD3 antibody, no IL-2 could be detected in the supernatants after overnight culture (Fig. 2 b). Stimulation
with anti-CD3 plus anti-CD28 antibodies, which dramatically enhances IL-2 production from wild-type cells, resulted in detectable, but still reduced, IL-2 secretion from
Itk
/
T cells. This signaling defect is proximal to the
TCR, as stimulation with PMA plus ionomycin, which
bypasses the early stages of TCR signaling, elicits comparable IL-2 secretion by Itk
/
and wild-type T cells.
|
The functional defect in IL-2 production after stimulation of Itk/
T cells suggested a defect in the TCR signaling pathway leading to IL-2 transcription. Since IL-2 promoter activity is highly dependent on TCR-induced
sustained increases in cytoplasmic free calcium (23), we
examined calcium responses after TCR stimulation of
CD4+ T cells. In wild-type cells, TCR cross-linking elicits
the expected large, transient peak of increased calcium in
the cytoplasm, followed by a sustained elevation in intracellular calcium (Fig. 3 a). In contrast, Itk
/
T cells have a
substantially reduced initial peak of elevated cytoplasmic
calcium and do not sustain elevated calcium levels. This response requires high concentrations of the stimulating anti-CD3
antibody (10 µg/ml); when lower concentrations of
antibody are used, wild-type T cells display a slower and
more flattened elevation in intracellular calcium, whereas
Itk
/
T cells fail to respond altogether (data not shown).
These differences are not due to defective dye loading in
the mutant cells, as ionomycin induces equivalent large increases in cytoplasmic calcium in both cell types (Fig. 3 a).
Although the defective calcium influx observed with high
concentrations of stimulating antibody is novel, the more
severe defect observed with lower concentrations of stimulating antibody parallels the defect observed upon surface
immunoglobulin cross-linking of Btk
/
chicken B cells
(18). It is not clear if the partial defect observed at high
concentrations of stimulating antibody would also be observed in the B cell system.
|
In normal cells, the overall pattern of calcium elevation
results from two components of the calcium response. The
initial transient peak is due to the release of calcium from
intracellular stores (26); the resulting depletion of these
stores triggers the opening of the plasma membrane calcium channels, generating a sustained elevation in intracellular calcium (27). To determine whether Itk/
T cells are
defective in one or both of these components of the calcium response, we chelated the extracellular calcium with
EGTA, eliminating the influx of calcium and thereby revealing the release of calcium from intracellular stores. In
the presence of EGTA, the transient calcium increase induced by TCR cross-linking is similar in both wild-type
and mutant cells (Fig. 3 b), suggesting that the intracellular
calcium stores are released (and therefore depleted) to a
similar extent in both cell types. Nonetheless, when extracellular Ca2+ is restored, allowing the influx of calcium
across the plasma membrane, wild-type cells sustain a substantially higher level of intracellular calcium than do mutant cells. These data indicate that the signaling pathways
opening plasma membrane calcium channels in response to
store depletion are defective in Itk
/
T cells, resulting in
the reduced level of calcium elevation after TCR stimulation. Since sustained calcium elevation is required for the
sustained nuclear translocation of nuclear factor of activated T cells (NFAT) and for IL-2 transcription (23), the failure of Itk
/
cells to sustain intracellular calcium levels is
likely to be a direct cause of the reduced IL-2 production
observed after TCR stimulation.
To determine whether the depletion of intracellular calcium stores is competent to induce the opening of plasma
membrane calcium channels, wild-type and mutant T cells
were treated with the drug thapsigargin. Thapsigargin, a
specific inhibitor of the Ca2+-ATPase in the endoplasmic
reticulum membrane, prevents the uptake of cytosolic calcium and results in a leak that depletes intracellular calcium
stores and triggers calcium entry (28, 29). In contrast to
anti-CD3 antibody cross-linking, thapsigargin treatment
initiates a substantial calcium influx in both wild-type and
mutant T cells, demonstrating that store-operated calcium
entry is normal in Itk/
T cells (Fig. 3 c). It is possible that
distinct depletion-sensitive signaling pathways are induced
depending on the mechanism of store depletion; however,
we favor a model in which the channel-operating calcium
stores are depleted to a lesser extent in the Itk
/
cells. This
latter hypothesis is consistent with the reduced production
of IP3 observed after TCR stimulation of Itk
/
T cells
(see below).
In the presence of extracellular EGTA, thapsigargin induces similar levels of calcium elevation in mutant and
wild-type T cell (Fig. 3 d), suggesting that the sizes of the
thapsigargin-sensitive intracellular calcium stores are similar
in both cell types. Notably, the thapsigargin-stimulated
calcium release observed in these naive T cells is unexpectedly small, yet capable of stimulating calcium influx. In
light of this result, it is surprising that the large calcium release observed upon TCR stimulation in Itk-deficient T
cells fails to trigger calcium influx. This apparent contradiction could be explained in at least three ways. First, the extent of calcium elevation in the presence of thapsigargin
may be a poor measure of store depletion, as rates of store
refilling may differ between wild-type and Itk/
T cells.
Second the similar extent of store release observed between
wild-type and Itk
/
cells may underestimate the actual
difference in total calcium released if the rate at which calcium is cleared from the cyotosol increases as the concentration of cytosolic calcium increases; this phenomenon has
been documented previously in T cells (30). Third, it is
possible that the thapsigargin-sensitive intracellular stores
are a distinct and small subset of the TCR-coupled intracellular calcium stores, and are uniquely competent to trigger calcium influx in response to depletion. In this latter
case, Itk would be required to couple the TCR to these
specific channel-operating calcium stores.
It should be noted that the amplitude of the thapsigargin-induced calcium elevation is always slightly diminished
in Itk-deficient T cells (Fig. 3 c). When purified cell populations are gated to exclude contaminating CD4 cells, the
same difference is observed (data not shown); therefore, this difference is not due to disparities in the purities of the CD4+ T cell preparations from mutant and wild-type
lymph nodes. The absence of Itk could affect peak intracellular calcium levels by multiple mechanisms, potentially reducing the frequency of calcium channel opening, decreasing the electrochemical gradient driving calcium entry, or
by facilitating the upregulation of the plasma membrane
calcium pump. At present we do not know which, if any,
of these mechanisms result in the reduction in maximum intracellular calcium levels induced by thapsigargin.
TCR-induced calcium responses in T cells are initiated
by the second messenger, IP3. IP3 is generated by the
PLC-mediated hydrolysis of the membrane phospholipid,
phosphatidylinositol 4,5-bisphosphate (PIP2). After PIP2
cleavage, IP3 diffuses through the cytoplasm and binds to
IP3 receptors, inducing the release of calcium from internal
stores (for review see reference 31). In addition, IP3 is
thought to play a role in the subsequent calcium signal that
causes the opening of the plasma membrane calcium channels (32). Therefore, we assessed the levels of IP3 generated after TCR stimulation of wild-type and Itk/
T cells (Fig.
4 a). In wild-type cells, IP3 levels begin to rise by 45 s after
TCR stimulation, remain at peak levels between 1 and 2 min, and return to basal levels by 5 min (Fig. 4 a and data not shown). In contrast, no significant increase in IP3 levels over background can be detected in Itk
/
T cells at any
time point (Fig. 4 and data not shown). Parekh et al. have
demonstrated that calcium release can be triggered by IP3
concentrations 50-fold below those required to trigger calcium influx (32). The minimal calcium-releasing doses of IP3 identified by Parekh are at the limit of detection of our assay system, and it is likely that TCR stimulation in Itk
/
T cells actually induces small amounts of IP3 that activate
calcium release without triggering calcium influx. It is unclear why this threshold effect exists. As proposed by
Parekh et al., it could result either from a low affinity IP3-receptor present on a specialized channel-coupled calcium
store, or from the need for IP3 concentrations sufficient to
allow diffusion to outpace the catabolism of IP3 (32).
|
The PLC isoform PLC-1 is abundant in T cells and tyrosine phosphorylated after TCR stimulation. Since this
PLC isoform is activated by tyrosine phosphorylation (33),
we examined whether reduced tyrosine phosphorylation of
PLC-
1 could account for the defective TCR-induced IP3
production in Itk
/
T cells. As shown in Fig. 4 b, immunoblotting of PLC-
1 with an antiphosphotyrosine antibody indicated that the tyrosine phosphorylation of PLC-
1 from stimulated Itk
/
T cells is markedly reduced at
0.5 and 1 min. This reduced phosphorylation is not due to
decreased levels of PLC-
1, as demonstrated by anti-PLC-
1 immunoblotting (Fig. 4 b). In contrast, the TCR-induced
phosphorylation of the receptor-proximal signaling molecules ZAP-70 and TCR-
is similar in both wild-type and
Itk
/
T cells. Furthermore, the overall pattern of tyrosine
phosphorylation induced by TCR stimulation, with minor
exceptions, is not markedly different between Itk+/+ and
Itk
/
T cells (data not shown); in addition, some downstream functions of TCR signaling are intact (e.g., changes
in activation marker expression, see above). Since ZAP-70
activation is critical for TCR signaling (34, 35), our data
suggest that ZAP-70 activation is normal in the absence of
Itk. These data indicate that Itk is not upstream of either
TCR-
or ZAP-70 phosphorylation, but is required for
optimal PLC-
1 activation after TCR cross-linking. This
placement of Itk relative to ZAP-70 is consistent with that proposed by Kurosaki et al. for Btk and Syk (17).
Here we demonstrate that Itk/
T cells have a defect in
TCR signaling that leads to reduced IL-2 secretion after
TCR cross-linking. Although we have not ruled out additional signaling defects in Itk
/
T cells, the impaired influx of calcium across the plasma membrane is likely to be
responsible for the loss of TCR-induced IL-2 production. Since calcium influx enables the sustained increases in cytoplasmic calcium required for transcription factor activation
(23), this defect could explain the inability of Itk
/
T
cells to secrete IL-2. As calcium release from intracellular stores appears normal in the Itk
/
T cells, these data suggest that Itk might be involved in the signaling pathways
coupling store depletion to calcium influx. However, we
found this explanation unlikely because thapsigargin induces calcium influx in the absence of Itk. This finding
suggests that a functional depletion-sensing pathway can be
decoupled from calcium release. Defective coupling could
result from impaired PLC-
1 activation in Itk
/
T cells
by at least two mechanisms. The dramatically reduced production of IP3 after TCR stimulation in Itk
/
T cells
could fail to trigger a low affinity IP3 receptor on a channel-operating subset of intracellular calcium stores, while fully activating calcium release from the remaining stores.
Alternatively, the low levels of IP3 produced in the Itk
/
T cells could fail to deplete intracellular calcium stores to the extent required to trigger calcium influx. In this case,
we would be forced to conclude that the observed store release is a poor measure of actual store depletion, either because we are underestimating release in the wild-type T
cells, or because the rates at which the stores refill differ between the wild-type and Itk
/
T cells. We believe that
this is the first demonstration that the TCR-induced influx
of extracellular calcium can be uncoupled from the TCR-induced release of intracellular calcium, while preserving functional, depletion-sensitive calcium influx mechanisms.
Thus, these experiments suggest that Itk
/
T cells will provide an interesting model system for detailed studies of the
precise mechanisms regulating intracellular calcium levels.
![]() |
Footnotes |
---|
Address correspondence to Leslie J. Berg, Department of Pathology, University of Massachusetts Medical Center, 55 Lake Ave. North, Worcester, MA 01655. Phone: 508-856-8371; Fax: 508-856-8372; E-mail: leslie. berg{at}ummed.edu
Received for publication 3 March 1998.
Christine Gurniak's current address is Mouse Biology Programme/Monterotondo EMBL, European Molecular Biology Laboratory, Meyerhofstrasse, Postfach 10.2209, 69012 Heidelberg, Germany.We thank Dr. Arlene Sharpe and Dr. Elizabeth Robertson for embryonic stem cell lines and technical advice; Jennifer Noble for generating the chimeric mice; Dr. Brian Druker for providing 4G10 antibody; Dr. B. Tang for providing the anti-Tec antibody; Dr. Andrew McMahon for providing FIAU; and Dr. Daniel Thomis for helpful discussions and critical reading of the manuscript.
This work was supported by grants from the National Institutes of Health (AI-37584) and the Council for Tobacco Research-USA, Inc.
Abbreviations used in this paper ES, embryonic stem; PI3, inositol 1,4,5 tris-phosphate; PLC, phospholipase C.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Wange, R.L., and L.E. Samelson. 1996. Complex complexes: signaling at the TCR. Immunity. 5: 197-205 [Medline]. |
2. |
Weiss, A.,
R.L. Wiskocil, and
J.D. Stobo.
1984.
The role of
T3 surface molecules in the activation of human T cells: a
two-stimulus requirement for IL 2 production reflects events
occurring at a pre-translational level.
J. Immunol.
133:
123-128
|
3. | Heyeck, S.D., and L.J. Berg. 1993. Developmental regulation of a murine T cell-specific tyrosine kinase gene, Tsk. Proc. Natl. Acad. Sci. USA. 90: 669-673 [Abstract]. |
4. | Teng, J., X. Liu, G. Mills, and B. Dupont. 1996. CD28-mediated cytotoxicity by the human leukemic NK cell line YT involves tyrosine phosphorylation, activation of phosphatidylinositol 3-kinase, and protein kinase C. J. Immunol. 156: 3222-3232 [Abstract]. |
5. | Gibson, S., A. August, Y. Kawakami, T. Kawakami, B. Dupont, and G.B. Mills. 1996. The EMT/ITK/TSK (EMT) tyrosine kinase is activated during TCR signaling. J. Immunol. 156: 2716-2722 [Abstract]. |
6. |
Heyeck, S.D.,
H.M. Wilcox,
S.C. Bunnell, and
L.J. Berg.
1997.
Lck phosphorylates the activation loop tyrosine of the
Itk kinase domain and activates Itk kinase activity.
J. Biol.
Chem.
272:
25401-25408
|
7. |
Gibson, S.,
A. August,
D. Branch,
B. Dupont, and
G.B. Mills.
1996.
Functional Lck is required for optimal CD28-mediated activation of the Tec family tyrosine kinase Emt/
Itk.
J. Biol. Chem.
271:
7079-7083
|
8. | King, P.D., A. Sadra, A. Han, X.-R. Liu, R. Sunder-Plassmann, E.L. Reinherz, and B. Dupont. 1996. CD2 signaling in T cells involves tyrosine phosphorylation and activation of the Tec family kinase, Emt/Itk/Tsk. Int. Immunol. 8: 1707-1714 [Abstract]. |
9. | Conley, M., O. Parolini, J. Rohrer, and D. Campana. 1994. X-linked agammaglobulinemia: new approaches to old questions based on the identification of the defective gene. Immunol. Rev. 138: 5-21 [Medline]. |
10. | Vetrie, D., I. Vorechovsky, P. Sideras, J. Holland, A. Davies, F. Flinter, L. Hammarstrom, C. Kinnon, R. Levinsky, M. Bobrow, et al . 1993. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein tyrosine kinases. Nature. 361: 226-232 [Medline]. |
11. | Tsukada, S., D.C. Saffran, D.J. Rawlings, O. Parolini, R.C. Allen, I. Klisak, R.S. Sparkes, H. Kubagawa, T. Mohandas, S. Quan, et al . 1993. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell. 72: 279-290 [Medline]. |
12. | Rawlings, D.J., D.C. Saffran, S. Tsukada, D.A. Largaespada, J.C. Grimaldi, L. Cohen, R.N. Mohr, J.F. Bazan, M. Howard, N.G. Copeland, et al . 1993. Mutation of unique region of Bruton's tyrosine kinase in immunodeficient XID mice. Science. 261: 358-361 [Medline]. |
13. | Thomas, J.D., P. Sideras, C.I.E. Smith, I. Vorechovsky, V. Chapman, and W.E. Paul. 1993. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science. 261: 355-358 [Medline]. |
14. | Desiderio, S.. 1997. Role of Btk in B cell development and signaling. Curr. Opin. Immunol. 9: 534-540 [Medline]. |
15. |
Aoki, Y.,
K.J. Isselbacher, and
S. Pillai.
1994.
Bruton's tyrosine kinase is tyrosine phosphorylated and activated in pre-B
lymphocytes and receptor-ligated B cells.
Proc. Natl. Acad.
Sci. USA.
91:
10606-10609
|
16. | Rawlings, D.J., A.M. Scharenberg, H. Park, M.I. Wahl, S. Lin, R.M. Kato, A.-C. Fluckiger, O.N. Witte, and J.-P. Kinet. 1996. Activation of BTK by a phosphorylation mechanism initiated by Src family kinases. Science. 271: 822-825 [Abstract]. |
17. |
Kurosaki, T., and
M. Kurosaki.
1997.
Transphosphorylation
of Bruton's tyrosine kinase on tyrosine 551 is critical for B
cell antigen receptor function.
J. Biol. Chem.
272:
15595-15598
|
18. |
Takata, M., and
T. Kurosaki.
1996.
A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation
of phospholipase C-![]() |
19. | Liao, X.C., and D.R. Littman. 1995. Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk. Immunity. 3: 757-769 [Medline]. |
20. | Harding, F.A., J.G. McArthur, J.A. Gross, D.H. Raulet, and J.P. Allison. 1992. CD28-mediated signalling co-stimulates murine T cells and prevents induction of anergy in T cell clones. Nature. 356: 607-609 [Medline]. |
21. | Yelon, D., L.M. Spain, K. Lim, and L.J. Berg. 1996. Alterations in CD4 dependence accompany T cell development and differentiation. Int. Immunol. 8: 1077-1090 [Abstract]. |
22. |
Bunnell, S.C.,
P.A. Henry,
R. Kolluri,
T. Kirchhausen,
R. Rickles, and
L.J. Berg.
1996.
Identification of Itk/Tsk SH3
domain ligands.
J. Biol. Chem.
271:
25646-25656
|
23. |
Wacholtz, M., and
P. Lipsky.
1993.
Anti-CD3-stimulated
Ca2+ signal in individual human peripheral T cells. Activation correlates with a sustained increase in intracellular Ca2+.
J. Immunol.
150:
5338-5349
|
24. | Timmerman, L., N. Clipstone, S. Ho, J. Northrop, and G. Crabtree. 1996. Rapid shuttling of NF-AT in discrimination of Ca2+ signals and immunosuppression. Nature. 383: 837-840 [Medline]. |
25. | Dolmetsch, R., R. Lewis, C. Goodnow, and J. Healy. 1997. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature. 386: 855-858 [Medline]. |
26. | Berridge, M.. 1993. Inositol trisphosphate and calcium signaling. Nature. 361: 315-325 [Medline]. |
27. | Putney, J.W. Jr., and G.S. Bird. 1993. The signal for capacitative calcium entry. Cell. 75: 199-201 [Medline]. |
28. | Gouy, H., D. Cefai, S.B. Christensen, P. Debre, and G. Bismuth. 1990. Ca2+ influx in human T lymphocytes is induced independently of inositol phosphate production by mobilization of intracellular Ca2+ stores. A study with the Ca2+ endoplasmic reticulum-ATPase inhibitor thapsigargin. Eur. J. Immunol. 20: 2269-2275 [Medline]. |
29. | Thastrup, O., P.J. Cullen, B.K. Drobak, M.R. Hanley, and A.P. Dawson. 1990. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc. Natl. Acad. Sci. USA. 87: 2466-2470 [Abstract]. |
30. | Telford, W.G., and R.A. Miller. 1996. Detection of plasma membrane Ca(2+)-ATPase activity in mouse T lymphocytes by flow cytometry using fluo-3-loaded vesicles. Cytometry. 24: 243-250 [Medline]. |
31. | Berridge, M.J., and R.F. Irvine. 1989. Inositol phosphates and cell signalling. Nature. 341: 197-205 [Medline]. |
32. | Parekh, A., A. Fleig, and R. Penner. 1997. The store-operated calcium current I(CRAC): nonlinear activation by InsP3 and dissociation from calcium release. Cell. 89: 973-980 [Medline]. |
33. | Weiss, A., G. Koretzky, R. Schatzman, and T. Kadlecek. 1991. Functional activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-gamma 1. Proc. Natl. Acad. Sci. USA. 88: 5484-5488 [Abstract]. |
34. | Chan, A.C., T.A. Kadlecek, M.E. Elder, A.H. Filipovich, W.-L. Kuo, M. Iwashima, T.G. Parslow, and A. Weiss. 1994. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science. 264: 1599-1601 [Medline]. |
35. | Elder, M.E., D. Lin, J. Clever, A.C. Chan, T.J. Hope, A. Weiss, and T.G. Parslow. 1994. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science. 264: 1596-1599 [Medline]. |