| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
University of Massachusetts Medical School Department of Pathology, 55 Lake Avenue North, Worcester MA 01655; and
Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, CA 92121
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. These signals
eventually lead to the production of second messengers, such as
diacylglycerol and inositol trisphosphate, and to the activation of
small GTP-binding proteins, such p21ras
(1, 2). The ultimate outcome of TCR signaling is the
activation of transcription factors leading to new gene expression. In
primary resting T cells, a hallmark of activation is the induction of
the IL-2 gene (3). The Tec family of tyrosine kinases is now the second largest subgroup of cytoplasmic protein tyrosine kinases, consisting of Tec, Btk/Bpk/Atk/Emb, Itk/Tsk/Emt, Txk/Rlk, and Bmx/Etk in mammalian cells (4, 5). These kinases share a similar overall structure. At their amino terminus, all members of this family except Txk contain a pleckstrin homology (PH) domain; for Btk, this domain has been shown to bind inositol phosphates in vitro (6, 7, 8) and to be responsible for membrane localization in vivo (9). Tec kinases also share a Tec homology domain containing a region homologous to the GTPase-activating protein 1 family of Ras GTPases at its amino-terminal end and one or two proline-rich motifs at its carboxyl-terminal end (10). In addition, all Tec kinases have a single Src homology (SH) 3 domain, a single SH2 domain, and a kinase domain.
Only three of the Tec kinases are known to be expressed in T cells. Two
of them, Itk and Txk, are predominantly expressed in T cells, whereas
Tec is more broadly distributed among all hemopoietic cell types,
including T cells (11, 12, 13). Despite many biochemical
studies linking Tec kinases with numerous cell surface receptors
(14), the precise role(s) of Tec kinases in T cell signal
transduction are not well understood. Gene-targeting studies first
demonstrated that Itk plays a role in TCR signaling leading to
IL-2 gene induction, and indicated a role for Itk in the
activation of PLC-1 leading to calcium mobilization (15, 16). More recent studies have suggested that Txk may have a
similar function to Itk in T cells (17, 18). Additional
studies have also suggested a specific role for Itk in the
differentiation and function of Th2 effector cells (19)
and for Txk in Th1 effector cells (20). Although Tec has
been implicated in cytokine receptor signaling in nonlymphoid cells
(21), little is known about the role of Tec in T cells.
The data that has been generated, implicating Tec in both TCR and CD28
signaling, has predominantly relied on transient transfections and
overexpression systems (13, 22).
The Tec family of tyrosine kinases is the only known family of kinases
containing a PH domain. Numerous functions of this domain have been
proposed, involving binding to both protein and phospholipid ligands.
For instance, the PH domain of Btk has been shown to interact with
protein kinase C (23) and actin (24), that of
Bmx binds to the protein tyrosine phosphatase D1 (25), and
both Itk and Btk PH domains bind to the 
subunits of
heterotrimeric G proteins (26, 27). In addition, the Itk
and Btk PH domains have been shown to preferentially interact with
inositol compounds phosphorylated at the D-3 position, such as
phosphatidylinositol 3,4,5 trisphosphate
(PtdInsP3) and inositol 1,3,4,5 tetrakisphosphate
(InsP4) (6, 7, 8, 9, 28, 29).
In particular, the phospholipid interactions of the PH domain are thought to play an important role in recruiting Tec family kinases to the plasma membrane, thus facilitating their proximity to upstream activators (e.g., Src family kinases) and downstream substrates (5). Supporting the importance of the PH domain in Tec family kinase function, point mutations in the Btk PH domain lead to immunodeficiency diseases in both mouse and human (xid and X-linked agammaglobulinemia, respectively; Refs. 30, 31). In particular, substitution of the conserved arginine at residue 28 of the Btk PH domain, resulting in reduced PH domain binding to phospholipids, leads to a loss of Btk function in B cells (8, 28, 32). Unexpectedly, a gain-of-function Btk mutant with transforming activity in fibroblast cells also results from a point mutation in the Btk PH domain (E41K); interestingly, this substitution leads to increased binding of the PH domain to phosphoinositides, and to increased Btk kinase activity in NIH 3T3 cells (9). In contrast to these data on the Btk PH domain, a point mutation in the Bmx PH domain (E42K, comparable to E41K in Btk) has been shown to result in decreased Bmx kinase activity in vivo (33). These results suggest that the PH domains of individual Tec kinase family members may differ in their function and/or binding specificities.
In addition to the biochemical data addressing the binding interactions
of Tec kinase PH domains, functional data also support the importance
of the PH domain in regulating Tec-kinase activity in cells. As
mentioned, PH domain mutations can cause the loss of Btk function in B
cells. Furthermore, the PH domain of Itk recently has been shown to be
important for colocalization of Itk with the TCR complex
(34). The phospholipid modifying enzyme, PI3K, has also
been implicated in the regulation of Tec family kinases. PI3K is
capable of phosphorylating membrane phospholipids at the D-3 position
of the inositol ring, thus creating the preferred phospholipid ligand
for the Btk PH domain (35). In this regard, activation of
Itk by a Src family kinase has been shown to require the Itk PH domain
and PI3K activity (32). In addition, overexpression of
PI3K, together with a Src family kinase, can directly activate Btk
in fibroblast cells (36). Together, these data suggest
that PI3K plays a role in regulating Tec-kinase activity downstream of
an activated receptor.
To determine the role of Tec in primary T cells, we examined the consequences of Tec protein depletion on TCR-medicated IL-2 production. We show that reduced Tec leads to a diminished response to TCR stimulation. We also show that overexpression of Tec in Jurkat T cells leads to enhanced TCR signaling, and furthermore that Tec colocalizes with the activated TCR. This activity of Tec requires an intact Tec PH domain and depends on PI3K function. These data implicate Tec in the TCR signaling pathway and emphasize the role of the Tec PH domain in this functional activity.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The PY20 anti-phosphotyrosine mAb and anti-Txk antiserum (M20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Tec polyclonal antiserum and the 4G10 anti-phosphotyrosine mAb were purchased from Upstate Biotechnology (Lake Placid, NY). The anti-Itk mAb (2F12) has been reported previously (36). Anti-human CD3 mAb UCHT1, anti-mouse IL-2 mAbs, and peroxidase-conjugated streptavidin were purchased from PharMingen (San Diego, CA). The C305 Ab-producing hybridoma was a gift from Dr. A. Weiss (37). The OKT3 Ab-producing hybridoma was obtained from American Type Culture Collection (Manassas, VA). PE-conjugated goat-anti-rabbit IgG, FITC-conjugated streptavidin, and Texas Red-conjugated goat anti-mouse (GAM) IgG were purchased from Molecular Probes (Eugene, OR). Anti-FLAG and GAM antiserum were purchased from Sigma (St. Louis, MO). Murine splenocytes, human leukemic Jurkat cells, and SV40 T Ag-transfected Jurkat (Jtag) cells were grown in RPMI 1640 medium supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), 2-ME (50 µM), sodium pyruvate (1 mM), and glutamate (292 µg/ml). COS-7 cells were maintained in DMEM medium supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and glutamate (292 µg/ml).
Antisense oligonucleotide
Synthetic oligonucleotides (phosphorothioate-2'-O-methyl RNA chimera) were purchased from Oligos Etc. (Wilsonville, OR). The Tec anti-sense sequence is 5'-TGAAATTCATCTCGGTCT-3', and the control Tec sense sequence is 5'-AGACCGAGATGAATTTCA-3'. In addition to the sequence complementary to the ATG (common to all mRNAs), the Tec anti-sense oligonucleotide shares only 4 and 5 of 18 nucleotides in common with the sequences complementary to the Itk and Txk mRNAs, respectively.
Tec constructs
The full-length murine Tec cDNA was amplified by PCR using the
following primers; forward, 5'-CCAGAAGACCGAGATGAATTTCAAC3';
backward, 3'-AAACTGAGGCCACCATTCATCTTCC-3'. A Flag-epitope tag
(DYKDDDDK) was added to the 5' end of the Tec cDNA sequence, and
the clone was introduced into the PME18S vector, producing pME18S-WTTec
(34). pME18S-
PHTec, containing the Tec cDNA lacking the
PH domain, has a deletion of aa 1–110 of the Tec protein sequence, and
was derived from PME18S-WTTec by PCR. The plasmid pME18S-PHSH2*Tec
encodes a Tec protein lacking the PH domain, and including a
substitution of Ala for Arg at residue 272 of the SH2 domain.
pME18S-E42KTec encodes a Tec protein with a single amino acid
substitution at residue 42 in the PH domain (EgK), whereas
PME18S-R29CTec has a substitution at residue 29 in the PH domain (RgC).
Point mutations were generated with the Quickchange site-directed
mutagenesis kit (Stratagene, La Jolla, CA) according to the
manufacturer’s instructions. green fluorescence protein (GFP) fusion
protein expression vectors, pWTTec-GFP, pPHTec-GFP, pE42KTec-GFP,
pR29CTec-GFP, and pPHSH2*Tec-GFP, were generated by excising the Tec
cDNAs from pME18S-WTTec, pME18S-PHTec, pME18SE42K-Tec, pME18S-Tec,
and pME18S-PHSH2*Tec, respectively, and introducing them into the
vector, pEGFP-N3 (Clontech, Palo Alto, CA). GST fusion protein
expression vectors, pWTBtkPH, pR28CBtkPH, and p41KPH, contain wild-type
or mutant versions of the murine Btk PH domain (aa 6–217) in pGEX
(Amersham Pharmacia Biotech, Piscataway, NJ) and have been described
previously (8). GST fusion protein expression vectors,
pWTTecPH, pR29CTecPH, and pE42KPH, contain the murine Tec PH domain (aa
5–187) in pGEX. The sequences of all Tec cDNAs generated by PCR or
site-directed mutagenesis were verified by DNA sequencing. Plasmid
pIL-2-Luc is composed of 2 kb of the murine IL-2 promoter region
driving expression of the firefly luciferase reporter gene, and was a
gift from Dr. T. Yokota (38). The pRL-TK vector is
composed of the Herpes simplex virus thymidine kinase promoter driving
expression of the Renilla luciferase reporter gene (Promega,
Madison, WI).
Stimulation of primary T cells
Splenocytes from 5C.C7 TCR-transgenic mice (39) were isolated and subjected to RBC lysis. Cells were preincubated in medium for 2 h and then incubated in the presence or absence of 10 µM of synthetic oligonucleotide (anti-sense Tec or sense Tec) for 8 h. Cells were then stimulated with 1 µM moth cytochrome c (MCC) peptide (residues 92–103, the cognate Ag for the 5C.C7 TCR; Ref. 39) for an additional 24 h. The quantity of IL-2 present in the supernatants was measured by ELISA.
Jurkat cell stimulations, immunoprecipitations, and immunoblots
Jtag cells were stimulated at a concentration of
107 cells/ml at 37°C for 2 min with 10 µg of
UCHT1 (anti-human CD3
) followed by 15 µg of GAM antiserum for
5 min at 37°C. For unstimulated controls, 107
cells/ml were incubated in medium alone at 37°C for 2 min, followed
by 30 µg of GAM antiserum for 5 min at 37°C. Postnuclear lysates
were immunoprecipitated and/or immunoblotted as previously described
(13).
Transfections and luciferase assays
Jtag cells (107) were electroporated at 960 µF and 260 V (Bio-Rad Gene Pulser) with 10 µg of pIL-2-Luc, 1 µg of pRL-TK, and 10 µg of additional DNA expressing Tec or Tec mutants. After a 2-h recovery, the cells were cultured in medium alone or stimulated for 6 h with C305 mAb (anti-TCR, 1:100 dilution of ascites) plus PMA (50 ng/ml). Cells were then washed and lysed, and the protein concentration of the lysate was quantitated with the Bradford reagent (Bio-Rad, Hercules, CA). Ten micrograms of cell lysate was subjected to the dual luciferase reporter assay (Promega), according to the manufacturer’s instructions. The efficiency of transfection, as determined by Renilla luciferase activity in the lysate, was used to normalize the activity of firefly luciferase. The normalized firefly luciferase activities are presented in arbitrary units.
Confocal microscopy
To examine the colocalization of the TCR/CD3 complex with the Tec-GFP fusion proteins, Jtag cells (2 x 107) were electroporated with Tec-GFP expression vectors (20 µg) at 960 µF and 260 V. After 48 h, cells were incubated for 30 min on ice in 250 µl of culture medium containing 40 µg/ml OKT3 mAb. After washing, the cells were incubated for 30 min on ice in medium containing 20 µg/ml Texas Red-conjugated GAM IgG. The cells were then incubated at 37°C (stimulated) or on ice (nonstimulated) for 10 min. Following several washes, the cells were placed on poly-L-lysine-coated glass slides and fixed with 2% paraformaldehyde in PBS. The slides were then mounted with Prolong anti-Fade kit (Molecular Probes), according to the manufacturer’s instructions. Confocal images were generated as previously described (34).
In vitro binding to [3H]dioctanoyl PtdIns P3
GST fusion proteins were produced according to the
manufacturer’s instructions (Amersham Pharmacia Biotech). For in vitro
binding assays, 20 µg of GST fusion proteins bound to glutathione
beads were incubated with 20,000 cpm of
[3H]dioctanoyl PtdIns P3
(
0.4 nmol) for 1 h at room temperature (8).
Following several washes with HNE buffer (30 mM HEPES, pH 7.0, 100 mM
NaCl, 1 mM EDTA) containing 0.5% Nonidet P-40, the beads and
supernatants were separated by centrifugation and counted in a Beckman
scintillation counter in scintillation cocktail. The relative binding
activity was obtained by normalizing the cpm bound to the beads to GST
fusion protein quantitation as determined by Coomassie Blue
staining.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Three Tec family tyrosine kinase members are expressed in T cells: Itk, Txk, and Tec. Although the roles of Itk and Txk in T cells have been addressed (15, 17, 18, 19, 40, 41, 42), no data on the role of Tec in primary T cells has been reported. Based on the known roles of Itk and Txk in TCR signaling leading to IL-2 production, we examined whether depletion of Tec protein from primary T cells would affect this signaling pathway. For these experiments, we used T cells isolated from the 5C.C7 TCR-transgenic line (43). The 5C.C7 TCR is specific for a peptide (92–103) of MCC bound to the MHC class II molecule I-Ek. Total splenocytes isolated from H-2k 5C.C7-transgenic mice include both T cells as well as Ag-presenting cells capable of binding and presenting the MCC peptide.
To deplete the Tec protein, splenocytes from 5C.C7 TCR-transgenic mice
were incubated with a Tec anti-sense oligonucleotide complementary
to the 5' end of the mRNA. This sequence is quite specific for Tec, as
it shares only 7 and 8 of 18 nucleotides in common with the sequences
complementary to the Itk and Txk mRNAs, respectively (three of which
are the ATG, common to all mRNAs). As controls, cells were incubated in
medium alone, or in the presence of a Tec sense oligonucleotide.
Following oligonucleotide incubation, splenocytes were stimulated by
addition of the MCC peptide at a concentration of 1 µM, and IL-2
secreted into the supernatants was measured 24 h later. As shown
in Fig. 1
A, Tec anti-sense
oligonucleotide treatment reduced IL-2 production by activated splenic
T cells in comparison to untreated cells, or to cells treated with the
Tec sense oligonucleotide. This reduced IL-2 production was not due to
diminished cell viability after anti-sense oligonucleotide
treatment (data not shown), and instead, correlated with reduced levels
of the Tec protein (Fig. 1B). In contrast, no decrease was
observed in the levels of the related family members Itk and Txk (Fig. 1B).
|
, treatment of Jtag cells
with anti-TCR Ab plus PMA leads to a modest increase in IL-2
promoter activity compared with unstimulated cells. Interestingly,
overexpression of Tec results in a 30-fold increase in TCR-mediated
IL-2 promoter activity compared with cells transfected with the
reporter construct alone. Together, these data strongly support a role
for Tec in TCR-mediated IL-2 gene induction.
|
It is well known that point mutations in the PH domain of Btk lead
to immunodeficiency diseases in humans and mice, thus demonstrating the
important role of the Btk PH domain (44). However, several
studies have indicated differences in the functional properties of PH
domains among the Tec kinase family members, suggesting that PH domains
of individual Tec kinases may have distinct roles in signaling
(6, 9, 33). To determine the role of the Tec PH domain in
TCR signaling, we generated mutant forms of the Tec protein containing
alterations in the PH domain. In addition to a total deletion of the PH
domain (1–110), Tec proteins were generated with single amino acid
substitutions in the PH domain. One mutation, R29C, is based on the
mutation in the Btk PH domain found in xid mice.
Substitutions at this position abolish the binding of the Btk PH domain
to phosphoinositides (7, 8). A second mutation, E42K, is
also based on a mutation first described in the Btk PH domain. In this
latter case, substitution of lysine for glutamic acid at position 41 of
Btk results in increased binding of the PH domain to phosphoinositides
and to increased Btk kinase activity (9).
Each of these mutant forms of Tec was overexpressed in Jtag cells
together with the IL-2 promoter reporter construct. As shown in Fig. 2
, each of the mutations in the Tec PH domain interfered with the ability
of Tec overexpression to dramatically enhance TCR-mediated IL-2
promoter activity. Although point mutations in the Tec PH domain (R29C
or E42K) led to a severe reduction in Tec function, deletion of the PH
domain (PH) totally abolished Tec kinase-mediated IL-2 promoter
activity following TCR stimulation. Of note, the E42K substitution in
the Tec PH domain did not appear to increase Tec function as might have
been expected based on the known activity of this mutant form of Btk.
Overall, these results indicate the important role of the Tec PH domain
in enhancing Tec-mediated IL-2 promoter activity in response to TCR
signaling.
The Tec PH domain is required for TCR-induced tyrosine phosphorylation of Tec
As described above, mutation of the Tec PH domain interferes with
Tec function downstream of the TCR. To determine whether this loss of
function is associated with a lack of Tec activation following TCR
stimulation, we examined TCR-induced tyrosine phosphorylation of Tec.
Wild-type or mutant forms of Tec were expressed in Jtag cells following
transient transfection. Cells were then stimulated by anti-TCR
cross-linking, and the transfected Tec protein was immunoprecipitated
using an Ab specific for the epitope tag (anti-FLAG). As shown in Fig. 3, the tyrosine phosphorylation of
wild-type Tec is substantially increased after TCR stimulation. A
similar increase in tyrosine phosphorylation is also observed for Tec
proteins carrying single amino acid substitutions in the PH domain
(R29C and E42K). In contrast, complete deletion of the Tec PH domain
abolishes the TCR-induced increase in tyrosine phosphorylation.
|
Previous studies have examined the specificity and affinity of PH domain interactions with inositol lipids and soluble inositol phosphates. For Tec family kinases, a strong correlation has been established between protein function and PH domain binding to phosphatidylinositides phosphorylated at the D-3 position of the inositol ring (6, 7, 8, 9, 28, 29). These studies have strongly implicated the PH domain in membrane targeting of Tec kinases, thus establishing the importance of Tec kinase recruitment to the site of an activated receptor, and indicating the role of PI3K in Tec kinase activation (32, 36). The vast majority of these studies have been performed on Btk, owing in part to the fact that a three-dimensional structure of the Btk PH domain has been determined (45). These data have demonstrated that substitutions at residue R28 in Btk (comparable to R29 in Tec) dramatically reduce binding of the Btk PH domain to D3 phosphoinositides, whereas substitution of lysine at residue E41 (comparable to E42 in Tec) increases binding. In a recent report, Okoh and Vihinen have presented models of the other Tec kinase PH domains based on the structure of the Btk PH domain (46). Interestingly, these models predict striking differences in the binding pockets between different Tec family PH domains. Nonetheless, these authors predicted that substitutions at residue E41(E42) would also generate gain-of-function mutants in other Tec family kinases as a result of increased PH domain binding to the membrane. Given our functional data indicating a loss of Tec activity with both the R29C and E42K substitutions, we chose to directly assess binding of the wild-type and mutated Tec PH domains to phosphoinositides.
For these experiments we used a radioactively labeled water-soluble
form of PtdInsP3, as described previously
(8). This compound was incubated with GST fusion proteins
containing wild-type or mutated Tec kinase PH domains. To directly
compare the Tec PH domain binding to that of Btk PH domains, we also
tested three GST fusion proteins containing wild-type Btk PH, Btk PH
(R28C), or Btk PH (E42K). After incubation, the immobilized GST fusion
proteins were washed, and the amount of labeled compound binding to
each fusion protein was determined. Fig. 4 shows the results of a representative
experiment indicating binding activities relative to the wild-type Btk
PH domain. As has previously been reported, substitution of R28C in the
Btk PH domain reduces binding to PtdInsP3
(8), whereas substitution of E41K dramatically increases
binding. The wild-type Tec PH domain binds
PtdInsP3 but to a lesser degree than the
wild-type Btk PH domain, as was previously reported (6).
Interestingly, both the R29C and E42K substitutions in the Tec PH
domain substantially reduce binding to PtdInsP3.
These data correlate well with the functional data and indicate that
both of these mutations are likely to reduce membrane localization of
the Tec kinase.
|
To further assess the role of the Tec kinase in TCR signaling, we examined whether the subcellular localization of Tec is altered by TCR stimulation. Furthermore, because previous studies had indicated that Itk colocalizes with the TCR/CD3 complex after activation, we were interested to determine whether Tec is also recruited to the activated TCR. For these studies, wild-type Tec or individual Tec mutants were fused to GFP to allow monitoring of the subcellular localization of the protein by confocal microscopy. We first determined that fusion of Tec to GFP did not interfere with Tec function by confirming that tyrosine phosphorylation of the Tec-GFP fusion protein was induced normally after TCR stimulation (data not shown).
As shown in Fig. 5, wild-type Tec is
clearly recruited to the capped TCR/CD3 complex after TCR cross-linking
(Fig. 5, A and B). Interestingly, deletion of the
Tec PH domain impaired the colocalization of Tec-GFP with the activated
TCR, but did not completely abolish Tec recruitment (Fig. 5, C and D). The Tec R29C and E42K mutants were also
somewhat reduced in their ability to colocalize with the activated TCR
compared with wild-type Tec (Fig. 5, E–H). These results
indicate that while the PH domain is playing some role in Tec
recruitment to the activated TCR, an additional domain(s) of Tec must
also be contributing to TCR colocalization.
|
PHSH2*Tec-GFP). When introduced into Jtag
cells, the PH/SH2 domain double mutant completely failed to colocalize
with the activated TCR (Fig. 5, I and J). These
results indicate that the Tec PH domain, as well as the SH2 domain, is
involved in the recruitment of Tec to the activated TCR/CD3
complex. Inhibition of PI3K abolishes Tec signaling in response to TCR stimulation
PI3K has been implicated in TCR-mediated IL-2 gene
induction (49, 50) and in the activation of Tec family
kinases such as Btk, Itk, and Bmx (32, 33, 36). Our
functional and biochemical data indicated that the Tec PH domain is
crucial for Tec function, and that this domain may regulate Tec
activity by mediating binding to phosphoinositides in the plasma
membrane. For these reasons, we chose to investigate the role of PI3K
in regulating Tec kinase signaling. Wortmannin, a specific inhibitor of
PI3K, was used for these studies. As shown in Fig. 6, treatment of Jtag cells with
wortmannin completely abolished the Tec-mediated induction of the IL-2
promoter in response to TCR stimulation. These results suggested that
PI3K plays an important role in regulating Tec kinase activity after
TCR cross-linking.
|
). Finally, we examined whether
wortmannin treatment would interfere with Tec colocalization with the
activated TCR in Jtag cells because Tec recruitment is partially
dependent on the Tec PH domain. As shown in Fig. 8, we found that wortmannin treatment
substantially inhibited Tec colocalization with the activated TCR/CD3
complex. Taken together, these three lines of evidence strongly support
the conclusion that Tec is regulated by PI3K during TCR signaling.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this report, we demonstrate that the Tec kinase plays a role in TCR-mediated stimulation of IL-2 production in primary T cells. This result is consistent with previous studies that showed that overexpression of Tec in Jurkat T cells could enhance TCR-mediated IL-2 gene induction (13, 22). Furthermore, we show that the Tec PH domain is important for Tec signaling leading to IL-2 gene induction, Tec tyrosine phosphorylation, and colocalization of Tec with the activated TCR. Finally, we provide evidence implicating PI3K in regulating Tec signaling downstream of the TCR. Thus, to date, three Tec kinase family members, Itk, Txk, and Tec, have been shown to play a role in TCR signaling.
Despite these data, the precise role of Tec family kinases in T cells
is still not well understood. Itk and Txk have both been shown to be
downstream of the TCR, and to play a role in Ca2+
mobilization, PLC-1 phosphorylation and activation, and cytokine
(IL-2 and IFN-) production (15, 16, 18). Interestingly,
Itk appears to play a more dominant role in these signaling pathways
than Txk, as Txk-/- T cells showed more
modest defects than those observed in T cells from
Itk-/- or
Itk-/-/Txk-/-
mice (17, 18). Additional data have suggested that Itk and
Txk have different functions in T cell signaling. For example, when
Itk-/- mice are backcrossed to the BALB/c
background, Itk-deficient T cells from these mice are unable to produce
IL-4 but make normal levels of IFN- when stimulated
(19). In contrast, Txk was found to be present in
differentiated Th1, but not Th2 cells, and is involved in regulating
TCR-mediated IFN- production (20).
It still remains possible that additional Tec family kinases are important in T cell signaling, as even T cells from Itk-/-/Txk-/- doubly deficient mice show residual TCR signaling; furthermore, T cell development is only modestly affected in Itk-/-/Txk-/- mice (18). Because the comparison of Itk-/-, Txk-/-, and Itk-/-/Txk-/- indicates that these two Tec family members can compensate for each other’s absence in T cells, it is not difficult to imagine that Tec, the third member of this family in T cells, may also be compensating for the absence of Itk, Txk, or both. For these reasons, we felt it was important to assess whether Tec was involved in TCR signaling. On the basis of our data, both dependent on Tec depletion in primary T cells, and on Tec overexpression in Jurkat cells, we conclude that Tec plays a role in IL-2 gene induction downstream of the TCR.
The importance of the PH domain in Tec family kinases
The first evidence supporting the importance of PH domains in Tec kinases came from the identification of the xid defect as a single amino acid substitution in the Btk PH domain (30). Subsequent studies have shed some light on the function of the Tec kinase PH domains. Biochemical experiments have demonstrated the preference of Tec kinase PH domains for phosphoinositides phosphorylated at the D-3 position of the inositol ring (6, 7, 8), thus implicating the PH domain in recruiting Tec kinases to the plasma membrane. This in vitro binding activity correlates well with the functional data available. For Btk, Itk, and Bmx, an intact PH domain is essential for activation-induced tyrosine phosphorylation of the kinase, for kinase activity, as well as for recruitment of the kinase to the plasma membrane (32, 33, 34, 51, 52). However, a PH domain is clearly not essential for all Tec kinase functions. Txk, one of the Tec kinases present in T cells, possesses no PH domain, yet can almost completely restore Btk signaling in Btk-deficient DT40 B cells (53). In addition, Wen et al. demonstrated that a chimeric protein containing the extracellular domain of the estrogen receptor fused to PH-deleted Bmx could activate the downstream STAT1/5 pathway following estrogen stimulation (25). These results indicate that Tec kinase PH domains play an important role in recruiting the kinase to the site of an activated receptor, but are not essential for kinase activity per se. Our results showed that the Tec PH domain, like that of Itk and Btk, is important for its membrane localization and tyrosine phosphorylation.
One surprising finding in this study was the loss of Tec functional activity with the PH domain mutant, E42K. This amino acid substitution was first described for Btk, where it increases Btk activity and Btk recruitment to the plasma membrane (52). Furthermore, in vitro binding experiments indicate that the Btk PH domain mutant, E41K, has increased binding to phosphoinositides. Our own in vitro binding data correlate with our functional data and indicate that the Tec PH domain mutant, E42K, has reduced binding activity for phosphatidylinositol-3,4,5-trisphosphate (PtdInsP3). These results with Tec are also consistent with a previous study of Bmx that found that the E42K mutant of Bmx has reduced kinase activity in fibroblasts (33). One possible explanation for the discrepancy between Btk and Tec/Bmx is apparent from models of other Tec kinase PH domain structures (46) based on the x-ray crystal structure of the Btk PH domain (54). Specifically, the glutamic acid residue at position 41 in Btk (42 in other Tec kinases) is thought to be too far away from the inositol-binding pocket to directly interact with the inositol phosphate moiety of phosphatidylinositol-3,4,5-trisphosphate (PtdInsP3). Instead, this residue may enhance Btk binding to the membrane by binding other polar head groups of phospholipids in the membrane or, alternatively, may stabilize a putative dimerization of the PH domain by creating a more favorable interaction with the glutamic acid residue at position 45 in the dimer interface (45, 55). Interestingly, the other Tec family PH domains, including Tec and Bmx, have an arginine or lysine residue at position 44 or 45; thus, substitution of E42 with lysine may, in fact, destabilize the putative dimer structure. This interpretation is consistent with our observation that the E42K substitution in the Tec PH domain decreases phosphoinositide binding and nearly eliminates Tec functional activity.
Another surprising finding of this study was the observation that
mutation of, or complete deletion of, the Tec PH domain did not abolish
Tec colocalization with the activated TCR. This is in direct contrast
to the results with Itk, where mutation of the PH domain totally
eliminated Itk recruitment to the TCR/CD3 cap in activated Jurkat cells
(data not shown; Ref. 34). Interestingly, we also observed
a difference in the subcellular distribution of Tec vs Itk in Jurkat
cells. Whereas wild-type Tec is predominantly cytosolic (Fig. 5A, 1), with no visible partitioning to the plasma membrane
in resting T cells, Itk is predominantly membrane associated before TCR
cross-linking (34, 51, 56). These distinctions provide
further evidence highlighting the differences between the PH domains of
the individual Tec family members, and support the conclusions from
structural modeling studies (46).
We also observed a discordance between the tyrosine phosphorylation of Tec mutants (R29C and E42K) in response to TCR stimulation, their membrane localization, and their ability to enhance IL-2 promoter activity. Specifically, we could detect no decrease in tyrosine phosphorylation of Tec(R29C) or Tec(E42K) after TCR cross-linking in transfected Jurkat cells, whereas each of these mutations substantially reduced the ability of Tec to enhance IL-2 promoter activity. The failure of these point mutations in the Tec PH domain to abolish Tec colocalization with the activated TCR may, in part, account for this discrepancy. The anti-phosphotyrosine immunoblotting may be sufficiently sensitive to detect the increased tyrosine phosphorylation of a small fraction of the mutant Tec proteins that still colocalize with the activated TCR; however, activation of this small fraction of Tec may not be sufficient to generate robust downstream signals leading to substantial enhancement of IL-2 promoter activity.
The role of PI3K in regulating Tec kinase function
Phosphoinositides have recently been identified as second messengers with distinct binding properties for different PH domains. Several studies have documented the preference of Tec family PH domains for binding phosphoinositides phosphorylated at the D-3 position of the inositol ring (6, 7, 8). These observations suggested that Tec kinases would be regulated by the activity of PI3K, an enzyme that converts phosphatidylinositol 4,5 bisphosphate to PtdInsP3, thus creating the optimum ligand for the Tec family PH domains (35). Previous studies have indicated that several members of the Tec family, including Btk, Itk, and Bmx, but not Txk (which lacks a PH domain), are regulated by PI3K. Here we present evidence strongly supporting a role for PI3K in regulating Tec activity in T cells. These findings also suggest the intriguing possibility that Tec kinases will also be negatively regulated by the phospholipid phosphatase, the tumor suppressor, PTEN, which has a preference for dephosphorylating PtdInsP3 at the D-3 position of the inositol ring (57). This possibility could relate to the observation that PTEN+/- mice exhibit T cell hyperresponsiveness, and eventually succumb to a fatal T cell-mediated autoimmune disease (58).
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Leslie J. Berg, Department of Pathology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655.
3 Abbreviations used in this paper: PI3K, phosphatidylinositol 3-kinase; PH, pleckstrin homology; PLC, phospholipase C; SH, Src homology; PtdInsP3, phosphatidylinositol 3,4,5 trisphosphate; GAM, goat anti-mouse; GFP, green fluorescence protein; Jtag, SV40 T Ag-transfected Jurkat; MCC, moth cytochrome c.
Received for publication May 30, 2000. Accepted for publication October 10, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production in human T lymphocytes. J. Exp. Med. 190:1147. subunits of heterotrimeric G proteins to the PH domain of Bruton tyrosine kinase. Proc. Natl. Acad. Sci. USA 91:11256. subunits. Proc. Natl. Acad. Sci. USA 92:8601. activates Bruton’s tyrosine kinase in concert with src family kinases. Proc. Natl. Acad. Sci. USA 94:13820.
T cell receptors. Annu. Rev. Immunol. 16:523.[Medline]
This article has been cited by other articles:
|
|
 |
|
  C. L.-T. Chang, S.-L. Chang, Y.-M. Lee, Y.-M. Chiang, D.-Y. Chuang, H.-K. Kuo, and W.-C. Yang Cytopiloyne, a Polyacetylenic Glucoside, Prevents Type 1 Diabetes in Nonobese Diabetic Mice J. Immunol., June 1, 2007; 178(11): 6984 - 6993. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-P. Liu, Y.-C. Kuo, C.-C. Shen, M.-H. Wu, J.-F. Liao, Y.-L. Lin, C.-F. Chen, and W.-J. Tsai (S)-Armepavine inhibits human peripheral blood mononuclear cell activation by regulating Itk and PLC{gamma} activation in a PI-3K-dependent manner J. Leukoc. Biol., May 1, 2007; 81(5): 1276 - 1286. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Qi, N. Sahu, and A. August Tec Kinase Itk Forms Membrane Clusters Specifically in the Vicinity of Recruiting Receptors J. Biol. Chem., December 15, 2006; 281(50): 38529 - 38534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. P. Kane and S. C. Watkins Dynamic Regulation of Tec Kinase Localization in Membrane-proximal Vesicles of a T Cell Clone Revealed by Total Internal Reflection Fluorescence and Confocal Microscopy J. Biol. Chem., June 10, 2005; 280(23): 21949 - 21954. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Aoukaty and R. Tan Role for Glycogen Synthase Kinase-3 in NK Cell Cytotoxicity and X-Linked Lymphoproliferative Disease J. Immunol., April 15, 2005; 174(8): 4551 - 4558. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. Tomlinson, V. L. Heath, C. W. Turck, S. P. Watson, and A. Weiss SHIP Family Inositol Phosphatases Interact with and Negatively Regulate the Tec Tyrosine Kinase J. Biol. Chem., December 31, 2004; 279(53): 55089 - 55096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Garcon, G. Bismuth, D. Isnardon, D. Olive, and J. A. Nunes Tec Kinase Migrates to the T Cell-APC Interface Independently of Its Pleckstrin Homology Domain J. Immunol., July 15, 2004; 173(2): 770 - 775. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. P. Kane, M. N. Mollenauer, and A. Weiss A Proline-Rich Motif in the C Terminus of Akt Contributes to Its Localization in the Immunological Synapse J. Immunol., May 1, 2004; 172(9): 5441 - 5449. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Tomlinson, L. P. Kane, J. Su, T. A. Kadlecek, M. N. Mollenauer, and A. Weiss Expression and Function of Tec, Itk, and Btk in Lymphocytes: Evidence for a Unique Role for Tec Mol. Cell. Biol., March 15, 2004; 24(6): 2455 - 2466. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Lachance, S. Levasseur, and P. H. Naccache Chemotactic Factor-induced Recruitment and Activation of Tec Family Kinases in Human Neutrophils. IMPLICATION OF PHOSPHATIDYLINOSITOL 3-KINASES J. Biol. Chem., June 7, 2002; 277(24): 21537 - 21541. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Takesono, L. D. Finkelstein, and P. L. Schwartzberg Beyond calcium: new signaling pathways for Tec family kinases J. Cell Sci., January 8, 2002; 115(15): 3039 - 3048. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. F. Reynolds, L. A. Smyth, T. Norton, N. Freshney, J. Downward, D. Kioussis, and V. L.J. Tybulewicz Vav1 Transduces T Cell Receptor Signals to the Activation of Phospholipase C-{gamma}1 via Phosphoinositide 3-Kinase-dependent and -independent Pathways J. Exp. Med., May 6, 2002; 195(9): 1103 - 1114. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
