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*
Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, CA 92182;
Division of Allergy, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; and
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Emt/Itk plays a critical role in T cell-mediated immune responses. Mice
deficient in Emt/Itk display compromised TCR/CD3-induced events
including phospholipase C
1 (PLC1) phosphorylation, inositol
trisphosphate production, intracellular Ca2+
mobilization, IL-2 and IL-4 production, cellular proliferation, and
immunity against viruses and parasites (6, 7, 8, 9, 10).
Interestingly, early TCR/CD3-mediated events, such as 
chain and
ZAP-70 (-associated protein of 70 kDa) phosphorylation, are not
affected in Emt/Itk-deficient animals, indicating that this kinase
regulates intermediate and later events during T cell signal
transduction (9). Emt/Itk-deficient mice also have
significant defects in thymic development, particularly in development
of the Th2 CD4+ subset, decreased numbers of
mature thymocytes, and altered ratios and numbers of peripheral
CD4+ and CD8+ T cells
(6, 10).
Src and Lck are capable of phosphorylating and activating Emt/Itk
(3, 11, 12). However, the mechanism responsible for
recruitment of Emt/Itk to the appropriate cellular compartments to
become activated is not clearly understood. Recent studies from our
laboratory have shown that upon T cell activation, Emt/Itk localizes in
TCR/CD3-containing molecular clusters (13). In the course
of these studies, we detected a tyrosine phosphoprotein of 
36-kDa
relative molecular mass that coimmunoprecipitated with Emt/Itk upon
TCR/CD3-mediated stimulation. This molecular size is consistent with
that of the 36- to 38-kDa adapter protein linker of activated T cells
(LAT) (14).
LAT bridges proximal TCR signaling events with major downstream
pathways such as Ras activation and intracellular calcium flux
(14, 15). Upon TCR engagement, LAT becomes heavily
phosphorylated at multiple tyrosine residues and associates with many
important signaling molecules including CD4, CD8, Grb2, Grap, Gads,
Cbl, Vav, SLP-76, PLC1, and the p85 subunit of phosphatidylinositol
3-kinase (PI3-kinase) (14, 15, 16, 17). Mutant Jurkat T cells
lacking expression of LAT (JCaM2) have deficiencies in TCR/CD3-mediated
signaling events, including intracellular Ca2+
mobilization; inositol phospholipid generation; and AP-1, NF-AT, and
IL-2 promoter activity, as well as in the phosphorylation of proteins
that associate with LAT (15). However, JCaM2 cells display
normal early signaling events, such as phosphorylation of chain and
ZAP-70 (15). LAT is also critical to T cell development in
that mice bearing a disruption of the LAT gene are blocked in
intrathymic development within the
CD4-CD8- stage
(18).
The similarity in the defects in calcium flux and PLC-1
phosphorylation between LAT-deficient Jurkat T cells (15)
and T cells from Emt/Itk-deficient mice (8, 9) suggest
that LAT and Emt/Itk may function in the same pathway. Thus, in this
study we have investigated the physical and functional relationship
between LAT and Emt/Itk that is important for the activation of Emt/Itk
via the TCR/CD3 complex. The data demonstrate that the Src homology 2
(SH2) domain, but not the SH3 domain or kinase activity of Emt/Itk,
mediates the formation of Emt/Itk-LAT complexes, which allow Emt/Itk to
be activated upon TCR/CD3 stimulation. Furthermore, we show via
confocal microscopy that in vivo, LAT and Emt/Itk both colocalize with
clusters of the TCR/CD3 complex.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The wild-type Jurkat cells used were transfected with the SV40 T-Ag (JTAg). The LAT-deficient Jurkat cell line (JCaM2) was kindly provided by Dr. Arthur Weiss (University of California, San Francisco, CA). JCaM2 cells were stably transfected via electroporation (as described below) with LAT-green fluorescence protein (GFP), and stable transfectants were selected with G418 Sulfate (Mediatech, Herndon, VA). Subsequently, flow cytometry was utilized to sort cells expressing LAT-GFP into the brightest 5% (median fluorescence intensity = 278.29) of fluorescent cells (termed JCaM2 "bright") and into the next brightest 5% (median fluorescence intensity = 52.75) of fluorescent cells (termed JCaM2 "dim"). Cells were cultured at 37°C with RPMI 1640 (Irvine Scientific, Irvine, CA) containing 10 mM HEPES, 2 mM L-glutamine (Sigma, St. Louis, MO), and 8% FBS (HyClone, Logan, UT) in a humidified 5% CO2 atmosphere. Cells (20 x 106 in 400 µl) were transfected with 20 µg of the desired plasmid DNA by electroporation (gene pulser; Bio-Rad, Hercules, CA) in a 0.4-cm gap electrocuvette (DocFrugal’s, San Diego, CA) at 960 µF and 240 mV. They were used in experiments after culturing at 37°C for 48 h.
Abs
The following Abs were used: anti-CD3
mAb OKT3 (hybridoma
obtained from American Type Culture Collection, Manassas, VA), IgG2a
isotype control UPC-10 (Bionetics, Charleston, SC), rabbit
anti-mouse IgG (Jackson Immunoresearch, West Grove, PA),
anti-LAT (Upstate Biotechnology, Lake Placid, NY), monoclonal
anti-H902 Ab against aa 319–333 of HIV gp120 (hybridoma obtained
from National Institutes of Health AIDS Reference Reagent Program,
Rockville, MD), anti-murine and anti-human Emt/Itk (Santa Cruz
Biotechnology, Santa Cruz, CA), monoclonal antiphosphotyrosine Abs pY20
(hybridoma provided by Dr. Bartholomew Sefton, Salk Institute, La
Jolla, CA) and 4G10 (hybridoma provided by Dr. Tomas Mustelin, Burnham
Institute, La Jolla, CA), and anti-CD3 peptide Ab (Dako,
Carpinteria, CA).
cDNA constructs
Wild-type (wt) murine Emt/Itk (19) was cloned into
the EcoRI-SpeI sites of the pME18s expression
vector (20). Emt/Itk mutants were generated by a two-step
PCR amplification of mutated cDNA sequences before substituting the
mutated portion for the wild-type sequence in the pME18s vector. To
generate the 
SH3 mutant, two PCR reactions were performed using the
wild-type cDNA as template. In one reaction, an appropriate 5' primer
and the mutagenizing 3' primer
(5'-TGGAGATTTTTCTACAATGACCAGGGTTTC-3') were used, whereas in the
other reaction the mutagenizing 5' primer
(5'-GAAACCCTGGTCATTGTAGAAAAATCTCCA-3') and an appropriate 3' primer
were used. Subsequently, another set of PCRs was performed using the
products of the PCR reactions above as mixed templates and the 5' and
3' nonmutagenizing primers. The correct PCR product was cloned into the
pCRII vector (Invitrogen, Carlsbad, CA) and was confirmed by
sequencing. The 519-bp Eco47III fragment containing the
mutated portion was isolated from this clone and used to replace the
wild-type sequence in pME18s. Similar strategies were used to generate
the SH2 and K390R mutants. The resulting SH3 and SH2 mutants
encode proteins devoid of residues 178–226 and 239–337, respectively
(19). The description of oligonucleotides and restriction
sites utilized for the construction of the SH2 and K390R mutants
will be furnished upon request.
The H902 epitope tag, consisting of aa 319–333 of HIV gp120, was added
by PCR amplification of pME18s-wt-Emt/Itk as previously described
(13). The H902 epitope was also introduced to the Emt/Itk
mutants as follows. The EcoRI-ScaI fragment of
pME18s-SH2-Emt/Itk was replaced with the equivalent fragment from
pME18s-H902-wt-Emt/Itk. The same strategy was used to prepare the
H902-tagged pME18s-K390R-Emt/Itk. To prepare H902-tagged
pME18s-SH3-Emt/Itk, the Eco47III fragment of
pME18s-wt-Emt/Itk was replaced with the equivalent fragment from the
SH3 mutant. All constructs were confirmed by sequencing.
LAT-GFP was generated by PCR of the construct pEF-HA-LAT containing human LAT (cloned from a Jurkat T cell cDNA library) with sense primer 5'-AAGAATTCGCCACCATGGAGGAGGCCATCCTGGTC-3' and antisense primer 5'-GCGGTACCGGTTCAGCTCCTGCAGATTCTC-3' to generate a fragment containing LAT and in frame KpnI and EcoRI sites at the 3' and 5' ends, respectively. This PCR fragment was digested with EcoRI and KpnI and subcloned into the pEGFP-N2 vector (Clontech, Palo Alto, CA).
The SH2 domain point mutant R265K and SH3 point mutant W208K of Emt/Itk were created by site-directed mutagenesis of H902-tagged wild-type Emt/Itk in the pME18s vector using the QuikChange Site-Directed Mutagenesis Kit following the manufacturer’s instructions (Stratagene, La Jolla, CA). The sense primer 5'-GGAGCTTTCATGGTCAAAGATTCCAGG-3' and the antisense primer 5'-CCTGGAATCTTTGACCATGAAAGCTCC-3' (mutated codon underlined) were used for the construction of the R265K mutant, whereas the sense primer 5'-CCGAGATCCACAAGTGGAGGGTTC-3' and antisense primer 5'-GAACCCTCCACTTGTGGATCTCGG-3' (mutated codon underlined) were used for the construction of the W208K mutant. Both of the mutant constructs were confirmed by sequencing.
Immunoprecipitation, Western blotting, autophosphorylation, cell stimulation, fluorescence labeling, and laser scanning confocal microscopy
All of these assays were performed as previously described (13). Minor modifications in the autophosphorylation assay are noted in the figure legend.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Emt/Itk is a modular protein tyrosine kinase that is organized in
discrete domains (2). We wished to investigate the
domain(s) of Emt/Itk that might play a role in its TCR/CD3-induced
activation. JTAg cells that had been transfected with H902-tagged
wt-Emt/Itk or various mutants were stimulated by cross-linking with
anti-CD3 Ab OKT3 or isotype control Ab, and the phosphorylation
of the transgene products was assessed. wt-Emt/Itk becomes highly
tyrosine phosphorylated, as was assessed by immunoprecipitation with
anti-H902 Abs and Western blotting with antiphosphotyrosine Abs
(Fig. 1
A, top
panels). Similarly, mutants of Emt/Itk (K390R) with deficient
enzymatic activity (4) or mutants lacking the SH3 domain
(SH3) are also highly tyrosine phosphorylated upon anti-CD3
cross-linking (Fig. 1A, top panels). However, in
sharp contrast, mutants lacking the SH2 domain (SH2) or mutants with
an inactivating SH2 domain point mutation (R265K) display deficient
phosphorylation under the same experimental conditions (Fig. 1A, top panels). The differences in
transphosphorylation are not due to unequal loading, as is shown by the
anti-Emt/Itk blotting (Fig. 1A, bottom
panels). It is interesting that a mutation in the SH2 domain of
the related kinase Btk, analogous to the R265K mutation in Emt/Itk used
here, has been shown to disrupt Btk function (21, 22).
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A, the R265K mutant displays severe deficiency in enzymatic
activity (Fig. 1B, top panels). This is in
contrast with the wt-Emt/Itk and with the SH3 domain mutant (W208K),
both of which display OKT3-inducible kinase activity (Fig. 1B, top panels). It should be noted that the
conversion of tryptophan to lysine at amino acid position 208 has been
shown to represent an inactivating mutation at the predicted binding
pocket of the SH3 domain (23).
As expected, the kinase domain mutant K390R does not display any
enzymatic activity (Fig. 1B, top panels). In
addition, the data with K390R also exclude the possibility that the
kinase activity detected with wt- or W208K-Emt/Itk might be due to a
fortuitously coprecipitating kinase. Western blotting with
anti-Emt/Itk shows that the data cannot be explained because of
unequal loading of samples (Fig. 1B, bottom
panels). Furthermore, lack of detection of enzymatic activity
cannot be due to insufficient expression of the R265K transgene because
its expression is comparable to that of W208K, which displays
detectable kinase activity (Fig. 1B, bottom
panels).
Emt/Itk and LAT form a TCR/CD3-inducible complex
The mechanism responsible for the activation of Emt/Itk is not clearly understood. Convincing evidence exists that activation of Lck is critical for the transphosphorylation and subsequent activation of Emt/Itk (3, 11). Furthermore, evidence from Monks et al. (24) and from our own laboratory (13) indicates that activation of T lymphocytes through the Ag receptor induces colocalization of TCR/CD3 with Lck and with Emt/Itk. Thus, colocalization of these molecules could represent a possible mechanism through which these proteins could come into close proximity and interact. However, there is no evidence that Emt/Itk has a direct, stable physical interaction with either Lck or TCR/CD3. Therefore, associations among these molecules must be mediated through a different mechanism, perhaps interaction with an adapter protein.
During the course of our studies, we noticed that immunoprecipitation
of Emt/Itk after anti-CD3 stimulation coimmunoprecipitates a
36-kDa phosphoprotein. The size similarity between this coprecipitated
species and the adapter protein LAT, as well as the fact that cells
lacking Emt/Itk share deficiencies in certain signal transduction
pathways with LAT-deficient cells (8, 9, 15), prompted us
to investigate whether LAT and Emt/Itk interact. To this end, JTAg
cells were transiently transfected with murine wt-Emt/Itk and
stimulated with OKT3 or isotype control Ab, and cell lysates were
immunoprecipitated with anti-LAT Ab. Analysis of the immune
complexes by PAGE and Western blotting reveals a substantial amount of
murine Emt/Itk coimmunoprecipitating with LAT upon stimulation (Figs. 2A and
3). We also detected some basal
association between LAT and Emt/Itk in nonstimulated cells (Fig. 3).
Furthermore, Emt/Itk that inducibly associates with LAT is tyrosine
phosphorylated (Fig. 2B). The observed differences between
OKT3-stimulated and nonstimulated cells are not due to unequal sample
loading as indicated by blotting of the samples with anti-LAT Ab
(Fig. 2C). Endogenous Emt/Itk can also be detected in
anti-LAT immune complexes from OKT3-stimulated cells (data not
shown). In a reciprocal experiment, we also observed TCR/CD3-induced
coimmunoprecipitation of LAT in anti-Emt/Itk immune complexes (data
not shown). The coimmunoprecipitation of Emt/Itk and LAT cannot be due
to inadvertent immunoprecipitation of Emt/Itk with residual stimulating
OKT3 Ab because direct coimmunoprecipitation of Emt/Itk with Ab OKT3
under the present experimental conditions is not seen (data not shown).
This is in agreement with observations by other investigators (3, 25).
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Because LAT becomes phosphorylated on multiple tyrosines upon
TCR/CD3 engagement (14), we tested whether the SH2 domain
of Emt/Itk is involved in the interaction with LAT. We transiently
transfected JTAg cells with murine SH2-Emt/Itk and examined its
effect on the inducible association between Emt/Itk and LAT. Cells
expressing the 66-kDa SH2-Emt/Itk mutant (Fig. 2D) do not
increase SH2-Emt/Itk association with LAT upon stimulation with OKT3
(Fig. 2A and Table I). As
expected, in view of the data in Fig. 1, no tyrosine-phosphorylated
SH2-Emt/Itk was detected (Fig. 2B). The weak 72-kDa band
seen in Fig. 2B most likely represents phosphorylated
endogenous Emt/Itk, which is undetected by the murine-specific
anti-Emt Ab. The lack of SH2-Emt/Itk binding to LAT is not due
to lack of expression of the SH2-Emt/Itk construct, because it is
detected in the lysate (Fig. 2D). It should be noted that
the reduced amount of wt-Emt/Itk in the stimulated lane of Fig. 2D is due to fortuitous loss of sample. As loading controls,
we probed the immune complexes with anti-LAT (Fig. 2C).
The SH2 domain point mutant R265K-Emt/Itk was also deficient in
inducible LAT binding (Fig. 3 and Table I).
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SH2 and R265K mutants, deletion of
the SH3 domain (SH3-Emt/Itk) or ablation of catalytic activity
(K390R) did not disrupt the inducible association of the kinase with
LAT (Fig. 3 and Table I). Similar to wt-Emt/Itk, there is some basal
association between all the mutants tested and LAT (Figs. 2 and 3).
To obtain some quantitative comparison between the amounts of
wt-Emt/Itk and its mutants that inducibly associate with LAT, we
analyzed the Emt/Itk bands that were coprecipitated by anti-LAT Ab
and normalized them to the total amount of LAT obtained in each case
using densitometry. As shown in Table I, the amounts of wt- and
SH3-Emt/Itk that associate with LAT increase 1.85- and 2.33-fold
upon stimulation, respectively. In contrast, the amounts of SH2 and
R265K mutants do not significantly (p < 0.05)
increase their association with LAT (Table I). It should be noted in
the above studies that the reason we do not detect endogenous Emt/Itk
is because the anti-Emt/Itk Ab utilized in the Western blotting
analyses is reactive only against murine Emt/Itk, which is the species
of the transfected gene.
LAT is required for TCR/CD3-induced Emt/Itk activation
The association of tyrosine-phosphorylated Emt/Itk with LAT (Fig. 2B) suggests that either activated Emt/Itk is recruited to
LAT or that Emt/Itk is activated after it associates with LAT. To
distinguish between these two possibilities, we examined whether LAT is
involved in the activation of Emt/Itk through TCR/CD3 engagement.
Because stimulation through the TCR/CD3 induces tyrosine
phosphorylation and subsequent activation of Emt/Itk by Lck (3, 11), we compared the OKT3-mediated tyrosine phosphorylation of
Emt/Itk in LAT-deficient (JCaM2) and regular Jurkat T cells. In sharp
contrast to regular Jurkat, stimulation of JCaM2 cells displays no
measurable increase in Emt/Itk phosphorylation (Fig. 4A). This observation was also
confirmed in another experiment in which Emt/Itk was transfected into
the cells to increase the observable signal (Fig. 4B).
Interestingly, even though JCaM2 cells fail to phosphorylate Emt/Itk
upon OKT3 stimulation (Fig. 4, A and B), they
display normal CD3 phosphorylation (Fig. 4C).
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To demonstrate that the defect in TCR/CD3-induced activation of
Emt/Itk in JCaM2 cells was solely a result of the deficiency in LAT
expression, we reconstituted LAT expression in JCaM2 cells by stable
transfection of the LAT gene. We decided to express a LAT-GFP fusion
protein to be able to isolate JCaM2 transfectants according to their
relative levels of LAT expression using fluorescence-activated cell
sorting. LAT-GFP is expressed as a 66/68-kDa doublet with some baseline
tyrosine phosphorylation that increases upon stimulation with Ab OKT3
(Fig. 5A). To assess the
effects of LAT-GFP expression on TCR/CD3-induced phosphorylation of
Emt/Itk, we sorted the JCaM2 transfectants according to the intensity
of the LAT-GFP transgene product they expressed. Thus, JCaM2
transfectants were sorted into "bright" (mean fluorescence
intensity = 278.29) and "dim" (mean fluorescence
intensity = 52.75) subpopulations using FACS. To assess
TCR/CD3-induced phosphorylation of Emt/Itk in the LAT-reconstituted
cells, we stimulated both LAT-transfected and regular, nonreconstituted
JCaM2 cells with OKT3 or isotype control Ab and analyzed Emt/Itk immune
complexes by Western blotting with anti-pY Abs. In these
experiments, the JCaM2 cells were transiently transfected with a H902
epitope-tagged Emt/Itk transgene to increase expression of the kinase.
Interestingly, only the "bright" LAT-GFP JCaM2 cells displayed
detectable Emt/Itk phosphorylation, whereas the "dim" cells,
similar to the nonreconstituted JCaM2, showed no Emt/Itk
phosphorylation (Fig. 5B). Equal loading and expression of
Emt/Itk was confirmed by blotting with anti-Emt/Itk Ab (Fig. 5B).
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As mentioned above, the interaction of Emt/Itk with LAT may
represent a mechanism through which the kinase may be recruited to the
TCR/CD3 complex to come into proximity with other critical molecules
such as Lck. If this is the case, LAT must associate with TCR/CD3.
However, there is no evidence supporting a stable physical interaction
between LAT and the Ag receptor complex. We decided to address this
issue by determining whether LAT-GFP can colocalize with TCR/CD3. To
this end, JTAg cells transiently transfected with LAT-GFP were treated
with Ab OKT3 before incubation with Texas Red-conjugated goat
anti-mouse Ig, and the cellular distribution of LAT and CD3 was
assessed by confocal microscopy. When cells are kept at 4°C
(unstimulated), LAT-GFP remains distributed throughout the plasma
membrane (Fig. 6A), and CD3
is localized in a punctate distribution around the plasma membrane
(Fig. 6C). Upon overlay, the two signals do not colocalize
(Fig. 6B). However, when cells are incubated at 37°C
(stimulated), both LAT and CD3 form similarly shaped clusters (caps)
of identical cellular localization and polarity (Fig. 6, D
and F) that colocalize upon overlay of the images (Fig. 6E). It should be noted that each cell that formed a CD3
cap also displayed a colocalized LAT-GFP cluster. In experiments
similar to the above, we also analyzed the inducible colocalization
between Emt/Itk-GFP and CD3. Under resting conditions, a portion of
Emt/Itk associates with the cell membrane, whereas the rest of the
kinase is cytoplasmic (Fig. 6G). Emt/Itk does not colocalize
with CD3 under resting conditions (Fig. 6, H and
I). Upon stimulation, Emt/Itk and CD3 colocalize in
clusters having similar orientations (Fig. 6, J–L).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported on the colocalization of Emt/Itk with the Ag receptor complex upon TCR/CD3 engagement (13). This event presumably brings Emt/Itk into close proximity with Lck that phosphorylates and subsequently activates Emt/Itk (3, 11). Membrane targeting of Emt/Itk and colocalization with TCR/CD3 are dependent on the PH domain of the kinase, as evidenced by the fact that a PH domain deletion mutant of Emt/Itk fails to associate with the cell membrane and does not become transphosphorylated upon TCR stimulation (13). Interestingly, when the membrane localization signal found in the N terminus of Lck was substituted for the PH domain, Emt/Itk was targeted to the membrane and colocalized with TCR/CD3, but it did not become activated upon Ag receptor engagement (13). Thus, the PH domain appears to have both a membrane-targeting function, as well as an unknown role in the inducible activation of Emt/Itk.
The finding that the Lck membrane localization signal can substitute
for the PH domain for membrane targeting and colocalization of Emt/Itk
with TCR/CD3 suggests that other domains of Emt/Itk must be responsible
for colocalization. The data presented in this paper support the
contention that the SH2 domain of Emt/Itk may be responsible for this
interaction. The interaction between Emt/Itk and TCR/CD3 is probably
indirect, and it may proceed through the participation of intermediates
such as adapter proteins. LAT, which becomes phosphorylated on
tyrosines upon activation of T cells through the Ag receptor (14, 15), is a conceivable candidate because its interaction with
Emt/Itk is mediated through the SH2 domain of the kinase (Figs. 2 and 3), and LAT can inducibly colocalize with the TCR/CD3 complex (Fig. 6).
The interaction of LAT with a multiplicity of signaling molecules such
as Grb2, PLC1, and the p85 subunit of PI3-kinase (14)
supports its role as a platform for recruiting signal transducers such
as Emt/Itk to the Ag receptor complex.
In the present study we find that the domains of Emt/Itk that are
required for its activation also correlate with binding to LAT (
Figs. 1–3). Furthermore, the data suggest that the TCR/CD3-induced
interaction between LAT and Emt/Itk is critical for the tyrosine
phosphorylation and subsequent activation of Emt/Itk. Thus, the SH2
and R265K mutants of Emt/Itk lack inducible association with LAT and
also fail to display inducible tyrosine phosphorylation and kinase
activity (
Figs. 1–3). In sharp contrast, mutations in other domains of
Emt/Itk, such as SH3 and kinase domains, have no effect on either
association with LAT or activation of Emt/Itk (Figs. 1 and 3). Further
confirmation of the importance of LAT for Emt/Itk activation is
rendered by the fact that cells deficient in LAT expression are also
deficient in the inducible activation of Emt/Itk (Fig. 4). However,
they become capable of doing so upon expression of a LAT transgene
(Fig. 5). It is interesting that, despite the deficiency in Emt/Itk
activation, LAT-deficient cells display normal early signaling events
such as CD3 and phosphorylation, as well as ZAP-70 activation
(Ref. 15 and Fig. 4). Because Lck has been claimed to be
critical for these early events (26, 27), these
observations suggest that deficiency in LAT expression has no effect on
the activity of Lck. Therefore, Lck that is also important for the
phosphorylation and activation of Emt/Itk (3, 11) must be
unable to target and activate Emt/Itk outside the context of
LAT.
The importance of LAT in the activation of Emt/Itk is also supported by the data of Shan and Wange (25). These investigators demonstrated that in ZAP-70-deficient Jurkat cells (P116 cells), LAT is not tyrosine phosphorylated and Emt/Itk does not become activated upon TCR/CD3 engagement. Furthermore, it was shown that there exists an inducible association between LAT and Emt/Itk in regular Jurkat cells, but not in the P116 mutants (25).
Recently Bunnell et al. (28), utilizing GST fusion
proteins representing various domains of Emt/Itk, were able to capture
another adapter protein, SLP-76. SLP-76 is an important adapter protein
that is phosphorylated by ZAP-70 and is required for PLC1
activation, intracellular Ca2+ mobilization, IL-2
production, and development of T lymphocytes (29, 30, 31, 32, 33).
Bunnell et al. (28) showed that GST-SH2 fusion proteins
interacted only with phosphorylated SLP-76, whereas GST-SH3 constructs
reacted with SLP-76 regardless of its phosphorylation status. Because
of the inability of full-length Emt/Itk to interact with
unphosphorylated SLP-76, these authors suggested that the SH3 domain is
sequestered in the intact Emt/Itk molecule and, furthermore, that the
finding that SLP-76 possesses adjacent binding sites for the Emt/Itk
SH2 and SH3 domains suggested that the interaction of SLP-76 with
Emt/Itk may represent a synergistic effect between these two domains
(28). A fusion protein encompassing the proline-rich
region of Emt/Itk, but excluding the SH3 and SH2 domains, was able to
interact with LAT, but not with SLP-76. Because LAT does not contain a
complementary SH3 domain to bind to the proline-rich region of Emt/Itk,
we suggest that this interaction is due to an intermediate protein such
as Grb2, which contains both SH3 and SH2 domains, with the latter known
to interact with LAT (28).
The above data suggest that the interaction of Emt/Itk with LAT may be
mediated through more than one mechanism. One model, suggested by the
data of Bunnell et al. (28), may involve a molecular
complex in which SLP-76 and Grb2 serve as intermediates. However, the
putative significance of such a complex is not clear. The interaction
with SLP-76 via the SH3 domain of Emt/Itk cannot be critical for
interacting with LAT because mutations in the SH3 domain still allow
interaction with LAT (Fig. 3). Furthermore, the activation of Emt/Itk
cannot be dependent on the presence of SLP-76 because TCR/CD3-mediated
transphosphorylation of Emt/Itk was not affected in SLP-76-deficient
cells (34). Another possibility would be a direct
interaction between Emt/Itk and LAT in which the SH2 domain of Emt/Itk
plays a critical role. This is supported by our data where SH2 domain
mutants disrupt the interaction between Emt/Itk and LAT, an event that
correlates with the TCR/CD3-induced activation of Emt/Itk. Furthermore,
the data of Bunnell et al. (28) also show an interaction
between an Emt/Itk GST-SH2 fusion construct and LAT. However, either of
the above models may not necessarily be exclusive of each other.
In summary, Emt/Itk appears to be a critical component of the signaling
complex that is assembled by LAT for the downstream propagation of
TCR/CD3-induced signals, including the activation of PLC1 and the
initiation of subsequent intracellular Ca2+
mobilization. This signaling complex contains additional molecules,
among which are SLP-76, PI3-kinase, and Grb2. Cells deficient in the
expression of Emt/Itk, LAT, or SLP-76 do indeed display deficiencies in
PLC1 activation and Ca2+ mobilization
(8, 15, 34). However, the exact interaction among these
molecules and their biological significance are not clear at this
point. Additional experimentation utilizing cells deficient in these
proteins will help address some of the important issues.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Constantine D. Tsoukas, San Diego State University, Department of Biology, Molecular Biology Institute, 5300 Campanile Drive, San Diego, CA 92182-4614.
3 Abbreviations used in this paper: Emt/Itk, expressed in mast and T cells/inducible T cell tyrosine kinase; PLC1, phospholipase C1; ZAP-70, -associated protein of 70 kDa; LAT, linker of activated T cells; PI3-kinase, phosphatidylinositol 3-kinase; SH, Src homology; GFP, green fluorescence protein; JcaM2, mutant Jurkat T cells lacking expression of LAT; wt, wild type; JTAg, SV40 T-Ag.
Received for publication March 1, 2000. Accepted for publication April 19, 2000.
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References |
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1 and the Ras pathway. Immunity 9:617.[Medline]
/CD3 induction of interleukin-2. Immunity 6:155.[Medline]
1 in an SLP-76-deficient T cell. Science 281:413.This article has been cited by other articles:
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  Y. Bogin, C. Ainey, D. Beach, and D. Yablonski SLP-76 mediates and maintains activation of the Tec family kinase ITK via the T cell antigen receptor-induced association between SLP-76 and ITK PNAS, April 17, 2007; 104(16): 6638 - 6643. [Abstract] [Full Text] [PDF]
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 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]
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 C. Tkaczyk, V. Horejsi, S. Iwaki, P. Draber, L. E. Samelson, A. B. Satterthwaite, D.-H. Nahm, D. D. Metcalfe, and A. M. Gilfillan NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc{epsilon}RI aggregation Blood, July 1, 2004; 104(1): 207 - 214. [Abstract] [Full Text] [PDF]
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 S. Goda, A. C. Quale, M. L. Woods, A. Felthauser, and Y. Shimizu Control of TCR-Mediated Activation of {beta}1 Integrins by the ZAP-70 Tyrosine Kinase Interdomain B Region and the Linker for Activation of T Cells Adapter Protein J. Immunol., May 1, 2004; 172(9): 5379 - 5387. [Abstract] [Full Text] [PDF]
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 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]
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H. M. Wilcox and L. J. Berg Itk Phosphorylation Sites Are Required for Functional Activity in Primary T Cells J. Biol. Chem., September 26, 2003; 278(39): 37112 - 37121. [Abstract] [Full Text] [PDF]
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J. A. Grasis, C. D. Browne, and C. D. Tsoukas Inducible T Cell Tyrosine Kinase Regulates Actin-Dependent Cytoskeletal Events Induced by the T Cell Antigen Receptor J. Immunol., April 15, 2003; 170(8): 3971 - 3976. [Abstract] [Full Text] [PDF]
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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]
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A. T. Miller and L. J. Berg Defective Fas Ligand Expression and Activation-Induced Cell Death in the Absence of IL-2-Inducible T Cell Kinase J. Immunol., March 1, 2002; 168(5): 2163 - 2172. [Abstract] [Full Text] [PDF]
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S. I. Gringhuis, E. A. M. Papendrecht-van der Voort, A. Leow, E. W. N. Levarht, F. C. Breedveld, and C. L. Verweij Effect of Redox Balance Alterations on Cellular Localization of LAT and Downstream T-Cell Receptor Signaling Pathways Mol. Cell. Biol., January 15, 2002; 22(2): 400 - 411. [Abstract] [Full Text] [PDF]
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 M. L. Woods and Y. Shimizu Signaling networks regulating {beta}1 integrin-mediated adhesion of T lymphocytes to extracellular matrix J. Leukoc. Biol., June 1, 2001; 69(6): 874 - 880. [Abstract] [Full Text] [PDF]
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Y. Baba, S. Hashimoto, M. Matsushita, D. Watanabe, T. Kishimoto, T. Kurosaki, and S. Tsukada BLNK mediates Syk-dependent Btk activation PNAS, February 8, 2001; (2001) 51626198. [Abstract] [Full Text]
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W. Sun, K. Kesavan, B. C. Schaefer, T. P. Garrington, M. Ware, N. L. Johnson, E. W. Gelfand, and G. L. Johnson MEKK2 Associates with the Adapter Protein Lad/RIBP and Regulates the MEK5-BMK1/ERK5 Pathway J. Biol. Chem., February 9, 2001; 276(7): 5093 - 5100. [Abstract] [Full Text] [PDF]
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