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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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chain induces the membrane
colocalization of Emt/Itk with TCR/CD3. Removal of the Emt/Itk
pleckstrin homology domain (
PH-Emt/Itk) abrogates the association of
the kinase with the cell membrane, as well as its activation-induced
colocalization with the TCR complex and subsequent tyrosine
phosphorylation. The addition of a membrane localization sequence to
PH-Emt/Itk from Lck restores all of these deficiencies except the
activation-induced tyrosine phosphorylation. Our data suggest that the
PH domain of Emt/Itk can be replaced with another membrane localization
signal without affecting the membrane targeting and activation-induced
colocalization of the kinase with the TCR. However, the PH domain is
indispensable for the activation-induced tyrosine phosphorylation of
the kinase. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After TCR stimulation, inositol phospholipids generated by
phosphatidylinositol 3-kinase recruit Emt/Itk via PH domain
interactions to the plasma membrane (5) where it comes in
proximity with Lck, which phosphorylates and activates Emt/Itk
(6, 7). Emt/Itk can also be activated by ligation of CD2
or CD28 on T cells (8, 9, 10, 11, 12) and by engagement of FcRI on
mast cells (13).
Although the downstream targets of Emt/Itk are unknown,
Emt/Itk-deficient mice display compromised phospholipase C-
1
phosphorylation and reduced extracellular calcium flux in response to
TCR stimulation (2). These events, along with others such
as activation of Src tyrosine kinases, are important early signals that
follow the engagement of the TCR (14). The interaction of
the TCR with antigenic peptide-MHC on APC induces the formation of
molecular clusters that, in addition to TCR/CD3, contain additional
cell surface and intracellular proteins that are critical for the
propagation of T cell activation signals (15, 16, 17). A
number of proteins, including the CD4 and CD8 coreceptors and the Src
family kinases Lck and Fyn, have been shown to colocalize with the
TCR/CD3 complex and facilitate transduction of the antigenic signal
(17, 18, 19).
Stimulation of T cells through CD28 induces the physical association of
this coreceptor with Emt/Itk, as demonstrated by coimmunoprecipitation
analysis (11, 12). Similar experiments, however, where
stimulation through TCR/CD3 has been attempted, have failed to
reproducibly reveal an inducible association of Emt/Itk with the TCR
complex (Refs. (6, 9) and our own unpublished
observations). These negative data do not necessarily rule out an
inducible physical association between Emt/Itk and TCR/CD3, but instead
they may point out the possibility that such an interaction may not be
amenable to biochemical analysis utilizing detergents and
coimmunoprecipitation techniques. To assess this, we have utilized
laser confocal microscopy where the interaction of Emt/Itk and TCR/CD3
is studied in its natural milieu at the single cell level. The data
indicate that Emt/Itk colocalizes with TCR/CD3 molecular clusters upon
CD3 engagement in a manner similar to that for CD28-Emt/Itk
colocalization. This phenomenon depends on the membrane association of
Emt/Itk through its PH domain and correlates with TCR-induced
activation of Emt/Itk as assessed by tyrosine phosphorylation. Finally,
the PH domain is indispensable for the activation of Emt/Itk through
TCR/CD3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SV40 T Ag-transfected human leukemic Jurkat T cells (JTAg) were
cultured at 37°C with RPMI 1640 (Irvine Scientific, Irvine, CA)
containing 10 mM HEPES, 2 mM L-glutamine (Sigma,
St. Louis, MO), 8% FBS (HyClone, Logan, UT) in a humidified 5%
CO2 atmosphere. Jurkat cells, 20 x
106 in 400 µl, were transfected with 20 µg of
the desired plasmid DNA by electroporation (Bio-Rad gene pulser,
Hercules, CA) in a 0.4-cm-gap electrocuvette (DocFrugal’s, San Diego,
CA) at 960 µF and 240 mV. After 48 h of culturing, 
10% of
the cells expressed visible levels of the transfected protein, as
assessed by green fluorescent protein (GFP) fluorescence.
Cloning
Wild type (wt) murine Emt/Itk (20) was cloned into
the EcoRI-SpeI sites of the pME18 expression
vector (21). Truncation of the PH domain (PH) was
performed by deletion of the PvuII fragment, containing the
PH and part of the TH domain; religation; and subcloning into the
EcoRI-EcoRV sites of pME18s-wt-Emt/Itk.
The H902 epitope tag, consisting of aa 319–333 of HIV gp120, was added
by PCR amplification (30 cycles, 94°C, 1 min; 51°C, 2 min; 72°C,
2 min) of pME18s-Wt-Emt/Itk using the following primers: sense primer
5'-GGAAGCCATGGCCCGTATCCAGAGAGGACCAGGGAGAGCATTTGTTACAATAGGAAAAATGAACAACTTCATCCTCCT-3'
(codingfor aa 319–333 of gp120 and aa 1–7 of Emt/Itk) and
antisense primer 5'-TTCCCATATCTTAGCCCTGC-3' (coding for aa 348–354 of
Emt/Itk). Addition of H902 tag to PH-Emt/Itk was performed similarly
with the same sense primer and the antisense primer
5'-CTCATAGGTTTCAAGGT-3' (coding for aa 233–238 of Emt/Itk). The
PCR products were resolved on a 1% agarose gel, isolated and purified
with the Geneclean II kit (Bio 101, Vista, CA), and cloned into the
pCR2.1 vector with the use of the TA cloning kit (Invitrogen, Carlsbad,
CA) according to the manufacturer’s instructions. Finally, the
EcoRI-ScaI fragments of the pCR2.1 constructs
were subcloned into the EcoRI-ScaI sites of
pME18s-Wt-Emt/Itk.
GFP was added to the C terminus of Emt/Itk by removing the stop codon
and creating an in-frame KpnI site by PCR amplification of
H902-tagged pME18s-Wt-Emt/Itk or H902-tagged pME18s-PH-Emt/Itk. The
following primers were used: sense primer
5'-TTGAATTCCGTTGGAAGCCATGGCCCGTAT-3' (containing an
EcoRI site and aa 1–4 of the H902 epitope tag), antisense
primer 5'-TTTCTAGAGGTACCCAAGCCCAGCTTCTGCGATTT-3' (containing aa
613–619 of Emt/Itk, KpnI, and XbaI sites). The
PCR products were purified as described above and subcloned into the
EcoRI-KpnI sites of pEGFP-N2 expression vector
(Clontech, Palo Alto, CA).
Addition of the first 12 amino acids of Lck to the
pEGFP-PH-Emt/Itk-GFP was performed by PCR amplification with the
following primers: sense primer
5'-AAGAATTCGAAGCCATGGGCTGTGTCTGCAGCTCAAACCCTGAAGATGACGCCCGTATCCAGAGAGGACC-3'
(containing aa 1–12 of Lck and aa 1–7 of H902 tag) and the antisense
primer described in the previous paragraph for the addition of GFP. The
PCR product was cloned into pCR2.1, and the
EcoRI-ScaI fragment was subcloned to
pEGFP-Wt-Emt/Itk-GFP.
The above constructs were confirmed by sequencing.
Immunoprecipitation and Western blotting
JTAg cells, 30 x 106, were lysed with 1 ml of lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris (pH 7.3), 0.4 mM EDTA, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 1 mM sodium o-vanadate, and 1 mM PMSF) for 1 h at 4°C. Cell lysates were incubated for 2 h at 4°C with 5 µg monoclonal anti-H902 (IgG1) Ab (National Institutes of Health AIDS Reference Reagent Program, Rockville, MD), followed by overnight incubation with 30 µl protein G-Sepharose at 4°C. Immune complexes were washed three times with lysis buffer, resolved by 8% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Gelman Sciences, Ann Arbor, MI). Western blotting analysis was done as previously described (22). Monoclonal anti-phosphotyrosine Ab PY-20 was provided by Dr. Bartholomew Sefton (Salk Institute, La Jolla, CA), and anti-Emt/Itk antiserum was produced in rabbits immunized with a GST fusion protein as previously described (23).
Autophosphorylation assay
Emt/Itk immune complexes, prepared as described above, were washed once with kinase reaction buffer (50 mM HEPES (pH 7.4), 2 mM MnCl2, 200 µM ATP, 10 mM MgCl2, and 10 mM DTT) and then resuspended in 100 µl of the same buffer. The immune complexes were incubated for 1 h at 37°C, resolved by SDS-PAGE (8%), and analyzed by Western blotting with anti-phosphotyrosine Abs.
Stimulation and fluorescence labeling
For stimulation, JTAg cells, 30 x
106, were incubated (30 min on ice) in RPMI 1640
culture medium containing 20 µg/ml concentrations of the
anti-CD3 mAb OKT3 (hybridoma obtained from American Type Culture
Collection, Manassas, VA) or an isotype (IgG2a) control Ab UPC-10
(Bionetics, Charleston, SC). Cells were washed, resuspended in culture
medium containing 20 µg/ml of rabbit anti-mouse IgG
(cross-linking Ab, Jackson Immunoresearch, West Grove, PA), and
incubated at 37°C for 3 min. After incubation, the cells were placed
on ice until further analysis.
For fluorescence microscopy, 2 x 106 cells were incubated (30 min on ice) in 250 µl of culture medium containing 40 µg/ml Ab OKT3. After a washing, the cells were incubated similarly in culture medium containing 20 µg/ml Texas Red-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR). The cells were then exposed to 37°C for 10 min to induce capping and were immediately cooled with 1 ml ice-cold culture medium containing 0.1% sodium azide to block TCR internalization. After two washes in azide-containing medium, the cells were placed on poly-L-lysine-coated glass slides and fixed with 2% paraformaldehyde in PBS. The slides were mounted with Prolong Anti-fade (Molecular Probes) according to the manufacturer’s instructions.
Laser scanning confocal microscopy
Confocal imaging was performed using the Bio-Rad MRC1024 LSCM system. GFP and Texas Red were imaged separately to avoid channel bleed-through. GFP was excited at 488 nm and read through the 522–535 nm channel PMT, whereas Texas Red was excited at 568 nm and detected by the 598–640 nm channel PMT. Images were acquired with the Bio-Rad LaserSharp software, converted with GraphicConverter (Lemke Software, Peine, Germany), and pseudocolored and overlaid with Adobe Photoshop 4.0 (Adobe, San Jose, CA). The relative amount of membrane-associated Emt/Itk was calculated by measuring pixel intensity with ImageQuant software version 1.1 (Molecular Dynamics, Sunnyvale, CA). To this end, the total amount of GFP fluorescence was determined by defining a gate that included the whole cell. The intracytoplasmic GFP fluorescence was determined by setting a gate as above but excluding the cell membrane. The fraction of membrane-associated GFP was calculated as the total minus intracytoplasmic over the total GFP.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Emt/Itk is important in the propagation of signals initiated both
through the TCR/CD3 molecular complex and through the CD28 coreceptor
molecule (6, 11, 24). Cross-linking of CD28 with specific
Abs induces the coimmunoprecipitation of Emt/Itk and CD28 (11, 12). However, under the same experimental conditions, an
association between Emt/Itk and TCR/CD3 has not been demonstrated
(6, 9). In our own studies (unpublished observations), we
have observed association of CD3 and Emt/Itk after anti-CD3
Ab immunoprecipitation of Brij 97 cell lysates. However, this
observation is not consistently reproducible. A possible explanation of
such findings is that a putative association among these proteins may
not be amenable to analysis by detergent solubilization and
immunoprecipitation techniques.
Here, we have reexamined this issue by utilizing GFP-tagged Emt/Itk and
fluorescence confocal microscopy. We have prepared fusion Emt/Itk
constructs, where GFP has been cloned at the C terminus of the kinase,
and analyzed expression in transiently transfected cells by laser
scanning confocal microscopy. The presence of GFP increases the
relative molecular size of the protein to 102 kDa and does not
interfere with either the TCR/CD3-induced phosphorylation (Fig. 3
E) or the kinase activity (Fig. 3F) of Emt/Itk.
In transiently transfected JTAg cells, wt-Emt/Itk-GFP is primarily
extranuclear, and it is distributed diffusely throughout the cytoplasm
with a distinct perimembranous localization (Fig. 1A).
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Ab
OKT3 and cross-linked with Texas Red-conjugated rabbit anti-mouse
IgG. Cells were examined by confocal microscopy before (nonstimulated
cultures) or after (stimulated cultures) incubation at 37°C for 10
min. Before exposure to 37°C, CD3 (red) appears as a punctate
circumference around the plasma membrane (Fig. 1
C), whereas
the membrane-associated Emt/Itk (green) forms a contiguous
perimembranous ring (Fig. 1A). Under these conditions, the
two signals do not colocalize (Fig. 1B). In cells incubated
at 37°C, both Emt/Itk and CD3 form similarly shaped clusters
("caps") of identical cellular localization (Fig. 1, D
and F, respectively). Superimposition of the two images
confirms the OKT3-induced colocalization of Emt/Itk and CD3 (Fig. 1E). This colocalization event was verified in four
independent experiments where every examined cell displaying CD3
"capping" also displayed Emt/Itk coclustering with the features
described above. To obtain an estimate of the fraction of total Emt/Itk-GFP that is membrane associated, we quantified GFP fluorescence in both stimulated and nonstimulated cells as described in Materials and Methods. Analysis of nine individual nonstimulated cells revealed that 28.9 ± 4.3% (average ± SD) of total Emt/Itk-GFP was membrane associated. Similar analysis of 10 individual stimulated cells revealed a significant (p < 0.001) increase in the fraction of membrane-associated kinase to 40.4 ± 6.2%.
Other investigators have previously established the inducible
association of Emt/Itk and the CD28 coreceptor utilizing a biochemical
approach (11, 12). In experiments similar to those
described above, we were able to confirm this association at the
single-cell level using confocal microscopy. Thus, CD28 cross-linking
induces membrane redistribution of Emt/Itk and its colocalization with
CD28 (Fig. 1, G–L).
The Emt/Itk PH domain is important for association with TCR/CD3
To further explore the association of Emt/Itk with CD3, we
examined the role of the Emt/Itk PH domain in this event. PH
do- mains can interact with membrane phospholipids and thus
allow recruitment of the PH domain-containing protein to the cell
membrane for potential interaction with other membrane-associated
structures (25). We constructed a truncation mutant of
Emt/Itk-GFP where the PH domain was deleted and transiently transfected
it into JTAg cells. In contrast to wt-Emt/Itk-GFP, the
PH-Emt/Itk-GFP does not display enhanced membrane localization, and
it is rather diffusely cytoplasmic (Fig. 2A). In stimulated cells
(incubation with Ab OKT3 and cross-linking with Texas red-conjugated
anti-mouse IgG at 37°C), PH-Emt/Itk fails to localize in
distinct patches as those seen in cells transfected with wt-Emt/Itk
(compare Fig. 1D with Fig. 2D). This occurs
despite the "capping" of CD3 (Fig. 2F). Thus, no
colocalization of Emt/Itk with CD3 occurs (Fig. 2E). The
above experiments were also performed with CD28-induced activation
using Ab 9.3 with identical results. Thus, PH-Emt/Itk fails to
colocalize with CD28 also (Fig. 2, G–L). In the above
experiments, we cannot totally exclude the possibility that small,
undetectable amounts of PH-Emt/Itk associate with the cell membrane
through a non-PH domain-mediated mechanism. The PH-Emt/Itk-GFP
transfectants look uniformly brighter than their wt-Emt/Itk-GFP
counterparts not because of higher levels of expression but because the
sensitivity of the photomultiplier tube was calibrated to the strongest
signal emitted by the cell, caused by the relatively uniform and
diffuse cytoplasmic fluorescence of the PH-Emt/Itk-GFP.
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Lck has been shown to be critical for the TCR/CD3-induced
activation of Emt/Itk, in that it is needed for
trans-phosphorylating and subsequently activating Emt/Itk
(6, 7). Therefore, association with the cell membrane is
an important requirement for Emt/Itk to be targeted by Lck. Thus, as it
might be predicted, PH-Emt/Itk transiently transfected into JTAg
cells fails to become tyrosine phosphorylated on incubation with Ab
OKT3 (Fig. 3A,
top). In contrast, similarly transfected wt-Emt/Itk becomes
phosphorylated (Fig. 3A, top). This latter finding is in
agreement with previously published observations (6, 7).
Similar experiments performed with anti-CD28 stimulation indicate
that PH-Emt/Itk is not phosphorylated in response to Ab 9.3
stimulation either (data not shown). The lack of PH-Emt/Itk
phosphorylation is specific, given that other target proteins do become
tyrosine phosphorylated upon stimulation (indicated by
anti-phosphotyrosine Western blotting of total cell lysates; data
not shown).
Tyrosine phosphorylation of Emt/Itk correlates with its kinase activity
(6, 7, 11). However, when we tested kinase activity by
autophosphorylation assay, we were surprised to see that in spite of
its lack of anti-CD3-induced trans-phosphorylation (Fig. 3A, top), PH-Emt/Itk has a significantly
higher spontaneous kinase activity than did the wt control (Fig. 3C, top). Densitometric analysis of the signals indicates
that the baseline in vitro kinase activity of PH-Emt/Itk is 3.5-fold
higher than that of wt-Emt/Itk. It is interesting that increases in
basal kinase activity upon removal of the PH domain have been also
observed with PH mutants of protein kinase B (26) and
protein kinase D (27). The addition of GFP does not alter
the inducible trans-phosphorylation of Emt/Itk (Fig. 3E). Furthermore, GFP linkage does not interfere with
autophosphorylation of the PH mutant (Fig. 3F). In Fig. 3A, we do not detect autophosphorylation of the PH mutant
because these experiments are performed in vivo and they only reflect
trans-phosphorylation presumably by Src family kinases. The
results of the above experiments cannot be attributed to unequal
protein concentrations in each lane, because probing with
anti-Emt/Itk antiserum reveals equal loading (Fig. 3, A
and C, bottom).
The PH-Emt/Itk gene product migrates as two bands (Fig. 3, A and C, bottom). We do not know the reason, but
possibly it might be due to alternative splicing. The same is
true for PH-Emt/Itk-GFP (data not shown). In addition, the higher
m.w. form appears to be more intensely phosphorylated than the lower
form (data not shown).
Membrane targeting of PH-Emt/Itk restores its colocalization
with TCR/CD3, but not its inducible activation
The lack of PH-Emt/Itk membrane localization raised the
question whether the PH domain serves merely as a membrane targeting
signal or whether it is also necessary for other events related to
Emt/Itk activation. To address this, we further modified the
PH-Emt/Itk-GFP by adding to its N terminus a membrane localization
sequence consisting of the first 12 amino acid residues of Lck, which
provides a myristoylation and acylation target sequence
(28). JTAg cells were transiently transfected with this
construct (mPH-Emt/Itk) and stimulated as described above. The
addition of the Lck-membrane localization sequence allows the PH domain
truncation mutant to localize to the cell membrane (Fig. 4A), and to colocalize with
the TCR/CD3 complex on stimulation with Ab OKT3 (Fig. 4
D–F). Similar results were obtained
when transfectants were stimulated with Ab 9.3 (Fig. 4, G–L). To ensure that colocalization of the mPH-Emt/Itk
with TCR/CD3 and CD28 is specific, we added the Lck membrane
localization signal to GFP alone (mGFP) and tested its localization in
transiently transfected JTAg cells. Even though mGFP localized to the
membrane (Fig. 4M), there was no colocalization with TCR/CD3
on OKT3 stimulation (Fig. 4, M–R). Similar results were
obtained with 9.3 stimulation (data not shown). Surprisingly, however,
transiently transfected JTAg cells do not display tyrosine
phosphorylation of mPH-Emt/Itk after incubation with Ab OKT3 (Fig. 3B, top). Furthermore, mPH-Emt/Itk did not display the
high basal autokinase activity seen with the PH-Emt/Itk mutant (Fig. 3D, top). Stimulation of mPH-Emt/Itk transfected cells
with Ab 9.3 also failed to reveal trans-phosphorylation and
kinase activity of the transfected mutant protein (data not shown). The
possibility that lack of phosphorylation of mPH-Emt/Itk is caused by
inactivation of endogenous Lck is not likely. Lowin-Kropf et al.
(29) have demonstrated that TCR/CD3 clustering ("cap"
formation) and cytoskeletal reorganization during T cell and APC
interaction depend on the presence of active Lck. Lck-deficient Jurkat
T cells do not display these events, but they do so when the Lck gene
is reconstituted by transfection (29). Because expression
of the PH-Emt/It mutant allows "cap" formation, we conclude that
Lck must be active.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PH domains are important targeting signals that allow proteins containing them to be recruited to the cell membrane through interaction with membrane phospholipids (25). The biological importance of PH domains is underscored by observations with the tyrosine kinase Btk. Mutations in this Tec family kinase have been associated with human X-linked agammaglobulinemia (30, 31) and murine X-linked immunodeficiency (32, 33). Many of the identified mutations have been localized in the PH domain of Btk (34), and they interfere with the ability of the mutant kinase to interact with specific membrane phospholipids and become activated (35, 36, 37).
After TCR-mediated stimulation, inositol phospholipids recruit Emt/Itk
via PH domain interactions to the plasma membrane (5) and
presumably bring it to close proximity with Lck which in turn
phosphorylates and activates Emt/Itk (6, 7). Thus,
deletion of the PH domain would not allow Emt/Itk to localize to the
appropriate molecular clusters on the cell membrane upon TCR
engagement, come to proximity with Lck, and become phosphorylated. In
fact, the data presented here support this contention, in that
PH-Emt/Itk fails to localize to the cell membrane and it does not
become phosphorylated on tyrosines upon TCR stimulation. This result
raised the question of whether the PH domain simply plays a
membrane-targeting role or has additional functions related to the
activation of Emt/Itk. To address this issue, we modified our
PH-Emt/Itk construct by adding to its N terminus the first 12 amino
acids of Lck (mPH-Emt/Itk). These residues are targets for
myristoylation/acylation and membrane localization of Lck
(28). Indeed, the mPH-Emt/Itk was targeted to the cell
membrane, and it coclustered with TCR/CD3 and CD28 upon ligation of
these receptors. However, to our surprise, it did not become tyrosine
phosphorylated upon stimulation.
August et al. (5) targeted a PH-Emt/Itk mutant to the
cell membrane of COS-7 cells by the addition of the murine c-Kit
extracellular and transmembrane domains and found that upon
coexpression of Src, the targeted PH-Emt/Itk becomes tyrosine
phosphorylated and enzymatically active. This seems to be in
disagreement with our data where membrane-targeted mPH-Emt/Itk does
not display significant trans-phosphorylation or enzymatic
activity upon TCR/CD3 ligation (Fig. 3). However, there might be
several reasons to explain the discrepancy between the results of
August et al. and those presented here. These include the use of
different cellular systems (nonlymphoid COS vs lymphoid Jurkat cells),
different modes of Emt/Itk activation (Src overexpression vs TCR/CD3
stimulation), and different membrane localization signals
(c-kit vs Lck signal).
One explanation of why mPH-Emt/Itk, although colocalized with
TCR/CD3, cannot become trans-phosphorylated on stimulation
could be that the PH domain may be critical for endowing the
appropriate conformation for Emt/Itk to become phosphorylated by Lck.
This contention is supported by the data of Andreotti et al. who
analyzed a fragment of Emt/Itk by multidimensional nuclear magnetic
resonance and found an intramolecular association between the SH3
domain and a proline-rich sequence located between the PH and SH3
domains (38). Thus, intramolecular interactions may be
important in regulating the interaction of Emt/Itk with its
targets/substrates and its subsequent activation. If the absence of the
PH domain causes conformational changes that affect receptor-induced
phosphorylation of Emt/Itk, such changes are unlikely to be global so
to affect the conformation of the whole molecule, because
mPH-Emt/Itk can colocalize inducibly with both TCR/CD3 and CD28 in a
manner similar to that of the nonmutant protein (Fig. 4).
Another explanation for the lack of phosphorylation of mPH-Emt/Itk
could be the inability of the mutant kinase to associate with other
critical signaling molecules through an interaction that is dependent
on the PH domain. The interaction of PH domains with membrane
phospholipids has been well documented (25). For example,
the PH domain of Emt/Itk can bind to inositol D3 phosphates, most
notably inositol 1,2,3,4,5-hexaphosphate (5). The PH
domain of Btk can interact with IP4,
IP5, IP6 (35),
and PI(3, 4, 5)P3 in a manner dependent on the
activation of phosphatidylinositol 3-kinase (39, 40, 41).
Mutations found in the PH domain of Btk from X-linked
agammaglobulinemia patients impair lipid-PH interactions and hamper
enzymatic activity (35, 41).
Other signaling proteins with which Emt/Itk and Btk have been shown to
interact are heterotrimeric GTP-binding proteins. Thus, the
G
q, G12 and Gß
subunits of GTP-binding proteins can interact with and activate the
enzymatic activity of Emt/Itk and Btk both in vivo and in vitro
(42, 43, 44, 45). The interaction of Btk with
Gq and G12 is
mediated through distinct domains. Gq binds to
a region composed of the TH and SH3 domains (42), whereas
G12 interacts with the PH domain and the
adjacent Btk motif (43). In our own studies, a Jurkat cell
line expressing a "dominant-negative" mutant of
G16 (46) reveals deficient
TCR/CD3-induced tyrosine phosphorylation of Emt/Itk (unpublished
observations) consistent with the interpretation that heterotrimeric
GTP-binding proteins are involved in the activation of this Tec kinase.
In addition to the above, there are still other signaling proteins that
have been shown to interact with Emt/Itk and Btk including protein
kinase C (13, 47) and BAP-135 (48).
The inducible association between Emt/Itk and CD28, previously demonstrated by coimmunoprecipitation (11, 12), has been confirmed here at the single-cell level by laser scanning confocal microscopy. The fact that a similar association between Emt/Itk and TCR/CD3 can be demonstrated only by confocal microscopy, but not by biochemical means (Refs. 6, 9 and our own unpublished results), suggests that this association may be sensitive to the manipulations required (e.g., detergent solubilization) for coimmunoprecipitation analysis. Another association that has been refractory to coimmunoprecipitation analysis but revealed by confocal microscopy is the one between Btk and the B cell Ag receptor (49).
In our experiments, we noticed that about one-third of total cellular
Emt/Itk-GFP was associated with the cell membrane in nonstimulated
Jurkat T cells; this amount increased significantly on stimulation
through TCR/CD3. The association of Emt/Itk-GFP with the cell membrane,
under nonstimulating conditions, cannot be attributed to the fact that
the protein has been modified by the addition of GFP because
PH-Emt/Itk-GFP does not accumulate in the plasma membrane (Fig. 2).
Furthermore, other investigators using different techniques have seen
association of both Emt/Itk and Btk with membrane components. Thus, use
of a biochemical cell fractionation approach reveals that 20% of
the total Emt/Itk in resting Jurkat T cells is associated with light
vesicles (L. Berg, unpublished observations). Nisitani et al.
(49) studied the colocalization of Btk with the B cell
receptor complex in Ramos B cells using specific Abs and confocal
microscopy. It is clear from their data that under "no stimulation"
conditions there exists significant association of Btk with the cell
membrane that increases on Ag receptor cross-linking. Thus, it appears
that in the cell lines studied, although the majority of Btk and
Emt/Itk remains cytoplasmic under nonstimulating conditions, a
significant proportion associates with the cell membrane.
Monks et al. (17) have recently demonstrated that on T
cell activation the Ag receptor complex along with other signal
transduction proteins, including protein kinase C-
, Fyn, Lck, and
LFA-1, are clustered into segregated three-dimensional domains within
the contacts between T cells and APC. Monks et al. have termed these
clusters supramolecular activation clusters (SMAC), and they have
demonstrated that upon activation proteins in the SMACs are
compartmentalized into central and peripheral clusters in reference to
the TCR (17). Thus, Lck and Fyn kinases seem to colocalize
in the central SMACs along with TCR/CD3, whereas LFA-1 remains
peripherally confined in the peripheral SMACs. The data presented here
support a two-step model where, upon stimulation, Emt/Itk first becomes
recruited to the cSMACs and then, in the close proximity of Src
kinases, becomes trans-phosphorylated and activated. The PH
domain seems to serve a dual role in this process; it mediates
targeting of Emt/Itk to the cell membrane and it is also involved in
its activation.
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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, Department of Biology, San Diego State University, San Diego, CA 92131-4614. E-mail address:
3 Abbreviations used in this paper: Emt/Itk, expressed in mast and T cells/inducible T cell tyrosine kinase; PH, pleckstrin homology; TH, Tec homology; SH, Src homology; GFP, green fluorescent protein; JTAg, SV40 T-Ag-transfected Jurkat cells; mPH-Emt/Itk, membrane-targeted PH-Emt/Itk; mGFP, membrane-targeted green fluorescent protein; wt, wild type; SMAC, supramolecular activation clusters.
Received for publication May 7, 1999. Accepted for publication September 21, 1999.
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on its effector Bruton’s tyrosine kinase. Proc. Natl. Acad. Sci. USA 95:12197.12 stimulates Bruton’s tyrosine kinase and a rasGAP through a conserved PH/BM domain. Nature 395:808.[Medline]
subunits. Proc. Natl. Acad. Sci. USA 92:8601.This article has been cited by other articles:
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  Y. H. Huang, J. A. Grasis, A. T. Miller, R. Xu, S. Soonthornvacharin, A. H. Andreotti, C. D. Tsoukas, M. P. Cooke, and K. Sauer Positive Regulation of Itk PH Domain Function by Soluble IP4 Science, May 11, 2007; 316(5826): 886 - 889. [Abstract] [Full Text] [PDF]
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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]
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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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W.-C. Yang, K. A. Ching, C. D. Tsoukas, and L. J. Berg Tec Kinase Signaling in T Cells Is Regulated by Phosphatidylinositol 3-Kinase and the Tec Pleckstrin Homology Domain J. Immunol., January 1, 2001; 166(1): 387 - 395. [Abstract] [Full Text] [PDF]
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X. Shan, M. J. Czar, S. C. Bunnell, P. Liu, Y. Liu, P. L. Schwartzberg, and R. L. Wange Deficiency of PTEN in Jurkat T Cells Causes Constitutive Localization of Itk to the Plasma Membrane and Hyperresponsiveness to CD3 Stimulation Mol. Cell. Biol., September 15, 2000; 20(18): 6945 - 6957. [Abstract] [Full Text]
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K. A. Ching, J. A. Grasis, P. Tailor, Y. Kawakami, T. Kawakami, and C. D. Tsoukas TCR/CD3-Induced Activation and Binding of Emt/Itk to Linker of Activated T Cell Complexes: Requirement for the Src Homology 2 Domain J. Immunol., July 1, 2000; 165(1): 256 - 262. [Abstract] [Full Text] [PDF]
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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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A. J. Mohamed, L. Vargas, B. F. Nore, C.-M. Backesjo, B. Christensson, and C. I. E. Smith Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase J. Biol. Chem., December 15, 2000; 275(51): 40614 - 40619. [Abstract] [Full Text] [PDF]
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