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Phosphatidylinositol 3-Kinase Is Required for
CD28 But Not CD3 Regulation of the TEC
Family Tyrosine Kinase EMT/ITK/TSK:
Functional and Physical Interaction of EMT
with Phosphatidylinositol 3-Kinase
Yiling Lu, Bruce Cuevas, Spencer Gibson, Humera Khan,
Ruth LaPushin, John Imboden and Gordon B. Mills
J Immunol 1998; 161:5404-5412; ;
http://www.jimmunol.org/content/161/10/5404
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Copyright © 1998 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Phosphatidylinositol 3-Kinase Is Required for CD28 But Not
CD3 Regulation of the TEC Family Tyrosine Kinase
EMT/ITK/TSK: Functional and Physical Interaction of EMT
with Phosphatidylinositol 3-Kinase1
Yiling Lu,* Bruce Cuevas,* Spencer Gibson,*‡ Humera Khan,* Ruth LaPushin,*
John Imboden,† and Gordon B. Mills2*
F
unctional T cell activation requires at least two signals:
one is delivered by the interaction of the Ag-specific TCR
with peptide in the context of the MHC on APCs, and the
second is delivered by costimulatory T cell surface molecules
binding to their cognate receptors on APC (1–3). Engagement of
the TCR without concurrent ligation of appropriate costimulatory
molecules leads to T cell clonal anergy or programmed cell death
through apoptosis (1–3). CD28, a T cell surface glycoprotein, is a
potent costimulatory molecule for T cell activation (1– 4). Ligation
of CD28 by its natural ligands, CD80 and CD86, on APC or by
anti-CD28 Abs enhances T cell proliferation and cytokine production initiated via the TCR/CD3 complex (5–9). CD28 also plays a
critical role in preventing the induction of T cell unresponsiveness
and apoptosis (10 –13).
Ligation of CD28 induces an immediate and transient increase
in tyrosine phosphorylation of specific intracellular substrates including CD28 itself (14, 15). Because CD28 does not possess intrinsic kinase activity (16), an intracellular nonreceptor tyrosine
kinase(s) must be recruited to mediate this activity. Ligation of
*Department of Molecular Oncology, Division of Medicine, University of Texas
M. D. Anderson Cancer Center, Houston, TX 77030; †Rosalind Russell Arthritis
Research Laboratory, Department of Medicine, University of California, San Francisco,
CA 94143; and ‡National Jewish Medical and Research Center, Denver, CO 80206
Received for publication January 29, 1998. Accepted for publication July 21, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants CA74247 (to
G.B.M.) and A126644 (to J.I.).
2
Address correspondence and reprint requests to Dr. Gordon B. Mills, University of
Texas M. D. Anderson Cancer Center, Department of Molecular Oncology, 1515
Holcombe Blvd., Box 92, Houston, TX 77030.
Copyright © 1998 by The American Association of Immunologists
CD28 activates the SRC family tyrosine kinase members, LCK
and FYN, and their kinase activities can be detected in CD28 immune complexes (17, 18). Following ligation of CD28, the
EMT/ITK/TSK (EMT)3 TEC family protein tyrosine kinase is activated and recruited to CD28 in a LCK-dependent manner, placing LCK upstream of EMT in a tyrosine kinase cascade activated
by CD28 (19, 20). Further, LCK can induce EMT phosphorylation
following coexpression in Cos 7 cells (20), and LCK increases
EMT kinase activity when coexpressed in bacculovirus-infected
insect cells through phosphorylation of Tyr511 (21). The intracellular domain of CD28 contains four potential tyrosine phosphorylation sites (Y170, Y185, Y188, and Y197). At least in vitro, both
EMT and LCK are capable of phosphorylating Tyr170 in the
YMNM phosphatidylinositol 39 kinase (PI3K) and GRB2 consensus binding site in CD28, whereas EMT, but not LCK, appears
competent to phosphorylate the distal three tyrosines of CD28
(22). Phosphorylation of Tyr170 and recruitment of PI3K to CD28
is critical for optimal IL-2 production in some T cell lines but not
others (23–27). In contrast, phosphorylation of the distal three tyrosines of CD28 and in particular Tyr188 appears critical for optimal IL-2 production (27–29).
Knockout mice, lacking EMT, demonstrate decreased antiviral
CTL immune responses and responses mediated by transgenic
TCR, while maintaining normal B cell immune responses to viruses and mitogens (30, 31), indicating that EMT likely plays a
positive role in some but not all TCR-mediated activation events.
3
Abbreviations used in this paper: EMT, protein tyrosine kinase expressed mainly in
T cells; PI3K, phosphatidylinositol 3-kinase; ITK, inducible T cell kinase; TSK, T
cell-specific kinase; PH, pleckstrin homology; SH2, SRC homology 2; SH3, SRC
homology 3; HRP, horseradish peroxidase; PVDF, polyvinylidene difluoride; ECL,
enhanced chemiluminescence.
0022-1767/98/$02.00
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Ligation of the TCR or CD28 induces activation of phosphatidylinositol 3-kinase (PI3K), the TEC family protein tyrosine kinase,
EMT/ITK/TSK (EMT), and the SRC family tyrosine kinase, LCK. LCK is required for the activation and phosphorylation of
EMT induced by ligation of the TCR or CD28 placing LCK upstream of EMT in T cell signaling cascades. We report herein that
inhibition of PI3K activity with the specific inhibitors LY294002 and wortmannin markedly decreased EMT activation induced
by CD28 cross-linking but not by CD3 cross-linking. Further, inhibition of PI3K markedly decreased EMT in vitro autokinase
activity induced by activated LCK. In contrast, PI3K inhibitors did not alter CD28 or CD3 cross-linking or LCK-induced EMT
phosphorylation. Consistent with the requirement of PI3K activity for CD28 but not CD3-induced stimulation of the EMT in vitro
autokinase activity, a small but significant portion of cellular EMT associates with PI3K following CD28 cross-linking but not
following CD3 cross-linking. CD28-induced association of EMT with PI3K also requires functional expression of LCK. Fusion
proteins containing the SRC homology 2 domain of EMT interact with PI3K or a PI3K-associated molecule in a tyrosine phosphorylation-dependent manner. Taken together, the data suggest that EMT is differentially regulated and recruited to different
signaling complexes following ligation of CD28 or the TCR complex, perhaps contributing to the disparate roles that EMT appears
to play downstream of CD28 and the TCR. The Journal of Immunology, 1998, 161: 5404 –5412.
The Journal of Immunology
following CD28 ligation potentially places EMT in the context of
a different set of targets and regulatory molecules as compared
with CD3 ligation, which does not induce association of EMT
with PI3K.
Materials and Methods
Abs and reagents
The anti-human CD28 mAb (9.3, IgG2a) was a gift of Dr. J. Ledbetter
(Bristol-Myers Squibb Research Institute, Seattle, WA). The mAb against
the e-chain of the CD3 complex (UCHT1, IgG1) was purified from the
supernatants of hybridoma cells provided by Dr. Peter Beverly (University
College, London, U.K.). Rabbit anti-mouse IgG was purchased from Western Blotting (Toronto, Ontario, Canada). The rabbit polyclonal Ab against
PI3K p85 subunit and the anti-phosphotyrosine mAb (4G10, IgG1) as well
as the mAb against EMT (clone 2F12, used for immunoblotting) were
purchased from Upstate Biotechnology (Lake Placid, NY). A goat antiEMT Ab (used for immunoblotting) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The production and specificity of the rabbit
anti-EMT serum used for immunoprecipitation has been described previously (59). In some experiments, the polyclonal anti-EMT Abs were further affinity purified using bacterially expressed glutathione S-transferase
fusion proteins containing EMT, and the purified Ab was linked to biotin.
Anti-TrpE mAb was purchased from Oncogene (Cambridge, MA). Protein
A conjugated with horseradish peroxidase (HRP) and [g-32P]ATP were
purchased from Amersham (Arlington Heights, IL). Goat anti-mouse IgG
Ab conjugated with HRP was purchased from BioRad (Hercules, CA).
Protein A conjugated Sepharose 4B was purchased from Pharmacia Biotech (Piscataway, NJ). TrpE fusion proteins containing the SH2 domain
(amino acid 242 to 342) of EMT were derived from PCR products and
verified by sequencing. LY294002 was purchased form Calbiochem (La
Jolla, CA), and wortmannin was purchased from Sigma (St. Louis, MO).
Expression vectors
Full length wild-type EMT cDNA was cloned in pSG5. Constitutively
activated LCK Y505F, a gift from Dr. Andre Veillette (McGill, Quebec,
Canada), was cloned in pCDNA3.
Cell lines
The human leukemic T cell line Jurkat E6.1 and its LCK defective mutant
J.CaM1.6 cells as well as Cos 7 cells were from American Type Culture
Collection (Manassas, VA). HER cells are Rat1 fibroblasts engineered to
overexpress the human epidermal growth factor receptor. Cells were cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented
with 10% FBS (Sigma), 2 mM L-glutamine and gentamicin (10 mg/ml)
(Life Technologies). Jurkat cells were serum starved for 18 h and preincubated with or without PI3K inhibitors LY294002 or wortmannin in the
presence of 1% DMSO prior to stimulation as indicated. We have demonstrated that the incubation conditions and concentrations of LY294002
and wortmannin utilized were sufficient to inhibit anti-CD3 or anti-CD28
increases in phosphorylation of AKT (not shown), which provides a surrogate for inhibition of PI3K (49). Control cells were also incubated with
equivalent amounts of DMSO (1%). CD28 or CD3 were cross-linked by
incubating the cells in 1 ml volume with 5 mg of anti-CD28 or anti-CD3
mAb plus 10 mg of rabbit anti-mouse IgG. Cos 7 cells were transiently
transfected with lipofectamine/DNA (Life Technologies) complex at 37°C
for 2 h. Cells were cultured in the above medium for 24 h following transfection and then serum starved for another 24 h before harvesting.
Cell lysis and immunoprecipitation
Cells were lysed in 1 ml of 1% Nonidet P-40, 50 mM HEPES, pH 7.4, 150
mM NaCl, 50 mM ZnCl2, 50 mM NaH2PO4, 50 mM NaF, 2 mM EDTA, 1
mM Na3VO4, 2 mM PMSF, and 10 mg/ml aprotinin. Postnuclear detergent
lysate was precleared by protein A-Sepharose beads and then incubated
with 5 ml of rabbit anti-EMT serum in the presence or absence of PI3K
inhibitors for 90 min as indicated. The immune complexes were captured
by protein A-Sepharose beads. Immunoprecipitates were washed with
0.5% Triton X-100, 0.5% Nonidet P-40, 10 mM Tris/HCl, pH 7.4, 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, and 2 mM PMSF, and
mixed in Laemmli sample buffer.
Immunoblotting
Proteins from total cell lysates or eluted from immunoprecipitates were
separated by SDS-PAGE and transferred to polyvinylidene difluoride
(PVDF) membranes. The membranes were blocked with 3% BSA and then
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In contrast, EMT appears to negatively regulate at least some of
the effects of CD28 costimulation, with EMT knockout mice being
hyperresponsive to CD28 stimulation for cellular proliferation but
not for IL-2 production (32). The mechanism(s) whereby EMT
differentially regulates TCR and CD28 signaling events remains
unclear, as ligation of either the TCR or CD28 induces similar
LCK-dependent increases in EMT tyrosine phosphorylation and
activation (20, 33). Indeed, concurrent ligation of CD3 and CD28
does not induce a significant alteration in the magnitude or kinetics
of EMT activation (33). In contrast to the knockout mouse studies,
transfection studies with EMT suggest that EMT plays a critical
role in costimulation of the IL-2 promoter and probably also in
IL-2 production (34).
Structurally, EMT, which is expressed primarily in T cells, NK
cells, and mast cells, possesses pleckstrin homology (PH), TEC
homology, SRC homology 2 (SH2), and SRC homology 3 (SH3)
domains, which have the capacity to bind to specific protein motifs. Yeast two-hybrid, phage display, in vitro fusion protein, and
coimmunoprecipitation studies have identified a number of potential ligands and mediators of EMT function. The PH domain of
EMT has been demonstrated to bind to G protein b/g subunits, a
number of different protein kinase C isoforms, and inositol lipids
(35–37). Further, the PH domain targets EMT to the cell membrane (37). Membrane localization may be needed to regulate
EMT function in response to members of the SRC family of kinases (37). The proline-rich region in the TEC homology domain
can bind to the GRB2 and the SH3 domain of SRC family members including FYN, LYN, and HCK (38, 39). Furthermore, fusion
proteins containing the EMT SH3 domain associate with CD28,
SAM68, Wiskott-Aldrich syndrome protein (WASP), hnRNP-K,
FYN, and CBL (40, 41). However, many of these associations
have been difficult to detect in intact T cells (34, 38, 40, 41), and
their role in regulating EMT function or action remains to be
determined.
PI3K is involved in CD28 signaling, as indicated by the accumulation of its D-3 phosphoinositide products and recruitment of PI3K to
CD28 in CD28-stimulated cells (42– 46). These PI3K products act as
second messengers, eventually leading to the activation of some forms
of protein kinase C (47, 48), p70 ribosomal S-6 kinase, PDK1, and
AKT (48, 49). PI3K consists of a p110 catalytic subunit and a p85
regulatory subunit, which contains a proline-rich region, a SH3 domain, and two SH2 domains (50). The proline-rich region of the p85
subunit of PI3K binds to the SH3 domains of LCK and FYN resulting
in the activation of PI3K (51–54). The SH2 domain of PI3K binds to
the tyrosyl-phosphorylated YMNM motif (Y170 in mouse and Y173
in human CD28) in the cytoplasmic domain of CD28, recruiting PI3K
to CD28 (23, 27, 55, 56).
Since both EMT and PI3K are inducibly recruited and activated
by CD28 cross-linking, we evaluated the functional and physical
interaction between these molecules. Further, as LCK is required
for EMT activation and recruitment, we assessed the role of LCK
in these processes. In Jurkat T cells, CD28-induced but not CD3induced stimulation of EMT in vitro autokinase activity is sensitive to the PI3K inhibitors LY294002 and wortmannin (57, 58).
Coexpression of constitutively activated LCK Y505F with EMT in
Cos 7 cells results in increases in EMT in vitro autokinase activity,
a process that is partially dependent on PI3K activity, as indicated
by treatment of the cells with the PI3K inhibitors. CD28 ligation
but not CD3 ligation induces a LCK-dependent physical association of EMT with PI3K. This association appears to be mediated
by the SH2 domain of EMT binding to tyrosine phosphorylated
PI3K or a PI3K-associated linker molecule. Thus, ligation of
CD28, but not CD3, regulates EMT by a mechanism at least partially dependent on PI3K. Further, the recruitment of EMT to PI3K
5405
5406
PI3K IS REQUIRED FOR CD28- BUT NOT CD3-INDUCED EMT ACTIVATION
incubated for 2 h at room temperature with primary Ab: antiphosphotyrosine mAb 4G10 (1 mg/ml), rabbit antiserum against p85 subunit of PI3K (1:1000 dilution), or anti-EMT mAb (1:1000 dilution). The
membranes were washed in TBS-T (10 mM Tris/HCl, pH 7.4, 150 mM
NaCl, 0.1% Tween 20). HRP-conjugated goat anti-mouse IgG (1:5000 dilution) and HRP-protein A (1:3000 dilution) were used as secondary reagents for a 1-h incubation at room temperature. After extensively washing, membranes were developed by enhanced chemiluminescence
detection reagents (ECL; Amersham) and exposed to X-ray films.
EMT in vitro autokinase assay
EMT was immunoprecipitated from detergent cell lysate and washed with
1% Triton X-100, 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM Na3VO4,
and 1 mM PMSF. The immune complex was incubated in 50 ml of kinase
reaction buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MnCl2, 5
mM MgCl2, 5 mM ATP, and 10 mCi [g-32P]ATP) at room temperature for
30 min. The reaction was stopped by washing the immune complex twice
with the above wash buffer containing 1 mM EDTA. Proteins were eluted
by Laemmli sample buffer, resolved by SDS-PAGE, and autoradiographed.
with increases in EMT tyrosine kinase activity (20, 33). Strikingly,
as compared with the effects on EMT autokinase activity (Fig. 1A),
CD28-induced increases in EMT tyrosine phosphorylation were
much more resistant to the effects of LY294002 and wortmannin
(Fig. 1, B–D). Indeed, treatment with 10 mM of LY294002 or 100
nM of wortmannin virtually eliminated CD28-induced EMT in
vitro autokinase activity without significantly altering CD28-induced EMT tyrosine phosphorylation. These data further suggest
that phosphorylation of EMT, at least as induced by ligation of
CD28, is not sufficient for EMT in vitro autokinase activity. The
discordance between the effect of inhibition of PI3K kinase activity on CD28- and CD3-induced EMT in vitro autokinase activity
(Fig. 1, A, C, and D) and EMT phosphorylation (Fig. 1, B–D)
suggests that CD28- and CD3-mediated EMT phosphorylation
could be on different tyrosines.
Fusion protein binding assay
Results
CD28- but not CD3-induced increases in EMT activity are
sensitive to inhibition of PI3K
EMT and PI3K are activated and inducibly recruited to CD28 following CD28 cross-linking (19, 20, 42– 46). Further, both EMT
and PI3K are activated following ligation of the TCR complex (33,
48, 61). This suggests that PI3K may play a role in CD28- or
CD3-induced increases in EMT in vitro autokinase activity. Human Jurkat T leukemia cells were chosen for the exploration of the
relationship between EMT and PI3K due to their well-characterized responses to ligation of CD3 and CD28 and the well-characterized effects of ligation of CD28 or CD3 on EMT activation and
phosphorylation (19, 20, 33).
As indicated in Fig. 1, in human Jurkat T cells, cross-linking of
CD28 or CD3 induces a similar increase in EMT immune complex
kinase activity. Pretreatment of Jurkat cells with the relatively specific PI3K inhibitors LY294002 (IC50, 1.4 mM) (57) and wortmannin (IC50, 5 nM) (58) decreased CD28-induced EMT in vitro autokinase activity. Indeed, 10 mM of LY294002 or 100 nM of
wortmannin induced a 70% inhibition of EMT in vitro autokinase
activity (Fig. 1, A and C). This effect was selective for CD28induced increases in EMT in vitro autokinase activity, as preincubation with LY294002 or wortmannin did not alter CD3-induced
increases in EMT in vitro autokinase activity (Fig. 1, A and D).
This latter observation also indicates that the effects of LY294002
or wortmannin are not due to nonspecific toxicity but rather are
due to specific effects of these inhibitors on the pathway(s) leading
to CD28-induced increases in EMT activity. Further, this demonstrates that EMT is regulated by PI3K-dependent (CD28) and
PI3K-independent (CD3) mechanisms.
CD28 or CD3 cross-linking-induced tyrosine phosphorylation of
EMT is resistant to inhibition of PI3K
Ligation of both CD28 and CD3 results in a transient LCK-dependent increase in EMT tyrosine phosphorylation, which correlates
PI3K is involved in up-regulation of EMT in vitro autokinase
activity by LCK
Functional LCK is required for both anti-CD28- and anti-CD3induced activation of EMT, placing EMT downstream of LCK (20,
33). As indicated in Fig. 1, CD28-induced EMT activation is dependent on intact PI3K activity. To determine whether the effects
of LCK on EMT activity also required intact PI3K activity, EMT
was transiently transfected with or without constitutively activated
LCK (LCK Y505F; LCK containing a tyrosine to phenylalanine
mutation at the negative regulatory site in the carboxyl terminus of
LCK) (60, 61), into Cos 7 cells. Cos 7 cells do not express detectable levels of either EMT or LCK, providing an excellent recipient for transient transfection studies. Coexpression of activated
LCK and EMT resulted in an ;10-fold increase in EMT in vitro
autokinase activity as assessed by EMT autophosphorylation in in
vitro kinase assays (Fig. 2, A and C). The kinase activity in EMT
immunoprecipitates is probably not due to coimmunoprecipitation
of LCK, as we and others have been unable to demonstrate LCK
in anti-EMT immunoprecipitates from intact cells (19, 37, 38, 40),
and LCK protein (Western blotting) or autophosphorylating activity (kinase assay) was not detected in the EMT immunoprecipitates
from cotransfected Cos 7 cells, despite being readily detected in
anti-LCK immunoprecipitates (not shown).
As indicated in Fig. 2, LY294002 induced a marked decrease in
the ability of activated LCK to increase EMT in vitro autokinase
activity. In contrast, there was no effect of either LY294002 or
wortmannin on LCK immune complex autokinase activity. When
corrected for EMT protein levels (Fig. 2 C), preincubation with
LY294002 induced an ;50% decrease in LCK-induced increases
in the specific activity of EMT. It is important to note, however,
that even following a 4-h pretreatment with 10 mM of LY294002
(which is sufficient to completely inhibit PI3K; data not presented)
(57, 58), activated LCK induced a fourfold increase in EMT in
vitro autokinase activity (Fig. 2C). This indicates, once again, that
EMT is regulated by PI3K-dependent and -independent mechanisms and that LCK appears to be able to regulate EMT by both
PI3K-dependent and -independent mechanisms.
Interestingly, as in CD28- or CD3-induced increases in EMT
tyrosine phosphorylation in Jurkat cells, coexpression of activated
LCK with EMT in Cos 7 cells induced an increase in tyrosine
phosphorylation of EMT. This increase in EMT tyrosine phosphorylation was resistant to a 4-h treatment with 10 mM of LY294002
(Fig. 2, B and D), suggesting that LCK-induced tyrosine phosphorylation of EMT is not sufficient for optimal EMT autokinase
activity.
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TrpE fusion protein containing the SH2 domain of EMT was coated to
protein A-Sepharose beads by anti-TrpE mAb plus rabbit anti-mouse IgG.
Cos 7 cells were starved overnight in serum-free medium and treated with
50 mM pervanadate for 30 min. Cells were harvested and lysed. Jurkat cells
were serum starved, stimulated by CD28 cross-linking, and lysed as described above. Detergent cell lysates from Cos 7 or Jurkat cells were precleared with protein A-Sepharose and then incubated with the beads coated
with fusion proteins at 4°C for 2 h. The beads were washed, and the proteins were eluted by Laemmli buffer, as described above, for immunoprecipitation. Proteins were resolved by SDS-PAGE, transferred to PVDF
membranes, and immunoblotted by the Abs against p85 subunit of PI3K or
against phosphotyrosine as described as above.
The Journal of Immunology
5407
CD28 cross-linking but not CD3 cross-linking induces
association of EMT with PI3K
Based on the observation that both PI3K and EMT associate with
CD28 following the ligation of CD28 (19 –20, 42– 46) and on the
involvement of PI3K in CD28- but not CD3-induced EMT activation
(Fig. 1), we evaluated whether cross-linking of CD28 or CD3 would
induce a physical association of EMT with PI3K. CD28-induced association of EMT with PI3K was assessed by immunoprecipitation of
EMT followed by immunoblotting for the p85 subunit of PI3K. To
prevent precipitation of CD28 or CD28-associated PI3K by the anti-
CD28 Abs used to activate CD28, the EMT Ab was biotin-conjugated
and precipitated with avidin-Sepharose beads (beads alone were used
as the control, and anti-CD28 Abs were added to the control cells
postlysis to ensure that equal amounts of anti-CD28 Abs were present
in each immunoprecipitate). Following CD28 cross-linking, the
amount of EMT associated with PI3K underwent a time-dependent
increase, peaking at 30 min (Fig. 3). The recruitment of PI3K to CD28
was more rapid, peaking at 5 min following cross-linking (Fig. 3).
Furthermore, the time course of recruitment of EMT to PI3K was
markedly different from that of recruitment of PI3K to CD28 (Fig.
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FIGURE 1. CD28 cross-linking- but not CD3 cross-linking-induced EMT activity is decreased by PI3K inhibitors, whereas CD28 or CD3 cross-linkinginduced tyrosine phosphorylation of EMT is resistant to the inhibitors. A, Jurkat E6-1 cells were stimulated by CD28 cross-linking (CD28XL) for 15 min
or CD3 cross-linking (CD3XL) for 5 min (optimal stimulation time for CD28 and CD3 induced increases in EMT activity, respectively; data not presented).
Where indicated, cells were incubated with the indicated concentrations of LY294002 or wortmannin in the presence of 1% DMSO for 30 min before
stimulation. Control cells were incubated with an equivalent amount of DMSO. Cells were lysed and EMT was immunoprecipitated from detergent cell
lysates with rabbit anti-EMT serum. The immunoprecipitates were subjected to an in vitro immune complex kinase reaction. Inhibitors were present
throughout the immunoprecipitation. Proteins were eluted and separated by 8% SDS PAGE and transferred to PVDF membranes. Radiolabeled proteins
were visualized by autoradiography. The membranes were immunoblotted with a goat polyclonal anti-EMT Ab (Santa Cruz) and developed by ECL to
demonstrate equal levels of EMT protein in each kinase reaction. B, Jurkat E6-1 cells were pretreated with or without LY294002 or wortmannin and
stimulated by CD28 or CD3 cross-linking as described in A. Cells were lysed and EMT was immunoprecipitated from detergent cell lysates as described
in A. Proteins were electrophoresed and immunoblotted with anti-phosphotyrosine mAb (4G10). The membranes were developed by ECL and exposed to
X-ray film. The membranes were stripped and reprobed with murine monoclonal anti-EMT (Upstate Biotechnology) to demonstrate equal protein loading.
C, Densitometry readings of A and B corrected by EMT levels for CD28 cross-linking. D, Densitometry readings of A and B corrected by EMT levels for
CD3 cross-linking. The data are from a single result of four similar experiments.
5408
PI3K IS REQUIRED FOR CD28- BUT NOT CD3-INDUCED EMT ACTIVATION
3A). The amount of PI3K associated with EMT represents a small
portion of the PI3K present in the cell (,1%; not shown). However,
as indicated in Fig. 3A, the amount of PI3K associated with EMT is
;50% of the amount of PI3K associated with CD28.
Strikingly, CD3 cross-linking or PHA stimulation did not induce
a detectable increase in the association of EMT with PI3K (Fig.
3B). Thus, similar to the dependence of CD28-induced EMT activation on functional PI3K, CD28 but not CD3 cross-linking induces a physical association of EMT with PI3K.
Functional expression of LCK is required for CD28-induced
association of EMT with PI3K
LCK is required for optimal CD28-induced activation and phosphorylation of EMT (20). Cross-linking of CD28 in J.CaM1.6
cells, which lack functional LCK (62), failed to induce the association of EMT with PI3K (Fig. 4), suggesting that tyrosine phosphorylation initiated by LCK is involved in the association of EMT
with PI3K. Strikingly, cross-linking of CD28 in J.CaM1.6 cells
induced detectable association of PI3K with CD28 (cf Figs. 3 and
4). This indicates that CD28 is at least partially functional in
J.CaM1.6 cells and that functional LCK is not required for the
recruitment of PI3K to CD28. Thus, LCK is required for CD28induced increases in EMT phosphorylation, increases in EMT in
vitro autokinase activity, and induced association of EMT with
PI3K. Furthermore, the mechanisms regulating recruitment of
PI3K to CD28, probably phosphorylation of Y170 (23, 27, 55, 56),
must be different from those regulating the recruitment of EMT to
PI3K and to the activation of EMT by CD28.
The SH2 domain of EMT can mediate association of EMT with
PI3K
Since SH2 domains bind to tyrosine-phosphorylated protein domains and the association of EMT with PI3K is most likely dependent on tyrosine phosphorylation, we assessed whether the SH2
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FIGURE 2. PI3K is involved in up-regulation of EMT in vitro autokinase activity but not tyrosine phosphorylation of EMT induced by LCK. EMT was
transiently transfected with or without constitutively activated LCK Y505F into Cos 7 cells. Cells were incubated with or without LY294002 (10 mM) in
the presence of 0.5% DMSO for 4 h before lysis. EMT was immunoprecipitated from detergent cell lysates with rabbit anti-EMT serum. A, EMT
immunoprecipitates were subjected to immune complex kinase reactions. Proteins were eluted from beads, separated by 8% SDS-PAGE, and transferred
to PVDF membranes. Radiolabeled proteins were visualized by autoradiography. The membrane was then immunoblotted with murine anti-EMT mAbs
(Upstate Biotechnology) demonstrating similar levels of protein loading (not shown). B, EMT immunoprecipitates were subjected to an 8% SDS-PAGE
and immunoblotted with anti-phosphotyrosine mAb (4G10). The membrane was developed by ECL and exposed to X-ray film. C, The results from A were
quantified by densitometry, normalized for EMT expression level, and are shown as fold increase in EMT-specific activity over basal levels. D, The results
from B were quantified by densitometry, normalized for EMT expression level, and are shown as fold increase in tyrosine phosphorylation of EMT over
basal levels. The data are from a single result of two similar experiments.
The Journal of Immunology
5409
domains of EMT or PI3K were sufficient for formation of a PI3K:
EMT complex. EMT does not contain the YmxM consensus binding motif for PI3K SH2 domains (50, 59). We therefore first assessed whether the SH2 domain of EMT was capable of mediating
the association of EMT with PI3K. Cos 7 cells, which do not
express CD28 or EMT, were used as the PI3K donor. As noted in
Fig. 5A, a trpE-SH2 fusion protein containing the SH2 domain of
EMT was able to bind PI3K or a PI3K-associated protein from
pervanadate-treated but not resting Cos 7 cells, as assessed by
Western blotting with anti-p85 Abs. Further, as indicated in Fig.
5A, the PI3K that associated with the EMT SH2-trpE fusion protein was tyrosine phosphorylated, as demonstrated by Western
blotting with anti-phosphotyrosine Abs. Indeed, the degree of association of PI3K with EMT correlated with the degree of tyrosine
phosphorylation of PI3K. Once again, this suggests that EMT can
bind PI3K or a PI3K-associated protein in a tyrosine phosphorylation-dependent manner.
We also assessed whether the SH2 domain of EMT could bind
to PI3K derived from resting or CD28-activated Jurkat cells. The
FIGURE 4. Functional expression of LCK is required for CD28-induced association of EMT and PI3K. J.CaM1 cells (50 3 106) were stimulated by CD28 cross-linking for the indicated time periods. EMT (rabbit
polyclonal anti-EMT) or CD28 (9.3) was immunoprecipitated from detergent cell lysates and immunoblotted with anti-PI3K p85 (a and c) or polyclonal rabbit anti-EMT Ab (b). The data are from a single result of three
similar experiments.
interpretation of this approach is complicated by the presence of
CD28 in Jurkat T cells, which could act as an intermediary in the
formation of a ternary complex. Nevertheless, as indicated in Fig.
5B, the SH2 domain of EMT was able to form a complex with
PI3K from lysates of CD28-treated Jurkat T cells as compared
with resting Jurkat T cells.
As expected, since EMT does not contain a PI3K SH2 consensus-binding motif and tyrosine phosphorylation of the PI3K donor
cell appears to be required for the association of the SH2 domain
FIGURE 5. The SH2 domain of EMT binds to PI3K in a tyrosine phosphorylation-dependent manner. A, Cos 7 cells (3 3 106 cells) were treated
with 50 mM pervanadate fro 30 min. Cells were lysed and the lysate was
mixed with Sepharose beads precoated with TrpE fusion proteins containing the SH2 domain of EMT or with TrpE fusion protein only. The precipitated proteins were separated by 8% SDS-PAGE and immunoblotted
by anti-PI3K p85. The membrane was stripped and reprobed by antiphosphotyrosine mAb 4G10. B, Jurkat cells (50 3 106 cells) were left
unstimulated or stimulated by CD28 cross-linking. Cells were lysed and the
lysate was mixed with Sepharose beads precoated with TrpE fusion proteins containing the SH2 domain of EMT. The precipitates were separated
by 8% SDS-PAGE and blotted by anti-PI3K p85. The data are from a
single result of five similar experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 3. CD28 cross-linking but not CD3 cross-linking induces association of EMT with PI3K. Jurkat cells (50 3 106) were stimulated by CD28
cross-linking for the indicated times or stimulated by CD3 cross-linking or PHA (5 mg/ml) for 10 min. EMT was immunoprecipitated from detergent cell
lysates using biotin-conjugated polyclonal rabbit anti-EMT Abs and streptavidin-Sepharose beads (to prevent immunoprecipitation of CD28 or CD3 by the
anti-CD28 or anti-CD3 Abs used to activate cells). The CD28 receptor was immunoprecipitated using anti-CD28 mAb (9.3), rabbit anti-mouse IgG, and
protein A-Sepharose beads. Proteins were separated by 8% SDS-PAGE and immunoblotted. A, EMT immunoprecipitates were blotted with an Ab specific
to the p85 subunit of PI3K (Upstate Biotechnology) (a). The membrane was stripped and reprobed with polyclonal rabbit anti-EMT (b). The CD28
immunoprecipitates were blotted with anti-PI3K p85 (c). B, EMT immunoprecipitates were blotted with anti-PI3K p85 (a). The membrane was stripped
and reprobed with polyclonal rabbit anti-EMT (b). The data are from a single result of five similar experiments.
5410
PI3K IS REQUIRED FOR CD28- BUT NOT CD3-INDUCED EMT ACTIVATION
of EMT with PI3K, we were unable to detect an association of
EMT from resting or CD28-stimulated T cells with GST fusion
proteins containing the two SH2 domains of the p85 PI3K subunit,
as assessed by Far Western or protein overlay blotting (data not
presented). The SH2 domains of PI3K readily bound to a number
of proteins from epidermal growth factor- or pervanadate-treated
HER cells, demonstrating the validity of the approach.
Taken together, the data suggest that the SH2 domain of EMT
can associate with tyrosine-phosphorylated PI3K. However, the
possibility of indirect association between EMT and PI3K through
other adapter molecule(s) cannot be excluded. CD28 is not an
obligatory linker molecule for the formation of this complex.
Whether CD28 plays a role in the formation of a ternary complex
in T cells remains to be determined.
Discussion
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Ligation of both CD28 and CD3 induces tyrosine phosphorylation
and activation of EMT (19, 33). We demonstrate herein that
CD28-induced stimulation of EMT tyrosine autokinase activity is
due, in part, to activation of PI3K. Strikingly, anti-CD3-induced
activation of EMT autokinase activity does not require functional
PI3K, indicating that EMT enzyme activity is regulated by PI3Kdependent and -independent mechanisms. Further, LCK-induced
increases in EMT in vitro autokinase activity also demonstrate
P13K-dependent and -independent components (Fig. 2). The
PI3K-independent mechanism of EMT in vitro autokinase activation probably involves tyrosine phosphorylation of EMT mediated
by LCK, as stimulation of EMT kinase activity is dependent on the
presence of LCK (21, 33). Indeed, Berg and colleagues have recently demonstrated that LCK induces tyrosine phosphorylation of
EMT on Tyr511, a process that is required for the optimal activation of EMT (21). Even following overexpression of EMT and
LCK in bacculovirus infected insect cells, Berg and colleagues
were unable to demonstrate significant phosphorylation of other
tyrosines in EMT, indicating that Tyr511 is likely to be the major
target of LCK-mediated phosphorylation (21). Phosphorylation of
a serine, threonine, or tyrosine residue in the activation loop of
kinases appears to be a common mechanism for regulation of kinase activity (63).
The PI3K-dependent mechanism of EMT activation is detectable following immunoprecipitation of EMT, suggesting that PI3K
leads to a posttranslational alteration in EMT or an associated molecule, or alternatively, that the products of PI3K remain bound to
EMT under the immunoprecipitation conditions utilized. August et
al. (37) have demonstrated that the PH domain of EMT, similar to
several other PH domains (49), has the ability to bind products of
PI3K. This may target EMT to the cell membrane, a location potentially required for its phosphorylation by SRC family kinases
(37), or alternatively, may directly regulate EMT in vitro autokinase activity as it does other PH-containing kinases such as
AKT (49).
August et al. have demonstrated that coexpression of SRC and
EMT in Cos 7 cells results in activation of EMT, a process that is
likely to be dependent, in part, on PI3K, as it was inhibited by
LY294002 and wortmannin, albeit at very high concentrations
(37). The relevance of this observation to activation of EMT in T
lymphocytes is uncertain, as this same group had previously demonstrated that similar concentrations of LY294002 and wortmannin as used in Cos 7 cells augmented rather than inhibited CD28induced activation of EMT in Jurkat T cells (22). The studies
presented herein serve to clarify the role of PI3K in EMT activation by demonstrating that LCK activates EMT, at least partly, in
a PI3K-dependent manner. Further, in Jurkat T cells, inhibition of
PI3K activity by LY294002 or wortmannin at concentrations
likely to be relatively specific for PI3K (56, 57) induced a significant decrease in CD28-mediated activation of EMT. The reason
for the discrepancy between the results reported herein and those
of Dupont and colleagues (22) is currently unknown. However, the
results observed herein cannot simply be due to the toxicity of the
inhibitors, as they failed to alter anti-CD3-induced activation
of EMT.
Consistent with the observation of a role for PI3K in the activation of EMT by ligation of CD28 and not ligation of CD3, EMT
associates with PI3K following ligation of CD28 but not CD3. The
SH2 domain of EMT is competent to bind to PI3K in a tyrosine
phosphorylation-dependent manner. Whether the SH2 domain of
EMT binds to tyrosine-phosphorylated PI3K p85 or p110 directly
or to a phosphorylated PI3K-associated protein has not yet been
determined. PI3K has been demonstrated to bind to a number of
proteins including CBL, VAV, and FYN among others (19, 51–54,
64), any of which may mediate binding to EMT. Indeed, EMT has
been suggested in surrogate model systems to bind to VAV, CBL,
and FYN (19, 21, 38 – 40). Further, although EMT can interact
with PI3K in the absence of CD28, both EMT and PI3K are recruited to CD28 following cross-linking of CD28, suggesting that
CD28 may provide a framework for the recruitment of both PI3K
and CD28.
The association of EMT with PI3K may be important in placing
EMT in the context of upstream or downstream regulatory molecules or targets. If this is the case, the failure of ligation of CD3 to
induce a PI3K:EMT complex is compatible with EMT playing
differential roles downstream of CD28 and CD3. Genetic “knockout” experiments, which suggest that EMT plays a positive role in
some TCR-mediated signaling events (30, 31) and a negative role
in some CD28-mediated signaling events (32), support this
contention.
As with the activation and phosphorylation of EMT (19 –21,
33), LCK is required for the recruitment of EMT to PI3K. Surprisingly, although LCK is required for the recruitment of EMT to
PI3K (Fig. 4) and of EMT to CD28 (20) as well as for the activation of EMT (20, 33), LCK is not required for recruitment of
PI3K to CD28 (Fig. 4). Thus, phosphorylation of Y170, which
recruits PI3K to CD28, does not require functional LCK or activation of EMT; this despite the observation that, at least in vitro,
both EMT and LCK are capable of phosphorylating Y170 (22).
Hirano and colleagues have demonstrated that the SH2 domain
of TEC can bind to the p85 PI3K subunit as well to a 55-kDa
PI3K-associated molecule in a process dependent on the kinase
activity of TEC (65) in yeast two-hybrid systems and in surrogate
model systems. Further, the association of TEC with PI3K, similar
to the association of EMT with PI3K, is increased by activation of
cells by ligands that increase intracellular tyrosine phosphorylation, in this case, IL-3 and IL-6 (65). This finding suggests that
TEC family kinases, at least TEC and EMT, and potentially other
members of the family such as BTK, BMX, TXK, and DSRC28
(66), interact with PI3K. Determining whether PI3K plays a role in
the regulation of TEC family members other than EMT will require additional investigation.
In summary, EMT activity is regulated through at least two
different mechanisms, one of which requires PI3K. PI3K activity is required for activation of EMT following CD28 ligation
but not CD3 ligation. Consistent with the requirement of PI3K
activity for CD28- but not CD3-induced activation of EMT, a
small but significant portion of cellular EMT associates with
PI3K following CD28 cross-linking but not following CD3
stimulation. This association is most likely mediated by the
binding of the SH2 domain of EMT to tyrosine-phosphorylated
The Journal of Immunology
PI3K or a PI3K-associated protein. CD28-induced association
of EMT with PI3K requires functional expression of LCK as
does CD28-induced EMT activation and phosphorylation. Further, LCK-induced EMT activation is dependent, at least in
part, on PI3K activity. Thus ligation of CD28 and CD3 regulates EMT through different mechanisms and recruits EMT to
different signaling complexes, perhaps contributing to differences in the role of EMT downstream of CD28 and the TCR.
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