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CUTTING EDGE |
*
Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206;
Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115; and
Cancer Biology Program, Division of Hematology-Oncology, Department of Medicine, Beth Israel-Deaconess Medical Center, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B-directed
transcription. Inhibition is abrogated by mutating the Gab2 p85-binding
sites, by treatment with PI3K inhibitors or by cotransfection of
phosphatase homolog of tensin. Our findings provide the first evidence
of a negative function for a scaffolding adapter in T cells and
identify Gab2/PI3K-containing complexes as novel regulators of TCR
signaling. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (2). Appropriate TCR signal
transduction is critical for the generation of effective immune
responses and for the prevention of autoimmunity. One key signal relay molecule in multiple cellular signaling pathways is the enzyme phosphatidylinositol 3'-kinase (PI3K). PI3K exists as several isoforms, the best studied of which are composed of an 85-kDa regulatory subunit (p85) containing SH2 and SH3 domains bound, via its inter-SH2 domain region, to a 110-kDa catalytic domain (p110). PI3Ks can be recruited via p85 to specific tyrosyl phosphorylation sites. Most often, PI3Ks are positive signaling components; i.e., they help transmit signals leading to cell survival (antiapoptosis), cell proliferation, cell adhesion, and/or cell motility. Recent work has begun to unravel the mechanisms by which PI3K transmits such signals. The products of PI3K, the lipids phosphatidylinositol 3,4-diphosphate and phosphatidylinositol 3,4,5-triphosphate, bind to pleckstrin homology (PH) domains in several signaling intermediates, including the serine/threonine kinases phosphoinositide-dependent kinase 1 and Akt, as well as members of the nonclassical and atypical protein kinase C family (3).
However, the role of PI3K in T cell signaling on TCR engagement has remained confusing. Studies of Jurkat T cells treated with pharmacological inhibitors of PI3K (e.g., wortmannin, Ly294002) or transfected with dominant negative or activated mutants of PI3K suggest that PI3K inhibits TCR-evoked activation of the IL-2P (4, 5). Studies of the role of PI3K in TCR responses in primary human (4) and murine (6, 7) T cells are conflicting, with different investigators reporting no effect on, inhibition of, or stimulation of TCR-evoked IL-2 production.
Recently, we (8, 9) and others (10) identified another PI3K-binding protein in T cells and other cell types. Gab2 is a member of a subfamily of scaffolding adapters (hereafter, "scaffolds") that includes Drosophila Dos and mammalian Gab1. These proteins have an N-terminal PH domain followed by multiple potential tyrosyl phosphorylation sites and several proline-rich sequences. Initial characterization of Gab2 suggested that it functions in a variety of signaling pathways, including those emanating from receptor tyrosine kinases, cytokine receptors, and antigen receptors in hemopoietic and nonhemopoietic cells (8, 10, 11). Thus far, the function of Gab2 in cytokine signaling has been evaluated most thoroughly. In response to stimulation by a number of cytokines, Gab2 becomes rapidly tyrosyl phosphorylated and associates with Shc, Shp-2, and PI3K. In cytokine signaling, Gab2 (8, 10) and, in particular, Gab2/Shp-2 (8) and Gab2/PI3K5 associations play positive signaling roles. Likewise, most other families of scaffolds, including IRS family members (e.g., IRS-1, IRS-2) (12) and FRS-2/SNT (13), are positive signaling components.
Recently, we began to investigate the role of Gab2 in other signaling pathways. As in cytokine signaling, Gab2 becomes rapidly tyrosyl phosphorylated on TCR stimulation in Jurkat cells (8, 10). Surprisingly, however, we found that, in contrast to its positive role in cytokine signaling, Gab2 inhibits TCR-evoked activation of the IL-2P in Jurkat cell transfection assays. Gab2-mediated inhibition requires its interaction with PI3K, but not Shp-2. Our results identify Gab2 as a new type of negative regulator of T cell signaling, indicate that Gab2 can have positive or negative effects on different signaling pathways, and show that at least some signals delivered through PI3K inhibit T cell activation events.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs used in this study include: anti-mouse CD3
(145-2C11), anti-human CD3 (OKT3), anti-mouse CD28, rabbit
anti-mouse cross-linking Ab (PharMingen, San Diego, CA), and
anti-hemagglutinin (HA) Ab (clone 12CA5, Boehringer Mannheim,
Indianapolis, IN). Anti-phosphotyrosine Ab (4G10) was a generous gift
of Dr. Tom Roberts (Dana-Farber Cancer Institute, Boston, MA). The
anti-Gab2 rabbit polyclonal Ab has been described previously
(8). Anti-p85/PI3K Ab was kindly provided by Dr. Lewis
Cantley (Beth Israel-Deaconess Medical Center, Boston, MA). PMA was
from Sigma (St. Louis, MO), ionomycin, and Ly294002 were purchased from
Calbiochem (San Diego, CA). A dual luciferase assay kit was purchased
from Promega (Madison, WI).
Plasmids
The Gab2 constructs used in these studies were generated by PCR
and subcloned into the pEBB vector, as described previously
(8). All constructs were confirmed by DNA sequence
analysis. The human IL-2P (14), NF-AT binding site,
NF-B binding site (15), Fos promoter (16),
constitutively active CD2P110 PI3K (5), and Shp-2
(8) constructs have been described. Constructs encoding
wild-type and a phosphatase-inactive mutant phosphatase homolog of
tensin (PTEN) were generated by PCR and site specific mutagenesis.
Cells and transfections
Jurkat cells and DO11.10 cells were grown in RPMI 1640 and DMEM,
respectively, containing 10% heat-inactivated FCS, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine.
Cells (107) were transfected with the indicated
plasmids at 800 µF/250V using a BRL electroporator (Gaithersburg,
MD). All cells were cotransfected with 30 ng of a Renilla
luciferase construct (Promega) to normalize for transfection
efficiency. The transfected cells were grown for 15 h, and an
aliquot (106) was set aside for immunoblot
analysis to confirm expression of the transfected protein. The
remaining cells were divided into aliquots and either left untreated or
incubated with inhibitors or stimulators, as indicated in the legends
to
Figs. 1–3
. Cells were washed once with PBS and lysed. Luciferase
activities were determined with a Monolight 2010 luminometer
(Analytical Luminescence, Ann Arbor, MI).
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Unstimulated or stimulated transfected cells were washed and lysed in buffer containing 0.5% Triton-X100. Lysates were spun at 13,000 rpm for 10 min, and subjected to immunoprecipitation with anti-Gab2 Abs and protein A-Sepharose beads. Bound proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the appropriate Abs. The enhanced chemiluminescence detection system (Amersham, Arlington, Heights, IL) was used to visualize proteins.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown previously that Gab2 becomes tyrosyl
phosphorylated after anti-CD3 cross-linking in Jurkat
cells (8). Here, we also examined Gab2
phosphorylation in human PBL by immunoprecipitating
Gab2 followed by Western blot analysis with an anti-phosphotyrosine
Ab. We found that Gab2 becomes tyrosyl phosphorylated
during CD3 or CD28 cross-linking in PBL (Fig. 1
A), suggesting that Gab2 is
involved in TCR signaling in normal T cells.
To investigate the role that Gab2 plays in T cell signaling, we first
assayed the effect of overexpressing Gab2 on TCR-stimulated IL-2P
activity. WT-Gab2 was cotransfected with an IL-2P-luciferase reporter
construct into Jurkat cells. Cells were left unstimulated or were
stimulated with anti-CD3 alone or anti-CD3 plus PMA, and
luciferase activities were measured. Significant stimulation of the
IL-2P requires signals from the TCR and a costimulus. As expected,
stimulation with anti-CD3 alone stimulated little IL-2P activity,
whereas stimulation with anti-CD3 plus PMA resulted in robust
activation of IL-2P transcription in vector-transfected cells. In
contrast, coexpression of WT-Gab2 dramatically inhibited IL-2P
activation in Jurkat cells (p < 0.001).
Notably, Gab2 overexpression had no effect on IL-2P activity stimulated
by treatment with PMA plus ionomycin, which bypasses early T cell
activation events (Fig. 1B). Similarly, expression of
WT-Gab2 also blocked IL-2P activation by anti-CD3 plus CD28
costimulation (Fig. 1C). These data suggested that Gab2 is a
specific negative regulator of an early event(s) in the T cell signal
transduction pathway leading to IL-2 production in Jurkat cells.
Two critical elements in the IL-2P are the binding sites for the
transcription factors NF-AT and NF-B. Overexpression of Gab2
inhibited both NF-AT (Fig. 1D) and NF-B (Fig. 1E)-directed reporter constructs. Gab2 does not globally
inhibit transcription, however, because there was no effect of Gab2
expression on activation of the c-fos promoter (Fig. 1F). Moreover, the ability of Gab2 to inhibit TCR signaling
was not restricted to Jurkat T cells, given that similar effects were
observed in D011.10 hybridoma (Fig. 1G) and D10 Th2 cells
(data not shown).
Gab2 inhibition is mediated by PI3K
To begin to understand the mechanism by which Gab2 negatively
regulates IL-2P activation, we compared the effects of WT-Gab2 and
several Gab2 mutants, all bearing C-terminal HA tags, on TCR-evoked
IL-2P-luciferase activity in Jurkat cells (Fig. 2A). Tyrosyl-phosphorylated
Gab2 binds to Shp-2. Because SH2-containing tyrosine phosphatases can
mediate other inhibitory signaling pathways, it seemed possible that
Gab2 inhibition was mediated by Shp-2. However, mutation of both Shp-2
binding sites (Gab2-DMF) had no effect on the ability of Gab2 to
inhibit IL-2P activity, even when Gab2-DMF protein accumulated to
higher levels than WT Gab2 (Fig. 2B). Also, when
cotransfected with WT Gab2, dominant negative mutants of Shp-2 were
unable to abrogate Gab-2- mediated inhibition (data not shown). In
contrast, deletion of the Gab2 PH domain (Gab2
PH) or Y>F
mutation of the three YXXM motifs in Gab2 that constitute potential
binding sites for p85 (Gab2-3YF) completely eliminated the ability of
Gab2 to inhibit TCR signaling (Fig. 2, A and B).
Similar results were obtained when larger amounts of Gab2PH or
Gab2-3YF expression constructs were cotransfected with IL-2P-luciferase
reporter (data not shown). Anti-p85 immunoblots of anti-HA
immunoprecipitates from transfected cells confirmed that Gab2-3YF has
lost the ability to bind PI3K after TCR stimulation (anti-CD3),
whereas the other Gab2 proteins retain p85 binding (Fig. 2C).
These findings implicate PI3K as a potential mediator of the
Gab2-inhibitory signal. However, it remained possible that another
protein(s) that binds to the three tyrosyl residues mutated in
Gab2-3YF, rather than p85, was actually responsible for inhibition. To
rule out this possibility, we performed several types of experiments.
First, we asked whether an activated mutant of PI3K (CD2P110) had
inhibitory effects on IL-2P activation. Consistent with a previous
report (5), expression of this mutant inhibited TCR-evoked
IL-2P activity (p < 0.001) to an extent
similar to that observed with WT-Gab2 (Fig. 3A). Next, we monitored the effects of
the PI3K inhibitor Ly294002 on TCR-evoked IL-2P activity. As expected,
Ly294002 treatment reversed the inhibitory effects of CD2P110. More
importantly, such treatment also significantly rescues Gab2-mediated
inhibition of TCR-evoked IL-2P activity (Fig. 3B). Finally,
we found that coexpression of wild type PTEN/MMAC, a tumor suppressor
gene product that functions as a 3'phosphoinositide phosphatase
(17), but not catalytically inactive PTEN, also reverses
Gab-2 mediated inhibition (Fig. 3C). To ensure that the
effect of PTEN on Gab2 function was not due to nonspecific
overexpression of a phosphatase, we analyzed the effect of SHP2 on Gab2
inhibition of IL-2P activity (Fig. 3D). Coexpression of
wild-type shp-2 had no significant effect on the inhibitory effect
mediated by Gab2 (p = 0.92).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-directed
transcription. These findings identify Gab2 as a new negative regulator
of T cell signaling and lend support to the notion that at least some
pools of PI3K have inhibitory functions.
Our results indicate that the pool of PI3K associated with Gab2
inhibits TCR activation events. Several lines of evidence support this
contention. First, overexpression of Gab2 inhibits TCR-evoked
activation of the IL-2P, interfering with NF-AT and NF-B-directed
reporters (Fig. 1D, E). Three lines of evidence argue that Gab-2
mediated inhibition is mediated by its ability to associate with PI3K:
1) inhibition is abrogated in Gab2 mutants unable to bind PI3K (Fig. 2); 2) inhibition can be largely rescued by treatment with PI3K
inhibitors; and 3) cotransfection with the 3'-lipid phosphatase PTEN
also partially rescues Gab2-mediated inhibition. The latter two
observations further imply that it is the ability of Gab2 to recruit an
active lipid kinase that mediates its inhibitory actions, rather than
potential adapter functions of p85 or p110. Our results also show that
the PH domain of Gab2 is required for inhibition (Fig. 2A).
PH domains mediate binding to specific phospholipids. The binding
specificity of the Gab2 PH domain has not been determined. However, its
sequence is highly similar to the Gab1 PH domain, which binds
phosphatidylinositol 3,4,5-triphosphate specifically (18, 19); it is highly likely that the Gab2 PH domain also binds this
lipid.
Therefore, one likely possibility is that PH domain binding to PIP3 localizes Gab2/PI3K complex to a specific subcellular site and/or receptor, where Gab2 can exert its inhibitory effects. At this time, we cannot rule out the possibility that another molecule mediates part of the Gab2 inhibition of IL-2P activation by interacting with one of the three tyrosine residues (Tyr441, Tyr461, Tyr574) and PH domain of Gab2. This possibility may explain the inability of LY294002 or PTEN to completely rescue Gab2 inhibition of IL-2P.
Previous studies have shown that treatment of Jurkat T cells with pharmacological PI3K inhibitors led to increased IL-2 secretion in response to CD3 plus CD28 stimulation (4). Transfection of dominant negative mutants of p85 resulted in enhanced TCR-evoked activation of an NF-AT-driven reporter construct, whereas an activated mutant of PI3K suppressed NF-AT activity in Jurkat and EL4 T cells (5). Thus, there appears to be general agreement that PI3K inhibits at least some TCR-evoked transcriptional events. Our data are consistent with these earlier studies and suggest that Gab2-associated PI3K activity may be responsible for this negative signal.
In contrast to its inhibitory role in TCR signaling as described
herein, Gab2 acts as a positive component in cytokine signaling
(8, 10). Both Gab2/Shp-2 and Gab2/PI3K interactions are
required for normal cytokine signaling
(8).5 The inhibitory action of Gab2
in T cells appears to require only Gab2/PI3K interaction, because
Gab2-DMF retained full inhibitory potency (Fig. 2). Recently, it was
reported that overexpression of Gab2 in 293 cells blocks Ras activation
of Elk-driven transcription (20). The relevance of these
observations for Gab2-mediated inhibition of TCR signaling is unclear,
however, because we observed no effect of Gab2 expression on the
Elk-dependent c-fos promoter (Fig. 1F).
Nevertheless, these studies emphasize that Gab2 and its associated
protein complexes can have different roles in distinctly different cell
types.
The precise mechanism(s) by which the Gab2-associated pool of PI3K activity inhibits TCR signaling remains to be determined. Previous work showed that, unlike activated mutants of PI3K, neither activated Rac nor activated Akt inhibited TCR-evoked NF-AT activity in Jurkat cells (5). In T cells, inhibitory signaling by Gab2 may be particularly important for terminating IL-2 production in the later stages of the response to Ag. Consistent with this notion, we have recently shown that Gab2 protein levels increase markedly after TCR stimulation of human PBL and Jurkat cells (9). Further studies are required to elucidate the components of the Gab2-inhibitory pathway in T cells and its importance in the immune response.
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Acknowledgments |
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Footnotes |
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2 Current address: Franklin W. Olin College of Engineering, 1735 Great Plain Avenue, Needham, MA 02492.
3 Address correspondence and reprint requests to Dr. Haihua Gu, Cancer Biology Program, Beth Israel-Deaconess Medical Center, 77 Avenue Louis Pasteur, HIM 1043, Boston, MA 02115.
4 Abbreviations used in this paper: SH, Src homology; IL-2P, IL-2 promoter; PI3K, phosphatidylinositol 3'-kinase; PH, pleckstrin homology; HA, hemagglutinin; PTEN, phosphatase homolog of tensin.
5 H. Gu, H. Maeda, J. J. Moon, J. D. Lord, M. Yoakim, B. H. Nelson, and B. G. Neel. A new role for Shc in activation of the PI3K/Akt pathway. Submitted for publication.
Received for publication June 20, 2000. Accepted for publication August 22, 2000.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, anti-.
Top, Lysates were subjected to immunoprecipitation with
anti-HA Ab and protein A-Sepharose beads. Immunoprecipitates (IP)
were immunoblotted with anti-PI3K (p85) Ab. Bottom,
The same blot was reprobed with anti-HA Abs to visualize expression
of the indicated Gab2 proteins.