Phosphatidylinositol 3′-kinase (PI3K) is a key component of multiple signaling pathways, where it typically promotes survival, proliferation, and/or adhesion. Here, we show that in TCR signaling, the scaffolding adapter Gab2 delivers an inhibitory signal via PI3K. Overexpression of Gab2 in T cell lines inhibits TCR-evoked activation of the IL-2 promoter, blocking NF-AT- and NF-κB-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.

Engagement of the TCR initiates positive (signal-enhancing) and negative (signal-attenuating) cascades that ultimately result in cellular proliferation, differentiation, cytokine production, and/or activation-induced cell death. TCR stimulation leads to the tyrosyl phosphorylation of multiple proteins, catalyzed by the src family tyrosine kinases Lck and Fyn, as well as Syk and ZAP70 (1). These initial phosphorylation events lead to the assembly of multiprotein complexes comprised of signal relay molecules containing Src homology 2 (SH2)4 and/or phosphotyrosine-binding domains, which then transmit signals to the nucleus. Signals emanating from the TCR lead to transcriptional activation of the promoter for IL-2. The IL-2 promoter (IL-2P), in turn, is regulated by several transcriptional elements, among which are sites for NF-AT and NF-κB (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.

Abs used in this study include: anti-mouse CD3ε (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).

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-κB 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.

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).

FIGURE 1.

Overexpression of WT-Gab2 inhibits TCR-mediated IL-2P transcriptional activation. A, Purified human PBL were stimulated through anti-CD3 and anti-CD28 cross-linking for indicated amount of time. Lysates were immunoprecipitated with anti-Gab2 Abs followed by anti-phosphotyrosine Western blot. Jurkat cells were transfected with Gab2 expression plasmids or expression vector alone (5 μg) in combination with 5 μg of IL-2P-luciferase (B and C) NF-AT-luciferase (D), NF-κB-luciferase (E), or Fos promoter-luciferase (F) construct and 30 ng Renilla luciferase normalization plasmid. G, D011.10 cells were transfected with Gab2 expression constructs or vector alone together with the NF-AT-luciferase and Renilla luciferase constructs. Eighteen hours after transfection, cells were left unstimulated or were stimulated with plate-bound anti-CD3 (1 μg/well), a combination of plate-bound anti-CD3 plus PMA (10 ng/ml), plate-bound anti-CD3 plus soluble anti-CD28 (1 μg/ml; C), and PMA plus ionomycin (0.5 μM), as indicated. Cells were incubated for an additional 6 h before lysis, and luciferase activity was measured as described in Materials and Methods. Data represent the results from at least three independent experiments.

FIGURE 1.

Overexpression of WT-Gab2 inhibits TCR-mediated IL-2P transcriptional activation. A, Purified human PBL were stimulated through anti-CD3 and anti-CD28 cross-linking for indicated amount of time. Lysates were immunoprecipitated with anti-Gab2 Abs followed by anti-phosphotyrosine Western blot. Jurkat cells were transfected with Gab2 expression plasmids or expression vector alone (5 μg) in combination with 5 μg of IL-2P-luciferase (B and C) NF-AT-luciferase (D), NF-κB-luciferase (E), or Fos promoter-luciferase (F) construct and 30 ng Renilla luciferase normalization plasmid. G, D011.10 cells were transfected with Gab2 expression constructs or vector alone together with the NF-AT-luciferase and Renilla luciferase constructs. Eighteen hours after transfection, cells were left unstimulated or were stimulated with plate-bound anti-CD3 (1 μg/well), a combination of plate-bound anti-CD3 plus PMA (10 ng/ml), plate-bound anti-CD3 plus soluble anti-CD28 (1 μg/ml; C), and PMA plus ionomycin (0.5 μM), as indicated. Cells were incubated for an additional 6 h before lysis, and luciferase activity was measured as described in Materials and Methods. Data represent the results from at least three independent experiments.

Close modal
FIGURE 2.

Gab2-mediated inhibition of IL-2P requires binding sites for PI3K. Jurkat cells were transfected with 0.2 (lanes 2, 4, 6, and 8) or 0.05 μg (lanes 3, 5, 7, and 9) of the indicated Gab2 constructs, along with IL-2P and Renilla luciferase reporters and vector DNA to maintain the same total DNA concentration. Eighteen hours after transfection, cells were left unstimulated or were stimulated with anti-CD3 plus PMA. A, Luciferase activities were determined as described. B, Expression of transfected and endogenous Gab2 proteins was determined by anti-Gab2 immunoblot analysis of whole cell lysates from the transfected cells. C, Association of Gab2 proteins with p85 was determined after transfection of Jurkat cells with the indicated of Gab2 constructs (5 μg). Transfected cells were left unstimulated or were stimulated with anti-CD3 for 2 min at 37°C. α, 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.

FIGURE 2.

Gab2-mediated inhibition of IL-2P requires binding sites for PI3K. Jurkat cells were transfected with 0.2 (lanes 2, 4, 6, and 8) or 0.05 μg (lanes 3, 5, 7, and 9) of the indicated Gab2 constructs, along with IL-2P and Renilla luciferase reporters and vector DNA to maintain the same total DNA concentration. Eighteen hours after transfection, cells were left unstimulated or were stimulated with anti-CD3 plus PMA. A, Luciferase activities were determined as described. B, Expression of transfected and endogenous Gab2 proteins was determined by anti-Gab2 immunoblot analysis of whole cell lysates from the transfected cells. C, Association of Gab2 proteins with p85 was determined after transfection of Jurkat cells with the indicated of Gab2 constructs (5 μg). Transfected cells were left unstimulated or were stimulated with anti-CD3 for 2 min at 37°C. α, 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.

Close modal
FIGURE 3.

Inhibitory signaling by Gab2 requires PI3K. A, Jurkat cells were transfected with constructs encoding WT-Gab2 or the activated PI3K mutant CD2P110 (5 μg). Cells were left unstimulated or were stimulated with anti-CD3 plus PMA, and luciferase activities were determined. B, Jurkat cells, transfected and stimulated as in A, were incubated with the PI3K inhibitor, LY294002 (15 μM), for 20 min before stimulation. C, Jurkat cells were transfected with constructs encoding WT-Gab2, alone or in combination with WT-PTEN, or catalytically inactive PTEN (5 μg each), in addition to IL-2P and Renilla luciferase reporters. Cells were stimulated and analyzed as described for Fig. 1. D, Jurkat cells were transfected with the indicated Gab2 and/or Shp-2 expression constructs, together with IL-2P and Renilla luciferase constructs and stimulated and analyzed as above. Data represent the results from at least four pooled experiments.

FIGURE 3.

Inhibitory signaling by Gab2 requires PI3K. A, Jurkat cells were transfected with constructs encoding WT-Gab2 or the activated PI3K mutant CD2P110 (5 μg). Cells were left unstimulated or were stimulated with anti-CD3 plus PMA, and luciferase activities were determined. B, Jurkat cells, transfected and stimulated as in A, were incubated with the PI3K inhibitor, LY294002 (15 μM), for 20 min before stimulation. C, Jurkat cells were transfected with constructs encoding WT-Gab2, alone or in combination with WT-PTEN, or catalytically inactive PTEN (5 μg each), in addition to IL-2P and Renilla luciferase reporters. Cells were stimulated and analyzed as described for Fig. 1. D, Jurkat cells were transfected with the indicated Gab2 and/or Shp-2 expression constructs, together with IL-2P and Renilla luciferase constructs and stimulated and analyzed as above. Data represent the results from at least four pooled experiments.

Close modal

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.

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. 1,B). Similarly, expression of WT-Gab2 also blocked IL-2P activation by anti-CD3 plus CD28 costimulation (Fig. 1 C). 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-κB. Overexpression of Gab2 inhibited both NF-AT (Fig. 1,D) and NF-κB (Fig. 1,E)-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. 1,F). 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. 1 G) and D10 Th2 cells (data not shown).

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. 2,A). 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. 2,B). 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 (Gab2ΔPH) 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 Gab2ΔPH 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. 2 C).

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. 3,B). 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. 3,C). 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. 3 D). Coexpression of wild-type shp-2 had no significant effect on the inhibitory effect mediated by Gab2 (p = 0.92).

Although PI3K functions as a positive signal in most cell types, promoting signaling pathways for cell survival, proliferation, adhesion, and/or migration, its role in T cell signaling has been unresolved. Here, we have found that the scaffold Gab2, acting via a PI3K-dependent pathway, negatively regulates TCR-evoked activation of the IL-2 promoter by suppressing both NF-AT- and NF-κB-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-κB-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. 2 A). 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. 1 F). 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.

We thank Drs. Sansana Sawasdikosol (Dana-Farber Cancer Institute) and Lewis Cantley (Beth Israel-Deaconess Medical Center) for helpful discussion and comments and Joanne Hahn and Philip Papst for technical assistance.

1

This work was supported by Grants R01 DK50693, R01 CA66600, and P01 DK50654 (to B.G.N.), R01 CA70758 (to S.J.B.), and R01 AI-42246 (to E.W.G.); The Cancer League of Colorado; and the Association for International Cancer Research (to J.C.P.). H.G. was supported by National Research Service Award Grant CA72144 and by an Anna D. Barker fellowship in basic science from the American Association for Cancer Research, Inc.

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.

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