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The Journal of Immunology, 2001, 167: 2714-2723.
Copyright © 2001 by The American Association of Immunologists

Phosphatidylinositol 3-Kinase Potentiates, but Does Not Trigger, T Cell Proliferation Mediated by the IL-2 Receptor1

James J. Moon and Brad H. Nelson2

Department of Immunology, University of Washington, Seattle, WA 98195; and Virginia Mason Research Center, Seattle, WA 98101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proliferative signaling by the IL-2R can occur through two distinct pathways, one mediated by Stat5 and one by the adaptor protein Shc. Although Stat5 induces T cell proliferation by serving as a transcription factor, the mechanism of proliferative signaling by Shc is poorly defined. We examined the roles of two major signaling pathways downstream of Shc, the p44/p42 mitogen-activated protein kinase (extracellular signal-related kinase (Erk)) and phosphatidylinositol 3-kinase (PI3K) pathways, in promitogenic gene induction and proliferation in the IL-2-dependent T cell line CTLL-2. Using IL-2R mutants and specific pharmacologic inhibitors, we found that the PI3K, but not Erk, pathway is required for maximal induction of c-myc, cyclin D2, cyclin D3, cyclin E, and bcl-xL by Shc. To test whether the PI3K pathway is sufficient for proliferative signaling, a tamoxifen-regulated form of PI3K (mp110*ER) was expressed in CTLL-2 cells. Activation of the PI3K pathway through mp110*ER failed to up-regulate expression of the c-myc, cyclin D2, cyclin D3, cyclin E, bcl-2, or bcl-xL genes or down-regulate expression of p27Kip1, even when coactivated with the Janus kinases (Jak) or the Raf/Erk pathway. Moreover, mp110*ER induced modest levels of thymidine incorporation without subsequent cell division. Although insufficient for mitogenesis, mp110*ER enhanced Stat5-mediated proliferative signaling through a mechanism independent of Stat5 transcriptional activity. Thus, in addition to serving a necessary, but insufficient role in Shc-mediated promitogenic gene expression, the PI3K pathway contributes to T cell proliferation by potentiating mitogenic signaling by Stat5.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Clonal expansion of T cells after Ag stimulation is driven by cytokines such as IL-2. Although signaling by the TCR complex causes resting T cells to exit the G0 phase of the cell cycle, IL-2R signaling promotes progression through the G1 phase cell cycle checkpoint, thus enabling proliferation to occur (1). According to current models of growth factor receptor signaling, the IL-2R promotes G1 to S phase progression in part by inducing expression of promitogenic genes such as the D- and E-type cyclins, which bind and up-regulate the activity of cyclin-dependent kinases (cdk)3 2, 4, and 6 (2, 3). In addition, signals from the IL-2R down-regulate expression of cdk inhibitors, in particular, p27Kip1 (4, 5). Once activated, cyclin-cdk complexes phosphorylate Rb family pocket proteins, resulting in the release of E2F transcription factors, which in turn transactivate critical S phase target genes (6). Thus, the induction of promitogenic genes in early G1 is a central component of cell cycle control by the IL-2R. However, the biochemical pathways that link the IL-2R to these genes remain poorly defined.

The IL-2R is comprised of three distinct transmembrane subunits, {alpha}, {beta}, and {gamma}c (1). Signaling is initiated by the ligand-induced heterodimerization of the cytoplasmic domains of IL-2R{beta} and {gamma}c, which activates the preassociated tyrosine kinases, Janus kinase (Jak) 1 and Jak3 (1, 7, 8, 9). Activated Jaks phosphorylate key tyrosines on IL-2R{beta}, which serve as docking sites for downstream signaling molecules, including Shc and Stat5 (9, 10, 11). Shc recruits at least two important protein complexes: Grb-2/Sos, which activates the Ras/extracellular signal-related kinase (Erk) pathway, and Grb-2/Gab-2, which activates the phosphatidylinositol 3-kinase (PI3K) pathway (12, 13, 14, 15). In contrast, Stat5 translocates to the nucleus and directly regulates transcription of target genes by binding enhancer elements (9, 16). Despite the fundamental differences in their mechanisms of signaling, Shc and Stat5 are both capable of inducing multiple promitogenic genes, including c-myc, bcl-2, and bcl-xL, thereby promoting T cell proliferation (13, 16).

Unlike Stat5, the molecular mechanism responsible for Shc-mediated proliferation remains undefined. Shc-mediated activation of the Ras/Erk pathway presents an appealing hypothesis, due to the well-established role of Ras in the regulation of cell proliferation and transformation. Many of the mitogenic signaling properties of Ras are attributed to the downstream mitogen-activated protein kinase (Erk) signaling cascade, which phosphorylates a wide range of transcription factors, including Elk-1, Ets-1, Fos, AP-1, NF-AT, and c-Myc (17, 18, 19). Extensive studies in nonlymphoid cells have identified a number of proliferative gene targets, including c-fos, c-myc, and cyclin D1 (20, 21, 22). However, the role of the Ras/Erk pathway in IL-2R proliferative signaling is not well understood.

Alternatively, the major Shc-mediated pathway to proliferation may be through PI3K and downstream effectors such as Akt(PKB), mTOR(FRAP), and p70S6 kinase (23). Akt is known to promote cell survival via activation of the NF-{kappa}B pathway as well as inactivation of the proapoptotic proteins Bad, caspase 9, GSK-3, and the Forkhead-related transcription factors FKHR, FKHRL1, and AFX (24). mTOR promotes cell growth by inactivating 4E-BP1(PHAS), an inhibitor of the translation initiation factor eIF4E, while p70S6 kinase phosphorylates the S6 subunit of the 40S ribosome, thus promoting translation of growth-related mRNA species distinguished by a unique 5' terminal oligopyrimidine tract (25, 26). In various cell types, the PI3K pathway has been linked to the up-regulation of cyclin D1 by growth factor receptors (27, 28). In T cells, the PI3K pathway has been implicated in the up-regulation of E2F transcriptional activity through a mechanism purportedly involving the induction of cyclin D3, the down-regulation of p27Kip1, and the phosphorylation of Rb pocket proteins (29).

Because both the Erk and PI3K pathways represent potential mechanisms for Shc-mediated proliferative signaling, we directly investigated the role of each pathway in regulating the promitogenic genes c-myc, cyclin D2, cyclin D3, cyclin E, bcl-2, and bcl-xL in the murine IL-2-dependent T cell line CTLL-2. Our results demonstrate the dispensability of the Erk pathway, and a necessary, yet insufficient role for the PI3K pathway in the maximal induction of these genes. Furthermore, we define a new role for the PI3K pathway in the enhancement of Stat5-mediated proliferative signaling through a G1 cyclin-independent mechanism, thus suggesting that PI3K potentiates, but does not directly induce proliferative signaling by the IL-2R.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction

The chimeric receptor chains {beta}{beta}wt and {alpha}{gamma}wt (formerly denoted GM{beta}/2{beta} and GM{alpha}/2{gamma}), and {beta}{beta}{Delta}325+Y510 have been described (8, 16). To generate {beta}{beta}325Shc{Delta}P, the phosphotyrosine-binding domain of Shc was removed from the previously described {beta}{beta}325Shc (13) via splice-overlap extension (SOE) PCR (30). To generate {beta}{beta}325Shc{Delta}PFFF, tyrosines 239, 240, and 317 of Shc in {beta}{beta}325Shc{Delta}P were point mutated to phenylalanine by SOE PCR. To generate mp110* estrogen receptor (mp110*ER), mAktER (31) was ligated to the C-terminal end of mp110* (32), and the mAkt region was then removed by SOE PCR. All cDNAs were sequenced and cloned into a human {beta} actin promoter-driven expression vector containing either a neomycin or hygromycin resistance gene (8, 13).

Cell culture

The murine IL-2-dependent T cell line CTLL-2 was obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 25 mM HEPES, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 mM sodium pyruvate, 25 mM 2-ME, and 50 U/ml rIL-2 (Chiron, Emeryville, CA). Linearized plasmids were electroporated into cells, and stably transfected subclones were selected at limiting dilution for G418 resistance. Receptor expression was assessed by flow cytometry with Abs to human GM-CSFR{alpha} or {beta}c (SC-458, SC-457; Santa Cruz Biotechnology, Santa Cruz, CA). Expression of mp110*ER and {Delta}RafER was assessed by Western blot with an Ab to the murine ER (sc-542; Santa Cruz Biotechnology). PD98059 (PD), LY294002 (LY), and 4-hydroxytamoxifen (4-OHT) were purchased from Calbiochem (San Diego, CA).

Western blots

Cytoplasmic and nuclear extracts of CTLL-2 cells were prepared and immunoblotted as described (33). Abs to phospho-Erk (9101S), Erk (9102), phospho-Akt (9271S), Akt (9272), and phospho-Stat5 (9351S) were purchased from New England Biolabs (Beverly, MA). Abs to p70S6 kinase (sc-230), Stat5 (sc-835), c-myc (sc-764), cyclin D2 (sc-593), and p27Kip1 (sc-528) were purchased from Santa Cruz Biotechnology. Ab to phospho-Jak1 44–422(44–422) was purchased from BioSource International (Camarillo, CA).

Northern blots

Northern blots were performed as described (13, 16) using probes from the murine genes c-fos (1.5-kb EcoRI/PstI), c-myc (400-bp PstI), cyclin D2 (1.2-kb EcoRI), cyclin D3 (800-bp SmaI), cyclin E (1.8-kb EcoRI), bcl-2 (900-bp PstI), bcl-xL (1-kb EcoRI), CIS (400-bp BamHI/HindIII), and GAPDH (1.2-kb PstI).

Proliferative assays

Thymidine incorporation, cell growth, and cell viability assays were performed as described (13, 16). Briefly, cells were pelleted, washed three times with PBS to remove all traces of IL-2 from the culture medium, and counted. Thymidine incorporation assays were conducted in triplicate wells containing 104 cells in 200 µl medium plus the appropriate stimulus. At 8 or 20 h, cells were pulsed with [3H]thymidine (2.5 µCi/well) for 4 h, harvested onto glass fiber filter mats, and analyzed for [3H]thymidine incorporation by liquid scintillation counting. For cell growth and viability assays, cells were plated at 2 x 105/ml in medium plus the appropriate stimulus. At each time point, aliquots were taken for enumeration and trypan blue staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Shc-mediated proliferative signaling requires the Grb-2 binding site

Experiments were performed in the IL-2-dependent murine CD8+ T cell line CTLL-2 using a chimeric receptor consisting of the extracellular domains of GM-CSFR{beta}c and GM-CSFR{alpha} fused, respectively, to the transmembrane and cytoplasmic domains of IL-2R{beta} and {gamma}c, to generate the chimeric subunits {beta}{beta}wt and {alpha}{gamma}wt (Fig. 1Go). GM-CSF-induced heterodimerization of {beta}{beta}wt and {alpha}{gamma}wt results in an intracellular signal that is indistinguishable from that induced by the native IL-2R (8, 16, 33, 34). To generate Shc-mediated signals in the absence of Stat5 activity, we modified {beta}{beta}wt to create {beta}{beta}325Shc{Delta}P, in which the membrane-proximal Jak1-binding region of IL-2R{beta} is covalently fused to the collagen homology and Src homology 2 domains of Shc (Fig. 1Go). In contrast to the previously described receptor mutant {beta}{beta}325Shc (13), which contained full-length Shc, {beta}{beta}325Shc{Delta}P lacks the phosphotyrosine-binding domain of Shc. The phosphotyrosine-binding domain normally mediates binding of Shc to tyrosine Y338 of IL-R{beta} (35), and therefore should be dispensable when Shc is covalently fused to the receptor. Indeed, comparisons between {beta}{beta}325Shc and {beta}{beta}325Shc{Delta}P have revealed no differences in signaling properties (data not shown).



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  		FIGURE 1. Schematic diagram of wild-type and mutant GM-CSF/IL-2 chimeric receptor chains. S, Serine-rich domain; A, acidic domain; H, carboxyl "half" domain.

 
GM-CSF-induced heterodimerization of {beta}{beta}325Shc{Delta}P and {alpha}{gamma}wt induces signaling events characteristic of Shc, including activation of the Ras/Erk pathway (as detected by Erk phosphorylation and c-fos gene expression) and the PI3K pathway (as detected by phosphorylation of the downstream effectors Akt and p70S6 kinase) (13, 15, 23) (Fig. 2Go, A and B). The phosphorylation of Erk and Akt occurred within minutes of stimulation, and was sustained through at least 3 h (data not shown). In addition, {beta}{beta}325Shc{Delta}P induced expression of the promitogenic genes c-myc, cyclin D2, cyclin D3, cyclin E, bcl-2, and bcl-xL, and promoted cell proliferation, as evidenced by thymidine incorporation (Fig. 2Go, B and C).



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  		FIGURE 2. Shc-mediated proliferative signaling requires the Grb-2 binding site. CTLL-2 subclones stably expressing chimeric receptors were deprived of cytokines for 8 h and then stimulated with IL-2 (100 U/ml) or GM-CSF (100 ng/ml) for the indicated times. A, Western blot analysis of cytoplasmic proteins revealing activation of the Erk and PI3K pathways. The phosphorylation of Erk and Akt was detected with phospho-specific Abs, whereas the phosphorylation of p70S6 kinase was evidenced by decreased electrophoretic mobility. B, Northern blot assessment of promitogenic gene induction by each indicated receptor. For c-fos, x = 1 h; c-myc and bcl-xL, x = 3 h; cyclin D2, cyclin D3, cyclin E, bcl-2, and GAPDH, x = 6 h. C, [3H]Thymidine incorporation assay showing the ability of each receptor to induce proliferation. Subclones expressing each receptor mutant were washed, stimulated with GM-CSF (100 ng/ml) for 20 h, and then pulsed with [3H]thymidine for 4 h. Values indicate [3H]thymidine incorporation as a percentage of IL-2-stimulated controls minus background levels from medium controls. Error bars represent 1 SD of the mean value for triplicate samples per subclone. Data shown in A–C are representative results from multiple independent subclones for each receptor mutant.

 
To test whether the Grb-2 binding site of Shc is required for proliferative signaling, we created {beta}{beta}325Shc{Delta}PFFF, a modified form of {beta}{beta}325Shc{Delta}P in which tyrosines 239, 240, and 317 comprising the Grb-2 binding site of Shc were mutated to phenylalanine (36) (Fig. 1Go). Unlike {beta}{beta}325Shc{Delta}P, this receptor failed to activate the Ras/Erk or PI3K pathways, induce expression of c-fos, c-myc, cyclin D2, cyclin D3, cyclin E, bcl-2, or bcl-xL, or promote cell proliferative events (15) (Fig. 2Go). Thus, the Grb-2 binding site is essential for Shc-mediated promitogenic gene expression and cell proliferation.

The PI3K, but not Erk, pathway is required for maximal promitogenic gene expression by Shc

Pharmacologic inhibitors were used to determine the relative contributions of the Erk and PI3K pathways to Shc-mediated promitogenic gene expression. Treatment of cells with the mitogen-activated protein/Erk kinase (MEK) 1/2 inhibitor PD (37) blocked IL-2-induced Erk phosphorylation in a dose-dependent manner with complete inhibition at 25 µM (Fig. 3GoA, and data not shown). However, PD did not inhibit the PI3K pathway, even at concentrations as high as 100 µM. Conversely, treatment with the PI3K inhibitor LY (38) blocked the PI3K pathway in a dose-dependent manner with complete inhibition at 25 µM, but did not inhibit the Erk pathway at concentrations up to 100 µM (Fig. 3GoA, and data not shown). Subsequent experiments used PD and LY at doses of 50 µM each to ensure complete inhibition of Erk or PI3K, respectively, over prolonged time courses.



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  		FIGURE 3. Shc-mediated promitogenic gene induction requires PI3K, but not Erk. CTLL-2 subclones expressing {beta}{beta}325Shc{Delta}P were treated as in Fig. 2Go. Where indicated, PD (50 µM) or LY (50 µM) was added to cells 30 min before cytokine stimulation. A, Western blot analysis of cytoplasmic proteins showing the effect of inhibitors on {beta}{beta}325Shc{Delta}P-mediated activation of the Erk and PI3K pathways. B, Northern blot analysis showing the effect of inhibitors on Shc-mediated gene expression. C, Western blot analysis of nuclear extracts showing the effect of inhibitors on Shc-mediated c-Myc and cyclin D2 protein expression. Data shown are representative results for multiple independent subclones.

 
As expected, PD blocked induction of c-fos, a Ras/Erk target gene, by {beta}{beta}325Shc{Delta}P (20) (Fig. 3GoB). However, it had no effect on the expression of c-myc, cyclin D2, cyclin D3, cyclin E, bcl-2, or bcl-xL. Consistent with this, PD also did not impact expression of the c-Myc or cyclin D2 proteins (Fig. 3GoC).

In contrast, LY significantly impaired promitogenic gene induction by {beta}{beta}325Shc{Delta}P. Induction of cyclin D2 and cyclin E was completely eliminated, while the induction of c-myc, cyclin D3, and bcl-xL was partially reduced (Fig. 3GoB). Induction of c-fos and bcl-2, however, was not inhibited. These effects of LY were observed in a dose-dependent manner that correlated with inhibition of Akt and p70S6 kinase phosphorylation (data not shown). Treatment of cells with both PD and LY did not increase the level of inhibition achieved with LY alone for any of the genes except c-fos (data not shown). LY also completely inhibited the induction of c-Myc and cyclin D2 proteins by {beta}{beta}325Shc{Delta}P (Fig. 3GoC). Thus, whereas the Erk pathway is dispensable, the PI3K pathway plays a major role in regulating Shc-mediated promitogenic gene expression. Furthermore, additional unidentified pathways must exist downstream of Shc to mediate the induction of bcl-2, and to some extent, c-myc, cyclin D3, and bcl-xL, because the expression of these genes was not fully blocked by PD and LY.

Activation of the PI3K pathway promotes limited proliferative activity

The foregoing results with the inhibitor LY indicate that the PI3K pathway plays a major role in proliferative signaling by Shc. To address whether activation of the PI3K pathway is sufficient for proliferative signaling, we used mp110*ER, a conditionally active PI3K construct regulated by the estrogen analogue 4-OHT. mp110*ER consists of a myristoylated, constitutively active PI3K p110 subunit (mp110*) (32) fused to a modified ER hormone-binding domain (39) (Fig. 4GoA). Association between the ER domain and the cytoplasmic chaperone heat shock protein 90 keeps mp110*ER in an inactive state. Upon addition of 4-OHT, which competitively binds ER, mp110*ER is released from heat shock protein 90, and via its myristoyl group localizes at the surface membrane, where it phosphorylates phospholipid substrates (32, 40). As expected, 4-OHT treatment of CTLL-2 cells stably expressing mp110*ER induced phosphorylation of Akt and p70S6 kinase, and this was inhibited by treatment with LY (Fig. 4GoB). Moreover, activation of mp110*ER increased the viability of cells undergoing cytokine withdrawal, in accordance with the known antiapoptotic properties of the PI3K pathway (23, 24) (Fig. 4GoC).



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  		FIGURE 4. Activation of the PI3K pathway generates a partial proliferative signal. A, Schematic diagram of mp110*ER. iSH2, intra-Src homology 2 domain; ER-HBD, ER hormone-binding domain. B, Western blot analysis of cytoplasmic proteins showing tamoxifen-regulated activation of the PI3K pathway in CTLL-2 subclones expressing mp110*ER. Cells were washed, deprived of cytokine for 8 h, and stimulated for the indicated times with IL-2 (100 U/ml) or 4-OHT (1 µM) in the presence or absence of LY (50 µM). C, Activation of mp110*ER increases cell viability during cytokine withdrawal. Three independent mp110*ER-expressing subclones (represented by three different symbols) were washed and stimulated with either medium, IL-2 (100 U/ml), or 4-OHT (1 µM). Cell viability was assessed at the indicated times by trypan blue exclusion. D, Activation of mp110*ER causes modest levels of thymidine incorporation. Six independent mp110*ER-expressing subclones were washed, stimulated with medium, IL-2 (100 U/ml), or 4-OHT (1 µM) for 8 h, and then pulsed with [3H]thymidine for 4 h. Values indicate [3H]thymidine incorporation as a percentage of IL-2-stimulated controls minus background levels from medium controls. Error bars represent 1 SD of the mean value for triplicate samples per subclone. E, Activation of mp110*ER does not cause cell expansion. Four independent mp110*ER-expressing subclones were washed, stimulated with medium, IL-2 (100 U/ml), or 4-OHT (1 mM), and counted 72 h later. Values for the IL-2-stimulated samples reflect a 1:3 split made at 48 h to maintain cell density at ~2 <5>x 105 cells/ml. Data shown in B–E are representative results from multiple independent subclones.

 
Activation of the PI3K pathway via mp110*ER induced thymidine incorporation equivalent to 30–60% of IL-2-stimulated controls (Fig. 4GoD), which is slightly below the range observed with {beta}{beta}325Shc{Delta}P (Fig. 2GoC). However, in contrast to signals mediated by Shc or the endogenous IL-2R, mp110*ER did not cause cells to divide (13) (Fig. 4GoE), as there was no overall increase in the number of cells, either alive or dead, at 12, 24, 48, or 72 h after stimulation with 4-OHT (Fig. 4GoE, and data not shown). By contrast, cells stimulated with IL-2 underwent one to three rounds of cell division during this time period (Fig. 4GoE). Hence, isolated activation of the PI3K pathway generates an incomplete proliferative signal that promotes DNA synthesis without subsequent cell division.

Activation of the PI3K pathway is not sufficient to induce promitogenic gene expression

Based on the ability of the PI3K inhibitor LY to impair expression of c-myc, cyclin D2, cyclin E, and bcl-xL, we speculated that mp110*ER might induce expression of one or more of these promitogenic genes. However, activation of mp110*ER failed to induce expression of c-myc, cyclin D3, cyclin E, bcl-2, or bcl-xL (Fig. 5GoA). A slight to moderate induction of cyclin D2 expression was observed in some CTLL-2 subclones, but this was not a consistent result overall. Moreover, mp110*ER failed to induce expression of the c-Myc or cyclin D2 proteins (Fig. 5GoB). Therefore, despite the requirement of the PI3K pathway for Shc-mediated expression of multiple promitogenic genes, activation of this pathway is not sufficient to up-regulate expression of these genes.



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  		FIGURE 5. Activation of the PI3K pathway fails to induce promitogenic genes. CTLL-2 subclones expressing mp110*ER were washed, deprived of cytokines for 8 h, and then stimulated with IL-2 (100 U/ml) or 4-OHT (1 µM) for the indicated times. A, Northern blot assessment of mp110*ER-mediated gene induction. For c-myc and bcl-xL, x = 3 h; cyclin D2, cyclin D3, cyclin E, bcl-2, and GAPDH, x = 6 h. B, Western blot analysis of nuclear extracts showing c-Myc and cyclin D2 protein expression. C, Activation of the PI3K pathway does not down-regulate p27Kip1 expression. Subclones expressing mp110*ER were washed and stimulated with medium, IL-2 (100 U/ml), or 4-OHT (1 µM) for 9 h, at which point nuclear extracts were Western blotted to assess p27Kip1 expression. Data shown in A–C are representative results from multiple independent subclones.

 
Accordingly, mp110*ER failed to down-regulate expression of the cdk inhibitor p27Kip1, which normally occurs near the end of G1 phase as a result of increased cyclin expression and subsequent cdk activity (5) (Fig. 5GoC). Thus, the modest proliferative response induced by mp110*ER in CTLL-2 cells does not appear to involve the conventional G1 cyclin pathway.

Activation of Jak kinases or the Raf/Erk pathway fails to complement proliferative signaling by PI3K

Because activation of the PI3K pathway alone was insufficient to generate a complete proliferative response, we sought to identify the additional components of the Shc-dependent IL-2R signal that are necessary to mediate proliferation. To determine whether the PI3K pathway could be complemented by elements of IL-2R signaling upstream of Shc:Grb-2 binding, mp110*ER was coexpressed with {beta}{beta}325Shc{Delta}PFFF in CTLL-2 cells. Simultaneous activation of mp110*ER and {beta}{beta}325Shc{Delta}PFFF resulted in the activation of the PI3K pathway as well as Jak1 and Jak3 (assessed by tyrosine phosphorylation of Jak1) (41) (Fig. 6GoA), but failed to induce expression of c-myc, cyclin D2, cyclin D3, cyclin E, bcl-2, or bcl-xL (Fig. 6GoB). Likewise, the combination of signals from mp110*ER and {beta}{beta}325Shc{Delta}PFFF did not significantly increase thymidine incorporation or cell expansion above the levels attained with mp110*ER alone (Fig. 6GoC, and data not shown).



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  		FIGURE 6. Jak kinase activation fails to complement PI3K pathway-mediated proliferative signaling. CTLL-2 cell subclones stably expressing mp110*ER and {beta}{beta}325Shc{Delta}PFFF were washed, deprived of cytokines for 8 h, and then stimulated with IL-2 (100 U/ml) or both GM-CSF (100 ng/ml) and 4-OHT (1 µM). A, Western blot analysis of cytoplasmic proteins showing activation of the Jak kinases (by anti-phospho-Jak1/Ab) and the PI3K pathway (by anti-phospho-Akt/Ab). B, Northern blot assessment of promitogenic gene induction. For c-fos, x = 1 h; c-myc and bcl-xL, x = 3 h; cyclin D2, cyclin D3, cyclin E, bcl-2, and GAPDH, x = 6 h. C, Thymidine incorporation. Subclones were washed, stimulated as indicated for 8 h, and then pulsed with [3H]thymidine for 4 h. Values indicate [3H]thymidine incorporation as a percentage of IL-2-stimulated controls minus background levels from medium controls. Data shown in A–C are representative results from multiple independent subclones.

 
We next determined whether concurrent activation of the Raf/Erk pathway could complement PI3K pathway-mediated proliferative signaling. This was addressed by coexpressing mp110*ER with {Delta}RafER, a conditionally active form of Raf (42). Similar to mp110*ER, {Delta}RafER consists of the kinase domain of Raf fused to a modified ER hormone-binding domain (39). Like mp110*ER, {Delta}RafER is normally inactive, but becomes activated upon the addition of 4-OHT. As expected, simultaneous activation of both mp110*ER and {Delta}RafER with 4-OHT resulted in the activation of both the PI3K and Erk pathways (Fig. 7GoA), as well as the induction of c-fos expression, but did not induce expression of the promitogenic genes c-myc, cyclin D2, cyclin D3, cyclin E, bcl-2, or bcl-xL (Fig. 7GoB). Furthermore, these concurrent signals did not induce thymidine incorporation or cell division to a greater extent than achieved with mp110*ER alone (Fig. 7GoC, and data not shown). Thus, neither the Jak kinases nor the Raf/Erk pathway complement the PI3K pathway to restore full proliferative signaling, indicating that an unidentified pathway downstream of Shc is essential for inducing cell proliferation.



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  		FIGURE 7. Activation of the Raf/Erk pathway fails to complement PI3K pathway-mediated proliferative signaling. CTLL-2 cell subclones stably expressing mp110*ER and {Delta}RafER were washed, deprived of cytokines for 8 h, and then stimulated with IL-2 (100 U/ml) or 4-OHT (1 µM). A, Western blot analysis of cytoplasmic proteins showing activation of the PI3K and Erk pathways. B, Northern blot assessment of promitogenic gene induction. For c-fos, x = 1 h; c-myc and bcl-xL, x = 3 h; cyclin D2, cyclin D3, cyclin E, bcl-2, and GAPDH, x = 6 h. C, Thymidine incorporation. Subclones stably expressing these constructs were washed, stimulated as indicated for 8 h, and then pulsed with [3H]thymidine for 4 h. Values indicate [3H]thymidine incorporation as a percentage of IL-2-stimulated controls minus background levels from medium controls. Data shown in A–C are representative results from multiple independent subclones.

 
Activation of the PI3K pathway complements proliferative signaling by Stat5

Collectively, our data indicate that activation of the PI3K pathway is required for maximal promitogenic gene induction by Shc, but is insufficient to trigger a full proliferative response. These results are consistent with the fact that PI3K is also activated by many nonmitogenic receptors, suggesting that it may play a general, permissive role in intracellular signaling (23). We therefore hypothesized that the primary role of PI3K in IL-2R-proliferative signaling may be to potentiate signals provided by other pathways, particularly those that up-regulate G1 cyclin expression. At present, the pathways downstream of Shc that trigger G1 cyclin expression remain unidentified, which precludes a direct test of this hypothesis in the context of Shc signaling. However, the transcription factor Stat5 constitutes an independent IL-2R-mediated proliferative pathway that induces G1 cyclin expression in parallel with the Shc pathway (16). Therefore, we addressed the issue of whether activation of the PI3K pathway could enhance proliferative signaling by Stat5.

mp110*ER was coexpressed with the previously described receptor mutant {beta}{beta}{Delta}325+Y510, which is a truncated version of {beta}{beta}wt containing a single tyrosine residue that activates Stat5, but not Shc or its downstream Erk and PI3K pathways (16) (Figs. 1Go and 8GoA, and data not shown). As previously demonstrated, {beta}{beta}{Delta}325+Y510 induces a proliferative response in CTLL-2 cells manifest by G1 cyclin up-regulation, thymidine incorporation, and cell expansion (16, 43). However, the magnitude of Stat5-mediated proliferation, as measured by thymidine incorporation, is only 50–70% of that achieved with a full IL-2R signal incorporating both Stat5 and Shc activity (16) (Fig. 8GoB). This diminished level of proliferative activity cannot be attributed to suboptimal Stat5 activation by {beta}{beta}{Delta}325+Y510, as the levels of active Stat5 attained with this receptor and the full-length {beta}{beta}wt receptor are indistinguishable (16).



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  		FIGURE 8. The PI3K pathway complements Stat5-mediated proliferative signaling. CTLL-2 cells were transfected with both mp110*ER and {beta}{beta}{Delta}325+Y510. A, Subclones stably expressing these constructs were washed, deprived of cytokines for 8 h, and then stimulated with either IL-2 (100 U/ml), GM-CSF (100 ng/ml), or both GM-CSF and 4-OHT (1 µM) for the indicated times. Cytoplasmic proteins were Western blotted to verify stimulus-induced activation of Stat5 and the PI3K pathway. B, Activation of the PI3K pathway complements Stat5-mediated thymidine incorporation to levels near that of a full IL-2R response. mp110*ER/{beta}{beta}{Delta}325 +Y510 expressing subclones were washed, stimulated as indicated for 20 h, and then pulsed with [3H]thymidine for 4 h. C, Complementation by the PI3K pathway occurs throughout a range of Stat5 activity. Three independent subclones (represented by three different symbols) were treated as in B and then stimulated throughout the GM-CSF dose-response range of {beta}{beta}{Delta}325+Y510. D, Shc- and Stat5-mediated signals complement each other to levels near that of a full IL-2R response. CTLL-2 subclones expressing the indicated constructs were washed, stimulated with GM-CSF (100 ng/ml), or both GM-CSF and 4-OHT (1 µM) if ER constructs are involved, for 20 h, and then pulsed with [3H]thymidine for 4 h. For B–D, values indicate [3H]thymidine incorporation as a percentage of IL-2-stimulated controls minus background levels from medium controls. Data shown in A–D are representative results from multiple independent subclones.

 
As expected, coactivation of mp110*ER with {beta}{beta}{Delta}325+Y510 resulted in the activation of both the PI3K pathway and Stat5 (Fig. 8GoA). With both signals active, thymidine incorporation was restored to 80–100% of IL-2-stimulated controls (Fig. 8GoB), thus demonstrating a complementation of proliferative signals by Stat5 and PI3K. This enhancement of proliferative signaling by mp110*ER was observed throughout the GM-CSF dose-response range of {beta}{beta}{Delta}325+Y510 (Fig. 8GoC). In contrast, mp110*ER activation did not enhance the level of thymidine incorporation induced by {beta}{beta}325Shc{Delta}P, which activates the PI3K pathway through Shc (Fig. 8GoD), or a wild-type IL-2R signal containing both Shc and Stat5 components (Fig. 8GoB). Enhancement of thymidine incorporation was also observed when {beta}{beta}{Delta}325+Y510 was coexpressed and coactivated with {beta}{beta}325Shc{Delta}P, but not {Delta}RafER, further demonstrating that the ability of Shc to complement Stat5-mediated proliferative signaling is an effect specific to the PI3K pathway (Fig. 8GoD).

The PI3K pathway does not modulate Stat5 transcriptional activity

Finally, we asked whether PI3K potentiates Stat5 signaling through a general enhancement of Stat5 transcriptional activity, or by activating a parallel signaling pathway that complements Stat5. {beta}{beta}{Delta}325+Y510 was triggered with a suboptimal dose of GM-CSF (1 ng/ml) so that target genes of Stat5, such as CIS, c-myc, cyclin D2, and bcl-xL, were induced to submaximal levels (16, 43, 44, 45). This created a situation in which a general enhancement of Stat5 transcriptional activity, if applicable, would be readily detectable. Concurrent activation of mp110*ER with {beta}{beta}{Delta}325+Y510 did not increase the magnitude or duration of Stat5 phosphorylation in response to GM (Fig. 9GoA), and the expression levels of Stat5 target genes were not enhanced (Fig. 9GoB). Thus, the PI3K pathway does not promote general enhancement of Stat5 transcriptional activity, but rather appears to induce cell proliferative events parallel to or downstream of Stat5 target genes.



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  		FIGURE 9. Activation of the PI3K pathway does not affect Stat5 transcriptional activity. CTLL-2 subclones expressing mp110*ER/{beta}{beta}{Delta}325+Y510were washed, cytokine deprived for 8 h, and then stimulated with IL-2 (100 U/ml), GM-CSF (1 ng/ml), or GM-CSF (1 ng/ml) and 4-OHT (1 µM). A, Western blot analysis showing the absence of mp110*ER-mediated enhancement of Stat5 phosphorylation. B, Northern blot analysis of Stat5 target genes showing the absence of enhanced expression by mp110*ER. Data shown in A and B are representative results from multiple independent subclones.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we investigated the roles of the Erk and PI3K pathways in the Shc-mediated induction of key promitogenic genes downstream of the IL-2R in T cells. Using the pharmacologic inhibitors PD and LY, we demonstrated that the Erk pathway is dispensable for the Shc-mediated expression of all the genes analyzed, while the PI3K pathway is required for maximal induction of c-myc, cyclin D2, cyclin D3, cyclin E, and bcl-xL. However, activation of the PI3K pathway is not sufficient for induction of these genes, as a tamoxifen-regulated PI3K construct failed to induce promitogenic gene expression, even with concurrent activation of the Jak or Erk kinases. Therefore, we conclude that the PI3K pathway potentiates Shc-mediated promitogenic gene induction, but that other unidentified signals downstream of Shc are essential for triggering this process. This role for PI3K is supported by the finding that activation of this pathway also potentiates Stat5-mediated proliferative signaling by promoting events parallel to the conventional G1 cyclin pathway.

Several reports have linked the Erk pathway to the induction of c-myc and cyclin D1 by growth factor receptors in nonlymphoid cells. These studies involved the obstruction of the Ras/Erk pathway with pharmacologic inhibitors or dominant-negative versions of these proteins, as well as the use of constitutively or conditionally active versions of Raf and MEK (22, 46, 47, 48). Our results indicate that these findings do not apply to IL-2-stimulated T cells, as we observed no effects of the MEK inhibitor PD on any of the genes analyzed, with the expected exception of c-fos. In addition, a conditionally active Raf construct was unable to induce expression of these genes (data not shown), even when activated in concert with the PI3K pathway (Fig. 7GoB). Hence, while the Ras/Erk pathway may be instrumental for the induction of promitogenic genes by certain growth factor receptors such as the CSF-1 receptor, this signaling pathway is neither necessary nor sufficient for Shc-mediated proliferative signaling by the IL-2R.

In contrast to PD, the PI3K inhibitor LY had a striking effect on the induction of several promitogenic genes, in particular cyclins D2 and E. This correlates with several reports in which PI3K inhibitors or dominant-negative PI3K constructs have been shown to block certain aspects of proliferative signaling, including the expression of cyclin D1, to various serum-derived stimuli (27, 28, 49). In T cells, Brennan and colleagues (29) showed that LY inhibited expression of cyclin D3 protein, down-regulation of p27Kip1, Rb/p130 hyperphosphorylation, and the inducible activity of an E2F reporter gene in response to IL-2. Based on these effects, Brennan and others have proposed that the activation of PI3K by growth factor receptors may trigger promitogenic gene expression and cell proliferation, thereby providing an attractive hypothesis to explain the Shc-mediated proliferative signal by the IL-2R.

Indeed, overexpression of wild-type p110 has been shown to elevate levels of cyclin D1 expression in fibroblasts (28). Moreover, Brennan et al. showed that a constitutively active version of PI3K could induce expression of an E2F reporter gene (29), further implicating this pathway as a driver of cell proliferation. Unexpectedly, however, we found that activation of the PI3K pathway with the conditionally active mp110*ER construct fails to induce expression of any of the promitogenic genes analyzed, even though many of these genes are sensitive to inhibition by LY. Furthermore, activation of the PI3K pathway failed to induce late G1 events such as cyclin E expression or down-regulation of p27Kip1, indicating that PI3K alone is unable to trigger the conventional G1 cyclin pathway in T cells.

Despite the inability of the PI3K pathway to induce promitogenic genes on its own, it can promote a partial proliferative response, characterized by increased thymidine incorporation without subsequent cell division. This partial proliferative response may reflect a general property of PI3K signaling in mammalian cell cycle regulation, as a similar result was observed in fibroblasts. In this case, the increased thymidine incorporation was attributed to small increases in cdk2 and cdk4 activity (40). Our results in CTLL-2 cells suggest the additional possibility that the PI3K pathway promotes events that are independent of and parallel to the conventional G1 cyclin pathway, as the proliferative response observed occurred in the absence of D- or E-type cyclin induction or down-regulation of the cdk inhibitor p27Kip1. Recent studies involving c-myc and cyclin E overexpression provide evidence for the existence of nonconventional, E2F-independent pathways leading to G1 to S phase cell cycle progression (50, 51, 52). It is tempting to speculate that elements of the PI3K pathway may intersect this or similar processes. Alternatively, other studies of c-myc function have highlighted the intimate relationship between cell growth and cell proliferation (53, 54), which raises the possibility that the cell growth-promoting capacity of the PI3K/mTOR/p70S6 kinase pathway may be sufficient for partial cell cycle progression in the absence of G1 cyclin induction.

Given the necessary, but insufficient role of the PI3K pathway in driving Shc-mediated promitogenic gene induction and proliferation in T cells, we propose that the primary role of PI3K in cell proliferation may be to potentiate mitogenic signals from other pathways. In such a case, cells with high levels of PI3K activity would be rendered more sensitive to mitogenic stimuli, which might account for the reported oncogenic nature of PI3K and several of its downstream constituents. Conversely, inhibition of PI3K by compounds such as LY would make cells less sensitive to the effects of these stimuli, as we observed for several promitogenic genes normally induced by Shc.

This model is supported by the finding that activation of the PI3K pathway can potentiate proliferative signaling by Stat5. This potentiation was not the result of an increase in the amount of Stat5 activated, nor an increase in the efficiency of Stat5 transcriptional activity. Indeed, in the case of CIS and c-myc, activation of the PI3K pathway diminished Stat5-mediated transcription, which may reflect competition between these pathways for some transcriptional components. Thus, rather than enhancing the Stat5 proliferative response through a transcriptional mechanism, PI3K appears to act on one or more proliferative events parallel to or downstream of Stat5. One possibility is that PI3K may simply provide ample levels of phosphorylated phosphatidylinositol species that are necessary for the membrane localization of pleckstrin homology domain containing signaling molecules, which are then activated by other independent signals (55). Another possibility is that components of the PI3K pathway may mediate posttranslational modification of cell cycle control proteins such as cdk inhibitors. Finally, PI3K may activate transcription factors that mediate events downstream of the G1 cell cycle checkpoint. Indeed, many Akt substrates are transcription factors or directly involved in the regulation of gene transcription (24). Any of these scenarios might explain the necessary, but insufficient role of PI3K in promitogenic signaling.

Finally, because inhibition of the Erk and PI3K pathways does not diminish cyclin D3 or bcl-2 expression, and only partially diminishes c-myc and bcl-xL expression, one or more unidentified pathways must also contribute to proliferative signaling by Shc. These signals appear to act downstream of Grb-2 because disruption of the Grb-2 binding site completely blocked the ability of {beta}{beta}325Shc{Delta}P to induce expression of these genes or induce cell proliferation. Candidate molecules are Ras effectors other than Erk, such as Ral-GDS and Rho family GTPases, many of which have been implicated in proliferative signaling (56, 57). In addition, the adaptor protein Gab2, which binds to Shc via Grb-2, contains consensus motifs for the potential binding of SHP-2, CrkL, Nck, and phospholipase C{gamma}, in addition to PI3K (58, 59, 60). Many of these candidate effectors are also involved in mitogenic signaling by the TCR and B cell Ag receptor as well as the receptor tyrosine kinase family, suggesting that Shc may link the IL-2R to a more generic, well-conserved proliferative signaling mechanism that complements Stat5-mediated proliferation.


   Acknowledgments
 
We thank Mark Benson, Bryan McIntosh, and Meghan Parsons for technical assistance; James Lord for chimeric receptor constructs; Anke Klippel for the mp110* construct; Richard Roth for the mAktER construct; Martin McMahon for the {Delta}RafER construct; Michael Greenberg and Mark Goldsmith for c-fos cDNA; Charles Sherr for cyclin D2 and cyclin D3 cDNA; and Jeff Singer for cyclin E cDNA.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants GM57931 and CA09537, by the M. J. Murdock Charitable Trust, and by the William Randolph Hearst Foundation. Back

2 Address correspondence and reprint requests to Dr. Brad H. Nelson, Virginia Mason Research Center, 1201 Ninth Avenue, Seattle, WA 98101-2795. E-mail address: bnelson{at}vmresearch.org Back

3 Abbreviations used in this paper: cdk, cyclin-dependent kinase; ER, estrogen receptor; Erk, extracellular signal-related kinase; Jak, Janus kinase; LY, LY294002; MEK, mitogen-activated protein/Erk kinase; 4-OHT, 4-hydroxytamoxifen; PD, PD98059; PI3K, phosphatidylinositol 3-kinase; SOE, splice-overlap extension. Back

Received for publication March 26, 2001. Accepted for publication June 22, 2001.


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