New Role for Shc in Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway (original) (raw)

Abstract

Most, if not all, cytokines activate phosphatidylinositol 3-kinase (PI-3K). Although many cytokine receptors have direct binding sites for the p85 subunit of PI-3K, others, such as the interleukin-3 (IL-3) receptor beta common chain (βc) and the IL-2 receptor beta chain (IL-2Rβ), lack such sites, leaving the mechanism by which they activate PI-3K unclear. Here, we show that the protooncoprotein Shc, which promotes Ras activation by recruiting the Grb2-Sos complex in response to stimulation of cytokine stimulation, also signals to the PI-3K/Akt pathway. Analysis of Y→F and “add-back” mutants of βc shows that Y577, the Shc binding site, is the major site required for Gab2 phosphorylation in response to cytokine stimulation. When fused directly to a mutant form of IL-2Rβ that lacks other cytoplasmic tyrosines, Shc can promote Gab2 tyrosyl phosphorylation. Mutation of the three tyrosyl phosphorylation sites of Shc, which bind Grb2, blocks the ability of the Shc chimera to evoke Gab2 tyrosyl phosphorylation. Overexpression of mutants of Grb2 with inactive SH2 or SH3 domains also blocks cytokine-stimulated Gab2 phosphorylation. The majority of cytokine-stimulated PI-3K activity associates with Gab2, and inducible expression of a Gab2 mutant unable to bind PI-3K markedly impairs IL-3-induced Akt activation and cell growth. Experiments with the chimeric receptors indicate that Shc also signals to the PI-3K/Akt pathway in response to IL-2. Our results suggest that cytokine receptors lacking direct PI-3K binding sites activate Akt via a Shc/Grb2/Gab2/PI-3K pathway, thereby regulating cell survival and/or proliferation.

The proliferation, differentiation, and survival of hematopoietic cells are controlled by multiple cytokines. Cytokines bind to cell surface receptors and activate receptor-associated Janus family tyrosine kinases (Jaks) (25). Activated Jaks are required for the phosphorylation of multiple sites on receptor cytoplasmic domains. These tyrosyl phosphorylation sites recruit signal relay molecules containing src homology-2 (SH2) and/or phosphotyrosine binding (PTB) domains (26). Signal relay molecules convert receptor-proximal events into the activation of downstream signaling pathways, including the Ras/Raf/mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI-3K)/Akt cascades. These pathways culminate in the phosphorylation of key transcription factors and other important cellular regulators (e.g., members of the cell survival/death machinery). Cytokine receptors also bind SH2 domain-containing transcription factors termed STATs, which, upon tyrosyl phosphorylation, activate transcription directly (11). Elucidating the molecular details of these signaling pathways is critical to understanding cytokine action.

Much is known about how cytokines activate the MAPK and PI-3K pathways. Most cytokine receptors have direct binding sites for the PTB domain of the adapter protein Shc (7, 26). Shc is recruited to activated receptors, where it becomes tyrosyl phosphorylated on as many as three sites (Y239, Y240, and Y317) (18). These phosphorylation sites conform to the consensus for binding to the SH2 domain of the adapter protein Grb2. Via its SH3 domain(s), Grb2 binds to the guanine nucleotide exchange protein Sos, which activates Ras and, consequently, the rest of the MAPK pathway (52). The protein-tyrosine phosphatase SHP-2 also binds via its SH2 domains to many cytokine receptors. Binding strongly enhances SHP-2 catalytic activity (3), and active SHP-2 is required for efficient MAPK activation by cytokines (58). Many cytokine receptors have direct binding sites for the SH2 domains of the p85 regulatory subunit of PI-3K. Engagement of these domains enhances PI-3K catalytic activity (2, 9). The 3′-phosphorylated lipid products of PI-3K lead to the activation of downstream kinases, including Akt (55).

Some cytokine receptors do not fully conform to this scheme. The receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 are heterodimers, comprised of a specific α chain and a β common (βc) chain. The α subunit determines ligand binding specificity, whereas βc is the signaling subunit. βc has a direct binding site for the Shc PTB domain (Y577) (46) and at least one direct binding site for Shp-2 (Y612) (5). Recent data suggest that the SH2 domain of Shc also may be able to bind to Y612 (6). However, βc lacks a p85 binding site. Likewise, the IL-2 receptor beta chain (IL-2Rβ), which functions as a signaling subunit for the IL-2 and IL-15 receptors, has a binding site for Shc (Y338) (48) but not p85.

Despite their lack of p85 binding sites, βc- and IL-2Rβ-containing receptors activate the PI-3K/Akt pathway (26), which has critical biological functions downstream of these receptors. PI-3K is required for optimal IL-3-induced cell proliferation in BaF3 cells (12) and cell survival in MC/9 (51) and 32D (54) cells. PI-3K also plays a central role in cell cycle progression in IL-2-dependent T-cell lines (8), and activation of Akt correlates with increased cell survival and optimal long-term proliferation of activated primary T cells in the presence of IL-2 (58).

Some clues to how these receptors activate PI-3K were provided by earlier work. Combined mutation of Y577 and Y612 in βc blocks cytokine-activated Akt activation (14), whereas the Y338F mutation in IL-2Rβ eliminates the ability of IL-2 to activate Akt (57). In both cases, mutation of the binding site for Shc correlates with diminished PI-3K/Akt activation. However, Shc is not known to bind to PI-3K. Instead, biochemical analyses implicated a phosphotyrosyl species of 80 to 110 kDa as the major PI-3K-binding protein in cells stimulated through the IL-3/GM-CSF/IL-5 (13) or IL-2/IL-15 receptors (17, 56).

Recently, we cloned the 97-kDa phosphotyrosyl protein (Gab2) that binds p85 in IL-3-stimulated BaF3 cells (20). Like its relatives Drosophila Dos and mammalian Gab1, Gab2 contains an N-terminal pleckstrin homology (PH) domain, several potential SH3 domain-binding motifs (PXXP), and multiple binding sites for SH2 domain-containing proteins. Gab2 becomes tyrosyl phosphorylated in response to multiple stimuli (including IL-2) in a wide range of hematopoietic cell types (including IL-2-responsive T-cell lines) and associates with Shc, Shp-2, and p85 (20, 43, 63). In addition, Gab2 is associated constitutively with Grb2. Although initial studies indicated that the Gab-2/Shp-2 interaction was important for IL-3-induced immediate-early gene induction, the consequences of Gab2-p85 interaction were not addressed. Since Gab1 immunoprecipitates contain PI-3K activity upon growth factor (22, 23) and B-cell receptor (BCR) (27) stimulation and Gab2 is a major cytokine-inducible p85-binding protein, we suspected that Gab2 might be a critical mediator of cytokine-evoked PI-3K/Akt activation.

We have investigated how Gab2 contributes to activation of the PI-3K/Akt pathway in IL-3/GM-CSF receptor (GM-CSFR) signaling. We show that the Shc binding site (Y577) on βc is the major site and Y612 is a minor site required for cytokine-evoked Gab2 tyrosyl phosphorylation. The Shc binding site (Y338) also is required for IL-2-evoked Gab2 tyrosyl phosphorylation. Using chimeric receptors, we show that Shc is sufficient for Gab2 tyrosyl phosphorylation; this action of Shc requires its tyrosyl phosphorylation. Cytokine-induced Gab2 tyrosyl phosphorylation also requires Grb2, indicating that Gab2 is recruited to activated cytokine receptor complexes by means of an Shc-Grb2-Gab2 complex. Gab2 is the major tyrosyl-phosphorylated binding protein for p85 and accounts for over 50% of IL-3-induced PI-3K activity. Moreover, inducible expression of a Gab2 mutant that cannot bind p85 inhibits IL-3-induced Akt activation and cell proliferation. Our results identify Gab2 as a critical regulator of the PI-3K pathway downstream of cytokine receptors that lack p85-binding sites and establish a novel role for Shc in mediating activation of the PI-3K/Akt pathway via Dos/Gab family scaffolds. An analogous Shc→PI-3K pathway may also operate downstream of other types of receptors, such as receptor tyrosine kinases (RTKs) and antigen receptors.

MATERIALS AND METHODS

Cell culture.

Parental BaF3 cells and BaF3 cell lines expressing different human granulocyte-macrophage colony-stimulating factor (hGM-CSF) αβ mutant receptors (generously provided by Sumiko Watanabe, Tokyo University Medical School, Tokyo, Japan) were grown in RPMI plus 10% fetal calf serum (FCS) and 10% WEHI-conditioned medium (as a source of IL-3), starved in RPMI with 0.8% bovine serum albumin (BSA) for 4 to 6 h, and stimulated with recombinant murine IL-3 (5 ng/ml) (Invitrogen) or recombinant hGM-CSF (10 ng/ml) as indicated. NIH-3T3 hGM-CSFR, which are NIH 3T3 cells expressing the hGM-CSFR, were generously provided by J. Griffin (Dana Farber Cancer Institute) and were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS.

Plasmids.

The hemagglutinin (HA)-tagged Gab2 ΔPH and 3YF mutants were generated by PCR mutagenesis and subcloned into pBluescript (pBS) plasmid. Primers used to generate this mutant are available from the authors upon request. Gab2 WT and 3YF-HA inserts were excised from pBS by digestion with _Hin_dIII and _Xba_I, blunted with Klenow fragment, and ligated into the vector pTet-splice (Gibco-BRL), generating pTet-splice-Gab2 WT-HA and pTet-splice-Gab2 3YF-HA, or ligated into the vector pEBB (a gift of Bruce Mayer, Children's Hospital, Boston), generating pEBB Gab2 WT HA and pEBB Gab2 3YF HA. Detailed information on these constructs can be obtained upon request. The Grb2 expression constructs Grb2 WT and Grb2 (W39K/W193K) in pEBG, which directs the expression of glutathione-_S_-transferase (GST)-tagged Grb2 proteins, as well as the _myc_-tagged Grb2 (R86→K) construct in pEBB, were kindly provided by Bruce Mayer.

Stable and transient transfections.

Stable BaF3 cells lines that inducibly express Gab2 WT and 3YF were generated by electroporation. The vector pTet-splice Gab2 WT-HA or pTet-splice Gab2 3YF-HA plasmid (20 μg) was cotransfected with pBabe-puro (4 μg) into BaF3 cells, which constitutively express the Tet activator (31). Thirty-six hours posttransfection, cells were seeded into 96-well plates (104 cells/well) in medium containing puromycin (0.5 μg/ml). Fourteen days later, puromycin-resistant clones were expanded and screened for inducible expression of Gab2-HA. Doxycycline (1 μg/ml) was added for 12 h, and cell were then lysed and subjected to anti-HA and anti-Gab2 immunoblotting to assess the expression of exogenous and endogenous Gab2 proteins.

Transient transfections of BaF3 were performed essentially as described (19). For transfection of NIH-3T3 hGM-CSFR cells, 0.5 μg of pEBB Gab2HA and 4 μg of pEBG Grb2 plasmid were mixed with Superfect (Qiagen) reagent and added to cells cultured in a 60-mm dish. Twenty-four hours posttransfection, cells were starved in DMEM containing 1% BSA for 18 h and stimulated with hGM-CSF (10 ng/ml) for 10 min prior to lysis and immunoprecipitation, as indicated.

Antibodies, immunoprecipitations, and immunoblotting.

Anti-Grb2 and anti-Erk2 rabbit polyclonal antibodies (for immunoblotting) were purchased from Santa Cruz. Anti-p97/Gab2 and anti-p85 rabbit polyclonal antibodies (for immunoblots and immunoprecipitations) and anti-HA monoclonal antibodies (12CA5) were described previously (20). Antiphosphotyrosine monoclonal antibodies 4G10 and PT66 were obtained from UBI and Sigma, respectively. Anti-Akt, anti-phospho-Akt (S473), and anti-phospho-MAPK monoclonal antibodies were purchased from New England Biolabs.

Cells were starved and stimulated with the appropriate cytokine, as indicated. Total cell lysates (TCLs) were prepared in 1% NP-40–50 mM Tris (pH 7.5)–150 mM NaCl–10 mM NaF–2 mM NaVO4 and a protease inhibitor cocktail, as described previously (19). Lysate from 107 cell equivalents was incubated with anti-Gab2 (4 μl) or anti-p85 (2 μl) rabbit serum. Immune complexes were recovered onto protein A-Sepharose and washed. Immunoprecipitates and TCLs were boiled in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membranes (Immobilon), immunoblotted with appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies, and detected by using enhanced chemiluminescence.

PI-3K assays.

Immune complex lipid kinase assays were carried out using PI as the substrate, and reaction products were resolved by thin-layer chromatography (TLC) as described previously (10). Quantification was carried out using a PhosphorImager (Molecular Dynamics).

RESULTS

Specific tyrosines on βc mediate Gab2 tyrosyl phosphorylation.

We first investigated the mechanism by which Gab2 becomes tyrosyl phosphorylated in response to cytokine stimulation. Conceivably, the Gab2 PH domain might promote its recruitment to activated cytokine receptors, since PH domains bind membrane phospholipids (32). To assess whether the PH domain is required for Gab2 tyrosyl phosphorylation, HA-tagged wild-type (WT) and PH domain-deleted mutant (ΔPH) Gab2 proteins were expressed transiently in BaF3 cells. The transfected cells were starved, stimulated with IL-3 or left unstimulated, lysed, immunoprecipitated with anti-HA antibodies, and subjected to antiphosphotyrosine immunoblotting. Upon IL-3 stimulation, both WT and ΔPH Gab2 appeared as a broad but comparably intense band (Fig. 1A, left panel); thus, the PH domain of Gab2 is dispensable for its tyrosyl phosphorylation. The PH domain of Gab1 is highly similar to that of Gab2 (20, 43); in particular, residues known to be important for binding specificity of phospholipids to PH domains (47) are conserved in these two molecules. Recent studies have shown that the Gab1 PH domain binds phosphatidylinositol 3,4,5-triphosphate (PIP3) preferentially (38, 49). PIP3 production, and therefore Gab2 recruitment via its PH domain, would be blocked by treatment with the PI-3K inhibitor wortmannin. However, wortmannin treatment had no effect on cytokine-induced Gab2 tyrosyl phosphorylation (Fig. 1B, right panel). These data strongly suggest that the Gab2 PH domain is dispensable for its tyrosyl phosphorylation, although it is likely to be important for other functions involving Gab2 (see Discussion). Interestingly, although wortmannin treatment did partially antagonize the Gab2 mobility shift evoked by IL-3 stimulation. These data, as well as studies using other inhibitors (data not shown), indicate that the mobility shift is most likely due to serine phosphorylation that is at least partly dependent on PI-3K activation.

FIG. 1.

FIG. 1

Structural requirements for Gab2 tyrosyl phosphorylation in response to βc cytokine stimulation. (A) The PH domain is dispensable for Gab2 tyrosyl phosphorylation. (Left) HA-tagged Gab2 WT and PH domain-deleted (ΔPH) Gab2 expression constructs were transiently transfected into BaF3 cells. BaF3 cells were starved (—), stimulated with IL-3 (+), and lysed. HA-tagged Gab2 proteins were immunoprecipitated (IP) with anti-HA antibodies and subjected to immunoblotting with anti-pTyr and anti-Gab2 antibodies, as indicated. (Right) BaF3 cells were starved and either treated with wortmannin (Wort, 100 nM) for 20 min or left untreated, stimulated with IL-3, lysed, immunoprecipitated with Gab2 antibodies, and subjected to anti-pTyr immunoblotting. (B and C) βc Y577 is the major site required for Gab2 tyrosyl phosphorylation in response to βc cytokine stimulation. A schematic of βc is shown at the top of the figure, indicating the positions of potential tyrosyl phosphorylation sites. Gab2 tyrosyl phosphorylation in BaF3 cells expressing Y→F (B) and add-back (C) mutants of human βc. BaF3 cells were starved (—), stimulated with human GM-CSF (+), and lysed. Gab2 immunoprecipitates were immunoblotted with anti-pTyr and anti-Gab2 antibodies, as indicated.

Next, we asked whether specific βc tyrosines are required for Gab2 tyrosyl phosphorylation. To address this question, we utilized BaF3 cell lines that express WT or mutant forms of human βc, along with hGM-CSFRα. Addition of hGM-CSF to such cells activates the heterodimeric hGM-CSFR without activating the endogenous murine IL-3R significantly. We employed two different groups of cell lines (Fig. 1B and C). The first set (F series mutants) express βc mutants in which single tyrosines within βc are mutated to phenylalanine. In the second set (Y series mutants), individual tyrosyl residues are added back onto an F-all mutant (a mutant in which all eight tyrosines within the βc cytoplasmic domain are mutated to phenylalanine) (28). Cells from each line were starved, stimulated with GM-CSF, lysed, and subjected to anti-Gab2 immunoprecipitation, followed by immunoblotting with antiphosphotyrosine antibodies. Analyses of F series mutant lines revealed that, compared to WT βc-expressing cells, Gab2 tyrosyl phosphorylation was diminished markedly (by ∼70 to 80%) in F3 cells (Fig. 1B), but not in any other cell line. These results indicate that Y577 is the major site in βc required for Gab2 tyrosyl phosphorylation. However, Y577 is not the only site capable of promoting Gab2 phosphorylation, since there is residual phosphorylation in the F3 cells. Consistent with this interpretation, Gab2 was tyrosyl phosphorylated strongly upon stimulation of the Y3 cell line, in which only Y577 was present on the F-all backbone. There was also significant tyrosyl phosphorylation of Gab2 in Y4 cells, although to a much lower extent than in Y3 cells (Fig. 1C). Importantly, these cells lines express comparable surface levels of the hGM-CSFR, as shown by flow cytometry (data not shown; see also reference28). Based on these data, we conclude that Y577 is the major site within βc required for Gab2 tyrosyl phosphorylation, whereas Y612 is a minor site.

Y577 also is the binding site for the PTB domain of Shc and is required for Shc tyrosyl phosphorylation in response to IL-3/GM-CSF (29, 46, 47). Likewise, Y612 is a binding site for Shp-2 (5) and possibly for the SH2 domain of Shc (6). Thus, our findings lead to two mutually exclusive models for cytokine-evoked Gab2 tyrosyl phosphorylation: either Shc (and Shp-2) competes with Gab-2 for binding to βc, or Shc (and to a lesser extent Shp-2) mediates recruitment of Gab-2 to βc. Consistent with the latter possibility, we showed previously that Shc coimmunoprecipitates with Gab2 upon cytokine stimulation, but we were unable to detect βc in Gab2 immune complexes prepared from cells expressing endogenous levels of all of these components. These findings were consistent with the possibility that Shc might function as an adapter to bring Gab2 to βc.

To test whether Shc can mediate Gab2 phosphorylation, we took advantage of a set of chimeric receptors comprised of the ectodomains of the hGM-CSFR α and β chains linked to the transmembrane and cytoplasmic domains of the WT IL-2R γ chain and WT or mutant IL-2R β chains, respectively (35). The chimera expressing WT IL-2β (ββWT) as well as chimeras containing two different IL-2Rβ chain mutants were assayed initially. In one, the Shc binding site in IL-2Rβ, Y338, is mutated to phenylalanine (ββY338F); the other is a truncation of IL-2Rβ at position 325 (ββΔ325), which deletes all six tyrosyl phosphorylation sites in the IL-2Rβ cytoplasmic domain (Fig. 2A). These chimeras were expressed stably and at comparable cell surface levels in the IL-2-dependent cell line CTLL-2 (Fig. 2B). In such lines, the chimeric receptors can be activated by treatment with hGM-CSF or the cells can be stimulated through their endogenous IL-2Rs. Previous studies established that the chimeric receptor ββWT exhibits normal IL-2 responses, including Shc tyrosyl phosphorylation, when stimulated with GM-CSF (35).

FIG. 2.

FIG. 2

Shc can mediate Gab2 tyrosine phosphorylation. (A) Schematic diagram of GM-CSFRα/IL-2Rβ and GM-CSFRα/IL-2Rβ/ShcΔPTB chimeras. (B) Surface expression of chimeric receptors. CTLL-2 cell lines expressing the indicated chimeras were analyzed by flow cytometry using anti-GM-CSF βc antibodies. Note that surface expression of the indicated chimeras was similar. (C) Gab2 tyrosyl phosphorylation in CTLL-2 cell lines expressing chimeric receptors. Cell lines expressing the indicated chimeras were starved for 4 h(—)and then stimulated for 4 min with hGM-CSF (+). Gab2 immunoprecipitates (IP) were prepared and subjected to immunoblotting with anti-Tyr antibodies, followed by anti-Gab2 antibodies, as indicated.

We examined Gab2 tyrosyl phosphorylation in cell lines expressing these chimeric receptors. Upon stimulation with GM-CSF, Gab2 was tyrosyl phosphorylated in cells expressing the WT chimera, but not in cells expressing either ββY338F or ββΔ325 (Fig. 2C). Thus, as in βc signaling, the Shc binding site in IL-2Rβ (Y338) is required for cytokine-evoked Gab2 tyrosyl phosphorylation within the context of these chimeric receptors. In other studies, we have shown that Y338 is also required for Gab2 tyrosyl phosphorylation in the context of the native IL-2Rβ chain (M. Gadina, H. Gu, B. G. Neel, and J. O'Shea, submitted for publication), further validating the use of these chimeras. To see whether Shc is sufficient to mediate Gab2 tyrosyl phosphorylation, we made use of additional CTLL2 cell lines. These express chimeras in which the collagen homology-2 (CH2) and SH2 domains of Shc (ββ325ShcΔPTB) are fused to the ββΔ325 chimera by means of a flexible polyglycyl polylinker (Fig. 2A). Cells expressing comparable surface levels of these chimeras (compared to ββWT-expressing cells) were chosen for further study (Fig. 2B). Upon GM-CSF stimulation, Gab2 from cells expressing ββ325ShcΔPTB became tyrosyl phosphorylated to the same extent as Gab2 from ββWT cells (Fig. 2C). In contrast, Gab2 tyrosyl phosphorylation was eliminated completely in cells expressing a chimera (ββ325ShcΔPTBFFF) in which all three Shc tyrosyl phosphorylation (and Grb2-binding) sites (Y239, Y240, and Y317) are mutated to phenylalanine. We conclude that, when tethered to a surface receptor, Shc is sufficient to mediate Gab2 tyrosyl phosphorylation upon cytokine stimulation. Moreover, this function of Shc requires that it be tyrosyl phosphorylated.

Grb2 mediates Gab2 tyrosyl phosphorylation.

The apparent requirement that Shc Y239, Y240, and/or Y317 be phosphorylated for cytokine-evoked Gab2 phosphorylation suggests that these sites bind an SH2 or PTB domain-containing protein. Gab2 has no apparent phosphotyrosine-binding motif. However, as indicated above, all of these sites conform to the consensus for binding by the SH2 domain of Grb2. Moreover, Grb2 is associated basally (i.e., in the absence of stimulation) with Gab2 (20), most likely via interaction of one or both Grb2 SH3 domains with the PXXP motif(s) in Gab2.

We hypothesized that, upon cytokine stimulation, the Grb2-Gab2 complex interacts with newly tyrosyl-phosphorylated Shc (via the SH2 domain of Grb2), thereby recruiting Gab2 to the activated cytokine receptor complex, where it can be tyrosyl phosphorylated. This model predicts that overexpression of mutant forms of Grb2 defective in either the SH2 or SH3 domain to levels sufficient to compete with endogenous Grb2 should block cytokine-evoked Gab2 tyrosyl phosphorylation. Grb2 is expressed at high levels in BaF3 cells, and we were unable to achieve significant overexpression of the mutant protein in these cells. Therefore, we utilized NIH 3T3 cells expressing the hGM-CSFR to test our hypothesis. Such cells do not express Gab2. Accordingly, we transiently cotransfected an epitope-tagged Gab2 expression construct with WT or mutant Grb2 expression plasmids into these cells and monitored Gab2 tyrosyl phosphorylation upon GM-CSF stimulation. Indeed, Gab2 tyrosyl phosphorylation was inhibited in cells cotransfected with either the Grb2 SH2 mutant (R→K) or SH3 double mutant (W39K/193K; W→K) compared to WT Grb2 (Fig. 3). These data suggest that Grb2 is required for Gab2 tyrosyl phosphorylation. Our results strongly support a model in which the major route to Gab2 tyrosyl phosphorylation is via formation of an Shc-Grb2-Gab2 complex.

FIG. 3.

FIG. 3

Grb2 SH2 and SH3 domains are required for Gab2 tyrosine phosphorylation upon GM-CSF stimulation. NIH 3T3 cells engineered to express the hGM-CSFR α and β chains were transiently cotransfected with HA-tagged Gab2 expression plasmid and different Grb2 expression constructs (WT, R86K, and W39/193K). Twenty-four hours posttransfection, cells were starved in DMEM with 1% BSA for 18 h and then stimulated with hGM-CSF (10 ng/ml) (+) or left unstimulated (—). Cell lysates were immunoprecipitated (IP) with anti-Gab2 antibodies, resolved by SDS-PAGE, and immunoblotted with anti-Tyr (top panel) and anti-Gab2 (middle panel) antibodies. The bottom panel shows a blot of TCL with anti-Grb2 antibodies.

Tyrosyl-phosphorylated Gab2 is a major contributor to PI-3K activation by βc cytokines.

Gab2 associates with p85 upon stimulation with IL-3 and other cytokines (20, 43). However, the functional significance of this association has not been determined. To begin to address the role of Gab2 in PI-3K activation by βc cytokines, we first asked whether Gab2 is a major PI-3K-binding protein in IL-3-stimulated BaF3 cells. We immunoprecipitated p85 from a BaF3 TCL or a TCL depleted of Gab2 by prior immunoprecipitation with anti-Gab2 antibodies (Fig. 4A). In BaF3 cells, the major IL-3-evoked p85-associated tyrosyl phosphoprotein is a 97-kDa species. This protein is absent in p85 immunoprecipitates from the Gab2-depleted lysates. Thus, Gab2 is the major tyrosyl-phosphorylated p85-binding protein in these cells.

FIG. 4.

FIG. 4

Gab2 is the major activator of the PI-3K/Akt pathway in IL-3 signaling. (A) Gab2 is the major tyrosyl-phosphorylated p85-binding protein in BaF3 cells. BaF3 cells were starved, stimulated with IL-3 for the indicated times, and lysed. Whole cell lysates were immunoprecipitated (IP) with preimmune serum (PI) or anti-Gab2 or anti-p85 antiserum, or cell lysates first immunodepleted with anti-Gab2 antibodies (see text) were immunoprecipitated by anti-p85 antibodies. Immune complexes were resolved by SDS-PAGE and subjected to anti-pTyr immunoblotting, followed by reprobing with anti-p85 antibodies, as indicated. (B) Gab2 contributes to the majority of PI-3K activity upon IL-3 stimulation. Immune complexes prepared as in panel A were subjected to PI-3K assay using PI as a substrate. Reaction products were resolved by TLC and quantified using a PhosphorImager. pTyr*, lysate predepleted of Gab2 protein was immunoprecipitated with anti-pTyr antibody.

Next, we asked whether Gab2 is associated with PI-3K activity. We assayed Gab2- and phosphotyrosine (pTyr)-associated PI-3K activity using PI as the substrate. Upon IL-3 stimulation of BaF3 cells, there is a marked increase in PI-3K activity in Gab2 and pTyr immunoprecipitates (Fig. 4B), whereas no PI-3K activity was detectable in immune complexes prepared with Gab2 preimmune antiserum. When Gab2 was depleted prior to anti-pTyr immunoprecipitation, there was a substantial (∼50%) decrease in pTyr-associated PI-3K activity (Fig. 4B). Thus, Gab2 accounts for a major fraction of the cytokine-evoked PI-3K activity in IL-3-stimulated BaF3 cells.

Gab2 association with PI-3K is required for optimal IL-3-induced Akt activation and cell growth.

Since Gab2 immunoprecipitates contain the majority of the IL-3-induced PI-3K activity, formation of the Gab2/PI-3K complex might be critical for activation of the PI-3K/Akt pathway. To test this possibility, we generated an HA-tagged Gab2 mutant that cannot bind p85, reasoning that, upon overexpression, such a mutant might have dominant negative effects. Y441, Y465, and Y574, each of which falls within a consensus p85 SH2 domain-binding sequence (YXXM), were converted to phenylalanine, creating Gab2 3YF. Expression of dominant negative p85 inhibits the growth of BaF3 cells (12); we suspected that constitutive expression of Gab2 3YF might have similar deleterious effects. Therefore, we established BaF3 cell lines that express Gab2 inducibly, using the tetracycline-on expression system. Multiple clones that show inducible expression of HA-tagged WT or 3YF Gab2 were generated (data not shown). Two 3YF (clones 5X and 7X), one WT, and one cell line transfected with vector alone (vector) were subjected to further analysis. Both 3YF clones and the WT Gab2-expressing lines show low basal expression of HA-tagged Gab2 and strong induction upon addition of the tetracycline analog doxycycline. Immunoblotting revealed that, when induced maximally, WT or mutant Gab2 is expressed at 5- to 10-fold-higher levels than endogenous Gab2 in these lines, whereas expression is less than 10% of the endogenous level in the absence of doxycycline (Fig. 5A and data not shown). As expected, whereas immunoprecipitations with anti-HA antibodies revealed p85 coimmunoprecipitating with Gab2 from the WT Gab2-expressing lines (in the presence of doxycycline), no p85 was recovered in HA immunoprecipitates prepared from doxycycline-treated 3YF cells (Fig. 5B). These data confirm that mutation of the three presumptive p85-binding sites in Gab2 eliminates its ability to bind p85.

FIG. 5.

FIG. 5

Inducible expression of a Gab2 mutant unable to bind PI-3K inhibits IL-3-evoked Akt phosphorylation. (A) Generation of BaF3 cell lines inducibly expressing WT Gab2 and Gab2 3YF. Shown are anti-Gab2 immunoblots of lysates prepared from a vector alone, a single WT Gab2-expressing clone, and two Gab2 3YF clones and grown in the presence or absence of doxycycline (DOX), as indicated. (B) Gab2 3YF mutant cannot bind p85. Appropriate BaF3 cell lines were induced to express HA-tagged WT or 3YF Gab2 protein by the addition of doxycycline (Dox) for 12 h. HA-tagged Gab2 proteins were immunoprecipitated (IP) from starved (—) or IL-3-stimulated (+) cells and subjected to SDS-PAGE and immunoblotting with anti-p85 antibodies, followed by anti-HA antibodies. (C) The indicated BaF3 cell lines (vector control, Gab2 WT and Gab2 3YF 5X and 7X) were induced to express HA-tagged WT or mutant Gab2 by the addition of doxycycline for 12 h, starved and stimulated with IL-3 for the indicated times or left unstimulated (lanes 0). TCLs were subjected to immunoblotting with anti-p-Akt (S-473), followed by reprobing with anti-Akt antibodies, as indicated.

We next examined the effect of Gab2 3YF expression on Akt activation. Vector alone, WT, and 3YF cell lines were incubated with doxycycline, starved, and then stimulated with IL-3. TCLs were immunoblotted with anti-phospho-Akt (S473) antibodies, which monitor phosphorylation of one of the two sites required for Akt activation. As expected, there was no detectable S473 phosphorylation in any of the cell lines in the absence of IL-3, and all of the lines exhibited robust Akt activation when stimulated with IL-3 in the absence of doxycycline induction. In vector and WT Gab2 cells, addition of doxycycline had no effect on IL-3-induced Akt phosphorylation. In marked contrast, inducing expression of the 3YF mutant (in either clone 5X or 7X) inhibited IL-3-induced Akt phosphorylation by more than 50% (Fig. 5C). Since Akt activation requires generation of the 3-phosphorylated inositol lipid products of PI-3K (16), our data establish Gab2 as a major regulator of the PI-3K/Akt pathway in IL-3 signaling in BaF3 cells.

Depending on the particular cell system and/or stimulus tested, activation of the PI-3K/Akt pathway can promote cell survival or increase cell proliferation. We examined the effect of Gab2 WT and 3YF overexpression on the growth of BaF3 cells. Addition of doxycycline to vector or WT Gab2-expressing BaF3 cells had no effect on cell growth. In contrast, induction of Gab2 3YF in either clone 5X or 7X markedly decreased growth in IL-3 (Fig. 6). Decreased growth in this assay could reflect diminished cell proliferation (caused by a prolonged cell cycle) and/or increased apoptosis. BaF3 cells were incubated with and without doxycycline for 48 and 72 h, and apoptosis was assessed by annexin V binding. Gab2 3YF expression did not affect the size of the apoptotic cell population, as monitored by this assay (data not shown). These data suggest that Gab2/PI-3K complex formation is necessary for optimal cell proliferation but not for survival in BaF3 cells (see Discussion).

FIG. 6.

FIG. 6

Expression of Gab2 3YF mutant inhibits BaF3 cell proliferation in response to IL-3. BaF3 vector control, Gab2 WT, and two independent Gab2 3YF cell lines were grown in the absence (—) or presence of doxycycline (Dox) for 72 h in 10% FCS and 10% WEHI-conditioned medium. Every 24 h, cell number was determined using a Coulter counter.

Shc signals to the PI-3K/Akt pathway in other response to other cytokines.

To explore whether our observations extend to other Gab2-expressing cell types and other cytokine receptor systems, we monitored Akt phosphorylation in CTLL2 cells expressing the GM-CSFR/IL-2Rβ chimeras (Fig. 2A). As expected, when stimulated through their endogenous IL-2R (I lanes), Akt was activated in all of these cell lines, indicating that their Akt activation machinery was intact. Likewise, there was an increase in Akt phosphorylation in cells expressing the WT GM-CSFR/WT IL-2Rβ chimera (ββWT). Consistent with recently published data (57), Akt phosphorylation was not increased in cells expressing IL-2Rβ Y338F, suggesting that Y338, the Shc binding site, is required for Akt phosphorylation. Remarkably, however, Akt phosphorylation was restored in cells expressing ββ325ShcΔPTB but not ββ325ShcΔPTBFFF (Fig. 7). Thus, tyrosyl-phosphorylated Shc (in the context of these chimeric receptors) is sufficient to evoke Akt activation. Moreover, consistent with our data on cytokine-evoked Gab2 tyrosyl phosphorylation (Fig. 1B and C and Fig. 2) and our finding that Gab2 association with p85 is required for optimal IL-3-evoked Akt activation (Fig. 5C), there is a precise correlation between the ability of these chimeras to evoke Gab2 tyrosyl phosphorylation and Akt activation, respectively. These findings suggest that, as in IL-3/GM-CSF signaling, Shc also may signal via a Grb2-Gab2 complex in IL-2-induced Akt activation.

FIG. 7.

FIG. 7

Shc mediates Akt activation in IL-2 signaling. CTLL-2 cells expressing different GM-CSFR/IL-2R and GM-CSFR/IL-2R/ShcΔPTB chimeras (see Fig. 2A) were starved and stimulated with IL-2 (I) or GM-CSF (G) or left unstimulated (—). TCLs were resolved by SDS-PAGE and subjected to immunoblotting with anti-pAkt (S473), followed by reprobing with anti-Akt. Note that, via their endogenous IL-2Rs, all of the cell lines respond to IL-2 stimulation by activating Akt. However, only ββ325ShcΔPTB-expressing cells (the same cells that can tyrosyl phosphorylate Gab2 in response to GM-CSF) activate Akt upon GM-CSF stimulation.

DISCUSSION

The PI-3K/Akt pathway is important for cytokine-induced cell survival and proliferation. Although some cytokine receptors bind p85 directly, others lack such sites; how these receptors activate PI-3K has remained unclear. We have provided evidence that cytokines (IL-3, GM-CSF, and IL-5) that signal from IL-3Rβc and, most likely, IL-2Rβ (IL-2 and IL-15) employ a novel route to PI-3K activation in which the recently cloned scaffold Gab2, when tyrosyl phosphorylated, plays a critical role (Fig. 8). Moreover, our data indicate that tyrosyl phosphorylation of Gab2 in response to βc and IL-2Rβ activation occurs primarily via an Shc-Grb2 complex. These findings suggest that Gab2, and possibly other Dos/Gab family scaffolds, constitutes a major pathway to activation of the PI-3K/Akt cascade in response to multiple cytokines and potentially other types of cell stimuli. Our results also suggest a new role for Shc in addition to its function in MAPK activation.

FIG. 8.

FIG. 8

Shc/Grb2/Gab2/PI-3K Akt pathway. (A) Shc signals to the PI-3K pathway via a Grb2-Gab2 complex. Shown are routes from the IL-3/GM-CSF/IL-5 receptors, which utilize βc, and the IL-2/IL-15 receptors, which signal via IL-2Rβ. For βc, tyrosyl-phosphorylated Shc provides the major route to the Grb2-Gab2 complex; tyrosyl-phosphorylated Shp-2 may provide an alternate route. In IL-2Rβ signaling, Shc provides the only route to PI-3K/Akt. (B) Model for Gab2 phosphorylation by βc cytokines. Following receptor activation, βc is phosphorylated on multiple sites. Y577, which is the Shc binding site, provides the major route to Grb2 tyrosyl phosphorylation, whereas Y612, the major Shp-2 binding site, as well as a minor binding site for Shc, is a minor route. Both Shc and Shp-2 become tyrosyl phosphorylated upon receptor stimulation and can bind Grb2, which is constitutively associated with Gab2, most likely via one or more proline-rich domains. This results in recruitment of Gab2 to the receptor complex and its tyrosyl phosphorylation. (C) Shc may integrate the Ras and PI-3K pathways. Shown are two potential means by which Shc might simultaneously nucleate complexes containing both Grb2/Sos and Grb2/Gab2/PI-3K. Such multicomplexes might facilitate Ras activation of PI-3K. For details, see the text.

Gab2 is the major regulator of PI-3K/Akt activation in βc signaling.

Several lines of evidence support our conclusion that Gab2 provides a major route to the PI-3K/Akt pathway. First, Y577 and, to a lesser extent, Y612 are capable of evoking GM-CSF-induced Gab2 tyrosyl phosphorylation in the absence of any other βc tyrosyl residue (Fig. 1B and C). In the context of full-length βc, Y577 is necessary for strong tyrosyl phosphorylation of Gab2, although some Gab2 phosphorylation is retained in Y3 mutant-expressing cells (Fig. 1B). Most likely, this residual phosphorylation is mediated through Y612 (Fig. 1C). Consistent with our finding that Y577 and (to a lesser extent) Y612 mediate βc-evoked Gab2 tyrosyl phosphorylation is a recent report that Y577 and Y612 are required for Akt activation in response to another βc cytokine, IL-5 (14). Second, Gab2 is the major tyrosyl-phosphorylated p85-binding protein in BaF3 cells stimulated by IL-3 (Fig. 4A), is associated with PI-3K activity (Fig. 4B), and accounts for more than 50% of IL-3-induced anti-pTyr-associated PI-3K activity (Fig. 4B). Third, and most convincingly, induced overexpression of a Gab2 mutant that cannot bind p85 blocks the majority of IL-3-induced Akt activation (Fig. 5). Consistent with this conclusion, our preliminary data indicate that IL-3-stimulated Akt phosphorylation is decreased by about 75% in bone marrow-derived mast cells with targeted disruption of the Gab2 gene compared to mast cell culture with WT Gab2 allele (H. Gu and B. G. Neel, unpublished observation). Our results suggest that Gab2 or other Dos/Gab family members may have a similar role in signaling by cytokines that utilize IL-2Rβ (Fig. 2 and 7) and possibly other pathways. Interestingly, the related scaffold Gab1 has been shown to associate with PI-3K activity in response to B-cell antigen receptor activation (27) and is implicated in PI-3K-mediated cell survival pathways in response to nerve growth factor (23).

Importantly, our results indicate that the Gab2 PH domain is dispensable for Gab2 tyrosyl phosphorylation. These findings are consistent with recent studies of Gab1 (37). The Gab1 PH domain is, however, required for Gab1 signaling to downstream kinase cascades (49) and biological responses (37). Likewise, we have recently found that the Gab2 PH domain is essential for its effects on T-cell antigen receptor signaling (J. Pratt, S. Burakoff, B. G. Neel, and H. Gu, submitted for publication).

Although our results implicate Gab2 as a major intermediary in cytokine-induced PI-3K activation, they do not exclude the possibility that other molecules contribute, since there is residual anti-pTyr-associated PI-3K activity in Gab2-depleted lysates (Fig. 4B) and residual Akt activation in cells expressing a 5- to 10-fold excess of Gab2 3YF (Fig. 5C). For example, Cbl becomes tyrosyl phosphorylated and associates with p85 upon IL-3 stimulation of 32D cells (24) as well as in response to other cytokines and growth factors (36). There is a tyrosyl-phosphorylated protein of around 120 kDa present in our p85 immunoprecipitates from BaF3 cells (Fig. 4A), which may be Cbl. However, in contrast to our results in cells expressing Gab2 3YF, there are no data indicating that the Cbl/PI-3K complex functions in Akt activation. Indeed, genetic evidence that Cbl family members (1, 39, 40) and their Caenorhabditis elegans ortholog Sli-1 (62) are negative regulatory molecules, along with recent biochemical studies showing that Cbl is a ubiquitin ligase that regulates RTK degradation (30, 33), raises the possibility that association of p85 with Cbl functions to inactivate the PI-3K/Akt pathway. Since p85 can bind to the SH3 domains of Grb2 (59), it also is conceivable that some PI-3K activity can be recruited to Shc via Grb2 itself (i.e., by means of an Shc-Grb2-p85 complex).

Novel role for Shc in cytokine receptor signaling.

Previous studies argued that, by binding to Grb2/Sos, Shc promotes activation of the Ras-MAPK pathway (7). We show here that tyrosyl-phosphorylated Shc also binds the Grb2-Gab2 complex and thereby can access the PI-3K/Akt pathway (Fig. 8A). Upon receptor activation, Shc is recruited to the receptor complex, where it becomes tyrosyl phosphorylated. For βc cytokines, recruitment of Shc is mediated by Y577 of βc (Fig. 1 and 2); Y338 has a similar function in IL-2Rβ signaling (Fig. 2C). Y577 is required for the majority of Gab2 tyrosyl phosphorylation in βc signaling (Fig. 1B), whereas Y338 is required for all Gab2 phosphorylation in response to IL-2Rβ activation (Fig. 2C). Most convincingly, directly tethering the CH2 and SH2 domains of Shc to a chimeric GM-CSFR/IL-2Rβ receptor devoid of receptor tyrosyl phosphorylation sites can promote cytokine-induced Gab2 tyrosyl phosphorylation, and this requires tyrosyl phosphorylation of Shc (Fig. 2C). The SH2 domain-containing protein that Shc must bind to promote Gab2 tyrosyl phosphorylation is almost certainly Grb2, since either SH2 or SH3 domain mutants of Grb2 block cytokine-induced Gab2 phosphorylation (Fig. 3). Grb2 is associated with Gab2 constitutively (20, 43). This interaction may be mediated by one or both SH3 domains of Grb2 and proline-rich (PXXP) motifs on Gab2 (e.g., at position 314 and/or 352). Alternatively, some evidence suggests a noncanonical interaction between Grb2 SH3 domains and Gab2, since a GST fusion protein containing Gab2 amino acids 435 to 515 precipitated Grb2 from cell lysates (50).

βc cytokines evoke a small amount of Gab2 phosphorylation even in the absence of Y577. Experiments with βc add-back mutants (Fig. 1C) strongly suggest that this phosphorylation is mediated by Y612. Previous studies revealed that Y612 is the major βc binding site for Shp-2 (5), although Y577 and Y695 may contribute (28, 44). Shp-2 is also tyrosyl phosphorylated in response to βc stimulation, and tyrosyl-phosphorylated Shp-2 binds Grb2 (41). Thus, residual Gab2 tyrosyl phosphorylation in Y577F (i.e., Y3) cells may be mediated via an Shp-2/Grb2/Gab2 complex analogous to the Shc/Grb2/Gab2 complex. Alternatively, after completion of this work, Bone and Welham reported that the SH2 domain of Shc can also bind to Y612 (6); hence, the small amount of Gab2 phosphorylation observed in the Y4 mutant may be due to Shc SH2 binding to this site (Fig. 8B). Notably, the latter workers also reported that Shc SH2 domain may bind directly to Gab2. Although this interaction may occur subsequent to Gab2 tyrosyl phosphorylation, it obviously cannot be important for initial recruitment of Gab2, because SH2 interactions are pTyr dependent.

Although its role in Ras/MAPK activation has been studied thoroughly, earlier work suggests that Shc has other functions. Expression of an Shc mutant in which both Y239 and Y240 are converted to phenylalanine was reported to block IL-3 induction of c-myc and to inhibit cell survival. Mutation of Shc Y317 had no effect on myc activation but was required for Shc to signal to the Ras/MAPK pathway; Y239 and Y240 are dispensable for the latter function (18). These findings raise the possibility that, by using different tyrosyl phosphorylation sites, Shc could bind simultaneously to Grb2/Sos and Grb2/Gab2 (Fig. 8C). An analogous complex containing Shp-2 bound to Grb2/Sos and Grb2/Gab2 also might exist. Alternatively, the same Shc/Grb2 complex might associate with both Sos and Gab2 via different SH3 domains of Grb2 (Fig. 8C). Gab1 interacts with the C-terminal SH3 domain of Grb2 (42), whereas the Grb2 N-SH3 binds preferentially to Sos (61). Conceivably, the same Grb2 molecule can bind Gab2 via its C-SH3 and Sos via its N-SH3. Given that activated Ras potentiates PI-3K activation (15), such multicomplexes might allow spatiotemporal coupling of the Ras/MAPK and PI-3K/Akt pathways. Shc also binds, via its PTB domain, to the 5′ inositol phosphatase SHIP (34). It is not clear whether such a complex could coexist with Shc/Grb2/Gab2. Nevertheless, it is intriguing that the same adapter, Shc, can form complexes that contain both activating (PI-3K) and inactivating (SHIP) enzymes for 3-phosphorylated phosphoinositides. It will be important to determine which phosphorylation sites on Shc are required for Gab2 tyrosyl phosphorylation and which, if any, of these higher-order complexes can form in response to cytokine signaling.

Downstream consequences of the Shc / Grb2 / Gab2 / PI-3K pathway.

Gab2 3YF blocks IL-3-evoked cell proliferation without affecting cell survival (Fig. 6 and data not shown). These observations are consistent with the inhibitory effects of dominant negative p85 on IL-3-induced BaF3 cell growth (12) and with the finding that a Y577F Y612F βc mutation results in diminished IL-5-induced proliferation (14). Likewise, the GM-CSFR/ββ Shc chimera is required for optimal cell proliferation in CTLL-2 cells, but not for cell survival (35). It seems likely, however, that the precise downstream effects of the Shc/Grb2/Gab2/PI-3K pathway will differ depending on the cellular context. For example, recent studies of primary T cells derived from IL-2Rβ−/− mice indicate that IL-2Rβ Y338 is essential for IL-2-evoked Akt activation, survival, and, accordingly, long-term T-cell expansion, but is dispensable for short-term proliferation (57). Most likely, Akt activation in T cells is mediated by the Shc/Grb2/Gab2/PI-3K pathway, but in primary T cells (as opposed to T-cell lines such as CTLL-2), this pathway is more important for cell survival than proliferation.

Does the Shc/Grb2/Gab2/PI-3K pathway function in other receptor signaling pathways?

Although we only analyzed signaling from βc and IL-2Rβ, our results suggest that Shc, acting via complexes between Gab2 and/or other Dos/Gab family members, may provide a general route to activate the PI-3K/Akt pathway for other receptors and oncogenes. Transformation of fibroblasts by the MEN2A-RET receptor tyrosine kinase requires Y1062, an Shc binding site that is necessary for activation of the PI-3K/Akt pathway (53). Interestingly, the nerve growth factor receptor TrkA contains a p85-binding site (Y751) (4). Nevertheless, mutation of this site has no effect on nerve growth factor-induced PI-3K activation, whereas mutation of the Shc binding site (Y490) in TrkA eliminates activation of both the Ras/MAPK and PI-3K pathways (4, 21). Nerve growth factor treatment of PC12 cells leads to increased Gab1-associated PI-3K activity (23); Gab2 is also tyrosyl phosphorylated under these conditions (H. Gu and B. G. Neel, unpublished observations). Most likely, upon TrkA activation, Shc signals to PI-3K via Grb2-Gab1 and/or Grb2-Gab2 complexes. Shc and Gab2 are tyrosyl phosphorylated upon stimulation of other receptors with p85-binding sites (e.g., RTKs, including CSF-1R and c-Kit, and cytokine receptors, such as EpoR) and/or receptors (e.g., antigen receptors) that utilize other surface molecules to activate PI-3K (20, 43, 60). Shc may be required for PI-3K activation in these receptor systems as well. Alternatively, the Shc/Grb2/Gab2/PI-3K pathway may amplify receptor signals and/or target a pool of activated PI-3K to specific intracellular locations. Further work is needed to test the generality of this pathway in PI-3K activation and its importance in intracellular signal transduction.

ACKNOWLEDGMENTS

We thank T. Itoh and S. Watanabe (Tokyo University) for generously providing the Y and F series mutant cell lines, D. Liu and J.-Q. Shen for technical assistance, L. Cantley (BIDMC) and J. Griffin (DFCI) for helpful comments on the manuscript, and S. Cohen for assistance with preparing the figures.

This work was supported by N.I.H. R01 DK50693 to B.G.N. and GM57931 to B.H.N. H.G. was the recipient of NRSA CA72144 from the N.I.H. and holds the Anna D. Barker Fellowship in Basic Science from the American Association for Cancer Research.

H. Gu and H. Maeda contributed equally to this work.

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