The tyrosine phosphatase SHP-2 is required for mediating ... - Nature

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Oncogene (2001) 20, 6018 ± 6025 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

The tyrosine phosphatase SHP-2 is required for mediating phosphatidylinositol 3-kinase/Akt activation by growth factors Chuan-Jin Wu1,2, Donald M O'Rourke1,3, Gen-Sheng Feng4, Gibbes R Johnson5, Qiang Wang1,2 and Mark I Greene*,1,2 1

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, PA 19104, USA; 2Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, PA 19104, USA; 3Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, PA 19104, USA; 4The Burnham Institute, La Jolla, California, CA 92037, USA; 5Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, MD 20892, USA

SHP-2 is a ubiquitously expressed non-transmembrane tyrosine phosphatase with two SH2 domains. Multiple reverse-genetic studies have indicated that SHP-2 is a required component for organ and animal development. SHP-2 wild-type and homozygous mutant mouse ®broblast cells in which the N-terminal SH2 domain was target-deleted were used to examine the function of SHP-2 in regulating Phosphatidylinositol 3-Kinase (PI3K) activation by growth factors. In addition, SHP2 and various mutants were introduced into human glioblastoma cells as well as SHP-27/7 mouse ®broblasts. We found that EGF stimulation and EGFR oncoprotein (DEGFR) expression independently induced the co-immunoprecipitation of the p85 subunit of PI3K with SHP-2. Targeted deletion of the N-terminal SH2 domain of SHP-2 severely impaired PDGF- and IGFinduced Akt phosphorylation. Ectopic expression of SHP-2 in U87MG gliobastoma cells elevated EGFinduced Akt phosphorylation, and the eect was abolished by mutation of its N-terminal SH2 domain. Likewise, the reconstitution of SHP-2 expression in the SHP-27/7 cells enhanced Akt phosphorylation induced by EGF while rescuing that induced by PDGF and IGF. Further lipid kinase activity assays con®rmed that SHP2 modulation of Akt phosphorylation correlated with its regulation of PI3K activation. Based on these results, we conclude that SHP-2 is required for mediating PI3K/Akt activation, and the N-terminal SH2 domain is critically important for a `positive' role of SHP-2 in regulating PI3K pathway activation. Oncogene (2001) 20, 6018 ± 6025. Keywords: SHP-2; phosphatidylinositol 3-kinase (PI3K); Akt; growth factors; signaling

*Correspondence: MI Greene, 252 John Morgan Building, 36th and Hamilton Walk, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA; E-mail: [email protected] Received 7 March 2001; revised 1 June 2001; accepted 6 June 2001

Introduction SHP-2 (also known as PTP1D, PTP2C, SHPTP2, or Syp) is a family member of the SH2 domain containing phosphatases (SHPs), which are characterized by two SH2 domains at the N-terminus and a phosphatase domain at the C-terminus. In contrast to its close relative, SHP-1, which is primarily detected in hematopoietic cells, SHP-2 is ubiquitously expressed (Neel, 1993; Feng and Pawson, 1994). SHP-2 was found to bind directly to a variety of receptor tyrosine kinases (RTKs) in response to stimulation by growth factors or cytokines (Vogel et al., 1993; Feng et al., 1993; Lechleider et al., 1993; or see review by Feng, 1999). Multiple reverse-genetic studies suggest that SHP-2 is a required positive component of growth factor and cytokine signal transduction pathways. Microinjection of mutant SHP-2 mRNA molecules has revealed that SHP-2 is required for Xenopus mesoderm induction and completion of gastrulation (O'Reilly and Neel, 1998). Corkscrew (csw), the Drosophila SHP-2 homolog, is needed for RTK signaling involved in early development (Allard et al., 1996). More recently, genetic evidence has been presented that SHP-2 enhances signaling from the epidermal growth factor receptor (EGFR) in mouse growth and development (Qu et al., 1999; Chen et al., 2000). Gene-targeted deletion of SHP-2 led to early mouse embryonic lethality, which is a common outcome for biologically important cell signaling components (Saxton et al., 1997). A variety of studies indicate that SHP-2 positively regulates the activation of MAPK (mitogen activated protein kinase) by growth factors and cytokines (Bennett et al., 1996; Deb et al., 1998; Shi et al., 1998; O'Reilly and Neel, 1998). However, the important physiological functions of SHP-2 may not solely result from its involvement in mediating MAP kinase pathway activation. For example, SHP-2 has been demonstrated to play an important role in the regulation of cell spreading and cell migration (Yu et al., 1998; Oh et al., 1999; Manes et al., 1999). However, SHP-2 modulation of IGF-induced cell migration was found to be independent of MAPK activation (Manes

SHP-2 mediates PI3K pathway activation C-J Wu et al

et al., 1999). Our recent work has implicated that SHP2 might be involved in regulating phosphatidylinositol 3-kinase (PI3K) signaling; we found that signalregulatory protein (SIRP) regulated EGF-induced PI3K activation through its binding to SHP-2 in human glioblastoma cells. Disruption of SIRP and SHP-2 association by mutation of SIRP eliminated SIRP modulation of PI3K activation (Wu et al., 2000). PI3Ks constitute a family of evolutionarily conserved lipid kinases. PI3K and phospholipid-regulated signaling have been implicated in regulating an array of fundamental cellular responses, including proliferation, superoxide production, cell adhesion and cell migration (Toker and Cantley, 1997; Rodriguez-Viciana et al., 1997). In particular, PI3K signaling mediates cell survival through activation of the serine/threonine protein kinase Akt (Hemmings et al., 1997). On the other hand, the mechanisms by which PI3K is activated in response to RTK activation have not been well characterized. PI3K is comprised of a 110KD regulatory subunit and a 85KD catalytic subunit. Interestingly, the p85 subunit was found to associate with SHP-2 in response to certain cytokines, such as interleukin-2, in hematopoietic cells (Gesbert et al., 1998; Craddock and Welham, 1997). To explore the potential involvement of SHP-2 in the PI3 kinase pathway, we ectopically introduced wildtype SHP-2 and a series of SHP-2 species mutated at various domains into human glioblastoma cells and mouse SHP-27/7 embryonic ®broblasts, in addition to conducting comparative analysis using SHP-2 wild-type and homozygous mutant ®broblasts. Our studies reveal that SHP-2 acts as a positive regulator, and is a common requirement for PI3K/Akt activation induced by several important growth factors. Furthermore, we show that the NH2-terminal SH2 domain is absolutely essential for this function of SHP-2.

Results and discussion EGF stimulation as well as DEGFR expression induces the association of the PI3 kinase p85 subunit with SHP-2 SHP-2 has been found to be associated with the PI3K p85 subunit in response to some cytokines in hematopoietic cells (Gesbert et al., 1998; Craddock and Welham, 1997). To determine whether the association between p85 and SHP-2 is induced by growth factor stimulation, the presence of p85 was examined in anti-SHP-2 immunoprecipitates from U87MG human glioblastoma cells. These cells have been used as a model system to characterize epidermal growth factor receptor (EGFR) related signaling (Nishikawa et al., 1994; Nagane et al., 1998; Myers et al., 1998; O'Rourke et al., 1997). As shown in Figure 1, p85 can be identi®ed in the anti-SHP-2 immunoprecipitates by immunoblotting analysis (lane 1), and the band intensity was elevated by EGF stimulation (lane 2). The association between p85 and SHP-2 was also markedly enhanced by the expression of DEGFR (lane

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Figure 1 EGF stimulation as well as DEGFR expression enhances the co-immunoprecipitation of the p85 subunit of PI3K with SHP-2. Human glioblastoma U87MG and U87/ DEGFR cells were treated with or without 50 ng/ml of EGF for 5 min, following serum-starvation for 24 h. Equal amounts of whole cell extracts as determined by protein concentration analysis were then immunoprecipitated with anti-SHP-2 antibody and the immunoprecipitates were resolved by SDS ± PAGE. Association of p85 with SHP-2 was examined by immunoblotting with anti-p85 antibody (upper panel, high reactive speci®city of the antibody has been proven by its provider as well as our experiments). Immunoblotting with anti-SHP-2 antibody was performed to con®rm consistent immunoprecipitation of SHP-2 (lower panel)

3). DEGFR, which is the partial truncation of EGFR at its extracellular domain, is the most commonly observed EGFR mutant in human tumors. The mutation leads to a weak, but unattenuated constitutive kinase activity in the intracellular domain, and introduction of DEGFR into the U87MG cell line greatly enhanced its tumorigenicity without increasing the rate of cell growth (Nishikawa et al., 1994; O'Rourke et al., 1997). Interestingly, it has been found that DEGFR preferentially activates the PI3 kinase pathway rather than the MAP kinase pathway (Moscatello et al., 1998; and data not shown). EGF stimulated Akt phosphorylation is enhanced by SHP-2 expression in human glioblastoma cells, and the N-terminal SH2 domain is essential for the regulation Akt is downstream of PI3K and linked to cell cycle progression, proliferation and protection from cell death (Hemmings, 1997). Akt activation has been shown to depend on phosphorylation of a speci®c serine (Ser473) (Alessi et al., 1996). Evaluation of Akt Ser473 phosphorylation provides a surrogate assay for PI3K activity in intact cells (Cuevas et al., 1999), and is much easier to perform than direct lipid kinase activity analysis. To investigate SHP-2 regulation of EGF induced Akt phosphorylation, we ®rst introduced wildtype SHP-2 and its variants, each mutated at one of its three domains, into U87MG cells [R32E in the Nterminal SH2 domain and R138E in the C-terminal SH2 domain render the two SH2 domains incapable of Oncogene


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binding Tyr(P) residues; C459S and phosphatase domain deleted mutation (1 ± 244) render the phosphatase inactive]. We used the bicistronic expression vector pIRES/hyg, which contains an internal ribosome entry site (IRES) to ensure a high cell percentage of protein expression in antibiotics resistant cells. As seen in Figure 2, wild-type SHP-2 expression enhanced Akt phosphorylation upon treatment with EGF (lane 4), and the R32E mutation in the N-terminal SH2 domain essentially abolished the eect (lane 10), suggesting that the N-terminal SH2 domain is critical for SHP-2's positive role in regulating EGF-induced Akt phosphorylation. Unexpectedly, and in contrast to a dominantnegative function in regulating activation of extracellular signal-regulated kinase (ERK) (Bennett et al., 1996; Deb et al., 1998), the phosphatase inactivated mutants led to elevated but not maximal Akt phosphorylation in response to EGF stimulation (lanes 6 and 8). These data perhaps re¯ect an adaptor like activity of the SH2 domains of SHP-2 in mediating PI3K signaling initiated by EGFR activation, since SHP-2 may link EGFR and PI3K by associating with both of them in response to EGF stimulation (Vogel et al., 1993; and Figure 1). SHP-2 is a common requirement for mediating Akt phosphorylation induced by EGF, PDGF and IGF The roles of SHP-2 in regulating Akt phosphorylation induced by growth factors were also evaluated with SHP-2+/+ and SHP-27/7 mouse ®broblasts in which 65

amino acids within the SHP-2 NH2-terminal SH2 domain were target-deleted (Shi et al., 1998). The expression of Akt was similar in the wild-type and mutant cells (Figure 3a, middle panel). Downmodulation of EGF-induced Akt phosphorylation was not observed in homozygous SHP-2 mutant cells as expected (Figure 3a, lanes 2 and 5). However, PDGF-induced Akt phosphorylation was markedly reduced by the SHP-2 mutation (Figure 3a, lanes 3 and 6). Consistent with the results obtained with ERK kinase assay by Shi et al. (1998), phosphorylation of extracellular signal-regulated kinase (ERK) was attenuated in the mutant cells, after PDGF as well as EGF stimulation (Figure 3b). Next, stable expressing clones of SHP-2 were derived from the homozygous mutant cells. The reconstitution of SHP-2 expression did not change the expression of Akt in the mutant ®broblasts (data not shown). Dierences in signaling of distinct receptor species are seen in Figure 3. However, both EGF- and PDGFinduced Akt phosphorylation was enhanced upon reconstituting SHP-2 expression into the mutant cells (Figure 4a, lanes 4, 6 and 8; Figure 4b, lanes 4, 6 and 8). To broaden the observation, IGF induced Akt phosphorylation was also assessed with ®broblasts. As seen in Figure 5, Akt phosphorylation upon IGF-I stimulation was reduced in the SHP-27/7 cells (5a, lane 4), and this reduction was relieved by restoring SHP-2 expression (5b, lanes 4, 6 and 8). These data indicates that SHP-2 is often required for growth factor-induced Akt phosphorylation. More-

Figure 2 The eects of expression of SHP-2 or its mutants on EGF stimulated Akt phosphorylation in human glioblastoma cells. U87MG cells were transfected with either empty expression vector (pIRES), or the vector containing wild-type SHP-2 (WT), SHP-2 C459S (C459S), 1 ± 244 residues of SHP-2 (1 ± 244), SHP-2 R32E (R32E) or SHP-2 R138E (R138E) cDNA. The transfected cells were selected and pooled as described under `Materials and methods'. Serum starved cells after 24 h starvation were treated with or without 50 ng/ml of EGF for 10 min. An equal amount of whole lysates for each sample were resolved by SDS ± PAGE and transferred onto a nitrocellulose membrane. The membrane was then probed with anti-phospho-Akt, anti-His or anti-SHP-2 or antib-actin antibody as indicated in the ®gure Oncogene


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Figure 3 The eects of SHP-2 targeted deletion on EGF and PDGF induced Akt phosphorylation in mouse ®broblasts. SHP-2+/+ and SHP-27/7 mouse ®broblasts were starved for 24 h and then stimulated with or without 50 ng/ml of EGF or 40 ng/ml of PDGF for 10 min. Whole cell lysates normalized by protein concentration assay were resolved by SDS ± PAGE, and subjected to Western blotting analysis of phospho-Akt (a, upper panel), Akt (a, middle panel), b-actin (a, lower panel), phospho-ERK (b, upper panel) or ERK2 (b, lower panel)

Figure 4 Reintroduction of SHP-2 into SHP-27/7 cells enhances Akt phosphorylation induced by EGF and PDGF stimulation. SHP-27/7 mouse ®broblasts were transfected with empty expression vector (pIRES), or pIRES containing wild-type SHP-2 (WT) or SHP-2 C459S (C459S) cDNA. The derived pIRES transfected ®broblast colonies were pooled, and stable clones expressing wildtype SHP-2 (WT3, WT7 and WT8) or SHP-2 C459S (C459S1 and C459S3) were identi®ed by blotting with anti-His antibody, after antibiotic selection. These derived cells were plated and starved for 24 h before treatment with or without 50 ng/ml of EGF (a) or 40 ng/ml of PDGF (b) for 10 min. Equal amounts of protein were analysed by blotting with anti-phospho-Akt, anti-b-actin, antiphospho-ERK, anti-ERK2, anti-His or anti-SHP-2 antibody as indicated in the ®gure Oncogene


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Figure 5 IGF stimulated Akt phosphorylation is attenuated by targeted SHP-2 deletion, and rescued by reconstitution of SHP-2 expression. After treating serum-starved cells with or without 50 ng/ml of IGF-I for 10 min, equal amounts of lysates from SHP-2+/+ and SHP-27/7 cells (a), or SHP-27/7 cells containing pIRES, expressing wild-type SHP-2 or SHP-2 C459S (b) were evaluated for Akt phosphorylation by Western blotting analysis (upper panels). Immunoblotting with anti-b-actin antibody was also performed to con®rm equal sample loading (lower panels)

over, the results support the role of the N-terminal SH2 domain for SHP-2 mediation of PI3K activation since only the N-terminal SH2 domain was impaired in the mutant cells. Interestingly, introduction of the phosphatase inactivated mutant, SHP-2 C459S, into the mutant cells modestly enhanced EGF-induced Akt phosphorylation (Figure 4a, lanes 10 and 12, and supporting the above observation in U87MG cells), but downregulated PDGF- or IGF-induced Akt phosphorylation which has already been attenuated by targeted deletion of the N-terminal SH2 domain (Figure 4b, lanes 10 and 12; Figure 5b, lanes 10 and 12). These data suggest that there are shared as well as unique receptor signaling features involving the components involved in PI3K activation mediated by either EGFR, PDGFR or IGFR. SHP-2 mediates PI3K activation To determine whether SHP-2 regulation of Akt phosphorylation results from its mediation of PI3K activation, PI3 kinase activities were further assayed in the SHP-2 mouse embryonic ®broblasts. As shown in Figure 6a,b, PDGF-induced PI3K activity was impaired by SHP-2 targeted mutation, and the reduction was rescued by the reintroduction of SHP-2 into the mutant mouse ®broblasts. Likewise, the reconstitution of SHP-2 expression upregulated EGF-induced PI3K activity (Figure 6c). This study shows that SHP-2 participates in the regulation of PI3K activation induced by growth factors. SHP-2 has previously been suggested to be a positive regulator of MAPK activation based on the evidence that the expression of catalytically inactive mutants of SHP-2 attenuates ERK activation (Bennett et al., 1996; Deb et al., 1998). It is speculated that the SH2 domains of SHP-2, especially the N-terminal SH2 domain, also play an important role in regulating ERK activation (Deb et al., 1998; O'Reilly and Neel, 1998). However, it is unclear how SHP-2 regulates MAPK activation while SHP-2 is thought to act in signaling events proximal to RTKs. Compared with ERK, PI3K more closely approximates the action of SHP-2. Our data Oncogene

has demonstrated the association of the p85 subunit of PI3K with SHP-2, a process that is inducible by EGF stimulation or by DEGFR expression. Growth factorinduced physical association of SHP-2 and PI3K is supported by the observation that a large amount of PI3K activities were detected in anti-SHP-2 immunoprecipitates after EGF or IGF-I stimulation in human skin ®broblasts (Takahashi et al., 1999). SHP-1 has been shown to directly bind to p85, and the inducible binding has been suggested to be responsible for its negative regulation of Lck-induced PI3K activation (Cuevas et al., 1999). However, the association of SHP2 and p85 appears to occur in a much larger complex with Gab1 (Holgado-Madruga et al., 1996), Cb1 (Hakak et al., 2000; Fixman et al., 1997), as well as a 97-100KD protein (Gesbert et al., 1998; Craddock et al., 1997; Carlberg and Rohrschneider, 1997), possibly Gab2 (Gu et al., 1998) and/or SIRP (Kharitonenkov et al., 1997). This signaling complex likely plays a critical role in determining cell signaling. It will be of interest to de®ne what other proteins are components of the ensemble, and moreover, how the complex functions are coordinated. We have noticed cell morphological changes both in the human glioblastoma cells and SHP-2 mutated mouse ®broblasts after introducing SHP-2 and its mutants (C-J Wu and MI Greene, unpublished data). It will be interesting in the future to investigate how SHP-2 modi®es PI3K pathway mediated biological consequences, and the study may shed light on clarifying the mechanism that SHP-2 is critical for development.

Materials and methods Cell lines and reagents Human U87MG glioblastoma cells were obtained from the American Type Tissue Collection (ATCC, Rockville, MD, USA). The U87MG stable clone overexpressing DEGFR (U87/DEGFR) were kindly provided by Dr Webster Cavenee (Ludwig Cancer Institute, San Diego, CA, USA). Mouse


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Figure 6 SHP-27/7 ®broblasts display a defective PI3K activation by PDGF and reconstitution of SHP-2 expression enhances the PI3K activities in the mutant cells stimulated by EGF and PDGF. SHP-2+/+ and SHP-27/7 ®broblasts were stimulated with 40 ng/ ml of PDGF for 5 min after starvation. Equal amounts of cell lysates were subjected to immunoprecipitation with antiphosphotyrosine antibody (a). Likewise, serum-starved SHP-27/7 cells containing empty vector (pIRES) and subclones reconstituted with SHP-2 expression were stimulated with 50 ng/ml of EGF (c) or 40 ng/ml of PDGF (b), after which samples normalized for protein were immunoprecipitated with anti-phosphotyrosine mAb. The resulting immunoprecipitates were analysed for PI3K activities as described under `Materials and methods'. Representative data from four independent experiments for each assay is shown with thin layer chromatography autoradiograms. Cell lysate normalization for immunoprecipitation was con®rmed by Western blotting analysis with anti-b-actin antibody after each PI3 kinase assay

embryonic ®broblast SHP-2+/+ and SHP-27/7 cells have been described previously (Shi et al., 1998). Recombinant human epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AA and insulin-like growth factor (IGF)-I were from Gibco-BRL (Gaithersburg, MD, USA). Antiphospho-Akt (Ser473), anti-Akt and anti-phospho-ERK antibodies were purchased from New England Biolabs Inc. (Beverly, MA, USA). Antisera to SHP-2, His and ERK2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-phosphotyrosine mAb, 4G10, and the antibody reactive with the p85 subunit of PI3K were obtained from Upstate Biotechnology (Lake Placid, NY, USA). The anti-b-actin antibody was from Sigma. Expression constructs and cell transfections Wild-type and various mutant SHP-2 cDNAs were epitopetagged by subcloning into pcDNA3.1 myc/his (Invitrogen,

Carlsbad, CA, USA), and all have been described previously (Deb et al., 1998). The myc/his tagged cDNAs were ampli®ed by high-®delity polymerase chain reaction (PCR) and digested with HpaI. The generated blunt-end fragments were ligated into the expression vector pIRES/Hyg (Clontech, Palo Alto, CA, USA) previously linearized with BamHI and bluntended. The correct orientation of the constructs was determined and the subcloned cDNAs were sequenced. U87MG cells and mouse SHP-27/7 ®broblasts were transfected with empty vector (pIRES), or pIRES containing wild-type SHP-2 or various mutant SHP-2 cDNAs using lipofectamine (Gibco-BRL) (O'Rourke et al., 1998). The transfected U87MG cells were selected with 70 mg/ml of hygromycin for 2 weeks, and the cell colonies that survived from each transfection were pooled. The pIRES transfected ®broblast colonies were pooled and used as experimental control, and positive stable subclones expressing wild-type SHP-2 or SHP-2 C459S were identi®ed by Western blotting, Oncogene


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after selection with 200 mg/ml of hygromycin for 2 weeks. All the derived ®broblasts were maintained in medium containing 60 mg/ml of hygromycin. Immunoprecipitation and Western blotting After treatment with or without the indicated growth factors, cell lysates were prepared as described previously (Wu et al., 2000). Equal amounts of cell lysates, as determined by protein concentration assay with the Dc Protein Assay kit (Bio-Rad, Hercules, CA, USA), were incubated with anti-SHP-2 antibody at 48C for 2 h. Immunocomplexes were collected with Protein A conjugated to Sepharose 4B (Sigma). For Western blotting analysis, the resulting immunoprecipitates or whole cell lysates (20 mg) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and then transferred onto nitrocellulose membranes. The membranes were blotted with the indicated antibodies and signals were detected by enhanced chemiluminescence (ECL, Amersham Corp.)

2 h. The immunoprecipitates were washed twice with lysis buer, twice with 0.5 M LiCl in 100 mM Tris-HCl (pH 7.5) plus 100 mM sodium othovanadate, and twice with reaction buer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 6.25 mM MgCl2, 0.625 mM EDTA). The beads were resuspended in 40 ml of reaction buer, and 10 mg of substrate mixture (phosphatidylinositol and phosphatidylserine dispersed by sonication in 10 mM HEPES, 1 mM EGTA, pH 7.5) was added. The tubes were incubated at room temperature for 10 min and reactions were initiated by addition of 5 mCi [g-32P]ATP in 10 ml of 400 mM ATP and terminated by addition of 80 ml of CHCl3:CH3OH (1 : 1) after another 10 min at room temperature. Phospholipids were extracted, desiccated and redissolved in 12 ml of CHCl3:CH3OH (2 : 1), and samples were chromatographed on thin layer chromatography (TLC) plates in CHCl3:CH3OH:2.5 M NH4OH (9 : 7 : 2, v/v). Signal corresponding to phosphatidylinositol 3phosphate (PIP) was visualized after autoradiography.

PI3 kinase assay The PI3 kinase assay was carried out basically as previously described (Qian et al., 1999). Brie¯y, cells were lysed in Nonidet P-40 lysis buer (Qian et al., 1999). Equal amounts of cell lysate proteins were immunoprecipitated with antiphosphotyrosine 4G10 at 48C for 3 h. Protein A-Sepharose was then added and incubated at 48C with rotation for another

Acknowledgments This work was supported by grants from the National Institute of Health and the Abramson Family Cancer Research Institute to MI Greene. This work was also supported by grants from the National Institutes of Health and the Department of Veteran Aairs to DM O'Rourke.

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