brain-derived neurotrophic factor stimulates interactions of shp2 with phosphatidylinositol 3-kinase...

Brain-Derived Neurotrophic Factor Stimulates Interactions ofShp2 with Phosphatidylinositol 3-Kinase and Grb2

in Cultured Cerebral Cortical Neurons

Masashi Yamada, *Hiroshi Ohnishi, *Shin-ichiro Sano, Toshiyuki Araki, Atsushi Nakatani,Toshihiko Ikeuchi, and Hiroshi Hatanaka

Institute for Protein Research, Osaka University, Osaka, and*Mitsubishi Kasei Institute of Life Science, Tokyo, Japan

Abstract: Shp2, a protein tyrosine phosphatase pos-sessing SH2 domains, is utilized in the intracellular sig-naling of various growth factors. Shp2 is highly ex-pressed in the CNS. Brain-derived neurotrophic factor(BDNF), a member of the neurotrophin family, which alsoshows high levels of expression in the CNS, exerts neu-rotrophic and neuromodulatory effects in CNS neurons.We examined how BDNF utilizes Shp2 in its signalingpathway in cultured cerebral cortical neurons. We foundthat BDNF stimulated coprecipitation of several tyrosine-phosphorylated proteins with anti-Shp2 antibody andthat Grb2 and phosphatidylinositol 3-kinase (PI3-K) werecoprecipitated with anti-Shp2 antibody in response toBDNF. In addition, both anti-Grb2 and anti-PI3-K anti-bodies coprecipitated Shp2 in response to BDNF. TheBDNF-stimulated coprecipitation of the tyrosine-phos-phorylated proteins, Grb2, and PI3-K with anti-Shp2 an-tibody was completely inhibited by K252a, an inhibitor ofTrkB receptor tyrosine kinase. This BDNF-stimulatedShp2 signaling was markedly sustained as well as BDNF-induced phosphorylation of TrkB and mitogen-activatedprotein kinases. In PC12 cells stably expressing TrkB,both BDNF and nerve growth factor stimulated Shp2signaling similarly to that by BDNF in cultured corticalneurons. These results indicated that Shp2 shows cross-talk with various signaling molecules including Grb2 andPI3-K in BDNF-induced signaling and that Shp2 may beinvolved in the regulation of various actions of BDNF inCNS neurons. Key Words: Neurotrophin—Neurotrophicfactor—Tyrosine phosphatase—Tyrosine kinase—Trk—PC12 cell.J. Neurochem. 73, 41–49 (1999).

Neurotrophin is a family of neurotrophic factors thatpromote differentiation, maturation, and survival of var-ious types of neurons in the PNS and the CNS (Snider,1994; Kubo et al., 1995; Nonomura et al., 1995; Abiru etal., 1996; Lewin and Barde, 1996). The neurotrophinfamily consists of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3(NT-3) and -4/5 (NT-4/5). The neurotrophins bind to andactivate Trk family proteins, receptor tyrosine kinases, to

exert their effects. NGF binds to TrkA, BDNF and NT-4/5 bind to TrkB, and NT-3 binds to TrkC (Barbacid,1995; Bothwell, 1995). BDNF and its receptor TrkB arehighly expressed in the brain (Hofer et al., 1990; Merlioet al., 1992). BDNF is involved in the regulation ofsynaptic plasticity as well as the promotion of differen-tiation and survival of the CNS neurons (Korte et al.,1995; Thoenen, 1995; Akaneya et al., 1996; Figurov etal., 1996; Kang et al., 1997; Takei et al., 1997).

The Trks activated by ligand binding are autophos-phorylated on tyrosine residues, and then they stimulateintracellular signaling, activating a variety of enzymesand effectors including phospholipase C-g, phosphati-dylinositol 3-kinase (PI3-K), Shc, Grb2, Ras, and mito-gen-activated protein (MAP) kinases (Kaplan and Ste-phens, 1994; Greene and Kaplan, 1995; Segal andGreenberg, 1996). Recently, studies using PC12 cellshave shown that NGF exerts a survival-promoting effectvia activation of PI3-K and promotes neuronal differen-tiation through activation of MAP kinases (Yao andCooper, 1995; Franke et al., 1997). In addition, NGFinduces sustained activation of MAP kinases in contrastto epidermal growth factor (EGF), which stimulates cellproliferation but does not induce neuronal differentiation

Received December 11, 1998; revised manuscript received February16, 1999; accepted February 16, 1999.

Address correspondence and reprint requests to Dr. M. Yamada atDivision of Protein Biosynthesis, Institute for Protein Research, OsakaUniversity, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

The present address of A. Nakatani is Takeda Chemical IndustriesLtd., 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan.

Abbreviations used:BDNF, brain-derived neurotrophic factor; BSA,bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium;DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; FGF, fi-broblast growth factor; FRS2, FGF receptor substrate 2; Gab1, Grb2-associated binder-1; GM-CSF, granulocyte/macrophage colony-stimu-lating factor; HS, horse serum; IL-3, interleukin-3; MAP, mitogen-activated protein; MEM, minimum essential medium; NGF, nervegrowth factor; NT-3 and -4/5, neurotrophin-3 and -4/5; PAO, phenyl-arsine oxide; PDGF, platelet-derived growth factor; PI3-K, phosphati-dylinositol 3-kinase; PMSF, phenylmethylsulfonyl fluoride; PNCS,precolostrum newborn calf serum; TBS, Tris-buffered saline.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 1999 International Society for Neurochemistry

in PC12 cells (Marshall, 1995; Yamada et al., 1996,1997a). This sustained activation of MAP kinases byNGF is thought to be important for induction of neuronaldifferentiation. However, these observations in intracel-lular signaling have been obtained mainly in transformedcell lines including PC12 cells, and the pathway respon-sible for neurotrophin-induced signaling in neurons isstill unclear.

Shp2, a protein tyrosine phosphatase (Matozaki andKasuga, 1996; Neel and Tonks, 1997), is activated byvarious growth factors including EGF, platelet-derivedgrowth factor (PDGF), and insulin (Feng et al., 1993;Kazlauskas et al., 1993; Lechleider et al., 1993). Shp2has two SH2 domains and is activated by associationwith tyrosine-phosphorylated proteins via its SH2 do-mains. In addition, Shp2 is tyrosine phosphorylated inresponse to EGF, PDGF, and insulin; then the phosphor-ylated Shp2 binds to Grb2, leading to Ras activation(Bennett et al., 1994; Li et al., 1994; Noguchi et al.,1994). Studies using phosphatase-inactive Shp2 as adominant negative mutant indicated that Shp2 works as apositive regulator in growth factor signaling pathways(Milarski and Saltiel, 1994; Yamauchi et al., 1995; Zhaoet al., 1995). Shp2 is expressed highly in the brain,although its expression is ubiquitous (Suzuki et al.,1995). Therefore, Shp2 is thought to have importantroles in the regulation of intracellular signaling in neu-rons. However, it remains unclear how Shp2 functions inthe signaling of neurotrophic factors, including neurotro-phins, in neurons.

Here, we found that Shp2 was a component of thesignaling pathway of BDNF in cultured cerebral corticalneurons. BDNF stimulated coprecipitation of Grb2,PI3-K, and several tyrosine-phosphorylated proteins withanti-Shp2 antibody. Shp2 might be involved in the reg-ulation of multiple signaling pathways including Ras–MAP kinase and PI3-K pathways, and it might be animportant signaling molecule in various effects of BDNFin neurons.

MATERIALS AND METHODS

Cell culturePrimary cultures of dissociated cerebral cortical neurons

were prepared from the brains of embryonic day 18 rats (WistarST, both sexes) as described previously (Yamada et al., 1995;1997b). The cells were cultured in a medium consisting of 5%precolostrum newborn calf serum (PNCS; Mitsubishi Kasei),5% heat-inactivated horse serum (56°C, 30 min; HS; Gibco),and 90% minimum essential medium (MEM; Gibco) contain-ing 3.5 mg/ml glucose, 30 nM selenium, and 0.5 mg/ml sodiumbicarbonate, at a final cell density of 53 105 cells/cm2 onpolyethylenimine-coated six-well plates. After culturing for 3days, the medium was changed to serum-containing MEM plus1 mM cytosine arabinoside (Sigma), and then the cells werecultured for 6 days. The medium was replaced with serum-freeMEM, and immunoprecipitation and in vitro PI3-K assays wereperformed after overnight incubation.

PC12 cells stably expressing TrkB (Nakatani et al., 1998)were maintained in 75-cm2 flasks using Dulbecco’s modifiedEagle’s medium (DMEM; Gibco) supplemented with 5% (vol/

vol) PNCS and 5% (vol/vol) heat-inactivated HS. The cellswere plated in 100-mm collagen-coated dishes and cultured inDMEM containing serum until near confluent. Then the assayswere performed after overnight serum starvation in DMEM.

Immunoprecipitation and immunoblottingCells were washed once with ice-cold Tris-buffered saline

(TBS) and lysed in a buffer containing 1% Triton X-100, 150mM NaCl, 5 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 20 mMTris-HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride(PMSF), 0.7mg/ml pepstatin, 1mg/ml leupeptin, and 1mg/mlantipain. Immunoprecipitation and immunoblotting were per-formed as described previously (Yamada et al., 1997b).

In vitro PI3-K assayPI3-K activity was measured as described previously

(Yamada et al., 1997b). In brief, cells were washed once withice-cold TBS and lysed in a buffer containing 1% NonidetP-40, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 2 mMNa3VO4, 20 mM Tris-HCl (pH 7.5), 1 mM PMSF, 0.7mg/mlpepstatin, 1mg/ml leupeptin, and 1mg/ml antipain. Immuno-precipitation from the lysates was performed as describedabove. The immunoprecipitates were washed three times with1% Nonidet P-40, 150 mM NaCl, 0.1 mM Na3VO4, and 20 mMTris-HCl (pH 7.5), twice with 0.5M LiCl, 0.1 mM Na3VO4,and 0.1M Tris-HCl (pH 7.5), twice with 100 mM NaCl, 1 mMEDTA, 0.1 mM Na3VO4, and 10 mM Tris-HCl (pH 7.5), andtwice with a reaction buffer containing 100 mM NaCl, 0.5 mMEGTA, 5 mM MgCl2, and 20 mM Tris-HCl (pH 7.5). Then, invitro kinase reaction was carried out with 10mg of phosphati-dylinositol as a substrate and the immunoprecipitate in 50ml ofreaction buffer plus 10mCi of [g-32P]ATP (10 mM ATP) at25°C for 10 min. Samples were developed on TLC plates, andthen the plates were exposed to x-ray films (Kodak).

Factors, antibodies, and reagentsBDNF, NT-3, and TrkB-IgG were kindly provided by Re-

generon Pharmaceutical Co. NGF (2.5S form) was preparedfrom male mouse submandibular glands (Yamada et al., 1994).The factors were dissolved at 50mg/ml in phosphate-bufferedsaline containing 1 mg/ml bovine serum albumin (BSA) as aworking solution and added to the cultures at 100 ng/ml.Anti-pan Trk polyclonal antiserum was raised in rabbits againsta synthetic peptide corresponding to the 14 carboxyl-terminalamino acids of human TrkA (Yamada et al., 1994). Anti-TrkBspecific monoclonal antibody was kindly provided by Dr. S.Koizumi (Novartis Pharma K. K.). Anti-phosphotyrosinemonoclonal antibody (4G10) was purchased from Upstate Bio-technology, anti-Shp2 and anti-Grb2 polyclonal antibodieswere from Santa Cruz Biotechnology, anti-p85 (the regulatorysubunit of PI3-K) polyclonal and anti-Shp2 and anti-Grb2monoclonal antibodies were from Transduction Laboratories,anti-MAP kinase and anti-phospho-MAP kinase polyclonal an-tibodies were from New England Biolabs, anti-p85 (the regu-latory subunit of PI3-K) monoclonal antibody was from MBL,and anti-Grb2-associated binder-1 (anti-Gab1) polyclonal anti-body was from Upstate Biotechnology. K252a was purchasedfrom Kyowa Hakko and dissolved at 2 mM in dimethyl sul-foxide (DMSO) as a stock solution.

RESULTS

Shp2 is activated by association with tyrosine-phos-phorylated proteins and is phosphorylated on tyrosineresidues in response to growth factors (Matozaki and

J. Neurochem., Vol. 73, No. 1, 1999

42 M. YAMADA ET AL.

Kasuga, 1996; Neel and Tonks, 1997). To examine whatkinds of tyrosine-phosphorylated proteins bind to Shp2,Shp2 was immunoprecipitated from lysates of culturedcerebral cortical neurons treated for 5 min with BSA (asa control), NGF, BDNF, or NT-3 and then immunoblot-ted with anti-phosphotyrosine antibody. As shown inFig. 1A, the anti-Shp2 immunoprecipitates containedseveral tyrosine-phosphorylated proteins in response toBDNF and NT-3 but not to BSA or NGF. We considerthat the 70-kDa tyrosine-phosphorylated protein is Shp2.TrkB and TrkC, but not TrkA, are expressed in cerebralcortical neurons (Merlio et al., 1992; Steininger et al.,1993). We observed that BDNF and NT-3 induced ty-rosine phosphorylation of Trks, thought to be TrkB andTrkC, respectively, when anti-Trk immunoprecipitatesfrom lysates of cultured cortical neurons were subjectedto western blotting analysis with anti-phosphotyrosineantibody (Fig. 1B). Recently, several reports have indi-cated that cerebral cortical neurons possess the ability toexpress TrkA (Lee et al., 1998; Sala et al., 1998; Seaboldet al., 1998). But we could not detect NGF-inducedtyrosine phosphorylation of TrkA (Fig. 1B). These re-sults suggest that only a small population of the culturedcortical neurons may express TrkA and/or the level ofTrkA expression may be very weak. On the other hand,tyrosine-phosphorylated Trks with a molecular mass of145 kDa could scarcely be detected in the anti-Shp2

immunoprecipitates (Fig. 1A). When the anti-Shp2 im-munoprecipitates were further immunoblotted with anti-TrkB specific monoclonal antibody, we detected veryweak signals only after long exposure (data not shown).Recently, Goldsmith and Koizumi (1997) reported thatShp2 associates with TrkA in response to NGF in PC12cells. However, in their case, the interaction of TrkAwith Shp2 could be detected only in the presence ofphenylarsine oxide (PAO), an inhibitor of protein ty-rosine phosphatase, in addition to Na3VO4 in lysisbuffer. When we used lysis buffer containing both PAOand Na3VO4, we could observe significant interaction ofShp2 with TrkB in response to BDNF in the culturedcortical neurons, although the intensities of other ty-rosine-phosphorylated proteins coprecipitated with theanti-Shp2 antibody did not change (data not shown).Therefore, Shp2 may interact with TrkB and then may beactivated and tyrosine phosphorylated in response toBDNF in cultured cortical neurons.

Several studies have indicated that Shp2 interacts withGrb2 (Bennett et al., 1994; Li et al., 1994; Noguchi et al.,1994) and PI3-K (Welham et al., 1994) in nonneuronalcells in response to various growth factors and cytokinesincluding PDGF, EGF, insulin, interleukin-3 (IL-3), andgranulocyte/macrophage colony-stimulating factor (GM-CSF). To examine whether Shp2 interacts with theseproteins in response to neurotrophins in neurons, the

FIG. 1. BDNF-stimulated Shp2 signaling in cul-tured cerebral cortical neurons. A and B: Severaltyrosine-phosphorylated proteins, PI3-K, and Grb2were coprecipitated with the anti-Shp2 antibody inresponse to neurotrophins, which induced tyrosinephosphorylation of Trks, in cultured cortical neu-rons. The cells were incubated with 2 mg/ml BSA(Con.) or 2 mg/ml BSA plus 100 ng/ml NGF (NGF),BDNF (BDNF), or NT-3 (NT-3) for 5 min and lysed.Shp2 (A) was immunoprecipitated with anti-Shp2polyclonal antibody and analyzed by western blot-ting using anti-phosphotyrosine, anti-PI3-K, anti-Grb2, and anti-Shp2 monoclonal antibodies. Ty-rosine-phosphorylated Shp2 is indicated by an ar-row on the right of the immunoblot. Trks (B) wereimmunoprecipitated with anti-pan Trk polyclonalantiserum and then immunoblotted with anti-phos-photyrosine monoclonal antibody. Molecular massesare shown on the left of the immunoblots. C and D:Inhibitory effects of K252a on the BDNF-stimulatedsignaling via Shp2 in cultured cerebral cortical neu-rons. Lysates were prepared from cells incubatedwithout (2 BDNF) or with (1 BDNF) 100 ng/mlBDNF for 5 min after treatment with 0.05% (vol/vol)DMSO (2 K252a) or 0.05% (vol/vol) DMSO plus 1mM K252a (1 K252a) for 10 min. Shp2 (C) and TrkB(D) were immunoprecipitated with anti-Shp2 poly-clonal antibody and anti-pan Trk antiserum, re-spectively, from the lysates. The anti-Shp2 immu-noprecipitates (ppt) were analyzed by westernblotting with anti-phosphotyrosine, anti-PI3-K, an-ti-Grb2, and anti-Shp2 monoclonal antibodies. Theanti-Trk immunoprecipitates were immunoblottedwith anti-phosphotyrosine monoclonal antibody.Molecular masses are shown on the left of theimmunoblots. We obtained similar results in threeindependent experiments (A–D).


43BDNF-INDUCED SIGNALING VIA Shp2

anti-Shp2 immunoprecipitates from lysates of culturedcerebral cortical neurons treated for 5 min with BSA (asa control), NGF, BDNF, or NT-3 were subjected towestern blotting analysis using anti-Grb2 and anti-p85(the regulatory subunit of PI3-K) antibodies. We foundthat the amounts of Grb2 and p85 proteins coprecipitatedwith anti-Shp2 antibody increased in response to BDNFand NT-3 but not in response to BSA or NGF (Fig. 1A).These results indicated that the neurotrophins stimulateinteractions of Shp2 with Grb2 and PI3-K in culturedcerebral cortical neurons.

To examine the effects of K252a, an inhibitor of Trktyrosine kinase (Knu¨sel and Hefti, 1992), on the BDNF-induced signaling via Shp2, immunoprecipitates withanti-Shp2 antibody from lysates of cultured cerebral cor-tical neurons treated for 5 min with BSA (as a control),NGF, BDNF, or NT-3 in the presence or absence of 1mM K252a were subjected to western blotting analysisusing anti-phosphotyrosine, anti-Grb2, and anti-p85 (theregulatory subunit of PI3-K) antibodies. K252a inhibitedthe BDNF-induced increase in tyrosine phosphorylationof proteins precipitated with anti-Shp2 antibody (Fig.1C). The BDNF-stimulated increases in the amounts ofGrb2 and p85 proteins coprecipitated with anti-Shp2antibody were also decreased to the levels in untreatedneurons by treatment with K252a (Fig. 1C). The BDNF-induced tyrosine phosphorylation of TrkB was com-pletely inhibited by K252a at the same concentration as

that which inhibited Shp2 signaling (Fig. 1D). The half-maximal effect of K252a was observed at a concentra-tion of ;200 nM in cultured cortical neurons (data notshown). These results indicated that BDNF-stimulatedsignaling via Shp2 requires the tyrosine kinase activity ofTrkB.

To confirm the interactions of Shp2 with Grb2 andPI3-K, we examined whether or not Shp2 protein iscontained in anti-Grb2 and anti-p85 (the regulatory sub-unit of PI3-K) immunoprecipitates in response to neuro-trophins. Immunoprecipitates with anti-Grb2 and anti-PI3-K antibodies from lysates of cultured cerebral corti-cal neurons treated for 5 min with BSA (as a control),NGF, BDNF, or NT-3 were subjected to western blottinganalysis using anti-Shp2 antibody. The levels of Shp2protein in both the anti-Grb2 and the anti-PI3-K immu-noprecipitates showed increases in response to BDNFand NT-3 but not in response to BSA or NGF (Fig. 2Aand B). The Shp2 bands on the immunoblots tended to bebroad, which may reflect tyrosine phosphorylation ofShp2 interacting with Grb2 and PI3-K. In addition, wefound that anti-Grb2 antibody coprecipitated PI3-K inresponse to BDNF and NT-3 (Fig. 2A) and that anti-PI3-K antibody coprecipitated Grb2 protein (Fig. 2B).These results indicated that Shp2, Grb2, and PI3-K in-teract with each other in response to neurotrophins incultured cortical neurons.

FIG. 2. Neurotrophin-stimulated interactions amongShp2, PI3-K, and Grb2 in cultured cerebral corticalneurons. A: Shp2 and PI3-K were coprecipitatedwith the anti-Grb2 antibody in response to neurotro-phins in cultured cortical neurons. The cells wereincubated with 2 mg/ml BSA (Con.) or 2 mg/ml BSAplus 100 ng/ml NGF (NGF), BDNF (BDNF), or NT-3(NT-3) for 5 min and lysed. Grb2 was immunopre-cipitated with the anti-Grb2 polyclonal antibody andanalyzed by western blotting with anti-phosphoty-rosine, anti-PI3-K, anti-Shp2, and anti-Grb2 mono-clonal antibodies. Molecular masses are shown onthe left of the immunoblots. B: Shp2 and Grb2 werecoprecipitated with anti-PI3-K antibody in responseto neurotrophins in cultured cortical neurons. Thecell lysates were prepared as described above.PI3-K was immunoprecipitated with anti-PI3-K poly-clonal antibody and analyzed by western blottingwith anti-phosphotyrosine, anti-Shp2, anti-Grb2,and anti-PI3-K monoclonal antibodies. Molecularmasses are shown on the left of the immunoblots. Cand D: PI3-K activities were coprecipitated withanti-Shp2 and anti-Grb2 antibodies in response toneurotrophins in cultured cortical neurons. The cellswere treated as described above and lysed. Shp2(C) and Grb2 (D) were immunoprecipitated with anti-Shp2 and anti-Grb2 polyclonal antibodies, respec-tively. The PI3-K activities in the immunoprecipitates(ppt) were measured as described in Materials andMethods. The arrows on the right indicate the origin(Ori.) and the position of phosphatidylinositol3-monophosphate (PIP), the product of PI3-K, esti-mated from the position of phosphatidylinositol4-monophosphate as a standard. We obtained sim-ilar results in three independent experiments (A–D).


44 M. YAMADA ET AL.

We found that BDNF and NT-3 stimulated the inter-action between the p85 regulatory subunit of PI3-K andShp2 or Grb2. To examine whether the p110 catalyticsubunit is involved in these interactions, PI3-K activitywas measured in the anti-Shp2 or Grb2 immunoprecipi-tates of lysates prepared from cultured cerebral corticalneurons treated for 5 min with BSA (as a control), NGF,BDNF, or NT-3. BDNF and NT-3 increased PI3-K ac-tivity in the anti-Shp2 or Grb2 immunoprecipitates, butBSA and NGF did not (Fig. 2C and D). These resultsindicated that BDNF and NT-3 stimulate the interactionsof Shp2 and Grb2 with functional PI3-K in culturedcortical neurons.

It has been reported that neurotrophic factors inducesustained intracellular signaling to exert their effects(Marshall, 1995; Yamada et al., 1997a). To examine theduration of BDNF-induced signaling via Shp2, the anti-Shp2 immunoprecipitates from lysates of cultured corti-cal neurons treated with BDNF for various periods wereimmunoblotted with anti-phosphotyrosine, anti-Grb2,and anti-p85 (the regulatory subunit of PI3-K) antibod-ies. The level of BDNF-stimulated coprecipitation oftyrosine-phosphorylated proteins with anti-Shp2 anti-body was markedly sustained, and these phosphorylatedproteins were detected even 5 h after addition of BDNF(Fig. 3A). In addition, we observed a tyrosine-phospho-rylated protein (with an apparent molecular mass of;68kDa) coprecipitated with anti-Shp2 antibody, whichshowed an increase in phosphorylation level 1.5 h after

BDNF exposure. The phosphorylation level of this pro-tein gradually increased until at least 5 h after BDNFexposure. The BDNF-stimulated increases in levels ofGrb2 and PI3-K coprecipitated with anti-Shp2 antibodywere also sustained, and these increases were observedeven 5 h after BDNF exposure (Fig. 3A). Consistent withthe observations with anti-Shp2 immunoprecipitation,we also observed sustained increases in the levels ofShp2 protein in the anti-Grb2 and anti-PI3-K immuno-precipitates (Fig. 3B and C). Furthermore, the BDNF-induced coprecipitation of Grb2 and PI3-K with anti-PI3-K and anti-Grb2 antibodies, respectively, alsotended to be sustained (Fig. 3B and C). In culturedcortical neurons, we found that BDNF-induced phos-phorylation of TrkB and MAP kinases was sustainedsimilarly to Shp2 signaling (Fig. 3D and E). Their phos-phorylation was obviously detected even 5 h after BDNFexposure. These results indicated that BDNF-inducedShp2 signaling including the interactions with Grb2 andPI3-K, as well as the activation of TrkB and MAPkinases, is markedly sustained in cultured cortical neu-rons.

To examine whether and how Shp2 signaling is alsostimulated by neurotrophins in PC12 cells, a model forstudying the mechanisms by which neurotrophic factorsact on neurons (Greene, 1978), in comparison withBDNF-induced signaling in cultured cortical neurons, weinvestigated BDNF- and NGF-induced signaling viaShp2 in PC12 cells stably expressing TrkB (Nakatani et

FIG. 3. Time course of BDNF-stimulated sig-naling via Shp2 in cultured cerebral corticalneurons. A–C: Time course of BDNF-stimu-lated interactions among Shp2, PI3-K, andGrb2 in cultured cortical neurons. Lysateswere prepared from cells incubated without(0m) or with 100 ng/ml BDNF for 5 min (5m),1.5 h (1.5h), 3 h (3h), and 5 h (5h). Shp2 (A)was immunoprecipitated with anti-Shp2 poly-clonal antibody. The immunoprecipitates(ppt) were analyzed by western blotting withanti-phosphotyrosine, anti-PI3-K, anti-Grb2,and anti-Shp2 monoclonal antibodies. Grb2(B) and PI3-K (C) were immunoprecipitatedwith anti-Grb2 and anti-PI3-K polyclonal an-tibodies, respectively, and then the immuno-precipitates were immunoblotted with anti-Shp2, anti-PI3-K, and anti-Grb2 monoclonalantibodies. Molecular masses are shown onthe left of the immunoblots. D: Time course ofBDNF-induced tyrosine phosphorylation ofTrkB in cultured cortical neurons. TrkB wasimmunoprecipitated with anti-pan Trk anti-serum from the lysates prepared as de-scribed above. The immunoprecipitates wereimmunoblotted with anti-phosphotyrosinemonoclonal antibody. Molecular masses areshown on the left of the immunoblot. E: Timecourse of BDNF-stimulated phosphorylationof MAP kinases in the cultured cortical neu-rons. The lysates, prepared as describedabove, were analyzed by western blottingwith anti-phospho-MAP kinase (apMAPK) and anti-MAP kinase (aMAPK) polyclonal antibodies. Molecular masses are shown on the leftof the immunoblots. We obtained similar results in three or four independent experiments (A–E).



al., 1998). The TrkB-expressing PC12 cells differenti-ated into neuron-like cells in response to BDNF as wellas to NGF and showed not only NGF-induced tyrosinephosphorylation of TrkA but also BDNF-induced ty-rosine phosphorylation of TrkB (Nakatani et al., 1998).Shp2 was immunoprecipitated with anti-Shp2 antibodyfrom lysates of TrkB-expressing PC12 cells exposed toBSA (as a control), NGF, or BDNF for 5 min. Then, theanti-Shp2 immunoprecipitates were immunoblotted withanti-phosphotyrosine, anti-Grb2, or anti-p85 (the regula-tory subunit of PI3-K) antibodies. The anti-Shp2 anti-body coprecipitated several tyrosine-phosphorylated pro-teins in response to NGF and BDNF but not to BSA,although the pattern of immunoblots with anti-phospho-tyrosine antibody in the TrkB-expressing PC12 cells wasdifferent from that in the cultured cortical neurons (Fig.4A). On the other hand, coprecipitation of Grb2 andPI3-K with anti-Shp2 antibody was also observed inTrkB-expressing PC12 cells as well as in the culturedcortical neurons (Fig. 4A). In addition, we examined theanti-Grb2 and anti-PI3-K immunoprecipitates in TrkB-expressing PC12 cells in comparison with those in cul-tured cortical neurons. Shp2 was coprecipitated withanti-Grb2 and anti-PI3-K antibodies in response to NGFand BDNF (Fig. 4B and C). As observed in culturedcortical neurons, the Shp2 proteins in the anti-Grb2 andanti-PI3-K immunoprecipitates showed broad or doublet

patterns, which may be due to tyrosine phosphorylationof Shp2 bound to Grb2 and PI3-K. In addition, PI3-Kwas detected in the anti-Grb2 immunoprecipitates, andGrb2 was also detected in anti-PI3-K immunoprecipi-tates in response to the neurotrophins in TrkB-expressingPC12 cells (Fig. 4B and C). Furthermore, we measuredPI3-K activities in the anti-Shp2 and anti-Grb2 immuno-precipitates in TrkB-expressing PC12 cells. Increases inthe PI3-K activities were observed in both of the immu-noprecipitates in response to NGF and BDNF (Fig. 4Dand E). These results indicated that neurotrophins stim-ulate similar Shp2 signaling with Grb2 and PI3-K inPC12 cells to that in cultured cortical neurons.

DISCUSSION

Here, we have reported that neurotrophins inducedinteractions of Shp2 with Grb2 and PI3-K in culturedcerebral cortical neurons and TrkB-expressing PC12cells. In addition, we showed neurotrophin-induced in-teraction between Grb2 and PI3-K. Therefore, neurotro-phins may stimulate formation of complexes includingShp2, Grb2, and PI3-K. These observations suggestedthat Shp2 is involved in the regulation of Ras–MAPkinase and PI3-K pathways in neurotrophin-induced sig-naling in neurons.

FIG. 4. Neurotrophin-stimulated Shp2signaling in PC12 cells stably express-ing TrkB. A–C: Neurotrophin-stimu-lated interactions of Shp2 with Grb2,PI3-K, and tyrosine-phosphorylatedproteins in the TrkB-expressing PC12cells. The cells were incubated with 2mg/ml BSA (Con.) or 2 mg/ml BSA plus100 ng/ml NGF (NGF) or BDNF (BDNF)for 5 min and lysed. Shp2 (A), Grb2 (B),and PI3-K (C) were immunoprecipitatedwith anti-Shp2, anti-Grb2, and anti-PI3-K polyclonal antibodies, respec-tively. Then the immunoprecipitates(ppt) were analyzed by western blottingwith anti-phosphotyrosine, anti-Shp2,anti-Grb2, and anti-PI3-K monoclonalantibodies. Molecular masses are shownon the left of the immunoblots. D and E:PI3-K activities were coprecipitated withanti-Shp2 and anti-Grb2 antibodies inresponse to neurotrophins in the TrkB-expressing PC12 cells. The cells weretreated as described above and lysed.Shp2 (D) and Grb2 (E) were immunopre-cipitated with anti-Shp2 and anti-Grb2polyclonal antibodies, respectively. ThePI3-K activities in the immunoprecipi-tates were measured as described inMaterials and Methods. The arrows onthe right indicate the origin (Ori.) and theposition of phosphatidylinositol 3-mono-phosphate (PIP), the product of PI3-K,estimated from the position of phospha-tidylinositol 4-monophosphate as astandard. We obtained similar results inthree independent experiments (A–E).


46 M. YAMADA ET AL.

We observed that BDNF and NT-3 induced tyrosinephosphorylation of Trks in the cultured cerebral corticalneurons. It was thought that BDNF activates TrkB andNT-3 activates TrkC. However, recent studies in vivoand in TrkB-expressing PC12 cells indicate that NT-3also activates TrkB (Farinas et al., 1998; Nakatani et al.,1998). We observed that TrkB-IgG inhibited NT-3-stim-ulated tyrosine phosphorylation of Trks and that ty-rosine-phosphorylated Trk in response to NT-3 was im-munoprecipitated with the anti-TrkB specific monoclo-nal antibody in the cultured cortical neurons (data notshown). These results indicate that NT-3 can stimulatetyrosine phosphorylation of TrkB in the cultured corticalneurons, although NT-3 seemed predominantly to acti-vate TrkC. Therefore, the NT-3-stimulated signaling thatwe observed may contain signaling through NT-3-in-duced TrkB activation in the cultured cortical neurons.

We demonstrated neurotrophin-induced interaction ofShp2 with Grb2. PDGF, EGF, and insulin induce ty-rosine phosphorylation of Shp2, and then the phosphor-ylated Shp2 binds to Grb2, leading to stimulation of theRas signaling pathway (Bennett et al., 1994; Li et al.,1994; Noguchi et al., 1994). BDNF may also stimulatetyrosine phosphorylation of Shp2 and binding of Shp2 toGrb2, activating Ras signaling. In PC12 cells, NGF ac-tivates the Ras pathway via tyrosine phosphorylation ofShc followed by its binding to Grb2 (Kaplan and Ste-phens, 1994; Greene and Kaplan, 1995; Segal andGreenberg, 1996). The interaction of Shp2 with Grb2may be an alternative pathway for activating Ras signal-ing. Shc was not expressed in cerebral cortical neurons,but N-Shc (also called ShcC), a homologue of Shc, wasexpressed in these cells (Nakamura et al., 1996; O’Bryanet al., 1996). We observed that N-Shc was tyrosinephosphorylated and bound to Grb2 in response to BDNF(data not shown). In cultured cortical neurons, both Shp2and N-Shc may mediate BDNF-stimulated activation ofthe Ras signaling pathway. On the other hand, it hasrecently been reported that fibroblast growth factor(FGF) receptor substrate 2 (FRS2) is tyrosine phosphor-ylated and associates with Shp2 and Grb2 in response toFGF and NGF in PC12 cells (Hadari et al., 1998). There-fore, we cannot exclude the possibility that Shp2 inter-acts indirectly with Grb2 through adaptor proteins in-cluding FRS2 in cultured cortical neurons and TrkB-expressing PC12 cells.

We showed that BDNF stimulated interaction of Shp2with PI3-K in cultured cerebral cortical neurons andTrkB-PC12 cells. There are no reports showing that Shp2can bind to PI3-K directly. In PC12 cells, Gab1 is ty-rosine phosphorylated and interacts with Shp2 and PI3-Kin response to NGF (Holgado et al., 1997). In addition,we observed that Gab1 was tyrosine phosphorylated inresponse to BDNF in the cultured cortical neurons (datanot shown). Shp2 may interact indirectly with PI3-Kthrough adaptor proteins including Gab1 in cultured cor-tical neurons and TrkB-expressing PC12 cells.

We demonstrated that interaction between Grb2 andPI3-K is stimulated by BDNF treatment in cultured cor-

tical neurons. Wang et al. (1995) reported that Grb2binds to PI3-K through direct interaction between SH3domains of Grb2 and proline-rich motifs of the p85regulatory subunit of PI3-K, although this interaction isnot enhanced by growth factor stimulation. In culturedcerebral cortical neurons, BDNF-stimulated interactionbetween Grb2 and PI3-K may be mediated by indirectrather than direct association. Gab1 may be involved inthis interaction. In PC12 cells, NGF stimulates interac-tions of Gab1 with Grb2 and PI3-K (Holgado et al.,1997).

Vambutas et al. (1995) have reported that NGF doesnot stimulate tyrosine phosphorylation and activation ofShp2 in PC12 cells. On the other hand, Goldsmith andKoizumi (1997) have demonstrated that Shp2 is acti-vated in response to NGF in PC12 cells. This contradic-tion may be due to differences between subclones ofPC12 cells that they used and/or between their experi-mental procedures. In the present study, in TrkB-ex-pressing PC12 cells, BDNF and NGF stimulated similarinteractions of Shp2 with Grb2 and PI3-K to thoseevoked by BDNF and NT-3 in cultured cortical neurons.These results indicate that neurotrophins stimulate Shp2signaling both in PC12 cells and in cultured corticalneurons. In addition, we consider that the neurotrophin-stimulated interactions of Shp2 with Grb2 and PI3-K is ageneral signaling mechanism of neurotrophins in neuro-nal cells and is involved in regulation of neurotrophiceffects including promotion of neuronal differentiation,maturation, and survival. IL-3 and GM-CSF also induceinteractions of Shp2 with Grb2 and PI3-K in a murinemyeloid progenitor-like cell line (Welham et al., 1994).In addition, we observed that EGF also stimulated sim-ilar interactions of Shp2 with Grb2 and PI3-K in PC12cells (data not shown). Various growth factors and cy-tokines may utilize interactions of Shp2 with Grb2 andPI3-K as their signaling mechanism.

We demonstrated that neurotrophins stimulated inter-actions of Shp2 with Grb2 and PI3-K and interactionbetween Grb2 and PI3-K in cultured cortical neurons andTrkB-expressing PC12 cells, although we could not de-termine whether these interactions were direct or indirector whether Shp2, Grb2, and PI3-K formed a ternarycomplex. In addition, we observed various neurotrophin-induced interactions among signaling proteins in neu-rons. For example, we have reported that neurotrophinsstimulate tyrosine phosphorylation of insulin receptorsubstrate-1 and -2 and their association with PI3-K incultured cerebral cortical neurons (Yamada et al.,1997b). We also observed that brain immunoglobulin-like molecule with tyrosine-based activation motifs(Ohnishi et al., 1996; Sano et al., 1997), also called SHPsubstrate-1 (Fujioka et al., 1996) and signal-regulatoryprotein (Kharitonenkov et al., 1997), is tyrosine phos-phorylated and binds to Shp2 in response to neurotro-phins in cultured cerebral cortical neurons and PC12cells (Ohnishi et al., 1999). To elucidate how theseneurotrophin-induced interactions are involved in vari-ous effects, we will examine where and when the inter-



actions occur in response to neurotrophins in neurons.These interactions may show differences at differentdevelopmental stages, in distinct populations of neurons,and in multiple subcellular structures.

Acknowledgment: We thank Regeneron PharmaceuticalCo. for the kind gifts of BDNF, NT-3, and TrkB-IgG; Dr. S.Koizumi (Takarazuka Research Institute, Novartis PharmaK. K.) for the generous gift of anti-TrkB monoclonal antibody;and Dr. M. Takahashi (Institute for Life Science, MitsubishiKasei) for his valuable advice and encouragement throughoutthis study. This work was supported in part by a Grant-in-Aidfor Scientific Research from the Ministry of Education, Sci-ence, Sports, and Culture of Japan and from the Japan Societyfor the Promotion of Science for Young Scientists.

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