Characterization of Intracellular Signals Via Tyrosine 1062 in RET Activated by Glial Cell Line-Derived Neurotrophic Factor

Oncogene (2000) 19, 4469 ± 4475 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor

Hironori Hayashi1,2, Masatoshi Ichihara1, Toshihide Iwashita1, Hideki Murakami1, Yohei Shimono1, Kumi Kawai1, Kei Kurokawa1, Yoshiki Murakumo1, Tsuneo Imai2, Hiroomi Funahashi2, Akimasa Nakao2 and Masahide Takahashi*,1

1Department of Pathology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; 2Department of Surgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

Glial cell line derived neurotrophic factor (GDNF) tively. However, interestingly, these four related signals through a multicomponent receptor complex neurotrophic factors appear to be able to cross-talk consisting of RET receptor tyrosine kinase and a through dierent GFRas in the presence of RET. For member of GDNF family receptor a (GFRa). Recently, example, GDNF and neurturin almost equally acti- it was shown that tyrosine 1062 in RET represents a vated RET in cells expressing both GFRa1 and RET binding site for SHC adaptor proteins and is crucial for (Baloh et al., 1997; Creedon et al., 1997; Jing et al., both RAS/mitogen activated protein kinase (MAPK) and 1997), and GDNF and artemin was also able to cause phosphatidylinositol 3-kinase (PI3-K)/AKT signaling RET phosphorylation through GFRa2 and GFRa1, pathways. In the present study, we characterized how respectively (Baloh et al., 1997, 1998; Jing et al., 1997; these two pathways diverge from tyrosine 1062, using Sanicola et al., 1997). In this respect, it is interesting to human neuroblastoma and primitive neuroectodermal note that EketjaÈ ll et al. (1999) recently reported that tumor cell lines expressing RET at high levels. In RET and GFRa1 pre-assemble at cell surface without response to GDNF stimulation, SHC bound to GAB1 GDNF stimulation. Based on the results from site- and GRB2 adaptor proteins as well as RET, and SHC directed mutagenesis of GDNF, they proposed the and GAB1 were highly phosphorylated on tyrosine. The presence of two distinct binding sites for GDNF, one complex formation consisting of SHC, GAB1 and GRB2 formed by GFRa1 alone and another formed by a pre- was almost abolished by replacement of tyrosine 1062 in assembled GFRa1-RET complex; the latter might lead RET with phenylalanine. Tyrosine-phosphorylated GAB1 to promiscuity in ligand-receptor speci®cities. was also associated with p85 subunit of PI3-K, resulting Gene knock out studies revealed that RET, GDNF in PI3-K and AKT activation, whereas SHC-GRB2-SOS and GFRa1 genes play crucial roles in the development complex was responsible for the RAS/ERK signaling of kidney and enteric nervous system, suggesting a pathway. These results suggested that the RAS and PI3- tight coupling of GDNF and GFRa1 in RET signaling K pathways activated by GDNF bifurcate mainly in vivo (Schuchardt et al., 1994; Moore et al., 1996; through SHC bound to tyrosine 1062 in RET. Pichel et al., 1996; Sanchez et al., 1996; Cacalano et al., Furthermore, using luciferase reporter-gene assays, we 1998; Enomoto et al., 1998). Neuturin7/7 and found that the RAS/ERK and PI3-K signaling pathways Gfra27/7 mice showed the drastic reduction of are important for activation of CREB and NF-kBin cholinergic innervation in the lacrimal and submandi- GDNF-treated cells, respectively. Oncogene (2000) 19, bular salivary glands as well as in the intestine 4469 ± 4475. (Heuckeroth et al., 1999; Rossi et al., 1999). The Gfra37/7 mice exhibited severe defects in the superior Keywords: RET; GDNF; SHC; CREB; NF-kB cervical ganglion (SCG), whereas other ganglia appeared normal (Nishio et al., 1999). These observa- tions suggested that, despite promiscuity in ligand- Introduction receptor speci®cities in culture cells mentioned above, each of the GDNF family ligands has a rather speci®c RET proto-oncogene codes for a receptor tyrosine function in vivo through GFRa-RET complex. kinase that represents a functional receptor for four Intracellular signaling downstream of RET has been related neurotrophic factors including glial cell line- extensively studied using culture cell lines and primary derived neurotrophic factor (GDNF), neurturin, arte- neurons (van Weering and Bos, 1997; Chiariello et al., min and persephin (see Airaksinen et al., 1999 for a 1999; Xing et al., 1998b; Feng et al., 1999; Melillo et review). In addition to RET, these factors require al., 1999; Murakami et al., 1999a,b; Soler et al., 1999; glycosylphosphatidylinositol-linked cell surface pro- Trupp et al., 1999). Binding of several adaptor proteins teins, called GDNF family receptor a1-4 (GFRa1-4), to phosphorylated tyrosine residues in its intracellular as ligand-binding components for intracellular signal- domain has been shown. For example, tyrosines at ing. In vitro binding study showed that GFRa1, codons 905, 1015, 1062 and 1096 were identi®ed as GFRa2, GFRa3 and GFRa4 bind GDNF, neurturin, docking sites for GRB7/GRB10, phospholipase Cg artemin and persephin with high speci®city, respec- (PLCg), SHC/Enigma and GRB2, respectively (Asai et al., 1996; Borrello et al., 1996; Durick et al., 1998; Liu et al., 1996; Arighi et al., 1997; Lorenzo et al., 1997; *Correspondence: M Takahashi Ohiwa et al., 1997; Alberti et al., 1998). Of these Received 2 May 2000; revised 29 June 2000; accepted 13 July 2000 tyrosines, we demonstrated that tyrosine 1062 is Signaling pathways via tyrosine 1062 in RET H Hayashi et al 4470 important for transforming activities of all multiple endocrine neoplasia (MEN) 2 mutant forms of RET examined so far (Asai et al., 1996; Ishiguro et al., 1999; Iwashita et al., 1999, 2000). SHC binding to RET through tyrosine 1062 leads to the activation of RAS/ MAPK signaling pathway. Interestingly, Segoun- Cariou and Billaud (2000) recently reported that the PI3-K/AKT pathway was also activated through tyrosine 1062 in RET with a MEN 2A mutation (RET-MEN2A). In addition, the PI3-K/AKT pathway was shown to be critical for transforming activity of the RET-MEN2A mutant protein, although it remains unclear how this signaling pathway is activated through tyrosine 1062 in RET. Here we show that the complex formation of GAB1 with SHC bound to tyrosine 1062, in response to GDNF, can lead to activation of PI3-K. In addition, we found that RAS/ERK and PI3-K/AKT signaling pathways activated by GDNF could be responsible for activation of CREB and NF-kB transcription factors, respectively.

Results

Activation of several signaling molecules depends on phosphorylation of tyrosine 1062 in RET activated by GDNF

We ®rst investigated whether GDNF stimulation can Figure 1 Activation of signaling molecules by GDNF. (a) TGW induce the activation of signaling molecules including neuroblastoma cells were serum-starved for 2 h and treated with AKT, ERK, JNK, p38MAPK and CREB in TGW GDNF (100 ng/ml) for 10 min. Their lysates (50 mg of proteins) human neuroblastoma cells expressing both RET and suspended in SDS-sample buer were subjected to Western blotting with anti-RET, anti-phosphotyrosine (pTyr), anti- GFRa1 (Nozaki et al., 1998). All of them were clearly phosphoAKT (pAKT), anti-phosphoERK1/2 (pERK1/2), anti- phosphorylated in response to GDNF stimulation phosphoJNK (pJNK), anti-phosphop38MAPK (pp38MAPK) or (Figure 1a). anti-phosphoCREB (pCREB) antibody. Anti-pTyr antibody To elucidate which tyrosine in the carboxyl-terminal detected the 175 kDa phosphorylated RET protein. (b) Tyrosines sequence of RET is important for activation of these at codons 981, 1015, 1029 and 1062 in human RET cDNA encoding a long isoform (1114 amino acids) were replaced with molecules, tyrosine at codon 981, 1015, 1029 or 1062 phenylalanine as previously described (Asai et al., 1996). Each was replaced with phenylalanine (Figure 1b). The mutant RET gene was inserted into the expression vector and sequences surrounding tyrosines 981, 1015 and 1062 transfected into SK-N-MC human primitive neuroectodermal match the consensus sequences for binding of p85 tumor cells by the calcium phosphate precipitation method. The lysates from established transfectants untreated or treated with subunit of PI3-K, PLCg and SHC, respectively. Each GDNF for 10 min were subjected to Western blotting with the mutant RET gene was transfected into SK-N-MC designated antibodies human primitive neuroectodermal tumor cell line expressing GFRa1 and GFRa2 (data not shown), and transfectants expressing high levels of mutant RET proteins were established (Figure 1b). Interestingly, SHC from lysates of TGW and SK-N-MC(RET) cells phosphorylation of AKT, ERK, JNK, p38MAPK and stimulated with GDNF. On the other hand, co- CREB was markedly impaired by a mutation of precipitation of p85 subunit of PI3-K (Figure 2a) and tyrosine 1062 but not a mutation of tyrosine 981, SOS (data not shown) with SHC was not found under 1015 or 1029 (Figure 1b), indicating the importance of our experimental conditions. When the lysate from SK- tyrosine 1062 for activation of all these signaling N-MC(RETY1062F) cells was used, co-precipitation of molecules. In addition, phosphorylation of PLCg was RET, GAB1 and GRB2 with SHC was almost impaired by a mutation of tyrosine 1015 as expected undetectable (data not shown), showing that this (data not shown). complex formation depends on phosphorylation of tyrosine 1062. Furthermore, GAB1 was highly phosphorylated on tyrosine in GDNF-treated cells and Characterization of complex formation of adaptor phosphorylated 52 and 46 kDa SHC proteins appeared proteins via tyrosine 1062 in RET to be eciently coprecipitated with GAB1 (Figure 2b). Since tyrosine 1062 in RET represents a docking site As we previously described, p85 subunit of PI3-K for SHC adaptor proteins (Asai et al., 1996; Arighi et was associated with GAB1 in GDNF-treated cells al., 1997; Lorenzo et al., 1997), we investigated which (Figure 2c), leading to activation of PI3-K (Murakami signaling molecules bind to SHC in response to et al., 1999b), whereas RET and SOS were not (data GDNF. As shown in Figure 2a, in addition to RET not shown). In addition, we found that GAB1 was and GRB2, GAB1 was clearly co-precipitated with associated with GRB2 in them (Figure 2c), indicating

Oncogene Signaling pathways via tyrosine 1062 in RET H Hayashi et al 4471 the complex formation of SHC, GRB2 and GAB1 in a increase by GDNF treatment (Figure 2d). These GDNF-dependent manner. Association of p85 and ®ndings thus suggested that GRB2-SOS complex and GRB2 with GAB1 was not observed in GDNF-treated GAB1 are recruited to SHC bound to tyrosine 1062 in SK-N-MC(RETY1062F) cells (data not shown). On RET after GDNF stimulation and could be responsible the other hand, the GRB2-SOS complex was detected for activation of RAS and PI3-K, respectively (Figure in unstimulated cells and its amount appeared to 5).

CREB activation by RAS/ERK pathway and NF-kB activation by PI3-K pathway We next investigated activation of transcription factors, CREB and NF-kB, by GDNF. A recent report suggested the involvement of RAS/ERK pathway for CREB activation induced by GDNF (Feng et al., 1999). Since it was shown that the PI3-K/AKT or p38MAPK signaling pathway was required for its activation by NGF, FGF or serum stimulation (Tan et al., 1996; Xing et al., 1998a; Du and Montminy, 1998), we tested whether these two pathways are also involved in CREB activation by GDNF. To inhibit each signaling pathway, we treated TGW cells with a MEK1 inhibitor, PD98059, two p38MAPK inhibitors, SB203580 and SB202190, or two PI3-K inhibitors, wortmannin and LY294002. Speci®city of each inhibitor was con®rmed by Western blotting, although LY294002 inhibited p38MAPK phosphorylation as well as AKT phosphorylation (Figure 3). CREB phosphorylation was inhibited by PD98059 but not Figure 2 Complex formation of adaptor proteins via tyrosine by SB203580, SB202190, wortmannin and LY294002 1062 in RET. (a) Association of RET, GAB1 and GRB2 with (Figure 3). SHC. TGW and SK-N-MC(RET) cells were serum-starved for In addition, transcription activity of CREB induced 2 h and treated with GDNF (100 ng/ml) for 10 min. The lysates by GDNF was determined by CREB reporter-gene (1 ± 1.5 mg of proteins) from GDNF-treated or untreated cells were immunoprecipitated with anti-SHC antibody, followed by assay. We made two constructs containing three immunoblotting with anti-RET, anti-GAB1, anti-p85 subunit of tandem repeats of CREB binding sequences (CRE) PI3-K (ap85), anti-SHC or anti-GRB2 antibody. (b) The lysates and herpes simplex virus thymidine kinase (TK) from GDNF-treated or untreated SK-N-MC(RET) and SK-N- minimal promoter or SV40 promoter, immediately MC(RETY1062F) cells were immunoprecipitated with anti-GAB1 upstream of the luciferase gene (Figure 4a). About antibody, followed by immunoblotting with anti-GAB1 or anti- pTyr antibody. (c) The lysates from GDNF-treated or untreated 1.6- and 2.5-fold increase of its activity was observed in SK-N-MC(RET) cells were immunoprecipitated with anti-GAB1 GDNF-treated TGW cells transfected with CRE-TK antibody, followed by immunoblotting with anti-GAB1, anti-p85 and CRE-SV40 constructs, respectively (Figure 4b,d). or anti-GRB2 antibody. (d) The lysates from GDNF-treated or As expected, this increase was almost completely untreated SK-N-MC(RET) cells were immunoprecipitated with anti-SOS antibody, followed by immunoblotting with anti-SOS or inhibited by PD98059 but not by SB203580 and anti-GRB2 antibody. IP, immunoprecipitation; IB, immunoblot- wortmannin, con®rming the importance of the RAS/ ting ERK signaling pathway on CREB activation.

Figure 3 RAS/ERK signaling pathway-dependent CREB phosphorylation. TGW cells were serum-starved for 2 h and incubated in the absence or presence of each kinase inhibitor at the designated concentrations for 15 ± 20 min. Then, the cells were treated with GDNF for 10 min. Inhibitors other than wortmannin were added together with GDNF. Wortmannin was removed before addition of GDNF because its action as an inhibitor was irreversible. The resulting cell lysates were subjected to Western blotting with the designated antibodies. Anti-pTyr antibody detected the 175 kDa phosphorylated RET protein

Oncogene Signaling pathways via tyrosine 1062 in RET H Hayashi et al 4472

Figure 4 Luciferase reporter gene assay. (a) Constructs used for dual luciferase reporter assay. CREB (CRE) and NF-kB binding sequences were tandemly repeated three and four times upstream of the herpes simplex virus thymidine kinase (TK) minimal promoter or SV40 promoter and the ®re¯y luciferase gene, respectively. These sequences were inserted into the pGL3-basic vector (Promega). (b±e) The plasmid vectors shown in (a) were cotransfected with pRL-TK plasmid carrying the renilla luciferase gene into TGW cells. After transfection, the cells were cultured for 48 h and incubated in the presence or absence of each kinase inhibitor for 15 ± 20 min. Then, the cells were treated with GDNF for 1 h. Inhibitors other than wortmannin were added together with GDNF as described in Figure 3. All values were normalized by the activities of the renilla luciferase gene. Results represent averages from at least three independent experiments and bars indicate the standard error. Averages of luciferase activities without GDNF treatment in each experiment were set at 1.0. (d) Degradation and phosphorylation of IkBa. Serum-starved TGW cells were incubated with GDNF for the indicated time and the resulting cell lysates were subjected to Western blotting with anti-IkBa and anti-phosphoIkBa (pIkBa) antibodies

Oncogene Signaling pathways via tyrosine 1062 in RET H Hayashi et al 4473 Romashkova and Makarov (1999) recently reported that, upon PDGF stimulation, IkB kinase (IKK) associates with AKT and plays a crucial role in NF- kB activation. Thus, we investigated whether GDNF induces NF-kB activation in a PI3-K-dependent manner. As shown in Figure 4f, Ik-Ba that is known to regulate NF-kB activity was rapidly phosphorylated and degraded in response to GDNF. NF-kB reporter- gene assay showed that GDNF induced about 1.4- and 3.5-fold increase of transcription activity of NF-kBin GDNF-treated TGW cells transfected with NF-kB-TK and NF-kB-SV40 constructs, respectively (Figure 4a,c and e). In contrast to the results from the CREB reporter-gene assay, wortmannin but not PD98059 and SB203580 almost completely inhibited the expression of the luciferase gene (Figure 4c,e). When we used SK-N- MC cells for the NF-kB reporter-gene assay, no activation was observed after GDNF stimulation (data not shown), suggesting that RET expression is necessary for NF-kB activation.

Discussion Figure 5 Schematic illustration of intracellular signals via SHC bound to tyrosine 1062 of RET Tyrosine residues in the intracellular domain of receptor tyrosine kinases represent docking sites for speci®c adaptor proteins. By site-directed mutagenesis, lanine did not impair AKT phosphorylation (Figure we demonstrated that a mutation of tyrosine 1062 in 1b). RET markedly impaired the transforming activity of On the other hand, GRB2-SOS complex was present all multiple endocrine neoplasia (MEN) 2 mutant in unstimulated cells at signi®cant levels. It has been forms of RET, suggesting its importance on signal well established that the recruitment of GRB2-SOS transduction for oncogenesis (Asai et al., 1996; complex to SHC is involved in the activation of RAS/ Iwashita et al., 1999, 2000). Consistent with the fact ERK signaling pathway. As expected, a mutation of that the amino acid sequence surrounding tyrosine tyrosine 1062 in RET markedly impaired activation of 1062 matches the consensus sequence for binding of ERK. Although tyrosine 1096 was also reported to be SHC SH2 and PTB domains, SHC binding to RET- a binding site for GRB2 (Alberti et al., 1998), deletion MEN2A and RET-MEN2B mutant proteins was of this tyrosine did not impair transforming activity of severely reduced by its mutation (Asai et al., 1996; RET-MEN2A and RET-MEN2B mutant proteins Arighi et al., 1997; Lorenzo et al., 1997; Ohiwa et al., (Asai et al., 1996) as well as phosphorylation of ERK 1997; Geneste et al., 1999; Ishiguro et al., 1999). SHC (our unpublished data). Thus, it seems likely that the bound to RET further complexes with GRB2 adaptor PI3-K and RAS pathways activated by GDNF proteins, leading to the activation of RAS/ERK bifurcate mainly through SHC bound to tyrosine pathway. Recently, it turned out that phosphorylation 1062 in RET (Figure 5), and that contribution of of tyrosine 1062 in RET is also crucial for activation tyrosine 1096 for the activation of RAS/ERK pathway of the PI3-K/AKT signaling pathway (Segoun- could be small. In addition, it is possible that PI3-K is Cariou and Billaud, 2000), although the sequence activated partially by RAS as previously described surrounding tyrosine 1062 does not match the (Rodriguez-Viciana et al., 1994; van Weering et al., consensus sequence for binding of p85 subunit of 1998). PI3-K. Several reports demonstrated CREB phosphoryla- In the present study, it was demonstrated that, in tion by GDNF and a possible involvement of the response to GDNF stimulation, SHC was associated RAS/ERK pathway for its activation (Xing et al., with GAB1 proteins in addition to GRB2 and RET. 1998b; Feng et al., 1999; Trupp et al., 1999). Since it Since we recently reported that PI3-K activity was has also been reported that the p38MAPK or PI3-K detected mainly in the GAB1 immunoprecipitate from pathway was involved in CREB activation by FGF, the lysate of GDNF-treated cells (Murakami et al., NGF or serum stimulation (Tan et al., 1996; Du et al., 1999b), association of SHC with GAB1 appears to be 1998; Xing et al., 1998a), we tested this possibility essential for the activation of PI3-K pathway through using kinase inhibitors. As a result we found that the tyrosine 1062 in RET. Moreover, we found that GAB1 RAS/ERK pathway but not the p38MAPK and PI3-K also complexes with GRB2 after GDNF treatment, pathways is essential for CREB phosphorylation by indicating the presence of the complex formation GDNF. A luciferase reporter-gene assay also con- consisting of SHC, GAB1 and GRB2 (Figure 5). ®rmed the importance of the RAS/ERK pathway for Although the sequence surrounding tyrosine 981 its CRE binding activity. Although the RAS/ERK matches the consensus sequence for binding of p85 pathway is believed to be necessary for cell growth and subunit of PI3-K, we were not able to detect its direct dierentiation, it is interesting to note that recent association with RET. This was consistent with the reports showed the importance of CREB for survival result that replacement of tyrosine 981 with phenyla- of neurons (Bonni et al., 1999; Riccio et al., 1999).

Oncogene Signaling pathways via tyrosine 1062 in RET H Hayashi et al 4474 In addition, we showed, for the ®rst time, NF-kB room temperature in 3% albumin in TPBS (phosphate activation in GDNF-stimulated cells. In response to buered saline containing 0.5% Tween-20) with gentle GDNF, Ik-Ba was rapidly phosphorylated and shaking and incubated with primary antibodies overnight at degraded. It is well known that phosphorylation and 48C. After washing the membranes with TPBS three times, degradation of IkB are required for NF-kB activation. they were incubated with the secondary antibodies con- jugated to horseradish peroxidase (swine anti-rabbit IgG- Taken together with the result from the luciferase HRP, rabbit anti-mouse IgG-HRP, Dako) for 1 h at room reporter-gene assay with kinase inhibitors, it is likely temperature. The reaction was examined by an enhanced that the PI3-K pathway is crucial for the transcrip- chemiluminescence detection kit (ECL, Amersham, UK) tional activity of NF-kB induced by GDNF (Figure 5). according to the directions of the supplier. Extensive studies revealed that the PI3-K/AKT pathway plays an important role in cell survival induced by Immunoprecipitation neurotrophic factors (see Datta et al., 1999 for a review). However, interestingly, Segoun-Cariou and Cells were grown subcon¯uently in 100 mm dishes and Billaud (2000) reported that the PI3-K/AKT pathway serum-starved for 2 h. Then the cells were stimulated with is also required for transforming activity of the RET- GDNF (100 ng/ml) for 10 ± 20 min, washed once with ice- cold phosphate-buered saline (PBS) and lysed in radio- MEN2A mutant protein, although it was not demon- immunoprecipitation assay (RIPA) buer (20 mM Tris-HCl, strated whether NF-kB activation is necessary for this pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P(NP)-40) activity. Thus, further analyses of the PI3-K/AKT containing 1 mM phenylmethylsulfonyl ¯uoride, 0.5 mM pathway activated by GDNF could provide a new sodium orthovanadate, 0.1 mM aprotinin and 1 mM leupeptin. insight into intracellular signals responsible for cell The cell lysates were centrifuged at 15 000 r.p.m. for 30 min transformation as well as survival of neurons. to remove cell debris and incubated with 2 mg of antibodies Finally, the fact that JNK and p38MAPK were also for 3 h at 48C. The resulting immunocomplexes were activated through phosphorylated tyrosine 1062 sug- collected with Protein A sepharose (Sigma, St. Louis, MO, gests the possibility that unknown complex formation USA) and washed four times with RIPA buer. The of adaptor proteins other than GRB2 and GAB1 may complexes were eluted in SDS sample buer by boiling for 5 min and subjected to SDS-polyacrylamide gel electrophor- be involved in activation of these signaling molecules. esis. Separated proteins were transferred to PVDF membranes and reacted with antibodies. The reaction was examined by ECL (Amersham). Materials and methods Dual luciferase reporter assay Cell line Cells were plated at 16105 in 12-well tissue culture plates and TGW human neuroblastoma and SK-N-MC human primitive incubated for 16 ± 18 h. Transfection was performed using neuroectodermal tumor cell lines were grown in RPMI lipofectamine (Gibco-BRL, Rockville, MD, USA) according medium supplemented with 10% fetal calf serum. to the manufacturer's instructions. In brief, 0.4 ml of opti- MEM (Gibco-BRL) containing 0.35 mg of the pGL3-basic Antibodies vector DNA (Promega, Madison, WI, USA) with herpes simplex virus thymidine kinase (TK) minimal promoter or Anti-RET antibody was developed against the carboxyl- SV40 promoter and tandemly repeated CREB or NF-kB terminal 19 amino acids of the long isoform of RET as binding sequences (Figure 4a) and 0.35 mg of pRL-TK vector described previously (Nozaki et al., 1998). Anti-phosphotyr- as internal standard for transfection eciency and 3.0 mlof osine monoclonal antibody and anti-GAB1 rabbit polyclonal lipofectamine was applied to one well and incubated at 378C antibody were purchased from Upstate Biotechnology Inc. for 5 h. After incubation, 0.4 ml of RPMI 1640 with 20% (Lake Placid, NY, USA). Anti-phosphoERK1/2, anti-phos- FBS was added to each well. The media were replaced with phop38MAPK, anti-phosphoAKT, anti-IkBa and anti-phos- fresh ones with 10% FBS 24 h after application of DNA phoIkBa polyclonal antibodies were from New England solution. After further 24 h culture, transfected cells were Biolabs Inc. (Beverly, MA, USA). Anti-SHC and anti- harvested by lysis. Lysis buer and reagents used in the GRB2 polyclonal antibodies and anti-p85 subunit of PI3- luciferase assay were supplied as dual luciferase assay kit kinase monoclonal antibody were from Transduction La- (Promega). Fire¯y and renilla luciferase activities were boratories (Lexington, KY, USA). Anti-SOS, anti-CREB and measured by Lumicounter 700 (MicroTech Niti-on, Saitama, anti-phosphoCREB antibodies were from Santa Cruz Japan) and transfection eciencies were normalized by the Biotechnology Inc. (Santa Cruz, CA, USA). activity of renilla luciferase derived from the cotransfected pRL-TK plasmid. Western blotting Cells were lysed in sodium dodecyl sulfate (SDS) sample buer (50 mM Tris-HCl (pH 6.8), 5 mM EDTA, 2% SDS, 10% glycerol, 20 mg/ml bromophenol blue (BPB), 10 mM Acknowledgments iodoacetamide) with 80 mM dithiothreitol (DTT). Equal We are grateful to K Imaizumi and M Kozuka for protein amounts of the lysates were subjected to SDS- technical assistance. This work was supported in part by polyacrylamide gel electrophoresis and transferred to poly- grants-in-aid for COE research and scienti®c research from vinylidene di¯uoride (PVDF) membranes (Nihon Millipore the Ministry of Education, Science, Sports and Culture of Kogyo, Tokyo). Membranes were blocked for 30 min at Japan.

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