PC12 cell activation by epidermal growth factor receptor: role of autophosphorylation sites

Int. J. Devl Neuroscience 21 (2003) 63–74

PC12 cell activation by epidermal growth factor receptor: role of autophosphorylation sites Darren R. Tyson, Selena Larkin1 , Yousuke Hamai, Ralph A. Bradshaw∗ Department of Physiology and Biophysics, University of California, Irvine, CA 92697-4560, USA Received 10 September 2002; received in revised form 27 November 2002; accepted 4 December 2002

Abstract PC12 cells have been used as a model system for neuronal differentiation due to their ability to alter their phenotype to a sympathetic neuron-like cell in response to nerve growth factor or fibroblast growth factor. Under some conditions, epidermal growth factor (EGF) can also induce PC12 cells to differentiate. To study signaling from the EGF receptor without the confounding effects of endogenous EGF receptors we generated a chimeric receptor comprised of the ectodomain of platelet-derived growth factor (PDGF) receptor in-frame with the transmembrane and cytoplasmic domains of EGF receptor, termed PER. Expression of PER in PC12 cells confers the ability of PDGF to induce differentiation whereas PDGF has no effect on untransfected PC12 cells. This response is kinase activity-dependent since a kinase-deficient mutant (K721M) fails to induce differentiation in response to PDGF. Mutation of five tyrosine residues that are autophosphorylated in response to EGF either individually or in combination had minimal effects on the ability of these receptors to induce morphological PC12 cell differentiation. The PER mutant with all five autophosphorylation sites mutated to phenylalanine (5YF) was equivalently capable of interacting with several important signaling molecules, including Shc, Grb2, Gab1, phospholipase C␥, and Cbl. Furthermore, both the phosphatidylinositol 3-kinase (PI3K)/Akt and Ras/Erk pathways were activated in a sustained manner when PER or 5YF-expressing cells were stimulated with PDGF. Our results show that the five autophosphorylation sites in the extra-kinase C-terminal domain of EGFR are not required for the ability of EGFR to induce morphological differentiation of PC12 cells. © 2003 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: PC12; Differentiation; Phosphorylation; Phosphotyrosine; Signal transduction

1. Introduction The rat pheochromocytoma PC12 cell line has been used extensively as a model for studying the function of neurotrophic factors and neuronal differentiation (Greene and Tischler, 1976). Interestingly, nerve growth factor (NGF) and fibroblast growth factor (FGF) can each induce these cells to assume a sympathetic neuron-like phenotype while epidermal growth factor (EGF) generally only promotes their proliferation even though many of the same signaling pathways are activated (see Vaudry et al., 2002). The most-accepted theory of the differential responses in PC12 cells involves the duration of Erk1/2 activation. NGF and FGF induce proAbbreviations: EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor; PER, PDGFR/EGFR chimeric receptor; 5YF, PER containing five tyrosine-to-phenylalanine mutations; NGF, nerve growth factor; FGF, fibroblast growth factor; PI3K, phosphatidylinositol-3-kinase; PLC␥, phospholipase C␥ ∗ Corresponding author. Tel.: +1-949-824-6236; fax: +1-949-824-8036. E-mail address: [email protected] (R.A. Bradshaw). 1 Present address: Applied Biosystems, 12309 Beechnut Ct., Woodbridge, VA 22192, USA.

longed activation (>2 h) and nuclear translocation of Erk1/2 whereas EGF induces only transient activity (<90 min) and minimal nuclear presence (Traverse et al., 1994). This theory is supported by observations that EGF can induce sustained activation of Erk and PC12 cell differentiation when exogenous receptors are overexpressed (Marshall, 1995) or when endogenous receptor expression is upregulated (Raffioni and Bradshaw, 1995). However, there is significant evidence that signaling events other than the Ras/Erk pathway are important for PC12 cell differentiation (Altun-Gultekin et al., 1998; Anneren et al., 2000; Boglari and Szeberenyi, 2001; Burry, 2001; Vaillancourt et al., 1995; Wu and Bradshaw, 1996). Two other pathways with potential roles in PC12 cell differentiation are the phosphatidylinositol 3-kinase (PI3K)/Akt and phospholipase C␥ (PLC␥)/protein kinase C (PKC) pathways. The PI3K/Akt pathway is required for PC12 cell survival (Klesse et al., 1999; Yao and Cooper, 1995); however, it may also affect cell morphology. Inhibition of PI3K in PC12 cells prevents NGF-induced neurite elongation (Jackson et al., 1996; Kimura et al., 1994; Posern et al., 2000; Yasui et al., 2001), and inhibits Shc (Sato et al.,

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1998) and Rap1 activation (York et al., 2000). Furthermore, overexpression of a constitutively active form of PI3K can induce process formation in PC12 cells suggesting that this pathway contributes to the morphological changes of PC12 cells (Kita et al., 1998; Kobayashi et al., 1997). Both PLC␥ and PKC also appear to have roles in PC12 cell differentiation. Mutant TrkA that is deficient in both Shc and PLC␥ binding cannot cause PC12 cell differentiation whereas mutants that interact with either Shc or PLC␥ are fully capable of inducing morphological PC12 cell differentiation suggesting these pathways are at least partially redundant (Obermeier et al., 1994; Stephens et al., 1994). Diacylglycerol, a product of PLC activity, is the physiological activator of PKC, and activation of the specific PKC isoforms have been shown to be important for PC12 cell differentiation induced by NGF. Furthermore, EGF-induced Erk activity appears to be dependent on PKC (Corbit et al., 2000). The extra-kinase C-terminal domain of EGFR contains five primary sites of tyrosine autophosphorylation sites (Fig. 1). These residues are phosphorylated to variable degrees following EGF binding and have been shown to act as binding sites for a number of signaling proteins, including Shc, Grb2, and PLC␥, among others (reviewed in Schlessinger, 2000). However, these tyrosine residues do not clearly define adaptor/effector protein binding sites of EGFR (Batzer et al., 1994; Soler et al., 1994b). When some or all of these autophosphorylation sites are mutated or deleted EGFR remains capable of activating the Ras/Erk pathway (Li et al., 1994; Soler et al., 1994a) and inducing mitogenesis in NIH 3T3 cells (Clark et al., 1988; Decker, 1993; Gotoh et al., 1994; Li et al., 1994; Soler et al., 1994a). NIH 3T3 cells were used for these studies because they were considered to be devoid of endogenous ErbB family members. It has since been shown that NIH 3T3 do express EGFR (Soler et al., 1994a) and ErbB2 (Olayioye et al., 1998; Vijapurkar et al., 1998), albeit at very low levels. Soler et al. (Soler et al., 1994a) determined that in parental NIH 3T3 cells (containing <1 × 104 receptors per cell) EGF induces the phosphorylation of Shc. This is significant since mutant EGFR expressed in these cells would likely dimerize with endogenous receptors making it impossible to determine the source of the signals leading to Shc activation

Fig. 1. Construction of chimeric receptor. A schematic diagram of the PER chimera is shown. The ectodomain consists of residues 1–531 of hPDGFR␤ fused in-frame with residues 620–1186 of hEGFR. SR and LVPR indicate the residues that were replaced to generate the XbaI and KpnI sites, respectively, as described in Section 2. The ATP binding motif within the kinase domain (KD) is indicated, as are the five sites of autophosphorylation within the C-terminus (C-term) of the receptor. Also indicated are the transmembrane (TM) and juxtamembrane (JM) domains.

(mutant versus endogenous receptors). Furthermore, EGFR can form heterodimers with and activate other EGFR family members, specifically ErbB2 (a.k.a. HER2 or p185Neu) and ErbB3 (HER3) providing another explanation as to how mutant EGFR retains its signaling capacity. PC12 cells express a relatively high number of EGFR (∼1.5 to 7 × 104 per cell) (Boonstra et al., 1985; Traverse et al., 1994) as well as a small number of ErbB2 (Neu) and ErbB3 (Soltoff and Cantley, 1996; Traverse et al., 1994); these endogenous receptors may play a significant role in PC12 cell differentiation when EGFR is overexpressed/upregulated. Expression of mutant EGFR in PC12 cells would lead to the heterodimerization (and activation) of endogenous receptors. Therefore, to study EGFR mutants in PC12 cells without the risk of heterodimerization with endogenous receptors, a chimeric receptor approach was devised. Since PC12 cells do not express PDGFR, fusing the human PDGFR␤ ectodomain in-frame with the transmembrane and cytoplasmic domains of the human EGFR (generating a chimeric receptor, PER) allows activation of mutant receptors without heterodimerization with endogenous EGFR. This approach has been used previously in this laboratory to study TrkA, p75-neurotrophin receptor, DDR1, and FGFR1, 3 and 4 in PC12 cells (Foehr et al., 2000a,b, 2001; Raffioni et al., 1999). In this report, we show that expression of PER in PC12 cells confers the ability of PDGF to induce differentiation. Furthermore, PER in which the autophosphorylation sites have been mutated to phenylalanine individually or in combination are fully capable of activating both the Ras/Erk and PI3K/Akt pathways and retain the ability to induce PC12 cell differentiation.

2. Materials and methods 2.1. Cell culture and neurite outgrowth assay PC12 cells expressing chimeric receptors were generally cultured in 75 cm2 tissue culture flasks (Corning) with PD medium (high-glucose DMEM (Invitrogen), containing 8% plasma-derived horse serum (Sigma), 4% plasma-derived newborn calf serum (Cocalico Biologicals), and 1% penicillin/streptomycin (pen/strep) solution (Invitrogen)) at 37 ◦ C in a humidified atmosphere of 5% CO2 . The use of plasma-derived (platelet poor) serum drastically reduces the amount of background activation of the chimeric receptors. GP + E86 and 293 cells were maintained in DMEM containing 10% fetal bovine serum and 1% pen/strep solution. For neurite outgrowth assays, PC12 cells were plated onto six-well plates (Corning) in PD medium at a density of 105 cells per well. After 16–24 h, the medium was replaced with DMEM containing 0.5% plasma-derived horse serum (without pen/strep) and various concentrations of PDGF-BB (Austral Biologicals). Cells were examined for the presence of neurites at various times. Responsive cells were defined as those bearing neurites at least two cell diameters in length.

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2.2. Construction of chimeric (PDGFRβ/EGFR) receptor and YF mutants A chimeric receptor of human PDGFR␤ and human EGFR was generated by altering a previously constructed chimeric receptor that consisted of the ectodomain of hPDGFR␤ and the transmembrane domain and endodomain of rat FGFR3 (PFR3) (Raffioni et al., 1998; Thompson et al., 1997). The junction of the two receptors was modified to facilitate the swapping of the FGFR3 domain with that of EGFR by adding a unique XbaI site. This was done by mutation of hPDGFR␤ residues 630 and 631 to Ser-Arg (SR) (Fig. 1). Site-directed mutagenesis was performed using the Quick-ChangeTM Mutagenesis kit (Stratagene) according to the manufacturer’s protocol. The human EGFR cDNA encoding residues 622–1186 (NM 005228, Ullrich et al., 1984) was amplified by PCR to contain an XbaI site at its 5 end and an XhoI site at its 3 end. Two internal EcoRI sites were eliminated and a HindIII site was added by silent mutagenesis to facilitate subcloning. Additionally, four residues at the junction of the juxtamembrane domain and kinase domain were mutated to generate a KpnI site (residues 682–685 to LVPR). The entire sequence was verified, and the cDNA of the EGFR transmembrane domain and endodomain was subcloned in-frame with the cDNA of hPDGFR␤ to create the PER construct. A schematic diagram of the construct is shown in Fig. 1. To eliminate the sites of tyrosine autophosphorylation, the cDNA was mutated to encode phenylalanine in their stead (YF mutations) by site-directed mutagenesis. The specific residues altered were Y992F, Y1068F, Y1086F, Y1148F and Y1173F. All mutations were verified by cycle sequencing to ensure no other mutations were generated. 2.3. Generation of stable cell lines To generate PC12 cells that express PER and mutants, the cDNA for each construct was first subcloned into pLXSN (Clontech), a vector for generating non-replicating retroviral particles. The pLXSN vectors were transfected into the retroviral packaging cell line GP + E86, a cell line derived from NIH 3T3 cells, on 100 mm dishes using LipofectAMINETM (Invitrogen) according to the manufacturer’s recommended protocol. The medium on the cells was replaced with low-serum PD (LSPD) medium (same as PD except 5% PD-HS and 2.5% PD-FCS) and incubated at 37 ◦ C for 24 h. The medium (containing retroviral particles) was then removed and spun at 3000 × g for 10 min and filtered through a 0.45 mm syringe filter. Three ml of cleared medium containing retroviral particles was added to approximately 7 × 105 PC12 cells on 100 mm dishes containing 2 ml of LSPD medium and 5 ␮l of 8 mg/ml polybrene (hexadimethrine bromide diluted in PBS and sterile filtered; Sigma). The cells were incubated at 37 ◦ C, 5% CO2 for 1 h, 5 ml of PD medium containing 5 ␮l of polybrene was added, and the cells were replaced into

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incubator for 16–20 h. The medium was replaced with 9 ml PD medium, and, after a 24 h incubation at 37 ◦ C, 5% CO2 , the medium was replaced with PD containing 700 ␮g/ml G418 (Invitrogen) to select for stable integration of the recombinant DNA. Individual clones were either picked as colonies or isolated by serial dilution. 2.4. Antibodies and inhibitors Mouse monoclonal anti-hPDGFR␤ for immunoprecipitations was from R&D Systems. Mouse monoclonal anti-hPDGFR␤ for immunoblotting was from Oncogene Research Products. Rabbit polyclonal antibodies to PLC␥1, Shc and Grb2 and goat polyclonal antibodies to Gab1 were from Santa Cruz Biotechnology. Rabbit polyclonal anti-Shc, mouse monoclonal antiphosphotyrosine (4G10), sheep polyclonal anti-EGFR, and mouse monoclonal anti-Gab1 were from Upstate Biotechnology Inc. Mouse monoclonal anti-Grb2 was from Transduction Laboratories. Phospho-specific anti-pErk1/2 (T202/Y204), anti-pAkt (S478), anti-pEGFR (Y1068), and anti-pEGFR (Y1045) were from Cell Signaling Technology. Horseradish peroxidase-conjugated secondary antibodies were from Jackson Immunochemicals. The EGFR-specific kinase inhibitor compound 56 and ErbB2-specific AG879 were from Calbiochem. 2.5. Immunoprecipitation and immunoblot analysis Clonal PC12 cell lines expressing chimeras or vector control cells were grown in plasma-derived complete medium on either 100 or 150 mm2 culture dishes (Corning) until approximately 70% confluence was reached. The cells were starved for at least 24 h in DMEM containing 0.2% plasma-derived horse serum and then treated with PDGF-BB (20 ng/ml) for various lengths of time at 37 ◦ C as specified. The cells were washed briefly with cold phosphate-buffered saline containing 0.5 mM sodium orthovanadate and lysed in ice-cold lysis buffer (10 mM Tris–HCl, pH 7.5; 50 mM NaCl; 1% Triton X-100; 5 mM EDTA; 0.2 mM sodium orthovanadate; 10 mM sodium pyrophosphate, 50 mM NaF, and protease inhibitors (Sigma)). Insoluble material was separated by microcentrifugation at 14000 × g for 10 min at 4 ◦ C. Protein concentration was determined with the Bradford colorimetric assay (Bio-Rad). For immunoprecipitations, lysates (500 ␮g) were incubated with appropriate antibodies for 2 h at 4 ◦ C followed by a 1 h incubation with protein A/G-Plus agarose (Santa Cruz Biotechnology). The immunocomplexes were washed twice with lysis buffer and then heated to 70 ◦ C for 10 min in 2 × LDS sample buffer (Invitrogen) containing 2.5% (v/v) ␤-mercaptoethanol. Proteins were separated by PAGE using 7% Tris–acetate NuPAGE gels (Invitrogen) and transferred to Immobilon P membrane (Millipore) by electroblotting at 100 V for 1 h using a Mini-Transblot apparatus (BioRad). The membranes were blocked with 5% BSA in Tris-buffered

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saline containing 0.1% (v/v) Tween-20 (TBST) for >1 h and incubated with the antibodies indicated. Localization of primary antibodies on the membrane was determined by using appropriate horseradish peroxidase-conjugated secondary antibodies and the ECL detection system (Amersham/ Pharmacia Biotech).

3. Results 3.1. Chimeric receptor expression and ligand-dependent neurite extension PC12 cells were infected with various replication-incompetent retroviruses engineered to express PER and various mutants (Table 1) and G418-resistant clones were selected for each receptor type. Receptor expression was determined by immunoprecipitation with PDGFR␤ ectodomain antibodies followed by immunoblot analysis using PDGFR␤ ectodomain (Fig. 2). Specific receptor levels were also kindly determined for PER and 5YF clones by Carol Sullivan and Neill Giese at COR Therapeutics using a sandwich Table 1 Chimeric receptor construct pLXSN (vector) PER PER K721M PER Y992F PER Y1068F PER Y1086F PER Y1148F PER Y1173F PER Y1068/1086F PER Y992/1086/1173F PER Y992/1068/1148/1173F PER Y992/1068/1086/1173F PER Y992/1068/1086/1148/1173F (5YF) Each construct was transfected into PC12 cells and selected for stable integration. Several clones of PC12 cells expressing each construct were isolated.

Fig. 2. Detection of PER and mutants expressed in PC12 cells. Lysates of stable transfectants of PC12 cells expressing PER or various mutants (500 ␮g each) were subjected to immunoprecipitation using antibodies that recognize the ectodomain of hPDGFR␤. Precipitated proteins were analyzed by immunoblotting using a different PDGFR␤ antibody. All clones were screened similarly, and all demonstrated similar levels of expression.

ELISA (Giese et al., 1999). Individual clones ranged in expression levels from ∼3.4 × 105 receptors per cell for one clone of PER-expressing cells to ∼1 × 106 receptors per cell for a clone expressing PER-5YF. These values correspond with the immunoprecipitation/immunoblotting data shown in Fig. 2. PC12 cells expressing PER extensively induced neurite extensions after 48 h treatment with 20 ng/ml PDGF, with similar morphology to cells treated with NGF (not shown). PER containing single or multiple tyrosine-to-phenylalanine mutations at autophosphorylation sites retained the ability to extensively induce neurite formation after 48 h exposure to PDGF; however, there were demonstrable differences at 24 h (Fig. 3). Generally, the receptors containing fewer mutations seemed to differentiate more rapidly. When Y1148 and Y1173 were singly mutated to phenylalanine, the PDGF-induced responses were nearly as low at 24 h (∼30% cells bearing neurites) as the mutants containing 3, 4 or 5 substitutions. This is in agreement with the fact that Y1173 and Y1148 are the primary sites of phosphorylation of EGFR and indicate that these sites are important for the initial PER response. Since the 5YF mutant retained the ability to induce PC12 cell differentiation, this moiety was studied further and compared to the unmodified construct. 3.2. Receptor activation The level of activation of the receptors before and after addition of ligand was examined. Whole cell lysates were analyzed for the presence of phosphotyrosine-containing bands (Fig. 4). Parental PC12 cells show no increase in phosphotyrosine when treated with PDGF, whereas, in both PER- and 5YF-expressing cells, PDGF caused an increase in phosphotyrosine incorporation into a number of proteins, most prominently around 170 and 120 kDa. The band of approximately 170 kDa contains the chimeric receptor. The drastic increase in levels of phosphotyrosine present in PER in response to PDGF demonstrates the ligand-dependent activation of this receptor. The phosphorylation detected in unstimulated cells is likely due to the overexpression of the chimera that causes some ligand-independent activation, a commonly observed phenomenon. Interestingly, the 5YF mutant has a relatively high level of tyrosine phosphorylation in the absence of ligand, and the level of phosphorylation still increases upon PDGF treatment. This is intriguing since all five of the primary sites of tyrosine autophosphorylation have been mutated in this construct. There appears to be a substantial amount of background phosphotyrosine formation in the both the PER and 5YF receptors; however, this does not lead to ligand-independent PC12 cell differentiation as the extension of neurites is clearly dependent on PDGF (Fig. 3). While the overall level of tyrosine phosphorylation is reduced in the 5YF-expressing cells compared to PERexpressing cells, nearly all of the phosphotyrosine-containing bands are detectable in both cell types. A wide band

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Fig. 3. Induction of neurite outgrowth of PC12 cells expressing PER and mutants. Stable clones of PER, mutant PER or untransfected PC12 cells were subjected to a neurite extension assay as described in Section 2. Shown here is the percent of differentiated cells (defined as cells possessing at least two neurites each of at least two cell body widths in length) at several time points after treatment with 20 ng/ml PDGF-BB. By 48 h, all of the cells had reached their maximal percent differentiation, and all cells expressing a PER construct had >75% of the cells differentiated by this time.

Fig. 4. Ligand-induced tyrosine phosphorylation PER- and 5YF-expressing PC12 cells. Stable clones of PC12 cells transfected with vector (pLXSN), PER or 5YF constructs were either left untreated (−) or treated (+) with 20 ng/ml PDGF-BB for 5 min. Whole cell extracts were separated by SDS–PAGE and subjected to immunoblotting using a phosphotyrosine-specific antibody, clone 4G10 (A). Blot was stripped and reprobed with antibodies to Akt (B). Molecular weight standards are indicated.

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of phosphotyrosine-containing proteins is clearly detectable within the range of 105–120 kDa in both PER and 5YF-expressing cells (Fig. 4, short). Several signaling proteins migrate within this region, including Cbl, Gab1, PLC␥, and RasGAP. Furthermore, after a longer exposure, phosphotyrosine-containing bands can be seen at approximately 42, 44, 46, and 52 kDa (Fig. 4, long). These bands comigrate with Erk2, Erk1, p46 Shc and p52 Shc, respectively. Although the specific residues phosphorylated in the 5YF construct have not been determined, alternative sites of tyrosine phosphorylation have previously been described for EGFR mutants (Li et al., 1994). One such site is Y1045, a residue that can act as a binding site for Cbl when it is phosphorylated (Levkowitz et al., 1999). To determine whether endogenous EGFR is transactivated by PER or 5YF in response to PDGF we examined the phosphorylation state of specific tyrosines within endogenous EGFR and exogenously expressed PER or 5YF in response to EGF or PDGF (Fig. 5). As expected, Y1068 is phosphorylated in endogenous EGFR only in response to EGF (Fig. 5, pY1068). PER is phosphorylated at Y1068 in the absence of ligand but is greatly enhanced in response to PDGF (Fig. 5, pY1068). The ligand-independent phosphorylation of Y1068 of PER corresponds to the higher level of phosphotyrosine-containing proteins in the absence of ligand (Fig. 4) and is due to the overexpression of the receptors. Endogenous EGFR is phosphorylated in PER-expressing PC12 cells in response to EGF but not PDGF, indicating that endogenous EGFR is not transactivated by PER (Fig. 5).

Fig. 5. Phosphorylation of specific tyrosines within EGFR, PER and 5YF. Parental PC12 cells or PC12 cells stably expressing PER or 5YF were left untreated (−), treated with 50 ng/ml EGF for 5 min (E) or treated with 20 ng/ml PDGF-BB for 5 min (P). Whole-cell extracts of these cells were then analyzed by immunoblotting with the indicated antibodies. The same blot was used for all antibody incubations performed in descending order. To detect the endogenous EGFR with the anti-EGFR antibody a longer exposure was required (EGFR longer). The blot was stripped as described in Section 2 before being analyzed by the next antibody. The migration of endogenous EGFR and PER or 5YF (both indicated as PER) are shown on the left.

The absence of Y1068 in 5YF (due to its mutation to Phe) prevents the phosphorylation of this residue, even when stimulated by PDGF. The lack of any reactive bands in the 5YF lanes when immunoblotted with the pY1068 antibodies clearly demonstrates that transactivation of endogenous EGFR by 5YF does not occur. Furthermore, phosphotyrosine immunoprecipitates from PER or 5YF-expressing PC12 cells (−/+ PDGF) did not contain any endogenous EGFR (not shown). Taken together, these data provide strong evidence that transactivation (or direct activation through heterodimerization) of endogenous EGFR by PER or 5YF does not occur. The tyrosine at position 1045 has not been mutated in these studies. This residue appears to be primarily involved in receptor degradation through the phosphorylationdependent interaction with Cbl (Ettenberg et al., 1999; Ettenberg et al., 2001; Levkowitz et al., 1999). As has been shown previously with endogenous EGFR in other cell types, Y1045 is phosphorylated in response to EGF in parental PC12 cells (Fig. 5, pY1045). Furthermore, PDGF treatment of PER or 5YF-expressing PC12 cells also leads to an increased phosphorylation of Y1045 (Fig. 5, pY1045). This suggests that the phosphorylation of this residue may be important for PDGF-induced differentiation of PC12 cells expressing PER-5YF. Interestingly, ErbB2 phosphorylation at tyrosine 1248 was detected in PER and 5YF-expressing cells treated with PDGF while no phosphorylation of this residue was detected in PDGF-treated parental PC12 cells (not shown). This phosphorylation event was inhibited by the addition of a specific inhibitor of EGFR (compound 56) but not an ErbB2-specific inhibitor (AG879) suggesting that ErbB2 is phosphorylated either directly by PER or 5YF or indirectly by another tyrosine kinase activated by PER or 5YF (but not ErbB2). The significance of this event is currently under investigation. Importantly, the kinase activity of ErbB2 does not appear to be required for PC12 cell differentiation since the addition of AG879 does not inhibit the differentiation of PER or 5YF-expressing PC12 cells in response to PDGF (not shown). Moreover, activation of endogenous EGFR and ErbB2 by the addition of EGF to PER or 5YF-expressing PC12 cells fails to cause differentiation, even though each receptor type is highly phosphorylated/activated (not shown). Therefore, the (trans)activation of endogenous ErbB family members is not important for the abilities of PER and 5YF to induce PC12 cell differentiation. 3.3. Coprecipitation of adaptor/effector proteins with chimeric receptors PER and 5YF each confer the ability of PDGF to induce differentiation of PC12 cells; however, it is not clear which signaling pathways are important for this. The major pathways that have been implicated in this response are those involving PI3K/Akt, Ras/Erk and PLC␥/PKC. It has been

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degradation as effectively as the PER moiety. It is also interesting to note that, upon ligand treatment, the 5YF construct fails to increase the level of phosphotyrosine in both PLC␥ and Cbl. In fact, tyrosine phosphorylation of these proteins appears to decrease in response to PDGF (Fig. 6). 3.4. Activation of adaptor/effector proteins

Fig. 6. Association of adaptor/effector proteins with activated receptors. Human embryonic kidney 293 cells were transfected with vector (pLXSN) or PER or 5YF constructs as described in Section 2. The cells were either left untreated (−) or treated (+) with 20 ng/ml PDGF-BB for 5 min. Lysates (1 mg each) were immunoprecipitated with PDGFR-specific antibodies and analyzed by immunoblotting using antibodies specific for Shc, Gab1 and PLC␥.

shown that EGFR can activate these pathways by direct association with effector proteins (e.g. PLC␥) or by indirect association of effectors using various adaptor proteins (e.g. Shc) (reviewed in Schlessinger, 2000). To determine which proteins can interact with the chimeric receptors, lysates from 293 cells expressing PER or 5YF (or vector controls) were treated with or without PDGF then subjected to immunoprecipitation by PDGFR antibodies and analyzed for the coprecipitation of various adaptor and effector proteins. As expected, Shc, Grb2, Cbl and PLC␥ were all detected in PDGFR immunoprecipitates from PER-expressing cells (Fig. 6). The ligand-independent association of these proteins was likely due to the overexpression of the receptors in 293 cells, known to cause oligomerization and activation of the receptors in the absence of ligand. While the interaction with PER did not depend on ligand, PDGF treatment still increased the level of phosphorylation of Shc, PLC␥, Cbl and Grb2 coprecipitated from PER-expressing cells, although Grb2 was least affected (Fig. 6). Previous studies failed to detect an interaction of Shc with a 5YF mutant of EGFR (Li et al., 1994; Soler et al., 1994b). In contrast, as shown in Fig. 6, a weak but detectable interaction can be seen between Shc and the 5YF mutant of PER indicating that Shc can interact with PER at sites other than the primary sites of autophosphorylation. Likewise, a weak but detectable interaction of 5YF with Grb2 was detected, and, surprisingly, PLC␥ appears to interact with 5YF nearly as well as unmodified PER. The association of Grb2 and PLC␥ with a 5YF mutant of the EGFR cytoplasmic domain has not previously been reported. Cbl has been shown to associate with EGFR at Y1045 (Levkowitz et al., 1999), a residue that has not been mutated in these studies. Interestingly, the 5YF construct has a reduced association with Cbl even though the putative binding site for this protein remains intact in the 5YF receptor. Since Cbl acts as a mediator of receptor degradation as an E3 ubiquitin ligase, the reduced binding of Cbl to 5YF may indicate that these receptors are not targeted for internalization and

The association of adaptor and effector proteins with EGFR in a ligand-dependent manner does not guarantee that the effectors are activated themselves. In the cases of Shc, Gab1 and PLC␥, incorporation of phosphotyrosine into the proteins is required for their activity. Shc exists as three distinct proteins of 46, 52 and 66 kDa, referred to as p46, p52 and p66 Shc. The 66 kDa moiety contains the entire 52 kDa protein and likewise the 52 kDa form contains the entire 46 kDa form (reviewed in Ravichandran, 2001). Therefore, phosphorylation occurring on p46 Shc would likely occur on the 52 and 66 kDa forms provided that the proteins are colocalized. Immunoprecipitation of Shc from PC12 cells expressing PER, followed by detection of phosphotyrosine, shows a significant amount of tyrosine phosphorylation in the absence of PDGF, but there is still a significant ligand-dependent response (Fig. 7A). A band of ∼170 kDa, which comigrates with the chimeric receptor, is more highly phosphorylated in response to PDGF treatment. The 5YF receptor shows a similar increase in phosphorylation of the 46, 52 and 66 kDa bands upon PDGF treatment. Curiously, the p66 form of Shc appears to be more highly expressed in the 5YF-expressing cells. As would be expected of receptors lacking five potential sites of phosphorylation, the band of ∼170 kDa is phosphorylated to a much lesser degree than for PER. EGFR has been shown previously to associate with and induce tyrosine phosphorylation of PLC␥. As shown in Fig. 7B, PER and all of the mutants are capable of causing the phosphorylation of PLC␥ in response to PDGF while PLC␥ in vector-transfected PC12 cells is unaffected by this ligand. The mutant forms of PER were also capable of inducing tyrosine phosphorylation of PLC␥ even though the primary sites of interaction are missing. The activation of PLC␥ without its association with EGFR has previously been shown by others to occur in the absence of binding sites for PLC␥ (Margolis et al., 1990), but the phosphorylation of PLC␥ alone was not sufficient to cause calcium flux and phosphatidylinositol hydrolysis (Li et al., 1991). Our results are at variance, however, with those of Sorkin et al. (Sorkin et al., 1991) who provided evidence that mutation of the Y1086, Y1148 and Y1173 to phenylalanine caused a significant reduction of PLC␥ phosphorylation. 3.5. Activation of downstream kinases While the mechanisms of activation of the individual pathways by the mutant receptors are important, the activation state of the downstream kinases remains the most relevant

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Fig. 8. Activation of downstream kinases. Whole cell extracts (40 ␮g each) from pLXSN-, PER-, and 5YF-PC12 cells (untreated or treated with PDGF for 5 min) were immunoblotted using a phosphoS473 Akt antibody (pAkt) or an antibody that recognizes dually phosphorylated (T202/Y204) Erk1 and 2 (pErk1/2). The amount of Akt and Erk1/2 in each lane was determined by stripping the blots and detecting the proteins using antibodies that recognize Akt or Erk1/2 regardless of their state of phosphorylation.

Fig. 7. Activation of adaptor/effector proteins upon ligand treatment. Lysates (500 ␮g each) from untreated and PDGF-treated clones pLXSN-, PER-, or 5YF-PC12 cells were subjected to immunoprecipitation using antibodies specific for different adaptor/effector proteins. (A) Shc immunoprecipitates were analyzed for the presence of phosphotyrosine using 4G10. The presence of Shc in all precipitates was verified using Shc-specific antibodies. The 46, 52 and 66 kDa isoforms of Shc are indicated. The uppermost band in the lanes containing PER and 5YF lysates comigrates with the receptors. (B) Immunoprecipitates generated using PLC␥ antibodies (rabbit polyclonal, Santa Cruz Biotechnology) were immunoblotted with a phosphotyrosine-specific antibody (PY99), then the blots were stripped and the amount of PLC␥ in each lane was detected using a mouse monoclonal antibody (Upstate Biotechnology). (C) A goat polyclonal Gab1 antibody (Santa Cruz Biotechnology) was used for immunoprecipitations and the presence of phosphotyrosine in Gab1 was detected using PY99. The amount of Gab1 in the precipitates was determined using a mouse monoclonal Gab1 antibody (Upstate Biotechnology).

indicator of signal flux through these pathways. As expected, PC12 cells expressing PER are able to activate Erk1/2 and Akt when induced with PDGF whereas mock-transfected cells are unaffected (Fig. 8). The equivalent phosphorylation of Erks and Akt is also detected in 5YF-expressing PC12 cells in response to PDGF indicating that both of these pathways are also strongly activated by the mutant receptor. Even though the activation of Erks and Akt appears similar in PER and 5YF-expressing cells treated with PDGF for 5 min, the kinetics of their activation may be significantly different. In order to examine this, the phosphorylation states of Erk1/2 and Akt were determined in cells treated with PDGF for various times. Since prolonged activation of Erk seems to correlate with the ability of growth factor receptors to induce PC12 cell differentiation, it is not surprising that

Fig. 9. Time course of downstream kinase activation. Lysates were prepared of PER- and 5YF-PC12 cells that had been treated with PDGF for various 0, 5, 30, 60, 120 or 180 min and analyzed as for Fig. 8.

the activation of Erk1 and 2 is both rapid and prolonged in cells expressing PER (Fig. 9). The duration of phosphorylation of Erk1 and 2 in PER-expressing cells is similar to that seen in PC12 cells overexpressing EGFR (Traverse et al., 1994). Erk activation induced by 5YF has a very similar pattern of phosphorylation, suggesting that the mechanisms by which PER induces Erk phosphorylation are retained in 5YF. In PC12 cells, NGF and EGF each induce a rapid and transient activation of PI3K (Raffioni and Bradshaw, 1992; Soltoff et al., 1992). In PER and 5YF-expressing PC12 cells the activation of Akt is also rapid, maximal at 5 min or less, but the phosphorylation of Akt is somewhat sustained as there is detectable phosphorylation even after 180 min (Fig. 8). Although the duration of activation of Akt appears longer in 5YF-expressing cells versus PER, phosphorylated Akt is detectably above basal levels in each cell type even after 180 min.

4. Discussion While EGF is predominantly considered a mitogen, numerous studies support a neurotrophic function for EGF in

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the central nervous system (CNS) (reviewed in Yamada et al., 1997). The expression of EGFR has been detected in cerebellar Purkinje cells, CA fields of the dentate gyrus, and a subpopulation of GABAergic neurons in the forebrain ventricular/subventricular zone and cerebellar external granule layer. EGF can act as a neurotrophic factor towards postmitotic neurons of the CNS (Morrison, 1993; Plata-Salamán, 1991). Furthermore, EGF can induce neuronal processes and enhance survival of cultured rat cerebellar cortical neurons (Kornblum et al., 1990; Morrison et al., 1988). PC12 cells have been used extensively to study the neurotrophic actions of NGF and FGF. However, there are certain conditions under which EGF can cause PC12 cells to differentiate, such as overexpression of exogenous EGFR or stimulation of endogenous EGFR synthesis by the use of chemical compounds. These studies demonstrate an inherent ability of EGFR to induce PC12 cell differentiation, albeit possibly by different mechanisms than those used by TrkA and FGFRs. One major difference is the ability of TrkA and FGFRs to bind and activate Frs2 whereas EGFR cannot. Frs2 has been shown to be important for the sustained activation of Erks in response to NGF and FGF (Ong et al., 2000). Since EGFR does not bind or activate Frs2, the sustained activation of Erks must be through an alternative mechanism. It is generally assumed that the overexpression of EGFR in PC12 cells merely overwhelms the ability of the cell to internalize and degrade all of the activated receptor. This, in turn, allows for the persistence of signals emanating from the activated EGFR remaining at the cell surface, leading to the sustained activation of Erk1/2 and ultimately their differentiation. Since PC12 cells express EGFR, ErbB2 and ErbB3 it is possible that each of these receptors play a role in EGF-induced PC12 cell differentiation. Thus, the use of a chimeric receptor that does not heterodimerize with endogenous ErbB family members is of particular importance. EGFR mutants have been extensively studied in NIH 3T3 cells. These cells were chosen because they were considered to be devoid of ErbB family members. Several of these reports demonstrated that EGF retains biological activity on cells overexpressing EGFR even when some or all of its autophosphorylation sites have been mutated (Decker, 1993; Gotoh et al., 1994; Helin and Beguinot, 1991; Li et al., 1991, 1994; Soler et al., 1994b). However, it has been shown that NIH 3T3 cells do express EGFR (Soler et al., 1994a) and ErbB2 (Olayioye et al., 1998; Vijapurkar et al., 1998). Since EGFR and ErbB2 can form functional heterodimers, it is possible that mutant EGFR heterodimerized with and activated the endogenous EGFR or ErbB2 in response to EGF. While it has not been shown that ErbB2 can be activated in mutant EGFR-overexpressing NIH 3T3 cells, it also has not been specifically ruled out as mechanism by which the mutant receptors signal. Furthermore, Soler et al. (Soler et al., 1994a) demonstrate that in parental NIH 3T3 cells endogenous EGFR levels are sufficient for the EGF-dependent phosphorylation of Shc. Therefore,

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avoiding the direct activation of endogenous receptors is particularly important when studying signaling from mutant receptors. The use of the PDGFR ectodomain should eliminate any heterodimerization of the chimeras with endogenous EGFR. We failed to detect any endogenous EGFR or ErbB2 coprecipitating with PER (or 5YF) before or after the addition of ligand indicating the inability of these receptors to heterodimerize. Furthermore, no direct interaction between PDGFR and ErbB family members has yet been reported. However, the transactivation of EGFR is a well-documented phenomenon. We have ruled out the possibility of endogenous EGFR becoming activated in response to PDGF treatment in cells expressing PER or 5YF either directly or indirectly, and therefore have excluded its role in the differentiation response. Interestingly, however, in both PER and 5YF-expressing cells, PDGF induces the phosphorylation of ErbB2 at tyrosine 1248. Since heterodimerization does not appear to occur between PER and ErbB2, the phosphorylation of Y1248 on ErbB2 is likely due to the activation of cytosolic tyrosine kinases. Importantly, inhibition of ErbB2 activity has no effect on the phosphorylation of Y1248 or of differentiation of PDGF-stimulated PER or 5YF-expressing PC12 cells. Moreover, EGF treatment of PER or 5YF-expressing cells activates endogenous EGFR and ErbB2 but does not induce neurite extension. Therefore, endogenous ErbB family members do not play a significant role in PDGF-mediated neurite extension in PER or 5YF-expressing PC12 cells. Our studies using the chimeric PER constructs appear to confirm a majority of the reports describing the ability of mutant EGFR to retain significant biological activity. First, others have reported that, when the five C-terminal autophosphorylation sites were mutated to phenylalanine, EGFR retained the ability to activate Shc in NIH 3T3 cells even in the absence of stable interaction between the two (Gotoh et al., 1994; Li et al., 1994; Soler et al., 1994a). The phosphorylation of Shc presumably leads to the activation of the Ras/Erk pathway since NIH 3T3 cells expressing 5YF-EGFR retain their mitogenic response to EGF. The detection of phosphorylated Shc as a coprecipitated protein with the 5YF receptors suggests that Shc interacts more stably with the mutant receptor than previously suspected. It is possible that Shc interacts with Y1045 (which has not been mutated in these studies) or with different tyrosines that only become phosphorylated if the primary sites of autophosphorylation have been removed, as has been suggested previously (Li et al., 1994). This could explain why the 5YF mutant still becomes tyrosine phosphorylated upon addition of ligand (see Fig. 2). There are fourteen additional tyrosines remaining in the EGFR cytoplasmic domain that may be phosphorylated. We are currently trying to determine the specific sites of phosphorylation that remain within the 5YF receptor and investigating the possible interaction of Shc with these sites within the context of an intact receptor.

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Gab1 has been shown to be important for RTK activation of both the PI3K/Akt and Ras/Erk pathways (reviewed in Liu and Rohrschneider, 2002), and Gab1 is a primary effector of TrkA for PC12 cell survival and differentiation (Holgado-Madruga et al., 1997; Korhonen et al., 1999). Since both PER and 5YF can lead to the phosphorylation of Gab1, the signaling pathways leading to its phosphorylation appear to be conserved even when the primary sites of interaction on EGFR have been mutated. This suggests that a strong association of EGFR with Gab1 is not required for its activation. Regardless of the mechanism of activation, the phosphorylation of Gab1 in response to PDGF in 5YF-expressing PC12 cells provides another potential mechanism by which the Erks become activated and possibly leads to the activation of the PI3K/Akt pathway. Several previous reports each indicated that the EGFinduced phosphorylation of PLC␥ in cells expressing C-terminal truncations or autophosphorylation site mutants of EGFR is severely inhibited (Decker, 1993; Gotoh et al., 1994; Li et al., 1994; Soler et al., 1994a). This is in clear contrast to our results that demonstrate no inhibition of the phosphorylation of PLC␥ with a 5YF construct. Even though the phosphorylation of PLC␥ appears unaffected by the mutation of the five autophosphorylation sites, the activation of this enzyme is likely significantly impaired. Li et al. demonstrated that in NIH 3T3 cells expressing EGFR or truncated mutants, the activation of any of these receptors led to the phosphorylation of PLC␥ but, significantly, only the full-length EGFR could induce phosphatidylinositol hydrolysis (Li et al., 1991). Therefore, even though the 5YF mutant is still capable of inducing the phosphorylation of PLC␥, the enzymatic activity of PLC␥ is likely unaffected, suggesting that PLC␥ enzymatic activity is not required for PC12 cell differentiation. This is in agreement with previous studies demonstrating that PLC␥ activity is not required for PC12 cell differentiation, although its activity can supplement the activity of other enzymes to lead to PC12 cell differentiation in situations where the overall signaling capacity of the receptors has been attenuated (Fanger et al., 1997; Obermeier et al., 1994). However, since we have not specifically determined whether the enzymatic activity of PLC␥ is increased by the PDGF-induced activation of 5YF, we cannot exclude a role for PLC␥ in PC12 cell differentiation caused by this mutant. Interestingly, PLC␥ has been shown to interact with Cbl in response to EGF (Tvorogov and Carpenter, 2002), and, since Cbl can bind 5YF-PER, this could explain how the mutant PER retains the ability to phosphorylate PLC␥. In addition to coupling PLC␥ to EGFR, Cbl has been shown to coprecipitate with Shc and Grb2 in EGFR-overexpressing NIH 3T3 cells (Galisteo et al., 1995) and can associate with p85 PI3K (Fukazawa et al., 1996; Soltoff and Cantley, 1996). This provides yet another mechanism by which PER can activate both the Ras/Erk and PI3K/Akt pathways. Since 5YF retains the ability to associate with Cbl, both of the Ras/Erk and PI3K/Akt pathways

could potentially be activated by 5YF through the activation of Cbl. This is the least likely mechanism by which 5YF activates the Ras/Erk pathways since Cbl does not associate with Shc or Grb2 when it is immunoprecipitated from 5YF-expressing NIH 3T3 cells (Galisteo et al., 1995). However, Cbl likely acts to couple the PI3K/Akt pathway to 5YF since EGF-induced PI3K activity is strongly associated with Cbl in PC12 cells (Soltoff and Cantley, 1996). As the 5YF mutant failed to prevent the activation of Akt, we cannot rule out a role for Akt in the differentiation of PC12 cells. In summary, we have begun the analysis of signaling pathways elicited by EGFR that are responsible for inducing the differentiation of PC12 cells. The use of a chimeric receptor in these cells has afforded us a unique opportunity to study the signals emanating from EGFR without the complication of activating other ErbB family members. We have shown here that the sites of autophosphorylation with the C-terminus of EGFR are dispensable for its ability to induce PC12 cell differentiation. Furthermore, while subtle differences between the signaling of the mutant versus unmodified receptor exist, the majority of the signaling events is retained. Clearly, other sites exist within the mutant receptors that allow the activation of these signaling pathways, and these sites are currently being investigated.

Acknowledgements We would like to thank Claudio De-Fraja for helpful discussions and critical review of the manuscript. This work was supported by NIH grants AG09735 (RAB), NS10981 (DRT) and MH20043 (YH). References Altun-Gultekin, Z.F., Chandriani, S., Bougeret, C., Ishizaki, T., Narumiya, S., de Graaf, P., Van Bergen en Henegouwen, P., Hanafusa, H., Wagner, J.A., Birge, R.B., 1998. Activation of Rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk adaptor protein. Mol. Cell. Biol. 18, 3044–3058. Anneren, C., Reedquist, K.A., Bos, J.L., Welsh, M., 2000. GTK, a Src-related tyrosine kinase, induces nerve growth factor-independent neurite outgrowth in PC12 cells through activation of the Rap1 pathway. Relationship to Shb tyrosine phosphorylation and elevated levels of focal adhesion kinase. J. Biol. Chem. 275, 29153–29161. Batzer, A.G., Rotin, D., Ureña, J.M., Skolnik, E.Y., Schlessinger, J., 1994. Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol. Cell. Biol. 14, 5192–5201. Boglari, G., Szeberenyi, J., 2001. Nerve growth factor in combination with second messenger analogues causes neuronal differentiation of PC12 cells expressing a dominant inhibitory Ras protein without inducing activation of extracellular signal-regulated kinases. Eur. J. Neurosci. 14, 1445–1454. Boonstra, J., Mummery, C.L., van der Saag, P.T., de Laat, S.W., 1985. Two receptor classes for epidermal growth factor on pheochromocytoma cells, distinguishable by temperature, lectins, and tumor promoters. J. Cell. Physiol. 123, 347–352. Burry, R.W., 2001. p21(Ras) stimulates pathways in addition to ERK, p38, and Akt to induce elongation of neurites in PC12 cells. J. Neurosci. Res. 63, 45–53.

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