A Minor Tyrosine Phosphorylation Site Located ... - Journal of Virology

JOURNAL OF VIROLOGY, Feb. 1995, p. 1172–1180 0022-538X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 69, No. 2

A Minor Tyrosine Phosphorylation Site Located within the CAIN Domain Plays a Critical Role in Regulating Tissue-Specific Transformation by erbB Kinase CHI-MING CHANG,1 HUI-KUO G. SHU,1 LAKSHMESWARI RAVI,1 ROBERT J. PELLEY,2 HUIDY SHU,1 AND HSING-JIEN KUNG1* Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio 44106,1 and Department of Hematology/Oncology, Division of Medicine, Cleveland Clinic Foundation, Cleveland, Ohio 441952 Received 23 March 1994/Accepted 8 November 1994

Avian c-erbB encodes a protein that is homologous to the human epidermal growth factor receptor. Truncation of the amino-terminal, ligand-binding domain of this receptor results in an oncogene product which is a potent inducing agent for erythroleukemias but not fibrosarcomas in chickens. Here we show that mutation of a single tyrosine residue, p5, in the carboxyl terminus of the erbB oncogene product allows it to become sarcomagenic in vivo and to transform fibroblasts in vitro. Mutations of other autophosphorylation sites do not generate comparable effects. The increased transforming activity of the p5 mutant is accompanied by an elevated level of mitogen-activated protein kinase phosphorylation. By analogy to the human epidermal growth factor receptor, p5 is a minor autophosphorylation site and is located in a domain known to be involved in regulating calcium influx and receptor internalization (CAIN domain). This area of the erbB product has been found to be repeatedly deleted in various sarcomagenic avian erythroblastosis virus isolates. We precisely deleted the CAIN domain and also made point mutations of the acidic residues within the CAIN domain. In both cases, fibroblast-transforming potential is activated. We interpret these data to mean that p5 and its surrounding region negatively regulate fibroblast-transforming and sarcomagenic potential. To our knowledge, this represents the first point mutation of an autophosphorylation site that activates erbB oncogenicity. translocation of MAP kinase and the p90rsk kinase to the nucleus and phosphorylation of transcriptional factors involved in cell growth (4, 13). Many of the direct substrates for the EGFR contain one or more SH2 domains, which recognize phosphorylated tyrosine residues. Their interactions with the EGFR are mediated by binding to the autophosphorylated tyrosines on the receptor molecules. There are five tyrosine autophosphorylation sites in the human EGFR, which are located at residues Y-1173 (p1), Y-1148 (p2), Y-1086 (p4), Y-1068 (p3), and Y-992 (p5) (15, 38, 65). Among these tyrosine residues, Y-1173 (p1), Y-1148 (p2), and Y-1068 (p3) are major phosphorylation sites and Y-1086 (p4) and Y-992 (p5) are minor ones. The binding sites of several signaling molecules to the EGFR have been mapped. Y-1173 (p1) is the anchor site for the SHC protein, and Y-1068 (p3) is the anchor site for the Grb2 protein (45, 53). PLC-g interacts with at least three autophosphorylation sites: Y-1173 (p1), Y-1068 (p3), and Y-992 (p5) (58, 61). Of particular relevance to this work is Y-992 (p5). Although a minor phosphorylation site, p5 apparently can interact with PLC-g and pp60c-src (35). In addition, it is located in a domain implicated in receptor internalization and calcium influx (CAIN domain) (12). Because of their functions as anchor sites for signal molecules, the autophosphorylation sites are thought to be crucial to the receptor’s ability to channel signals (44). However, the past reports based on studies of human EGFR mutants of the major autophosphorylation sites are not convergent on this point. In some cases, mutations of these sites lead to diminished transforming activity in rodent fibroblasts (27, 62), while in others, no or even enhanced effects were observed (14, 28, 65). The varied results are likely due to the use of cells of different types and with different receptor levels. In addition, the presence of minor phosphorylation sites, unknown at that

Binding of epidermal growth factor (EGF) to its receptor (EGFR) results in a series of early and late cellular responses that ultimately lead to mitogenesis (9, 53). Following ligand stimulation, the activated receptor molecule undergoes autophosphorylation on tyrosine residues located in its carboxyl terminus (15). These phosphorylated tyrosine residues are responsible for interaction between the receptor molecule and its substrate proteins (8). Cellular proteins phosphorylated by the EGFR include the g isozyme of phospholipase C (PLC-g), the GTPase-activating protein (GAP), the p85 subunit of the phosphatidylinositol-3 kinase (PI-3 kinase), cytoplasmic tyrosine kinase (pp60c-src), adaptor proteins with no catalytic activity such as Crk, Nck, SHC, and Grb2, transcription factor p91, and finally the syp phosphotyrosine phosphatase (1, 3, 17, 19, 20, 34, 39–41, 46, 64). The variety of these substrates illustrates the complexity and the multiplicity of the EGFR signaling cascade (10, 18). At least one of the major pathways, the Ras/Raf cascade, has recently been delineated (16). In this scheme, the activation of EGFR leads to association and tyrosine phosphorylation of the SHC protein, which binds to Grb2 through a phosphotyrosine-Src homology 2 (SH2) domain interaction (52). Alternatively, the autophosphorylated receptor can itself bind to Grb2 (34). While Grb2 itself is not phosphorylated by EGFR, its translocation to the membrane results in association with a Ras GTP-GDP exchange protein SOS and leads to activation of Ras (6, 7, 22, 33). The GTP-Ras, in turn, activates Raf, MEK and mitogen-activated protein (MAP) kinases, and p90rsk kinase (11, 29, 42, 54, 60). This process culminates in the * Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106. Phone: (216) 368-6655. Fax: (216) 368-3055. 1172

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time, may have complicated the interpretation. More recently, deletion mutants that removed the majority of the carboxylterminal sequence including all five phosphorylation sites were shown to give a higher transforming ability (12). Similar mutants with p1 to p4 deleted and p5 mutated yielded comparable results (25). The caveat to this analysis is that deletions remove not only autophosphorylation sites but other sequences as well. The studies described above were all conducted in rodent immortalized cell lines with the human EGFR. Very little is known about the roles of autophosphorylation sites in transformation mediated by avian c-erbB/EGFR, a system in which the oncogenicity of the EGFR was originally uncovered (21, 63, 66). As described below, the chicken erbB system is particularly suited to studies of cell-type-specific transformation by the EGFR both in vitro and in vivo. Nonacute retrovirus insertion within the c-erbB locus results in the generation of an amino-terminally truncated but otherwise intact receptor molecule which confers erythroleukemogenic potential (21, 36, 43). This insertionally activated c-erbB (IA c-erbB) when cloned into a retroviral vector is predominantly leukemogenic (47). Variants of IA c-erbB or v-erbB which are isolated from acute avian erythroleukemia viruses (AEVs) show expanded disease tropism in that they can also induce fibrosarcomas and angiosarcomas (23, 47, 49, 50). The expanded disease potential is attributed to the additional mutations in the cytoplasmic domain of erbB (47, 51, 56, 57). These mutations often include removal of the autophosphotyrosine sites and the CAIN domain (31). To date, there have been no reports that systematically examine the role of autophosphorylation in cell-type-specific transformation mediated by erbB. To this end, we have generated erbB mutants with all five tyrosines individually converted to phenylalanine. We found that none of them is essential for sarcomagenic potential. In fact, mutation of these sites tends to increase sarcomagenicity. Interestingly, this increase is mapped to the mutant of the minor phosphorylation site p5 and not the major sites, p1, p2, and p3. The increased transforming potential of the p5 mutation is accompanied by a slightly enhanced association with Grb2 and an increased phosphorylation of MAP kinase. As p5 is located in the CAIN domain, we also constructed mutants with precise deletion or mutations in the CAIN domain. These mutants also exhibit increased transforming potential. Our data are consistent with the notion that p5 and the surrounding sequence in the CAIN domain serve a negative regulatory role in sarcomagenic potential. MATERIALS AND METHODS Plasmid construction and mutagenesis. Mutations within the IA c-erbB carboxyl terminus were generated by site-directed mutagenesis. The IA c-erbB ClaI fragment was cloned into pBluescript M131 KS (Stratagene). The singlestranded DNA of IA c-erbB was modified by using a site-directed mutagenesis kit (Amersham). The oligonucleotides used to create the tyrosine-to-phenylalanine mutants are as follows: P1, 59-GAAAACCCAGAGTTTTTGAGGGTAGCA-39; P2, 59-GACAATCCTGACTTTCAGCAGGACTTT-39; P3, 59-CCTGCTCCAG AGTTTGTAAACCAGCTG-39; P4, 59-GACAACCCTGAGTTTCTCAACAC TAAC-39; and P5, 59-GATGCAGATGAGTTTCTTGTCCCACAC-39. The oligonucleotide 59-GAAAGGATGCACTTGACTCCTCTTCTGAGT-39 was used to construct the DCAIN mutant, and the oligonucleotide 59-TATCGCACCCTGAT GCAGCAGCAGAACATGGAAGACATTG-39 was used to construct the PCAIN mutant. The underlined sequences represent the mutated regions. The identity of each mutant was confirmed by dideoxy sequencing. Each mutant construct was cloned into an avian replication-competent retroviral vector, RCAN. This vector carries the gag-pol region of the Bryan high-titer virus, allowing two- to threefoldgreater levels of expression per integrated provirus than the original RCAN vector (30). Tissue culture and transfection. Early-passage line 0 chicken embryo fibroblasts (CEFs) were used in the studies of the truncated constructs. These cells do not have endogenous virus, which eliminates the risk of recombination between RCAN and the endogenous viral sequences (30). CEFs were cultured in mix

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medium (1:1 Dulbecco’s modified Eagle’s medium-medium M199) with 3% fetal bovine serum and 2% chicken serum (Gibco). Transfection was done by electroporation with 20 mg of plasmid DNA as described previously (56). After electroporation, the cultures were passaged for 7 days before being assayed for their biochemical and biological properties. Transformation assay. Transforming potential was assessed in vitro by a soft agar assay to measure anchorage-independent growth of CEFs. This assay was performed as described previously (56). The in vivo transforming potential was assessed after wing web injection of viral supernatant into 2-day-old 15I5 3 71 chickens (56). Peripheral blood smears were taken weekly after injection to monitor the presence of immature erythroblasts in the circulation. Advanced erythroblastosis usually results in death of the host 4 weeks postinjection. Sarcoma formation was assessed in the wing webs of the injected chickens up to 6 weeks postinjection. Immunoblotting. Cells grown to confluency on a P-100 dish were washed twice with phosphate-buffered saline (PBS) and then lysed in 1 ml of boiling sodium dodecyl sulfate (SDS) lysis buffer (50 mM Tris-HCl [pH 6.8], 1% SDS, 1% b-mercaptoethanol, 0.1 M dithiothreitol, 5% sucrose, 0.1 mM Na3VO4). Lysates containing 100 mg of protein were resolved by polyacrylamide gel electrophoresis (PAGE) on an SDS–7.5% or 10% polyacrylamide gel and then electrotransferred to an Immobilon membrane (Millipore). The membrane was probed with either a rabbit polyclonal antibody specific for the erbB carboxyl-terminal domain (37) or PY20, a mouse antiphosphotyrosine monoclonal antibody (ICN); 125Ilabeled protein A (Amersham) was used for detection. Autoradiography was performed with an intensifying screen at 2708C. Immunoprecipitation. Cells grown to confluency on a P-150 plate were washed with PBS and then lysed in 1 ml of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-Cl [pH 6.8], 150 mM NaCl, 0.5% deoxycholic acid, 1.0% Triton-100, 0.1% SDS, 1 mM EDTA, 100 mM NaF, 10 mg of aprotinin per ml, 10 mg of leupeptin per ml, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4). Lysates were incubated on ice for 10 min and then clarified by centrifugation at 13,000 rpm for 15 min at 48C. Antibodies for PLC-g, PI-3 kinase, GAP, SHC (Upstate Biotechnology, Inc.) and MAP kinase (Erk1) (Santa Cruz Biotechnology) were added to lysates and mixed for 2 h at 48C. Protein A beads (Pharmacia) were added for 1 h to precipitate the immune complexes. For PLC-g, PI-3 kinase, GAP, SHC, and MAP kinase, the immune complexes were washed three times with RIPA buffer, then resolved on duplicate SDS–7.5% gels, and blotted with either antisubstrate antibody or antiphosphotyrosine antibody. The bound antibody was detected by using enhanced chemiluminescence reagents (ECL kit; Amersham). For the Grb2 experiment, the same procedure was followed except that cells were lysed in HNTG buffer (20 mM HEPES [N-hydroxyethylpiperazine-N9-2-ethanesulfonic acid; pH 7.5], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mg of aprotinin per ml, 10 mg of leupeptin per ml, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4). The lysates were immunoprecipitated with an anti-erbB antibody specific for the carboxyl-terminal domain and protein A beads. The immune complexes were washed with HNTG buffer and resolved by SDS-PAGE (7.5% polyacrylamide gel). After transfer to Immobilon, the top portion of the membrane was probed with an antiphosphotyrosine antibody to quantitate the amount of erbB autophosphorylation and the bottom portion of the membrane was probed with an anti-Grb2 antibody (Upstate Biotechnology, Inc.). Pulse-chase experiment. For determinations of the erbB protein half-life, subconfluent cells grown on a six-well plate were first starved for 3 h in methioninefree Dulbecco’s modified Eagle’s medium (Gibco). After a wash with PBS, [35S]methionine (100 mCi/ml) was added and cells were incubated for 10 h. After the labeling, cells were grown in complete mix medium for 3 h before the first time point was taken to allow the labeled protein to be processed and transferred to the cell surface. Chase times were at 1, 3, 6, 12, and 18 h. At each time point, cells were washed with PBS and then lysed with RIPA buffer. The erbB proteins were immunoprecipitated with an antibody specific for the erbB kinase domain as described above for immunoprecipitation. After electrophoresis, the gel was treated with En3Hance (Du Pont) before autoradiography. The protein bands were quantitated with a Sci Scan 5000 densitometer (U.S. Biochemical). Phosphorylation of exogenous substrate. The erbB proteins were isolated by immunoprecipitation with an antibody against the erbB carboxyl-terminal domain and protein A beads (Pharmacia) in RIPA buffer. Two micrograms of enolase (Sigma) was incubated at 378C for 3 min in 50 mM acetic acid. The activated enolase was then added to the immune complex in 50 ml of kinase buffer (20 mM HEPES [pH 8], 10 mM MgCl2) with 20 mCi of [g-32P]ATP (3,000 Ci/mmol; Amersham). The reaction mixtures were incubated at room temperature for 20 min. The phosphorylated proteins were separated by SDS-PAGE (7.5% polyacrylamide gel) and visualized by autoradiography. Reverse transcriptase assay. The viral supernatant was pelleted in a microcentrifuge at 12,000 rpm at 48C for 1 h and resuspended in 10 ml of 1% Triton X-100. Then 50 ml of reverse transcription buffer {0.5 U of poly(rA-dT) (Pharmacia), 0.05 M Tris-Cl (pH 7.8), 7.5 mM KCl, 2 mM dithiothreitol, 5 mM MgCl2, 0.05% Nonidet P-40, 0.5 mCi of [a-32P]TTP (3,000 Ci/mmol)} was added to the viral lysate, and the mixture was allowed to incubate at 378C for 60 min. Five microliters of each reaction mixture was blotted on DE81 filter paper (Whatman). The filter was washed twice with 23 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and twice with 95% ethanol and was allowed to dry

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FIG. 1. Schematic diagram of IA c-erbB and the autophosphorylation site mutants. IA c-erbB is the amino-terminally truncated form of c-erbB/EGFR. Major phosphorylation sites (p1, p2, and p3) and minor phosphorylation sites (p4 and p5) are designated on the basis of sequence homology with the human EGFR. The numbering system is according to the IA c-erbB sequence and differs from the full-length chicken c-erbB/EGFR sequence by 549 residues. Each tyrosine residue was mutated to phenylalanine (F). TM, transmembrane domain; PK, protein kinase domain; dots, phenylalanine mutations.

under a heat lamp. Reverse transcriptase activity was determined by quantitating the radioactivity on the filter in scintillation counter. Tryptic phosphopeptide mapping. The erbB proteins were isolated by immunoprecipitation with an antibody against the erbB carboxyl-terminal domain in HNTG buffer. The proteins were labeled in kinase buffer containing 0.1 mM [g-32P]ATP (10 Ci/mmol; Amersham), 10 mM MgCl2, and 10 mM MnCl2 in HNTG buffer (56). Tryptic digestions of gel-purified proteins were performed as described previously (5). The tryptic phosphopeptide mappings were done with the HTLE-7000 system (CBS Scientific Co.). The trypsinized fragments were resolved in two dimensions with electrophoresis in pH 8.9 buffer (1% ammonium carbonate) at 1 kV for 27 min and then subjected to thin-layer chromatography in isobutyric acid buffer (isobutyric acid–pyridine–acetic acid–n-butanol–water [1:0.07:0.04:0.03:0.44]). Peptides were visualized by autoradiography with an intensifying screen after 1 week at 2708C.

RESULTS Construction of autophosphorylation site mutants. In the human EGFR, five autophosphorylated tyrosine residues, Y-1173, Y-1148, Y-1086, Y-1068, and Y-992, were mapped to the carboxyl-terminal domain of the molecule. Among these residues, Y-1173, Y-1148, and Y-1068 are within the context of a consensus NPXY peptide motif, whereas residues Y-1086 and Y-992 are not. Very little is known about the autophosphorylation sites in chicken c-erbB or the constitutively activated IA c-erbB. However, five putative autophosphorylation sites, Y-630 (p1), Y-605 (p2), Y-522 (p3), Y-571 (p4), and Y-447 (p5), could be assigned on the basis of sequence homology with the human EGFR and the consensus NPXY peptides. To understand the role of these tyrosine residues in the oncogenic transformation pathway of IA c-erbB, they were individually changed to phenylalanines by site-directed mutagenesis (mutants P1F to P5F; Fig. 1). Following mutagenesis, each construct was cloned into the replication-competent retrovirus vector RCAN, which allows efficient virus spread both in vitro and in vivo. The mutants were analyzed for in vivo autokinase activity, in vitro and in vivo transforming potentials, and ability to phosphorylate exogenous substrates. Autokinase activity of autophosphorylation site mutants. To understand the effect of the tyrosine mutation on the IA c-erbB autophosphorylation level, we examined the in vivo autokinase

FIG. 2. Autokinase activities of IA c-erbB and the tyrosine mutants. The erbB proteins were expressed in CEFs by using the RCAN retroviral vector. Total cell lysates were prepared from CEFs, resolved by SDS-PAGE (7.5% polyacrylamide gel), and transferred onto Immobilon filters. One filter was probed with an anti-erbB antibody specific for the carboxyl-terminal domain (A). A duplicate of the filter was probed with the antiphosphotyrosine antibody PY20 (B). Bound antibody was detected by using 125I-protein A. The diffuse erbB bands which migrate between 70 to 88 kDa is denoted by the bracket. Sizes are indicated in kilodaltons.

activity of IA c-erbB and the autophosphorylation site mutants. RCAN vectors with various IA c-erbB constructs were used to infect CEFs. The cell extracts were separated on an SDS–10% gel, and Western blotting (immunoblotting) was performed with anti-erbB and antiphosphotyrosine antibodies. The antierbB blot showed that various erbB proteins were expressed at comparable levels in cells infected by different erbB constructs (Fig. 2). The antiphosphotyrosine blot showed the level of tyrosine phosphorylation of each of the erbB mutants. IA cerbB demonstrated a low but significant amount of tyrosine phosphorylation. Mutants P1F to P5F did not show much reduction of the autophosphorylation level, and some of them actually exhibited a slight increase, presumably due to compensatory phosphorylations of remaining tyrosine sites. The in vitro kinase assay using enolase as an exogenous substrate also did not reveal a significant difference in kinase activity between IA c-erbB and the tyrosine site mutants (data not shown). Biological properties of IA c-erbB and its mutants. We then tested the in vivo tumorigenicity of the tyrosine mutants by injection of the virus supernatants into the wing web of newly hatched birds. Under these conditions, sarcoma induction usually occurs before the onset of leukemia, which results in the death of the host by 6 weeks postinjection. The control IA c-erbB virus did not induce sarcomas, consistent with previous results (48) (Fig. 3). Among the mutants tested, only P5F induced a significant level of sarcomas (Table 1), and the

FIG. 3. In vivo sarcomagenicity of IA c-erbB and P5F. Viral supernatants from IA c-erbB and P5F virus-infected CEFs were injected into 2-day-old chickens. Chickens were sacrificed 4 weeks postinjection. The wing webs of two representative birds are shown. The IA c-erbB virus-injected bird shows a relatively normal structure with no apparent fibrosarcoma induction. However, the P5F virus-injected wing web is significantly abnormal, showing a large tumor mass.

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TABLE 1. In vivo transforming potentials of the erbB mutants Construct

RCAN............................................................................... IA c-erbB .......................................................................... P1F .................................................................................... P2F .................................................................................... P3F .................................................................................... P4F .................................................................................... P5F .................................................................................... DCAIN.............................................................................. PCAIN .............................................................................. erbB VI .............................................................................

Sarcoma incidence (no. with sarcoma/ no. tested)a

0 0/7, 0/6, 0/3 1/6 0/6 1/6 0/6 6/9, 4/6, 2/3 6/6 0/6 6/6

a Injected chickens were scored for the development of wing web sarcomas for up to 6 weeks or before they succumbed to erythroleukemia. Sarcomas greater than 1 cm in diameter were counted as positive. The titers of the individual inocula, determined by reverse transcriptase assay and expressed in arbitrary units (counts per minute), were as follows: IA c-erbB, 3,440; P1F, 3,600; P2F, 4,660; P3F, 3,180; P4F, 3,860; P5F, 4,700; PCAIN, 2,620; DCAIN, 2,610; and erbB VI, 4,480.

sarcomas developed were among the most aggressive (Fig. 3). All virus-injected birds that were not sacrificed subsequently succumbed to leukemia, indicating that the failure of some virus isolates to induce a significant level of sarcomas is not due to the lack of infectious viruses. The positive control used in this experiment was the erbB VI virus, which in our hands was the most potent sarcomagenic virus and induced 100% sarcomas in injected birds (Table 1). The erbB gene in the erbB VI virus carries a point mutation (valine 157 to isoleucine) in the catalytic domain, previously shown to be responsible for the sarcomagenicity of the strain of AEV (55, 56). The tyrosine mutant constructs were also tested for the ability to transform CEFs in vitro. Transformation potency was assayed by allowing CEFs to grow in an anchorage-independent manner in soft agar. Here again, the P5F mutant induced significantly larger colonies than did the other mutants (Fig. 4). The other four mutants showed some variations in the potential to induce soft agar colonies, but the majority of the colonies were small, comparable to the background colonies induced by IA c-erbB. Thus, both the in vivo and in vitro transformation data point to p5 as the crucial tyrosine residue in controlling sarcomagenic potential. Phosphorylation of exogenous substrates. As the signal transduction pathway for avian c-erbB has not been defined, we examined the involvement of several substrates of the human EGFR in the sarcomagenic pathway. CEFs transfected with the erbB mutant constructs were lysed and then immunoprecipitated with antibodies against PLC-g, GAP, and PI-3 kinase. The immunoprecipitates were resolved on SDS–7.5% gels, transferred to membranes, and immunoblotted with antibodies against individual substrates and with antiphosphotyrosine antibody. In contrast to the ligand-stimulated EGFR, the levels of phosphorylation of exogenous substrates by truncated erbB are generally low, since only sustained phosphorylations are detectable. Like others (32, 51), we were unable to detect phosphorylation of PLC-g, GAP, and PI-3 kinase in any of the samples (data not shown). We also analyzed proteins involved in the Ras/Raf signaling cascade, including SHC, Grb2, and MAP kinase. Increased phosphorylation of SHC protein could be detected in all erbB virus-infected cells compared with the uninfected CEFs (Fig. 5A), although no correlation with transformation was demonstrated. The association of the Grb2 protein with the erbB molecules was measured by immunoprecipitation with anti-erbB antibody followed by Western blotting

FIG. 4. In vitro fibroblast transformation potentials of IA c-erbB and the tyrosine mutants. CEFs expressing various erbB constructs were analyzed for in vitro transformation potential by the soft agar colony assay as described in Materials and Methods. Pictures were taken on day 17 after the seeding of CEFs at a density of 5 3 103 cells per well.

with anti-Grb2 antibody. In this assay (Fig. 5B), Grb2 association was detected in all erbB virus-infected samples. After calibration against the amount of the phosphorylated erbB in the immunoprecipitates, a slight increase of association was seen with the P5F sample. Since one of the consequences of binding between Grb2 and the phosphorylated receptor molecule is the activation of MAP kinase, we also examined the phosphorylation of MAP kinase, which is generally low in erbB virus-infected CEFs (57). In CEFs infected by the P5F mutant, a higher level of MAP kinase phosphorylation could be detected (Fig. 5C). This level, however, was less than that of the sarcomagenic virus erbB VI. In vitro autophosphorylation of the P5F mutant. While the autophosphorylation level of P5F is quantitatively similar to those of other mutants, we sought to determine whether there may be a qualitative difference in the phosphorylation pattern, due to unequal compensatory phosphorylations at other sites or the creation of new sites. To this end, tryptic phosphopeptide mapping of IA c-erbB and P5F was performed. erbB proteins were immunoprecipitated by anti-erbB antibody, labeled by an in vitro autophosphorylation reaction, and subjected to tryptic digestion. The digested products were subjected to twodimensional tryptic peptide analysis (Fig. 6). The tryptic map of IA c-erbB showed three major spots, which are unaltered by the P5F mutation. This finding agrees with previous reports on the full-length EGFR (65), which showed that p5 is a minor phosphorylation site and its loss does not have a significant impact on the overall phosphorylation pattern. The observed transforming effect of P5F is therefore caused by either a more subtle phosphorylation indiscernible by the present method or a structural alteration unrelated to tyrosine phosphorylation. Involvement of a region encompassing p5 in IA c-erbB transformation. The results presented above showed that the p5 tyrosine is unique in its having a significant effect on the bio-

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FIG. 6. Tryptic peptide mapping of IA c-erbB and P5F. The IA c-erbB and P5F proteins were immunoprecipitated with anti-erbB antibody, labeled by in vitro autophosphorylation reaction with [g-32P]ATP, resolved by SDS-PAGE, and subsequently transferred to a nitrocellulose membrane. The membranebound erbB proteins were digested with trypsin, and the resulting tryptic fragments were eluted from the membrane and resolved in two dimensions on thin-layer chromatography plates. The horizontal direction represents resolution by high-voltage electrophoresis, and the vertical direction represents resolution by thin-layer chromatography.

FIG. 5. Tyrosine phosphorylation of cellular substrates by IA c-erbB and the tyrosine mutants. Cell lysates (350 mg of protein) of CEFs expressing each construct were subjected to immunoprecipitation with anti-SHC (A), anti-erbB (B), and anti-MAP kinase (MAPK) (C). The immunoprecipitates were run on SDS–7.5% gels and transferred to filters. The filters were immunoblotted with either antiphosphotyrosine antibody (a-P-tyr.) or antisubstrate antibodies as indicated. Bound antibody was detected with an ECL detection kit. SHC has three isomers (66, 52, and 46 kDa); only the phosphorylation of the 52- and 46-kDa bands is readily visualized by the antiphosphotyrosine antibody (PY20) used in this experiment. The molecular masses of the detected bands are as follows: SHC, 52 and 46 kDa; Grb2, 23 kDa; and MAP kinase, 42 kDa. Ig, heavy chain of rabbit immunoglobulin derived from the precipitating antibodies; CEF, uninfected CEFs; VI, CEFs infected by the erbB VI virus (positive control for fibroblast transformation).

logical and biochemical properties of IA c-erbB. Although we do not know whether the p5 site is phosphorylated in its native state, its presence seems to suppress the sarcomagenic potential. We hypothesize that the p5 tyrosine and its surrounding region may be involved in interacting with negative regulators that interfere with erbB kinase activity. The p5 tyrosine is located in the CAIN domain, which is a 42-amino-acid region involved in calcium influx and receptor internalization previously identified in the human EGFR (12). Interestingly, several naturally occurring AEVs carry v-erbB with deletions mapped to regions that overlap or are close to this domain (Fig. 7A). The erbB variants v-erbB S3, v-erbB 5005, v-erbB DY.1, and v-erbB ID-1, which have been extensively characterized by us and by others, all have acquired sarcomagenic potential and all have in-frame deletions in this region (31, 50, 51). These data together with those for the p5 mutation suggested to us that perturbation of this region may be a major underlying mechanism for unleashing fibroblast-transforming ability. To test this hypothesis, we made a DCAIN mutant in which the CAIN domain was precisely removed (Fig. 7A). This mutant construct displayed a higher autokinase activity than IA c-erbB (Fig. 8A) and induced very large soft agar colonies in vitro (Fig. 8B) and high incidence of sarcomas in vivo (Table 1). The data presented above suggest that the CAIN domain contains a negative regulatory region that includes p5. We tested whether other sequences in the CAIN domain may also

contribute to this regulatory function. There is an 18-aminoacid region in the amino-terminal half of the CAIN domain that is highly acidic and forms a helical structure postulated to be involved in protein-protein interaction (Fig. 7B) (12, 24). To study the relevance of this motif, we made a mutant with the four consecutive acidic residues replaced by neutral ones (dotted residues in Fig. 7). This mutant, designated PCAIN, again showed higher autokinase activity (Fig. 8A) and could transform CEFs in vitro (Fig. 8B); however, it did not cause fibrosarcomas in vivo (Table 1), thus displaying only a partial transformation phenotype. Half-lives of the CAIN domain mutants. The CAIN domain is functionally involved in ligand-dependent internalization and degradation of the full-length EGFR. The observed higher sarcomagenicity of the DCAIN mutant could be attributed to a longer half-life of the receptor. We therefore measured the half-life of the DCAIN mutant and compared it with the halflife IA c-erbB. CEFs expressing IA c-erbB, DCAIN, and PCAIN were metabolically labeled with [35S]methionine, subsequently chased with unlabeled methionine, and lysed at different time points. The erbB proteins were immunoprecipitated with anti-erbB antibody and resolved by SDS-PAGE (7.5% polyacrylamide gel). The rates of degradation of these products did not differ dramatically, with the half-lives being approximately 4 h (Fig. 8C and 9). This finding suggests that the CAIN domain probably is not critical to the downregulation of truncated erbB molecules and that the acquired oncogenicity of the DCAIN molecule cannot be explained solely by its prolonged half-life. DISCUSSION Insertional activation of the c-erbB locus by the nonacute retrovirus avian leukemia virus generates an oncogene IA cerbB which induces exclusively erythroleukemias (21, 48). The acute retroviruses, AEVs, acquired sarcomagenic potential during viral propagation. We and others have shown that this alteration is primarily due to point mutations in the catalytic domain or deletions in the carboxyl-terminal domain of the erbB gene (51, 56, 57). These structural alterations therefore hold keys to our understanding of the cell-type-specific signaling and transformation of the EGFR (26, 31, 36). Among them, the best studied is V157I, which has a valine-to-isoleucine change in the ATP-binding pocket of the kinase domain (55, 56). This mutation when introduced into IA c-erbB can increase the kinase activity and the sarcomagenic potential of

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FIG. 7. Schematic diagrams of IA c-erbB and the erbB internal deletion mutants. (A) IA c-erbB and six erbB mutants. Autophosphorylation sites are designated p1, p2, p3, p4, and p5. v-erbB S3, v-erbB DY.1, v-erbB ID-1, and v-erbB 5005 are derived from erbB sarcomagenic viruses which differed from IA c-erbB by internal deletion within the carboxyl-terminal domain. The v-erbB DY.1 mutant has an additional point mutation (dot) at the p1 tyrosine residue. The erbB DCAIN construct is derived from IA c-erbB by site-directed mutagenesis, which deletes the 42-amino-acid (a.a.) segment corresponding to the CAIN domain. The erbB PCAIN construct differs from IA c-erbB by alteration of four acidic amino acids in the CAIN domain to the corresponding neutral acids (oval). TM, transmembrane domain; PK, protein kinase domain. (B) Comparison of sequences of the CAIN domain of IA c-erbB and the human EGFR (h-EGFR). The predicted turn-acidic helix-turn motif in indicated. The conserved acidic amino acids are underlined. Dots denote amino acid substitutions in the PCAIN mutant. A space is inserted into the sequence of the human EGFR to maximize the homology.

the wild-type molecule, presumably by enhancing the phosphate transfer reaction. The mechanism by which the deletion mutations in the carboxyl-terminal domain activate the sarcomagenic potential is less clear. We therefore undertook studies to more precisely define the critical region involved. The carboxyl-terminal regulatory domain carries autophos-

phorylation sites and the CAIN domain. When the autophosphorylation sites are phosphorylated, they are postulated to be important for the association of molecules that connect the kinase signal to downstream pathways (8). In the unphosphorylated state, they have been postulated to serve as the competitive inhibitors for crucial substrates (2). Thus, the tyrosine

FIG. 8. Biochemical and biological properties of the CAIN domain mutants. (A) The PCAIN and DCAIN constructs were expressed in CEFs by using the RCAN vector. Total cell lysates were prepared, resolved on SDS–7.5% gels, and transferred onto Immobilon filters. One filter was probed with an anti-erbB antibody (aerbB) specific for the kinase domain (left panel). A duplicate filter was probed with an antiphosphotyrosine antibody (a-P-tyr.; right panel). Bound antibodies were detected by using 125I-protein A. (B) The CEFs expressing the PCAIN and DCAIN constructs were seeded into soft agar as described in Materials and Methods. (C) The erbB mutant proteins were labeled with [35S]methionine as described in Materials and Methods and resolved by SDS-PAGE (7.5% polyacrylamide gel). The gel was treated with En3Hance and subjected to autoradiography for 3 days with an intensifying screen.

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FIG. 9. Half-life analysis of the IA c-erbB, DCAIN, and PCAIN proteins. The erbB half-life was determined by pulse-chase analysis followed by immunoprecipitation with an erbB antibody against the kinase domain. The immunoprecipitates were resolved by SDS-PAGE (7.5% polyacrylamide gel). The chase times were 0, 1, 3, 6, 12, and 18 h. The first time point (0 h) was taken 3 h after the chase period started (see Materials and Methods). Following autoradiography, the amount of the labeled protein at each time point was quantitated by densitometry scanning. The percentage of labeled protein was calculated by the scanned value at each time point relative to the value at 0 h for each construct. F, IA c-erbB; E, DCAIN; ■, PCAIN.

residues can be both positive and negative modulators. Many of the erbB deletion mutants that have acquired sarcomagenic potential delete one or more major phosphorylation sites. We were therefore interested in examining the role of the phosphorylated tyrosine residues in the determination of disease tropism. By mutating individual tyrosine residues to phenylalanines, we found p5 (human Y-992 equivalent) to be the most relevant to the sarcomagenic potential of IA c-erbB. Our finding is echoed by a recent report which indicates that mutation of the p5 residue increases the potential of a human EGFR deletion mutant to transform rodent fibroblasts in a liganddependent manner (25). The p5 site is located in an acidic helical domain (Fig. 7B), a motif which has been postulated to a site for interaction of GAL4 and GCN4 yeast transcription factors with the transcriptional apparatus (12, 24). By analogy, p5 and its surrounding sequences may be a site for interaction with inhibitory protein(s) in fibroblasts; this protein would suppress the kinase activity toward certain substrates, thus hindering oncogenic transformation. In an attempt to define the immediate substrates that are modulated by p5, we tested the phosphorylations of a number of SH2 domain-containing molecules, including PLC-g, GAP protein, PI-3 kinase, and the SHC protein. None of the tested proteins showed heightened phosphorylation that correlated with transformation. The association of receptor with Grb2 has been shown to be linked to the activation of the Ras/Raf pathway (6). The P5F mutant showed a slightly enhanced ability to bind Grb2 compared with other nontransforming tyrosine mutants and induced a higher level of MAP kinase phosphorylation. These correlations notwithstanding, we are not sure that they can fully account for the dramatic in vivo sarcomagenic phenotype induced by the P5F mutant. In fact, some of the sarcomagenic erbB variants failed to show elevated MAP kinase phosphorylation (10a). Thus, there must be other pathways involved, and experiments are under way to explore this possibility. Further support of a negative regulatory role of p5 came from the deletion and mutation analysis of the surrounding CAIN domain. Prior to this study, we and others had repeat-

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edly isolated sarcomagenic erbB variants that carry deletions in this region. The mutant with precise deletion of the CAIN domain (i.e., DCAIN) reported here gave strong sarcomagenicity. Point mutants in the acidic residues (EEED) postulated to be sites of protein-protein interaction (12, 24) also yielded a partial transforming phenotype. In addition, mutation of two serine residues adjacent to the CAIN domain were shown by Theroux et al. (59) to activate sarcomagenic potential. These data suggest that the CAIN domain and its adjacent region with their negatively charged surface (E, D, phosphoserine, or tyrosine) may be structured in a particular conformation and involved in negative regulation. Neutralizing the negative charge by point mutations activates fibroblast-transforming potential. However, as exemplified by the PCAIN mutant, there must be other determinants in this region that contribute to the full sarcomagenic potential. Since the CAIN domain is involved in internalization and degradation (12), one mechanism to explain our result is that deletion of the CAIN domain prolongs the half-life of the erbB molecule, thereby increasing its transforming potential. However, we were unable to demonstrate a difference in half-life between DCAIN, PCAIN (Fig. 8C and 9), the P5F mutant (data not shown), and wild-type IA c-erbB. This may not be totally unexpected, as truncated erbB does not respond to ligand-induced dimerization, clustering, and internalization. The constitutive degradation pathway may not depend on the CAIN domain. We therefore favor the hypothesis stated earlier that p5 and the CAIN domain are involved in binding inhibitory molecules or form a structure that negatively regulates the kinase activity. Two proteins, PLC-g (61) and pp60c-src (35), have been shown to bind to p5 of the ligand-stimulated full-length receptor. The challenge ahead is to identify the putative inhibitory molecule that operates in a cell-type-specific manner. In summary, we have identified a mutation of one tyrosine residue in the IA c-erbB product that can activate its sarcomagenic potential. This mutation results in increased phosphorylation of MAP kinase. It should provide a useful tool with which to dissect the cell-type-specific transformation pathway mediated by IA c-erbB. ACKNOWLEDGMENTS We thank Elizabeth Walter for stimulating discussion. This work was supported by grant CA39207 from National Cancer Institute (to H.-J.K.) and Cancer Center Core Grant P30 CA43703 (to Case Western Reserve University). REFERENCES 1. Anderson, D., C. A. Koch, L. Grey, C. Ellis, M. F. Moran, and T. Pawson. 1990. Binding of SH2 domains of phospholipase Cg1, GAP and Src to activated growth factor receptors. Science 250:979–982. 2. Bertics, P. J., W. S. Chen, L. Hubler, C. S. Lazar, M. G. Rosenfeld, and G. N. Gill. 1988. Alteration of epidermal growth factor receptor activity by mutation of its primary carboxyl-terminal site of tyrosine self-phosphorylation. J. Biol. Chem. 263:3610–3617. 3. Bjorge, J. D., T. O. Chan, M. Antczak, H.-J. Kung, and D. J. Fujita. 1990. Activated type I phosphatidylinositol kinase is associated with the epidermal growth factor (EGF) receptor following EGF stimulation. Proc. Natl. Acad. Sci. USA 87:3816–3820. 4. Blenis, J. 1993. Signal transduction via the MAP kinases: proceed at your own RSK. Proc. Natl. Acad. Sci. USA 90:5889–5892. 5. Boyle, W., P. V. derGeer, and T. Hunter. 1991. Phosphopeptides mapping and phosphoamino acid analysis by two dimensional separation on thin-layer cellulose plates. Methods Enzymol. 201:110–149. 6. Buday, L., and J. Downward. 1993. Epidermal growth factor regulates p21 ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell 73:611–620. 7. Buday, I., and J. Downward. 1993. Epidermal growth factor regulates the exchange of guanine nucleotides on p21ras in fibroblasts. Mol. Cell. Biol. 13:1903–1910. 8. Cantley, L. C., K. R. Auger, C. Carpenter, B. Duckworth, A. Graziani, R.

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