©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Epidermal Growth Factor Receptor-Leukocyte Tyrosine Kinase Chimeric Receptor Generates Ligand-dependent Growth Signals through the Ras Signaling Pathway (*)

(Received for publication, February 13, 1995; and in revised form, May 23, 1995)

Hiroo Ueno Naoto Hirano Hiroyuki Kozutsumi Ko Sasaki Tomoyuki Tanaka Yoshio Yazaki Hisamaru Hirai (§)

From the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase that belongs to the insulin receptor family. LTK is mainly expressed in pre B cells and brain. Previously we cloned the full-length cDNA of human LTK, but no ligands have so far been identified, and hence, very little is known about the physiological role of LTK. To analyze the function of the LTK kinase, we constructed chimeric receptors composed of the extracellular domain of epidermal growth factor receptor and the transmembrane and the cytoplasmic domains of LTK and established cell lines that stably express these chimeric molecules. When cultured in medium containing EGF, growth of these cell lines was stimulated, and these fusion proteins became autophosphorylated and associated with Shc in vivo in a ligand-dependent manner. By treatment with EGF, Shc was associated with the Grb2/Ash-Sos complex. Our analyses demonstrate that LTK associates with Grb2/Ash through an internal adaptor, Shc, depending on a ligand stimulation. The LTK binding site for Shc was tyrosine 862 at the carboxyl-terminal domain and to a lesser extent tyrosine 485 at the juxtamembrane domain. Both of them are located in NP/AXY motif which is consistent with binding sites for Shc. These findings demonstrate that LTK can activate the Ras pathway in a ligand-dependent manner and that at least one of the functions of this kinase is involved in the cell growth.


INTRODUCTION

Leukocyte tyrosine kinase (LTK) (^1)is a receptor tyrosine kinase that belongs to the insulin receptor family. The LTK gene was initially cloned from a mouse pre B cell cDNA library. Northern blot analysis revealed that the LTK gene is expressed in pre B lymphocyte, brain, placenta, and several hematopoietic cell lines (Ben-Neriah and Bauskin, 1988; Maru et al., 1990). Initially obtained clones encoded transmembrane protein tyrosine kinase with only eight amino acids as an extracellular domain, but recent studies have suggested that LTK cDNA encodes a larger extracellular domain, including the signal sequence (Haase et al., 1991; Snijders et al., 1993). Finally, we cloned a cDNA of human LTK with 423 amino acids as an extracellular domain and 415 amino acids as a cytoplasmic domain, and this clone was supposed to be full-length (Toyoshima et al., 1993). This cDNA clone encodes a 100-kDa protein when transiently expressed in COS-1 cells.

Very little is known about the physiological role of LTK tyrosine kinase. The ligands for LTK have not been identified, and the expression level of LTK has been revealed to be relatively low. Therefore functional analyses of LTK has not been progressed. We showed previously that, in Northern blot analysis, the LTK gene is preferentially expressed in leukemias with no cell lineage specificity, but not in other neoplasms (Maru et al., 1990). Furthermore, by an in vitro kinase assay using anti-LTK antibody, a 100-kDa phosphoprotein was detected in human hematopoietic cell lines. These data indicate that native LTK gene product might have a critical role in malignant transformation in these cell lines (Kozutsumi et al., 1993).

Recently ALK tyrosine kinase, which has 64% of amino acids sequence similarity with LTK, was cloned by the positional cloning strategy. In the t(2;5)(p23;q35) chromosomal translocation observed in one-third of large cell lymphomas, a nuclear protein, nucleophosmin, is fused with the catalytic domain of ALK by the gene rearrangement (Morris et al., 1994). This fusion protein could contribute to malignant transformation in these lymphomas. From these observations, we supposed that the physiological role of LTK is concerned with the cell growth and malignant transformation.

Chimeric receptor molecules have been successfully used as a tool to study the signaling properties of ligand-orphan receptors in which the extracellular domain of an orphan receptor is replaced by the extracellular domain of another well characterized receptor tyrosine kinase whose ligand is available. This kind of approach enables us to analyze the molecular events involved in the signal transduction pathway of the tyrosine kinase of interest, even when its ligands are not yet identified.

We constructed EGF receptor (EGFR)-LTK chimeric receptor cDNAs composed of the extracellular domain of EGFR and the transmembrane and cytoplasmic domains of LTK and introduced them into human embryonic kidney 293 cells which express a relatively low level of endogenous EGFR. Using these cells, we analyzed biological and biochemical properties of LTK tyrosine kinase.

In this study, we report that EGFR-LTK chimeric receptors are correctly synthesized and transported to the cell surface. The growth of chimeric receptor-expressing transfectants are promoted in the presence of EGF. These chimeric receptors associate with Shc in vivo in a ligand-dependent manner, and Shc connects the cytoplasmic domain of LTK with Grb2/Ash-Sos complex. These findings indicate that the cytoplasmic domain of LTK generates growth signals upon ligand stimulation and that they may be transmitted through the Ras signaling pathway.


MATERIALS AND METHODS

Antibodies

Anti-LTK monoclonal antibody, KM912 that recognizes the extracellular domain and 1D3-1 that recognizes the carboxyl-terminal domain of LTK were produced as described previously (Kozutsumi et al., 1993; Toyoshima et al., 1993). Ab-1, the monoclonal antibody directed against the extracellular domain of EGFR, anti-Grb2/Ash monoclonal antibodies 3F2 and C23, the rabbit anti-GST antibody, the rabbit anti-Shc antibody, and the anti-phosphotyrosine monoclonal antibody 4G10 were purchased from Oncogene Science Inc., MBL Inc., Santa Cruz Biotechnology Inc., AMRAD Inc., and Upstate Biotechnology, Inc., respectively. The rabbit anti-Grb2/Ash polyclonal antibody was a gift from T. Takenawa (University of Tokyo, Tokyo). The rabbit anti-Sos antibody, which is specific for mSos1, was provided by Y. Kaburagi (University of Tokyo, Tokyo).

Plasmids

To generate an expression vector pSSRalphabsr, the EcoRI-PvuII fragment of pSV2bsr containing the blasticidin-resistant gene was blunted, linked with ClaI linker, and subcloned into the ClaI site of an expression vector, pSSRalpha (Toyoshima et al., 1993). The expression vector pUC-CAGGS was kindly provided by J. Miyazaki (University of Tokyo, Tokyo).

Construction of EGFR-LTK Chimeric Receptor and Mutant LTK cDNAs

Three chimeric receptor cDNAs were constructed by ligating the extracellular domain of human EGFR with the transmembrane and the cytoplasmic domains of LTK. These chimeric receptors differed in the joining regions (Fig. 1). To construct EL1, the NheI site of LTK was blunted, and the NotI-NheI fragment encoding the extracellular domain of human LTK was exchanged with the NotI-NaeI fragment encoding the extracellular domain of human EGFR. To construct EL2, a unique EcoRV site was introduced at position 2090 of the EGFR cDNA by site-directed mutagenesis (Kunkel, 1985) with a mutagenic primer 5`-TTGGACAGATATCAAGACC-3`. The NheI site of LTK was blunted and the NotI-NheI fragment of LTK was exchanged with the NotI-EcoRV fragment of EGFR. To generate EL3, a unique EcoRV site was introduced at position 1362 of the LTK cDNA, using a mutagenic primer 5`-CAGGTGACGATATCCACAGC-3`. The NotI-EcoRV fragment of LTK was exchanged with the NotI-EcoRV fragment of EGFR. These chimeric receptor cDNAs were subcloned into the expression vector pSSRalphabsr. Tyrosine-phenylalanine LTK mutants, Y485F, Y721F, Y753F, and Y779F, were generated by site-directed mutagenesis with mutagenic primers, the 30-mer 5`-GCCCCACCTGGCAAAAATAGGGATTGGGGG-3`, the 32-mer 5`-GATAGGGCATGAAGCCTAGGGAGAAGATCTCC-3`, the 29-mer 5`-CACTGGGTCATAATGCGGAACACAGGCCC-3`, and the 20-mer 5`-CTGAGTGCAGAACTGCAGAC-3`. Tyrosine 862-phenylalanine substitution was done by introducing a point mutation with polymerase chain reaction using 5`-GATGGCACGAGATATCTACC-3` and 5`-CTCAGGAGCGAAAAGTGGGATTCC-3` primers and verified by direct sequencing. The BglII-NotI fragment containing the introduced point mutation was substituted for the comparable fragment of wild-type LTK. All these mutant LTK cDNAs were subcloned into the expression vector pUC-CAGGS.


Figure 1: Schematic structures of EGFR-LTK chimeric receptors. The structures of human EGFR, wild-type LTK, and EGFR-LTK chimeric receptors are shown. These chimeric receptors have the EGFR extracellular domain fused to LTK transmembrane and cytoplasmic domains. Amino acid residues of EGFR and LTK at the joining region are shown. Stippled boxes, cysteine-rich motif; striped boxes, tyrosine kinase domain; black boxes, transmembrane domain; arrows, joining region.



Cell Culture and Transfection

Human embryonic kidney fibroblast 293 cells (Japanese Cancer Research Resources Bank; JCRB, CRL1573) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). Transfections were carried out according to the protocol of Chen and Okayama (Chen and Okayama, 1987). Eighteen hours after transfection, cells were washed once with DMEM and cultured in fresh medium containing 5% FCS. After 24 h, 5 µg/ml of blasticidin was added to the medium. Following 2 weeks of selection, resistant colonies were isolated and expanded.

Bacterial Fusion Proteins

Full-length cDNA of Grb2/Ash subcloned into pGEX expression vector was gifted by T. Takenawa (University of Tokyo, Tokyo). Bacterial cultures expressing Grb2/Ash were grown in Luria-Bertani (LB) medium containing 100 µg/ml ampicillin and induced with 1 mM isopropyl-1-thio-beta-D-galactopyranoside for 2 h at 37 °C. The induced bacteria were lysed in Escherichia coli lysis buffer (40 mM Tris-HCl, pH 7.5, 1% Triton X-100, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride), sonicated, and insoluble materials were removed by centrifugation.

Immunoprecipitation and Immunoblotting

Prior to stimulation, cells were starved in DMEM containing 0.1% FCS for 24 h. Cells were then stimulated with 200 ng/ml of EGF for 5 min at 37 °C, washed twice with ice-cold phosphate-buffered saline, and lysed in radioimmune precipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% (w/v) sodium dodecyl sulfate (SDS), 1% (w/v) deoxycolate, 1% (v/v) Triton X-100, 10 units/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM EDTA) or Triton lysis buffer (0.5% (v/v) Triton X-100, 50 mM Tris-HCl, pH 7.4, 2 mM phenylmethylsulfonyl fluoride, 10 units/ml aprotinin, 1 mM sodium orthovanadate, 1 mM EDTA). Cell lysates were centrifuged, and the supernatant was collected. For analysis of total cellular proteins, SDS sample buffer was added directly to lysate, and the mixture was denatured for 5 min at 95 °C and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoprecipitations were performed at 4 °C for 3 h with specific rabbit or mouse antibodies coupled to the protein A-Sepharose beads. Immunoprecipitates were washed five times in the wash buffer (0.1% (v/v) Triton X-100, 50 mM Tris-HCl pH 7.4), resuspended in SDS sample buffer, and denatured for 5 min at 95 °C prior to loading on the gel. Proteins separated on SDS-PAGE were transferred electrophoretically to a polyvinylidene difluoride membrane (Immobilon, Millipore). The filters were preincubated for an hour with 1% bovine serum albumin in Tris-buffered saline-Triton X-100 (TBST) buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20), incubated for 2 h at room temperature with the specific antibody, washed three times with TBST buffer, and incubated for another hour with the alkaline phosphatase-conjugated goat anti-mouse or goat anti-rabbit antibody. The color reaction was performed with the ProtoBlot system (Promega). For the assays of protein-protein interaction on filters, proteins were transferred to a polyvinylidene difluoride membrane, blocked with 1% bovine serum albumin in TBST buffer for an hour, incubated with bacterial lysates containing GST-Grb2/Ash or GST alone for an hour at room temperature, and washed four times in TBST buffer. The filters were subsequently incubated with the rabbit anti-GST antibody and then incubated with the alkaline phosphatase-conjugated goat anti-rabbit antibody and visualized by the ProtoBlot system as described above.

Cell Surface Labeling

One mg of 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycouril (IODO-GEN, Sigma) was dissolved in 100 µl of chloroform in an Eppendorf tube and dried under nitrogen. Approximately 1 10^7 cells expressing chimeric receptors were detached from the dish and transferred to the tube. Then 0.3 mCi of NaI (ICN) was added to the tube and incubated for 10 min at room temperature with gentle agitation. Then cells were washed three times with phosphate-buffered saline and lysed in 500 µl of radioimmune precipitation buffer. Cell lysates were centrifuged, and the supernatant was subjected to immunoprecipitation with anti-LTK antibody, 1D3-1. The immunoprecipitates were separated by SDS-PAGE as described above, and labeled proteins were detected by autoradiography.

Cell Growth Assays

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to quantitate the factor-induced proliferation of cell lines expressing chimeric receptors and EGFR as described previously (Mosmann, 1983). Briefly 1 10^4 cells were seeded in a 96-well plate and cultured in 90 µl of DMEM containing 0.1% FCS and various concentration of EGF for 72 h. Then 10 µl of MTT solution (MTT, 5 mg/ml) was added to the medium and incubated for 6 h at 37 °C. Next, 150 µl of acid isopropanol (0.04 N HCl in isopropyl alcohol) was added to all wells and mixed, and optical density was measured on a microelisa plate reader (Dynatech Laboratories Inc.) at 540 nm. To examine a time-dependent cell growth rate, 1 10^5 cells were seeded in a six-well dish in DMEM containing 0.1% FCS with or without EGF (20 ng/ml) and were counted every 2 days. Each experiment was repeated at least three times, and average values were presented.


RESULTS

Construction and Expression of EGF Receptor-LTK Chimeric Receptor cDNAs

Three chimeric receptor cDNAs, whose products differ in their joining regions upstream of the juxtamembrane domain, were constructed. All these chimeric receptor cDNAs encoded proteins in which the extracellular domain of human EGFR was joined to the transmembrane and the cytoplasmic domains of the human LTK. The joining region of EL3 is the nearest from the extracellular border of the LTK transmembrane domain (Fig. 1). All chimeric receptor cDNAs were subcloned into an expression vector, pSSRalphabsr. These constructs were then introduced into human embryonic kidney 293 cells which express a relatively low level of endogenous EGFR. Approximately 140-kDa polypeptides corresponding to chimeric receptors were detected by immunoblotting with anti-LTK antibodies (data not shown). To establish cell lines that stably express the chimeric receptors, 5 µg/ml of blasticidin was added to the medium, and after selection for 2 weeks, resistant colonies were isolated and expanded. Three to five clones were found to express chimeric receptors by immunoblotting of the whole cell lysates (data not shown). Among them, clones EL1-3, EL2-3, and EL3-3 expressed approximately the same level of fusion proteins and were used for further analyses. In addition, we established human EGFR and wild-type human LTK stable transfectants, clones E-5 and P-3, respectively. To confirm the expression of these fusion proteins, lysates from these stable transfectants were immunoprecipitated with Ab-1, a monoclonal antibody that recognizes the extracellular domain of human EGFR, and immunoprecipitates were then immunoblotted with 1D3-1, a monoclonal antibody that recognizes the carboxyl-terminal region of human LTK. As shown in Fig. 2A, approximately 140-kDa proteins were detected in the 293 cells transfected with the chimeric cDNAs, but not in those transfected with the expression vector alone (Fig. 2A).


Figure 2: Expression of EGFR-LTK chimeric receptors and LTK in 293 cells. A, cell lines which stably express wild-type human LTK (P-3) and chimeric receptors (EL1-3, EL2-3, EL3-3) were lysed and immunoprecipitated with 1D3-1 antibody specific for LTK carboxyl-terminal domain (P-3) or Ab-1 antibody which recognizes the EGFR extracellular domain (EL1-3, EL2-3, EL3-3) and immunoblotted with 1D3-1. B, cell surface proteins of EL1-3, EL2-3, and EL3-3 cells, and mock-transfected cells were labeled with I, lysed, and immunoprecipitated with 1D3-1. Immunoprecipitates were subjected to SDS-PAGE, and labeled proteins were detected by autoradiography.



To verify that these chimeric receptors are expressed on the cell surface, cell surface proteins were labeled with NaI. Then the cells were lysed, immunoprecipitated with 1D3-1 and subjected to the SDS-PAGE analysis. Labeled proteins were visualized by autoradiography. In this experiment, approximately 140-kDa proteins corresponding to chimeric receptors were detected (Fig. 2B). These data indicate that chimeric receptors are expressed on the cell surface.

Biological Activities of Chimeric Receptors

To investigate whether the EGFR-LTK chimeric receptors in fibroblast could affect their morphology or growth properties, the cells expressing the chimeric receptors were maintained in DMEM at 0.1% FCS, supplemented with various concentrations of EGF, and subjected to MTT assay. In this experiment, the growth of EL1-3, EL2-3, EL3-3, and E-5 cells was promoted in proportion to EGF concentration, compared with parental 293 cells and cells transfected with the expression vector alone (Fig. 3A). When these stable transfectants were cultured in DMEM with 0.1% FCS in the presence of EGF for 7 days, the cells that express the chimeric receptor (EL3-3) and EGFR (E-5) increased in number, whereas the cells transfected with the expression vector alone decreased (Fig. 3B). The growth of EL1-3 cells and EL2-3 cells showed a similar tendency to that of EL3-3 cells (data not shown). However, when cultured in the presence of EGF for 2 weeks, no morphological changes were observed in EL1-3, EL2-3, EL3-3, and E-5 cells (data not shown). These results suggest that the signal transduction pathway concerning their cell growth exists downstream of the cytoplasmic domain of LTK, depending on the ligand stimulation. Because 293 cells express endogenous EGFRs and, to some extent, the growth of parental 293 cells were stimulated in the presence of EGF, we expressed the chimeric molecules in Ba/F3 cells, which do not express endogenous EGFR and examined the growth rate in the presence of EGF. In this experiment, we have obtained similar results (data not shown).


Figure 3: Growth of cells expressing chimeric receptors and EGFR. A, the 293 cells expressing chimeric receptors (EL1-3, EL2-3, and EL3-3 cells), EGFR (E-5 cell), control mock-transfected cell (Vec), and parental 293 cells (293) were incubated for 72 h at 37 °C with various concentrations of EGF in DMEM containing 0.1% FCS and subjected to MTT assay. Average optical densities at 540 nm (repeated three times) were expressed as a ratio over the basal optical density at no EGF stimulation. B, growth of 293 cells expressing a chimeric receptor (EL3-3) and EGFR (E-5) and mock-transfected cells (Vec). These cells were cultured in DMEM containing 0.1% FCS and 20 ng/ml of EGF.



Ligand-induced Phosphorylation of Chimeric Receptors and Cellular Proteins

Ligand binding to the extracellular domains of receptor tyrosine kinase results in activation of the cytoplasmic kinase function. To investigate the ability of EGF to induce the autophosphorylation of chimeric receptors in 293 cells, EL1-3, EL2-3, EL3-3, E-5 cells, and mock cells were starved for 24 h in medium containing 0.1% FCS and stimulated with 200 ng/ml of EGF for 5 min at 37 °C. Cells were then lysed and subjected to immunoblotting with anti-phosphotyrosine monoclonal antibody 4G10. As shown in Fig. 4, each chimeric receptor became increasingly phosphorylated on tyrosine in a ligand-dependent manner. At the same time, approximately 70-kDa protein was phosphorylated on tyrosine. No differences of the pattern of phosphoproteins among chimeric receptors (EL1, EL2, and EL3) were observed, but this 70-kDa phosphoprotein was not detected in EGF-treated E-5 cell lysates. Instead, an 85-kDa phosphoprotein was detected in E-5 cell lysates depending on EGF stimulation. These differences might suggest the existence of the signal transduction pathways specific for LTK tyrosine kinase. When mock cells were stimulated with EGF, a 170-kDa tyrosine-phosphorylated protein was faintly detected. This phosphoprotein was thought to be endogenous EGFR, because the 293 cells express a relatively low level of EGFR. But compared with introduced chimeric proteins, the expression level of endogenous EGFR was relatively low. Because no differences were observed so far among the biological and biochemical properties of the chimeric receptors, we used EL3-3 cells for further analyses.


Figure 4: EGF-induced tyrosine phosphorylation of chimeric receptors and EGFR. Quiescent cells were treated with or without EGF (200 ng/ml) for 5 min at 37 °C and solubilized. The cell lysates were electrophoresed on SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody.



EGFR-LTK Chimeric Receptor Associates with Grb2/Ash in Vivo

Since EGF promoted proliferation of cells expressing chimeric receptors, the signaling pathway concerning cell growth should exist downstream of LTK. Recent studies have suggested that many receptor tyrosine kinases activate Ras through binding with the Grb2/Ash-Sos complex. Therefore, we examined the physiological role of Grb2/Ash in the signaling pathway of LTK.

Lysates from EL3-3 cells and E-5 cells stimulated with EGF were immunoprecipitated with the anti-Grb2/Ash polyclonal antibody and immunoblotted with the anti-phosphotyrosine monoclonal antibody. Major 140-, 56-, and 42-kDa phosphoproteins were detected in EL3-3 cell lysates, whereas major 170-, 115-, 100-, 56-, and 46-kDa phosphoproteins were detected in E-5 cell lysates (Fig. 5A). As it has been reported that EGF induces association between Grb2/Ash and EGFR or Shc (Rozakis-Adcock et al., 1993, 1994), the 170-kDa protein immunoprecipitated from E-5 cell lysates was considered to be EGFR, and 52- and 46-kDa proteins were considered to be Shc. We could not characterize the 115- and 100-kDa phosphoproteins co-immunoprecipitated with Grb2/Ash. The 140-kDa phosphoprotein immunoprecipitated from EL3-3 cell lysates was not detected from E-5 cell lysates and therefore considered to be the EGFR-LTK chimeric receptor. To verify this, lysates from EL3-3 cells stimulated with EGF were immunoprecipitated with anti-EGFR monoclonal antibody and immunoblotted with anti-Grb2/Ash antibody, and 27-kDa Grb2/Ash protein was detected (Fig. 5B). Furthermore, anti-Grb2/Ash immunoprecipitates from EL3-3 cells stimulated with EGF were immunoblotted with 1D3-1 anti-LTK monoclonal antibody. In this experiment, the 140-kDa chimeric receptor was identified (Fig. 5C). These results suggest that chimeric receptor is physically associated with Grb2/Ash in vivo in a ligand-dependent manner.


Figure 5: EGF-induced association of the chimeric receptor with Grb2/Ash. A, lysates from E-5 cells and EL3-3 cells treated with or without EGF were immunoprecipitated with anti-Grb2/Ash antibody and immunoblotted with anti-phosphotyrosine antibody. B, lysates from EL3-3 cells treated with or without EGF were immunoprecipitated with Ab-1 which is specific for the extracellular domain of EGFR and immunoblotted with anti-Grb2/Ash antibody. An arrow indicates Grb2/Ash. C, lysates from EL3-3 cells treated with or without EGF were immunoprecipitated with anti-Grb2/Ash antibody and immunoblotted with 1D3-1. An arrow indicates the chimeric receptor. TCL denotes total cell lysates from EL3-3 cells.



Chimeric Receptor Associates with Shc in Vivo

As shown in Fig. 5A, anti-Grb2/Ash immunoprecipitates from EGF-treated EL3-3 cells contained 52- and 46-kDa phosphoproteins, and we considered these proteins to be Shc. To verify this, anti-Grb2/Ash immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-Shc antibody, and as expected, p46, p52, and p66 Shc proteins were detected (Fig. 6A). Furthermore, anti-Shc immunoprecipitates from EGF-treated EL3-3 cells were revealed to contain the p27 Grb2/Ash protein (Fig. 6B). These data indicate that LTK kinase can induce tyrosine phosphorylation of Shc and its association with Grb2/Ash in vivo. These findings raised the possibility that LTK can directly associate with Shc. To examine this possibility, lysates from EL3-3 cells stimulated with EGF were immunoprecipitated with anti-Shc antibody. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-LTK monoclonal antibody, and then the 140-kDa chimeric receptor protein was detected. EGF-treated EL3-3 cells were then lysed and immunoprecipitated with anti-LTK monoclonal antibody, and immunoblotting with anti-Shc antibody was performed. In this experiment, p46, p52, and p66 Shc proteins were co-precipitated with LTK. (Fig. 7C). These data demonstrate that the chimeric receptor associates with Shc in vivo in a liganddependent fashion.


Figure 6: EGF-induced formation of the chimeric receptor, Shc, and Grb2/Ash complex. EGF-induced binding of Shc with Grb2/Ash in EL3-3 cells (A, B). EGF-induced binding of the chimeric receptor with Shc (C). A, lysates from EL3-3 cells treated with or without EGF were immunoprecipitated with anti-Grb2/Ash antibody and immunoblotted with anti-Shc antibody. Arrows indicate p66, p52, and p46 Shc. B, lysates from EL3-3 cells treated with or without EGF were immunoprecipitated with anti-Shc antibody and immunoblotted with anti-Grb2/Ash antibody. An arrow indicates Grb2/Ash. C, lysates from EL3-3 cells treated with or without EGF were immunoprecipitated with anti-Shc antibody or 1D3-1 and immunoblotted with 1D3-1 or anti-Shc. TCL denotes total cell lysates from EL3-3 cells.




Figure 7: EGF-induced complex formation involving the chimeric receptor, Sos, and Grb2/Ash. A, lysates from EL3-3 cells treated with or without EGF were immunoprecipitated with Ab-1 or C23 anti-Grb2/Ash polyclonal antibody and immunoblotted with anti-Sos antibody. An arrow indicates Sos. B, E-5 cells, EL3-3 cells, and mock-transfected cells were stimulated with indicated concentration of EGF at 37 °C for 5 min, solubilized, and immunoblotted with anti-Sos antibody.



Chimeric Receptor Phosphorylates Sos and Forms a Complex with Sos in Vivo

Recent studies have suggested that the SH3 domains of Grb2/Ash bind constitutively to proline-rich motifs within the carboxyl-terminal tail of Sos (Buday and Downward, 1993; Chardin et al., 1993; Egan et al., 1993; Li et al., 1993). Since the chimeric receptor associated with Grb2/Ash in vivo, Sos should also associate with this complex. To investigate this, EL3-3 cells stimulated with EGF were lysed and immunoprecipitated with anti-EGFR antibody and then subjected to the Western blot analysis by anti-Sos antibody, and the immunoprecipitates were revealed to contain Sos protein (Fig. 7A). Furthermore, when anti-Sos immunoprecipitates were immunoblotted with the anti-phosphotyrosine antibody, a 140-kDa phosphoprotein corresponding to the chimeric receptor was detected (data not shown). These data suggested that the EGFR-LTK chimeric receptor and Sos form a complex in vivo. To examine whether this association occurs through Grb2/Ash, lysates from EGF-stimulated EL3-3 cells were immunoprecipitated with anti-Grb2/Ash antibody and immunoblotted with anti-Sos antibody. In this experiment, Sos was shown to form a stable complex with Grb2/Ash, but EGF stimulation resulted in a mobility shift of Sos in SDS-PAGE (Fig. 7A). This shift in electrophoretic mobility is thought to be a result of phosphorylation of Sos on serine and threonine residues (Skolnik et al., 1993a). To test whether this phosphorylation results from the LTK kinase activity and not from activation of endogenous EGFR, lysates from E-5 cells, EL3-3 cells, and mock cells stimulated with EGF were subjected to Western blot analysis by anti-Sos antibody. In this experiment, mobility shift of Sos was observed in E-5 cells and EL3-3 cells, but little shift of Sos band in mock cells was observed (Fig. 7B). These data suggested that LTK can activate Sos through association with Grb2/Ash.

In summary, there were two possible models for association between the EGFR-LTK chimeric receptor and Grb2/Ash. One is that, like EGFR, LTK can directly bind both of Grb2/Ash and Shc. Another is that, like Trk tyrosine kinase, LTK can directly associate only with Shc, but not with Grb2/Ash, because Grb2/Ash indirectly binds LTK through Shc.

The EGFR-LTK Chimeric Receptor Indirectly Associates with Grb2/Ash through Shc

Recent studies have revealed that the consensus motif of binding sites for the SH2 domain of Grb2/Ash is YXNX motif (Skolnik et al., 1993b; Songyang et al., 1993, 1994). For example, the Shc binding site for Grb2/Ash is Tyr, and the peptide sequence containing this tyrosine is DPSYVNVNQ. Similarly, the Grb2/Ash binding motif on IRS-1 is PGEYVNIE, and the binding motif on EGFR is VPEYINQSV. This motif is strictly conserved among binding sites for Grb2/Ash, Drk, and Sem-5 (Songyang et al., 1993, 1994). But the YXNX motif does not exist in the cytoplasmic region of LTK. Therefore, association between the chimeric receptor and the Grb2/Ash-Sos complex was considered to be indirect. To examine this, the far-Western blot analysis using GST-Grb2/Ash fusion protein was performed (Mayer et al., 1991). EL3-3 cells and E-5 cells stimulated with EGF were lysed in Triton lysis buffer and immunoprecipitated with Ab-1, a monoclonal antibody directed against the extracellular domain of EGFR. Immunoprecipitates were subjected to SDS-PAGE and transferred to polyvinylidene difluoride filters. Replica filters were incubated either with a bacterial lysate containing the GST-Grb2/Ash fusion protein or with GST alone. Binding of the GST proteins to the transferred proteins on the filter was detected by incubation of the filters with the anti-GST antibody followed by the alkaline phosphatase-conjugated second antibody. In this assay, GST-Grb2/Ash bound to EGFR but did not bind to the EGFR-LTK chimeric receptor. However, GST-Grb2/Ash could bind to the 52- and 46-kDa proteins of immunoprecipitates from EL3-3 cells (Fig. 8). These proteins were also detected in E-5 cell lysates and therefore considered to be Shc proteins associated with the chimeric receptor. GST alone could not bind to any proteins of immunoprecipitates from EL3-3 cells and E-5 cells. These data indicate that Grb2/Ash indirectly binds the EGFR-LTK chimeric receptor via Shc as an internal molecule.


Figure 8: Grb2/Ash binds to EGFR directly but to EGFR-LTK chimeric receptor indirectly. Lysates from E-5 cells and EL3-3 cells treated with or without EGF were immunoprecipitated with Ab-1 and subjected to immunoblotting with anti-phosphotyrosine antibody or to the far-Western blot analysis using GST or GST-Grb2/Ash as indicated. Arrows indicate 52- and 46-kDa proteins recognized by GST-Grb2/Ash.



Tyrosine 862 of LTK Is the Binding Site for Shc

Recent studies have revealed that the consensus binding motif for Shc is NP/AXY (Prigent and Gullick, 1994). The cytoplasmic domain of LTK has two NP/AXY motifs, Tyr located on the juxtamembrane region and Tyr located on the carboxyl-terminal region. As shown in Fig. 10, the NP/AXY motif located on the juxtamembrane region is conserved among the insulin receptor family members. Therefore we hypothesized that the binding site for Shc is Tyr located on the juxtamembrane region of LTK.


Figure 10: Conserved NP/AXY motif at the juxtamembrane domain among the insulin receptor families. Comparison of the amino acid sequence of the human LTK protein (hLTK) (Toyoshima et al., 1993) with the corresponding domain of the murine LTK protein (mLTK) (Ben-Neriah and Bauskin, 1988; Snijders et al., 1993), the human ALK protein (ALK) (Morris et al., 1994), the human Trk protein (Trk) (Martin-Zanca et al., 1986), the human insulin receptor (IR) protein (Ebina et al., 1985), the human insulin-like growth factor receptor (IGF1R) protein (Ullrich et al., 1986), and the human c-ros protein (cROS) (Matsushime et al., 1986). Matching residues are boxed and shaded. Conserved NP/AXY motifs are indicated by asterisks.



We analyzed the binding sites of Shc on the LTK by using the wild-type LTK and various LTK mutants. The mutant LTKs used in this study include receptors containing point mutations at Tyr, Tyr, Tyr, Tyr, Tyr, or Tyr/Tyr (Tyr to Phe) or lysine 544 to methionine (LysM) (Fig. 9A). These wild-type and mutant LTK cDNAs were subcloned into the expression vector pUC-CAGGS and transiently introduced into 293 cells. In this experiment, the wild-type and all of the mutant LTKs except for K544M mutant were autophosphorylated (data not shown). While an approximately equal amount of Shc was associated with Y485F, Y721F, and Y779F LTK mutants, the amount of Shc associated with the Y862F mutant was markedly reduced, and no Shc protein could bind to Y485F/Y862F LTK mutant (Fig. 9B).


Figure 9: Tyrosine 862 is involved in association of Shc to the LTK. A, schematic structure of intracellular domains of the wild-type and mutant LTKs. LTK, wild-type LTK; Y485F, Y721F, Y753F, Y779F, Y862F, and Y485F/Y862F, mutant LTKs in which the indicated tyrosines were mutated to phenylalanine; K544M, lysine 544 was mutated to methionine. B, the 293 cells transiently expressing either wild-type or mutant LTKs were solubilized and immunoprecipitated with KM912 monoclonal antibody and immunoblotted with 1D3-1 monoclonal antibody and anti-Shc antibody. TCL denotes the total cell lysates from 293 cells transiently expressing wild-type LTK.



As shown in Fig. 10, the NP/AXY motif located on the juxtamembrane region is conserved among the insulin receptor family members. Therefore we hypothesized that the binding site for Shc is Tyr located on the juxtamembrane region of LTK. But, as shown in Fig. 9, the major LTK binding site for Shc is tyrosine 862, and the minor binding site for Shc is tyrosine 485. The Lys mutant is a kinase-inactive LTK mutant (Kozutsumi et al., 1994), and we confirmed that it is not autophosphorylated in this experiment by the immunoblot with anti-phosphotyrosine antibody (data not shown), but the binding of Shc protein was not completely canceled.

From these data, we summarized the signal transduction pathway of LTK as shown in Fig. 11and compared it with that of EGFR (Batzer et al., 1994) and Trk (Obermeier et al., 1993b). While EGFR directly binds both Shc and Grb2/Ash, Trk and LTK cannot bind Grb2/Ash directly. In this respect, LTK has a similarity to Trk tyrosine kinase.


Figure 11: Comparison of the Ras pathway of LTK with those of EGFR and Trk. Summary of signal transduction pathway of LTK and its comparison with those of EGFR (Batzer et al., 1994) and Trk (Obermeier et al., 1993b) are shown. Minor pathways are indicated by dashed arrows.




DISCUSSION

Many receptor tyrosine kinases have been shown to play a critical role in the cell growth and differentiation and to be implicated in generation of various human neoplasms. The evidence that, in Northern blot analysis, the LTK gene is preferentially expressed in human leukemias with no cell lineage specificity (Maru et al., 1990) suggests that LTK gene product might play an important role in human leukemogenesis. However, since LTK tyrosine kinase was cloned in 1988, the functional analyses of this kinase has not been advanced, mainly because its specific ligands have not been identified. Although it was reported that, when expressed in COS-7 cells, LTK resides in the endoplasmic reticulum, where its kinase activity might be controlled by changes in the redox potential, the cDNA clone used in this study was not full length and therefore devoid of the signal sequence (Bauskin et al., 1991). Furthermore, there is no evidence that native LTK protein was expressed in endoplasmic reticulum.

Chimeric receptor molecules have been successfully used to investigate the biological and biochemical properties of ligand-orphan receptors. This kind of approach is particularly useful to analyze the signaling molecules implicated in the signal transduction pathway of the kinase. EGFR is the well characterized receptor tyrosine kinase and therefore frequently used to construct such chimeric receptor molecules to analyze the function of ligand-orphan receptors. These include kit (Herbst et al., 1991; Lev et al., 1991), neu (Pandiella et al., 1989), Trk (Obermeier et al., 1993a, 1993b), insulin receptor (Ballotti et al., 1989), ret (Santoro et al., 1994), and elk (Lhotak and Pawson, 1993). In general, the biological properties of such molecules depend on their cytoplasmic catalytic domains. We therefore constructed EGFR-LTK chimeric receptor cDNAs composed of the extracellular domain of EGFR and the transmembrane and the cytoplasmic domains of LTK. When expressed in 293 cells, these chimeric receptors were correctly synthesized and transported to the cell surface (Fig. 2). EGF-induced tyrosine phosphorylation of chimeric receptors, as well as several cellular proteins, but the pattern of phosphoproteins was different from those of cells expressing EGFR (Fig. 3), suggesting the existence of the signal transduction pathway specific for LTK tyrosine kinase in the cytoplasm.

We analyzed the biological activities of cells expressing the chimeric receptors and found that growth of these cells are promoted in the presence of EGF (Fig. 4). These data indicated that LTK kinase can transmit signals concerning the cell growth. To examine the intracellular events, signaling molecules involved in the LTK signal transduction pathway were examined. Recent studies have suggested that many tyrosine kinases activate Ras through binding to the Grb2/Ash-Sos complex. Therefore we examined the roles of Grb2/Ash in the LTK signaling pathway. We found that EGF induced association between the chimeric receptor and Grb2/Ash in vivo. Furthermore, Shc was phosphorylated on tyrosine and associated with the chimeric receptor upon stimulation with EGF. Although the cytoplasmic domain of LTK has two NP/AXY motifs (Tyr and Tyr) which match the binding motif for Shc (Prigent and Gullick, 1994), Grb2/Ash binding motif, YXNX (Skolnik et al., 1993b; Songyang et al., 1993), does not exist on the cytoplasmic region of LTK. Therefore we hypothesized that association between the chimeric receptor and Grb2/Ash is indirect. The data of the far-Western analysis using GST-Grb2/Ash were consistent with our hypothesis (Fig. 8). The Shc binding motif, NP/AXY, which is located on the juxtamembrane domains, is conserved among the members of insulin receptor family (Fig. 9). This NP/AXY motif located in the Trk juxtamembrane domain has been shown to represent the binding site for Shc (Obermeier et al., 1993b). Interestingly, this motif is also conserved at the juxtamembrane domain of ALK tyrosine kinase (Morris et al., 1994), which might play a critical role in malignant transformation. Therefore we considered that LTK binding site for Shc is Tyr located at the juxtamembrane region, but in contrast to our anticipation, the major binding site for Shc was Tyr, which is located at the carboxyl-terminal region and Tyr was the minor binding site. The significance of this difference is unclear. For example, EGFR has three NP/AXY motifs on the carboxyl-terminal domain (Tyr, Tyr, and Tyr), but the major binding site for Shc is Tyr, whereas Tyr and Tyr exhibit weak or no interaction with Shc (Batzer et al., 1994). These findings may imply that additional residues are also required to generate a high-affinity binding site for Shc.

The activation of the Ras pathway is implicated in generation of various tumors. For example, Bcr-Abl protein tyrosine kinase, which is thought to play important roles in the development of Philadelphia chromosome positive (Ph+) chronic myelogenous leukemia and acute lymphoblastic leukemia, binds directly to Grb2/Ash and mediates Ras stimulation (Puil et al., 1994). We have shown that, in some human hematopoietic cell lines, a 100-kDa phosphoprotein is detected by an in vitro kinase assay using anti-LTK antibody (Kozutsumi et al., 1993). Therefore it is possible that LTK is implicated in the tumorigenesis in these cell lines through activating the Ras pathway.

Several kinds of adaptor molecules are known to connect phosphotyrosine of a tyrosine kinase with the SH2 domain of Grb2/Ash when the tyrosine kinases do not have the binding motif for Grb2/Ash. platelet-derived growth factor receptor, insulin receptor, and Trk tyrosine kinase do not have the YXNX motif on their cytoplasmic domains and therefore they cannot bind Grb2/Ash directly, but Syp (Li et al., 1994), IRS-1 (Giorgetti et al., 1994;Skolnik et al., 1993), and Shc (Obermeier et al., 1993b) have been shown to connect these receptors with Grb2/Ash. It is an intriguing idea that the adaptor molecule specify the biological activities of the kinase. For example, insulin induces tyrosine phosphorylation of IRS-1 and its association with the Grb2/Ash-Sos complex (Baltensperger et al., 1993), but at the same time IRS-1 associates with p85 subunit of phosphatidylinositol 3-kinase, Syp, phospholipase C, and Nck (Kuhne et al., 1993; Lee et al., 1993), and thus the signal transduction pathways through these molecules should also be activated. In the case of LTK, Shc is the adaptor molecule which couples the cytoplasmic domain of LTK and the SH2 domain of Grb2/Ash. In this respect, LTK may be referred to as a Trk-type receptor tyrosine kinase.

Many growth factors induce tyrosine phosphorylation of Shc and its association with Grb2/Ash (Cutler et al., 1993; Damen et al., 1993; Ravichandran and Burakoff, 1994; Ravichandran et al., 1993). Therefore it is obvious that Shc has a critical role in connecting various growth factor receptors with the Ras pathway. Furthermore, Shc itself has been shown to possess a transforming activity. Overexpression of Shc proteins induces transformation of mouse fibroblasts (Pelicci et al., 1992). Middle T-antigen, through binding to Shc, associates with Grb2/Ash and stimulates the Ras activity, and this leads to cell transformation (Dilworth et al., 1994). These data indicate that Shc proteins play an important role in generating the cell growth signals through binding to the Ras pathway.

On the other hand, when overexpressed in PC12 cells, Shc induces Ras-dependent neurite outgrowth in these cells (Rozakis-Adcock et al., 1992). PC12 cells express both EGFR and Trk and both of them can activate the Ras pathway, whereas only nerve growth factor, but not EGF, can induce neuronal differentiation in these cells (Chao, 1992). Obermeier et al.(1994) showed that, in PC12 cells, neuronal differentiation signals are mainly controlled by the Trk binding site for Shc. Recently, when stimulated with nerve growth factor, Shc is shown to associate with CRK in vitro (Matsuda et al., 1994) and microinjection of the CRK protein has been shown to induce neuronal differentiation of PC12 cells (Tanaka et al., 1993). These data suggest that Shc's neuronal differentiation signals are generated through the CRK protein. Northern blot analysis showed that the LTK gene is expressed in neuronal tissues (Maru et al., 1990) and therefore these findings raised the possibility that LTK might also generate neuronal differentiation signals through activating Shc-CRK pathway.

In this study, we analyzed the biological and biochemical activities of LTK tyrosine kinase, but our experiments were carried out with recombinant chimeric molecules expressed in fibroblasts in which native LTK protein is not expressed. Therefore the question remains whether these findings are true events when native LTK product is stimulated by its unknown ligands. To address these questions, its specific ligands should be identified.


FOOTNOTES

*
This work was supported in part by grant-in-aid from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Third Dept. of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3815-5411 (ext. 3102); Fax: 81-3-3815-8350.

(^1)
The abbreviations used are: LTK, leukocyte tyrosine kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; IRS-1, insulin receptor substrate-1; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.


ACKNOWLEDGEMENTS

We thank T. Takenawa for anti-Grb2/Ash antibody and Grb2/Ash pGEX. We also thank Y. Kaburagi for anti-Sos antibody.


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