(Received for publication, February 13, 1995; and in revised form, May 23, 1995)
From the
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.
Leukocyte tyrosine kinase (LTK) ()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.
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.
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.
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.
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.
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.
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.
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.
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.
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 (Lys
M) (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.
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.