An Epidermal Growth Factor Receptor-Leukocyte Tyrosine Kinase Chimeric Receptor Generates Ligand-dependent Growth Signals through the Ras Signaling Pathway

Hiroo Ueno,Naoto Hirano,Hiroyuki Kozutsumi,Ko Sasaki,Tomoyuki Tanaka,Yoshio Yazaki,Hisamaru Hirai

doi:10.1074/jbc.270.34.20135

Hiroo Ueno, Naoto Hirano + Show 5 more

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https://doi.org/10.1074/jbc.270.34.20135

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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.

Highlights

Leukocyte tyrosine kinase (LTK)l is a receptor tyrosine kinase that belongs to the insulin receptor family
All these chimeric receptor cDNAs encoded proteins in which the extracellular domain of human EGF receptor (EGFR) was joined to the transmembrane and the cytoplasmic domains of the human LTK
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

Summary

Introduction

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 She. Both of them are located in NP/AXY motif which is consistent with binding sites for She 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. 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-l cells. These data indicate that native LTK gene product might have a critical role in malignant transformation in these cell lines (Kozutsumi et al, 1993)

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