ALK-Activating Homologous Mutations in LTK Induce Cellular Transformation

J Devon Roll,Gary W Reuther,Antimo Migliaccio

doi:10.1371/journal.pone.0031733

J Devon Roll, Gary W Reuther + Show 1 more

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https://doi.org/10.1371/journal.pone.0031733

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Abstract

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase reported to be overexpressed in human leukemia. Though much regarding the function of LTK remains unknown, it shares a high degree of similarity with anaplastic lymphoma kinase (ALK), which is found mutated in human cancer. In order to determine if LTK has transforming potential, we created two LTK mutants, F568L and R669Q, that correspond to two well-characterized activating mutations of ALK (F1174L and R1275Q). LTK-F568L, but not wildtype LTK or LTK-R669Q, transformed hematopoietic cells to cytokine independence. LTK-F568L exhibited a stronger ability to induce loss of contact inhibition and anchorage-independent growth of epithelial cells compared to LTK-R669Q, while wildtype LTK was non-transforming in the same cells. Likewise, LTK-F568L induced greater neurite outgrowth of PC12 cells than R669Q, while wildtype LTK could not. Correlating with transforming activity, LTK-F568L displayed significantly enhanced tyrosine phosphorylation compared to wildtype LTK and LTK-R668Q and induced activation of various signaling proteins including Shc, ERK and the JAK/STAT pathway. Expression of wildtype LTK or LTK-R669Q generally led to weaker activation of signaling proteins than expression of LTK-F568L, or no activation at all. Thus, mutating LTK at residue F568, and to a lesser extent at R669, activates the receptor tyrosine kinase, inducing cell signaling that results in transforming properties. These studies suggest that aberrant activation of LTK may contribute to neoplastic cell growth.

Highlights

Receptor tyrosine kinases (RTKs) are cell signal transducers which span the plasma membrane, binding ligands with their extracellular domain [1]
In order to determine if mutations in the kinase domain of Leukocyte tyrosine kinase (LTK) possess a similar transforming potential as the known anaplastic lymphoma kinase (ALK) mutations, we generated mutations at the F568 and R669 residues of LTK, which correspond to ALK F1174 and R1275, respectively (Figure 1B)
Treatment with PNGase F resulted in a reduction in the size of the observed LTK protein, with the major band at,115 kDa shifting to an approximately 100 kDa band (Figure 2B), which is closer to the 92 kDa predicted molecular weight of the protein encoded by the cDNA that was expressed

Summary

Introduction

Receptor tyrosine kinases (RTKs) are cell signal transducers which span the plasma membrane, binding ligands with their extracellular domain [1]. Ligand-binding typically triggers receptor dimerization, which in turn, causes the intracellular kinase domain to become activated. Leukocyte tyrosine kinase (LTK) is a RTK reported to be expressed in pre-B lymphocytes, B lymphocytes, and other hematopoietic cells, as well as brain and placenta [2,3,4,5,6]. It shares significant homology with fellow insulinreceptor superfamily member anaplastic lymphoma kinase (ALK) [7]. Two decades after its cloning much remains unknown regarding this protein’s function, largely because a ligand for LTK has not been identified yet

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