Abstract
Activating mutations in full length anaplastic lymphoma kinase (ALK) have been reported in neuroblastoma and in anaplastic thyroid cancer. ALK-L1198F and ALK-G1201E mutations were originally identified in anaplastic thyroid cancer (ATC) and characterized as constitutively activating mutations. In this study, we employed in vitro cell culture assays together with biochemical and in vivo Drosophila analyses to characterize their sensitivity to either activation by the FAM150A (AUG-β) and FAM150B (AUG-α) ALK ligands or inhibition by ALK inhibitors. Here we report that neither ALK-L1198F nor ALK-G1201E mutations result in ligand independent gain-of-function (GOF) activity in either in vitro biochemical analysis or the various model systems employed. ALK-L1198F is activated by the FAM150 (AUG) ligands and its ligand-dependant activity is similar to the wild type full length ALK receptor. ALK-G1201E is only very weakly activated by the FAM150 (AUG) ligands, most likely due to impaired protein stability. We conclude that neither ALK-L1198F nor ALK-G1201E displays ligand independent kinase activity, with ALK-L1198F belonging to the class of ligand dependent ALK mutations which are not constitutively active but that responds to ligand activation, while the ALK-G1201E mutation generates an unstable receptor with very low levels of kinase activity.
Highlights
Anaplastic lymphoma kinase (ALK) belongs to the insulin receptor kinase subfamily of receptor tyrosine kinases [1], and was originally identified as a fusion protein with nucleophosmin (NPM) in anaplastic large cell lymphoma (ALCL) [2, 3]
We conclude that neither anaplastic lymphoma kinase (ALK)-L1198F nor ALK-G1201E displays ligand independent kinase activity, with ALK-L1198F belonging to the class of ligand dependent ALK mutations which are not constitutively active but that responds to ligand activation, while the ALK-G1201E mutation generates an unstable receptor with very low levels of kinase activity
ALK-L1198F and ALK-G1201E have been reported as mutations with constitutive ALK tyrosine kinase activity in anaplastic thyroid cancer (ATC) [12]
Summary
Anaplastic lymphoma kinase (ALK) belongs to the insulin receptor kinase subfamily of receptor tyrosine kinases [1], and was originally identified as a fusion protein with nucleophosmin (NPM) in anaplastic large cell lymphoma (ALCL) [2, 3]. The full length ALK receptor possesses an extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase domain (TKD), and is activated by the FAM150A (AUG-β) and FAM150B (AUG-α) ligands [4, 5]. Most neuroblastoma mutations are situated within the kinase domain of ALK, mainly located around the α-Chelix and the activation loop. The ALK mutations found in ATC are located in the vicinity of the ATP-binding site of the kinase domain/hinge region between the Nand C-lobes (Figure 1A). ALK-positive neuroblastoma mutants fall into three classes: gain-of-function (GOF) ligand independent mutations, ligand dependent mutations which are not constitutively active and require activation with either FAM150 (AUG) ligands or agonist antibodies, and kinase-dead mutations [13]. Since the FAM150 (AUG) ligands are able to drive further activation of ALK mutants from neuroblastoma, dysregulation of the ALK ligands may potentially play a role in neuroblastoma [4]
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