Functional Characterization of the Kinase Activation Loop in Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Using Tandem Affinity Purification and Liquid Chromatography-Mass Spectrometry

Abstract

Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of >1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK.

Fusion Tyrosine Kinase NPM-ALK Deregulates MSH2 and Suppresses DNA Mismatch Repair Function

“…NPM-ALK Y191 has not been identified as contributing to any previously reported NPM-ALK activated signaling pathway, thus minimizing the contribution of off-target effects, and the Y191F mutation does not result in reduced NPM-ALK conferred growth advantage. 31 Compared with native NPM-ALK, transient transfection of the NPM-ALK Y191F mutant conferred a significantly lower suppressive effect on MMR function ( Figure 3B), demonstrating that the binding between MSH2 and NPM-ALK is essential for mediating NPM-ALK-induced MMR suppression. The observed decrease in cell viability (thus, a partial restoration in MMR function) on mutation of NPM-ALK at tyrosine 191 ( Figure 3B) is in agreement with the 60% reduction in MSH2-binding observed for NPM-ALK Y191F ( Figure 3A).…”

“…9 Site-directed mutagenesis (Y¡F) was used to change one or more of the three tyrosine residues of NPM-ALK in kinase activation loop (including Y338, Y342, and Y343) to phenylalanine (F). 31 Specifically, mutation of all these three tyrosine residues (termed FFF in figures) resulted in a loss of i) NPM-ALK phosphorylation, ii) phosphorylation of many known NPM-ALK downstream targets, and iii) NPM-ALK-induced growth advantage on clonogenic assay. 31 …”

“…31 Specifically, mutation of all these three tyrosine residues (termed FFF in figures) resulted in a loss of i) NPM-ALK phosphorylation, ii) phosphorylation of many known NPM-ALK downstream targets, and iii) NPM-ALK-induced growth advantage on clonogenic assay. 31 …”

“…Because NPM-ALK is known to interact with other proteins primarily through its phosphorylated tyrosine residues, we hypothesized that mutation (ie, replaced by phenylalanine) of the one of the tyrosine residues involved in phosphorylation may decrease the NPM-ALK·MSH2 binding. Of the eight tyrosine residues that are outside the kinase activation loop of ALK and are known to be involved in phosphorylation, 31,39 only NPM-ALK Y191 showed an appreciable (60% reduction by densitometry) decrease in the NPM-ALK·MSH2 interaction ( Figure 3A). NPM-ALK Y191 has not been identified as contributing to any previously reported NPM-ALK activated signaling pathway, thus minimizing the contribution of off-target effects, and the Y191F mutation does not result in reduced NPM-ALK conferred growth advantage.…”

“…To address this question, we used a panel of NPM-ALK mutants in which one of more of the three tyrosine residues in the kinase activation loop had been replaced by phenylalanine (Y¡F mutations). 31 Mutation within the kinase activation loop alters the autophosphorylation of NPM-ALK, and mutation of all three residues (FFF mutant) abrogates NPM-ALK autophosphorylation and NPM-ALK-induced growth advantage. 31 As shown in Figure 6A, affinity purification and subsequent immunoblot analysis of various NPM-ALK mutants was performed.…”