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

• Published in: The Journal of biological chemistry Vol. 285; no. 1; pp. 95 - 103
• Main Authors: Wang, Peng, Wu, Fang, Ma, Yupo, Li, Liang, Lai, Raymond, Young, Leah C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Amino Acid Substitution - genetics; Cell Line; Cell Survival; Chromatography, Affinity - methods; Chromatography, Liquid; Enzyme Activation; Humans; Molecular Sequence Data; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutation - genetics; Neoplasms - pathology; Phosphorylation; Phosphotyrosine - metabolism; Protein Structure, Secondary; Protein Synthesis, Post-Translational Modification, and Degradation; Protein-Tyrosine Kinases - chemistry; Protein-Tyrosine Kinases - isolation & purification; Protein-Tyrosine Kinases - metabolism; Structure-Activity Relationship; Tandem Mass Spectrometry - methods
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.059758

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.