Functional characterization of the kinase activation loop in nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) using tandem affinity purification and liquid chromatography-mass spectrometry.
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 >or=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.
Wang
Peng
P
Department of Laboratory Medicine and Pathology, University of Alberta and Cross Cancer Institute, Edmonton, Alberta T6G 2Z2, Canada.
Wu
Fang
F
Ma
Yupo
Y
Li
Liang
L
Lai
Raymond
R
Young
Leah C
LC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000473:pathology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship; D053719:Tandem Mass Spectrometry / Q000379:methods*
D050505:Mutant Proteins; D019000:Phosphotyrosine; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases