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 >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.
Highlights
Residues) of the nucleophosmin (NPM) gene directly upstream of the DNA segment encoding the C-terminal/kinase portion (1058 –1620 amino acid residues) of the anaplastic lymphoma kinase (ALK) gene [4]
All of these 11 tyrosine residues were found in the ALK portion; all of the 3 tyrosine residues (Tyr338, Tyr342, and Tyr343) present in the kinase activation loop (KAL) were found to be phosphorylated, and 8 tyrosine residues found outside the KAL were phosphorylated
The Effects of a Single Tyr-to-Phe Mutation of Tyrosine Residues Outside the KAL—Because we observed that mutations of the KAL resulted in dramatic decreases in the phosphorylation of the 6 effector proteins, we investigated the possibility that 1 of these 8 tyrosine residues may serve as their docking sites, and a Tyr-to-Phe mutation of these sites is expected to be sufficient to abrogate the phosphorylation of these substrates
Summary
Residues) of the nucleophosmin (NPM) gene directly upstream of the DNA segment encoding the C-terminal/kinase portion (1058 –1620 amino acid residues) of the anaplastic lymphoma kinase (ALK) gene [4]. Once the autophosphorylation of the KAL tyrosine residues is achieved, NPM-ALK becomes activated, and subsequent phosphorylation of various tyrosine residues outside of the KAL follows Some of these phosphorylated tyrosine residues have been shown to serve as the docking sites for a number of effector proteins, many of which are known to be involved in cellular signaling. The interaction between NPM-ALK and Shc, an adaptor cellular signaling protein, provides an example to illustrate these concepts It has been demonstrated Shc co-immunoprecipitates with NPM-ALK and site-directed mutagenesis of Tyr567 of NPM-ALK was found to abrogate the Shc/NPM-ALK interaction and reduce the phosphorylation of Shc [1, 18]. We aimed to provide new insights into the functional role of the KAL of NPM-ALK that can serve as a proof of principle for future studies of related oncogenic tyrosine kinases
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