Multi-layered proteomic analyses decode compositional and functional effects of cancer mutations on kinase complexes.

Nature communications (2020-07-18)
Martin Mehnert, Rodolfo Ciuffa, Fabian Frommelt, Federico Uliana, Audrey van Drogen, Kilian Ruminski, Matthias Gstaiger, Ruedi Aebersold
RESUMEN

Rapidly increasing availability of genomic data and ensuing identification of disease associated mutations allows for an unbiased insight into genetic drivers of disease development. However, determination of molecular mechanisms by which individual genomic changes affect biochemical processes remains a major challenge. Here, we develop a multilayered proteomic workflow to explore how genetic lesions modulate the proteome and are translated into molecular phenotypes. Using this workflow we determine how expression of a panel of disease-associated mutations in the Dyrk2 protein kinase alter the composition, topology and activity of this kinase complex as well as the phosphoproteomic state of the cell. The data show that altered protein-protein interactions caused by the mutations are associated with topological changes and affected phosphorylation of known cancer driver proteins, thus linking Dyrk2 mutations with cancer-related biochemical processes. Overall, we discover multiple mutation-specific functionally relevant changes, thus highlighting the extensive plasticity of molecular responses to genetic lesions.

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Triton X-100, laboratory grade
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Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
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Protease Inhibitor Cocktail, for use in purification of Histidine-tagged proteins, DMSO solution
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L-(−)-Glucose, ≥99%
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Suberic acid bis(N-hydroxysuccinimide ester), ≥95%, powder
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Anti-DYRK2 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
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DYRK2 Active human, recombinant, expressed in baculovirus infected insect cells, ≥50% (SDS-PAGE)

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