1. A Split-Abl Kinase for Direct Activation in Cells.
- Author
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Diaz JE, Morgan CW, Minogue CE, Hebert AS, Coon JJ, and Wells JA
- Subjects
- Enzyme Activation drug effects, HEK293 Cells, Humans, Phosphorylation, Phosphotyrosine metabolism, Proto-Oncogene Proteins c-abl genetics, Sirolimus pharmacology, src-Family Kinases genetics, Protein Engineering, Proto-Oncogene Proteins c-abl metabolism
- Abstract
To dissect the cellular roles of individual kinases, it is useful to design tools for their selective activation. We describe the engineering of a split-cAbl kinase (sKin-Abl) that is rapidly activated in cells with rapamycin and allows temporal, dose, and compartmentalization control. Our design strategy involves an empirical screen in mammalian cells and identification of split site in the N lobe. This split site leads to complete loss of activity, which can be restored upon small-molecule-induced dimerization in cells. Remarkably, the split site is transportable to the related Src Tyr kinase and the distantly related Ser/Thr kinase, AKT, suggesting broader applications to kinases. To quantify the fold induction of phosphotyrosine (pTyr) modification, we employed quantitative proteomics, NeuCode SILAC. We identified a number of known Abl substrates, including autophosphorylation sites and novel pTyr targets, 432 pTyr sites in total. We believe that this split-kinase technology will be useful for direct activation of protein kinases in cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
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