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Development and Application of Technologies to Study Individual Kinase Substrate Relationships

  • Author(s): Allen, Jasmina Josie
  • Advisor(s): Shokat, Kevan M
  • et al.
Abstract

The ubiquitous nature of protein phosphorylation makes it challenging to map kinase substrate relationships, which is a necessary step towards defining signaling network architecture. To trace the activity of individual kinases, in complex biological contexts, we developed a semi-synthetic reaction scheme which affords affinity tagged substrates of a kinase in question. A kinase, engineered to utilize a bio-orthogonal ATPγS analog, catalyzes thiophosphorylation of its direct substrates. Alkylation of thiophosphorylated residues creates an epitope for thiophosphate ester specific antibodies. A primary sequence context independent, high affinity rabbit monoclonal antibody was elicited against a hapten which presented the modification on a simple alkyl linker. The generality of semi-synthetic epitope construction was demonstrated with diverse in collaboration with multiple researchers. Labelled kinase substrates were analyzed with a number immunoassays and could be immunopurified and identified. By incorporating an isotopic label, at the step of thiophosphate alkylation, we were able to quantify phosphorylation events with mass spectrometry. To improve this methodology and discover new biological targets of ATP analogs, which are naturally occurring derivatives of plant hormones, we identified casein kinase 2 as an endogenous ATP analog utilizing enzyme. The tools we developed and insights we discovered advance field of kinase-substrate and kinase-ATP interactions.

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