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Covalent Capture of Kinase Substrate Phosphopeptides for Analysis of Cellular Signaling Networks

  • Author(s): Blethrow, Justin David
  • Advisor(s): Shokat, Kevan M
  • et al.
Abstract

Kinase-mediated protein phosphorylation regulates nearly every cellular signaling pathway. Protein kinases function in the integration and modulation of information flow through the regulation of signaling components and act as direct effectors of cellular processes by controlling the behavior of protein machines and cellular structural components. Approximately one third of all cellular proteins are phosphorylated, and so it is thought that many individual kinases likely have numerous (>100) direct targets. A detailed understanding of this architecture will be required in order to accurately model complex cellular behaviors both normal and aberrant, to reliably predict cellular responses to therapeutic intervention in disease states, and to successfully implement cell or tissue based therapeutic strategies. Such an understanding will require a comprehensive elucidation of biologically relevant kinase-substrate interactions. At present, the discovery of such interactions represents a significant challenge.

In this work we describe a novel method for rapid identification of protein kinase substrates. In this method, a kinase of interest is engineered to accept an ATP analog that allows it to uniquely label its substrates with a bio-orthogonal phosphate analog tag. A highly specific, covalent capture-and-release methodology was developed for rapid purification of tagged peptides derived from labeled substrate proteins. Mass spectrometric analysis of the recovered peptides affords identification of the parent protein species, and often the exact site of phosphorylation. Our experiments show that tagged may be purified from complex mixtures with extremely high specificity. Importantly, we found the sensitivity of the method to be sufficient for the recovery of picomole scale targets from milligrams of cell extract digest. This is a regime suitable for the identification of novel kinase substrates after their specific tagging by as-kinases in cell extracts. Application of this approach to the discovery of Cdk1-Cyclin B substrates in cell extracts yielded identification of over seventy substrates and phosphorylation sites. Many of these sites are known to be phosphorylated in vivo, but most of the proteins have not been previously characterized as Cdk1-Cyclin B substrates. This approach has the potential to expand our understanding of kinase-substrate connections in signaling networks.

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