IDENTIFICATION OF KINASE SUBSTRATES BY COVALENT CAPTURE OF PHOSPHOPEPTIDES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We will optimize a method for rapid identification of protein kinase substrates. In this method a protein kinase is first engineered to accept an ATP analog that allows it to uniquely label its substrate with a bio-orthogonal phosphate analog. Then we will use a highly specific covalent capture-and-release methodology to rapidly purify tagged peptides derived from labeled substrate proteins. Initial work will focus on the optimization of this technique, followed by application to several kinases in several important pathways including the PI3K mTOR signalling pathway.