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Open Access Embargo

Date of Award


Degree Type


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First Advisor

Mary Kay Pflum


Post translational modifications regulate a variety of biological processes inside the cell.Protein phosphorylation is one such PTM modification catalyzed by protein kinases, which aid to transfer a signal from one place to another inside the cell. However, irregularities in kinase-mediated signaling are often implicated in many diseases, making kinases effective drug targets. To understand kinase-related disease formation and to discover drugs to treat these diseases, it is crucial to have a clear understanding on kinase-mediated cell signaling networks. A current gap in the kinase biology field is a lack of tools to identify which kinase phosphorylates which protein substrate inside the cell. To fill this gap in the kinase biology field, the Pflum Lab previously established kinasecatalyzed crosslinking to covalently link kinases and substrates using ɤ-modified ATPphotocrosslinkers, such as ATP-arylazide (ATP-ArN3). As the next step, we developed and validated kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP), a powerful tool to identify kinase-substrate pairs and interactors. K-CLIP with ATP-ArN3 was applied to the substrate p53 in cell lysates. Both known and unknown kinases of p53 were identified, demonstrating that K-CLIP is a valuable tool to discover kinases-substrate pairs. In addition, we used K-CLIP to discover both the kinases and substrates of BRAF kinase. Further, we developed a new affinity-based ATP analog, ATP-methylacrylamide (ATPMAc). We demonstrated that ATP-MAc selectively crosslinked cysteine-containing kinases with their substrates using, EGFR, FGFR4 and ERK2 kinases. We next applied ATP-MAc in lysate-based K-CLIP to identify novel substrates of EGFR kinase. Based on the LC-MS/MS analysis followed by secondary validation with in-vitro kinase assays, we discovered that SAM68 substrate of EGFR kinase. Finally, we performed K-CLIP in live cells using ATP-MAc to potentially identify kinases and substrates of EGFR in a physiologically relevant manner. Based on LC-MS/MS analysis followed by secondary validation with in vitro kinase assays, we discovered that MEK5 is a direct kinase of EGFR. The results demonstrate that K-CLIP is a viable method for kinase substrate identification. In summary, we have developed a powerful chemical tool to monitor phosphorylation events in both cell lysates and in living cells, which is a current need in the kinase biology field.