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Date of Award
Mary K. Pflum
Kinases and phosphatases are the key enzymes governing protein phosphorylation, which plays a major role in cell signaling. Unregulated cell signaling can cause diseases, such as cancer. Therefore, characterizing the function of kinase and phosphatase activities in cell signaling is vital. Unfortunately, limited methods are available to identify kinase and phosphatase substrates. To address this need, the Pflum lab developed a series of methods based on the ability of kinases to accept ɤ- phosphoryl modified ATP analogs as a cosubstrate to label cellular protein. Related to phosphatases, K-BIPS (Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates) was tested to identify substrates of tyrosine phosphatase PTP1B by combining ATP-biotin and RNA interference-mediated phosphatase inactivation. From the mass spectrometry analysis, eight high confident possible protein hits were identified including PKM (pyruvate kinase PK – a known substrate of PTP1B) strengthening the K-BIPS as a working method. LDHA also was validated as a new substrate of PTP1B. As a second application of kinase catalyzed labeling, K-CLASP (Kinase Catalyzed CrossLinking And Streptavidin Purification) was applied to identify the kinases of the CARP-1, HDAC1 and MrpC proteins using ATP-arylazide for collaboration projects. The K-CLASP method was able to identify kinases and associated proteins for CARP-1 and HDAC1, proving that K-CLASP is a valuable tool to identify kinases in known phosphosite. Interestingly, several uncharacterized proteins and bifunctional enzyme were identified for MrpC in Myxococcus xanthus lysate. As a third and final application of ATP analogs, ATP-biotin was tested to label human histidine kinases (NME1 and NME2), which confirmed that both kinases are successfully biotinylated. In summary, kinase-catalyzed labeling methods are empowering tools to characterize cell signaling events of kinases, phosphatases, and their substrates.
Punchi Naide Acharige, Nuwan Chinthaka Perera, "Kinase-Catalyzed Labeling To Study Phosphatase Substrates, Phosphosite-Specific Kinases, And Histidine Kinases" (2020). Wayne State University Dissertations. 2504.