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Updated: Jun 20, 2025
Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Kinase-catalyzed biotinylation for discovery and validation of substrates to multispecificity kinases NME1 and NME2
Chelsea R Gary1, Nuwan P N Acharige1, Tolulope O Oyewumi1
1Department of Chemistry, Wayne State University, Detroit, Michigan, USA.
Abstract:
Protein phosphorylation by kinases regulates mammalian cell functions, such as growth, division, and signal transduction. Among human kinases, NME1 and NME2 are associated with metastatic tumor suppression but remain understudied due to the lack of tools to monitor their cellular substrates. In particular, NME1 and NME2 are multispecificity kinases phosphorylating serine, threonine, histidine, and aspartic acid residues of substrate proteins, and the heat and acid sensitivity of phosphohistidine and phosphoaspartate complicates substrate discovery and validation. To provide new substrate monitoring tools, we established the γ-phosphate-modified ATP analog, ATP-biotin, as a cosubstrate for phosphorylbiotinylation of NME1 and NME2 cellular substrates. Building upon this ATP-biotin compatibility, the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates method enabled validation of a known substrate and the discovery of seven NME1 and three NME2 substrates. Given the paucity of methods to study kinase substrates, ATP-biotin and the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates method are valuable tools to characterize the roles of NME1 and NME2 in human cell biology.
Insights
Researchers developed new tools, ATP-biotin and Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (KIBIDS), to identify protein substrates for NME1 and NME2 kinases. These methods aid in understanding their roles in cell functions and tumor suppression.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Protein phosphorylation by kinases is crucial for mammalian cell functions, including growth, division, and signal transduction.
- NME1 and NME2 kinases are implicated in metastatic tumor suppression but are understudied due to limited tools for monitoring their cellular substrates.
- NME1 and NME2 are multispecificity kinases, phosphorylating various residues, with challenges in phosphohistidine and phosphoaspartate detection complicating substrate discovery.
Purpose of the Study:
- To develop novel tools for monitoring cellular substrates of NME1 and NME2 kinases.
- To overcome limitations in detecting specific phosphorylated residues, facilitating substrate discovery and validation.
Main Methods:
- Established a γ-phosphate-modified ATP analog, ATP-biotin, as a cosubstrate for phosphorylbiotinylation.
- Developed the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (KIBIDS) method utilizing ATP-biotin compatibility.
- Applied KIBIDS to validate known substrates and discover novel NME1 and NME2 substrates in cellular contexts.
Main Results:
- Successfully utilized ATP-biotin for phosphorylbiotinylation of NME1 and NME2 substrates.
- Validated a known substrate for NME1/NME2 using the developed method.
- Identified seven novel NME1 substrates and three novel NME2 substrates.
Conclusions:
- ATP-biotin and the KIBIDS method provide valuable tools for characterizing kinase substrates.
- These tools are essential for advancing the study of NME1 and NME2 kinases and their roles in human cell biology and cancer.
- The developed methodology addresses the paucity of methods for studying kinase-substrate interactions.
