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Identification of Upstream Kinases by Fluorescence Complementation Mass Spectrometry
Lingfei Zeng1, Wen-Horng Wang1, Justine Arrington2
1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, United States.
ACS Central Science
|November 7, 2017
Summary
A new proteomic strategy, fluorescence complementation mass spectrometry (FCMS), identifies kinase-substrate pairs. This method reveals new kinases for cAMP response element-binding protein (CREB), uncovering its roles in signaling pathways.
Area of Science:
- Molecular Biology
- Proteomics
- Cell Signaling
Background:
- Protein kinases regulate cellular functions through extensive signaling networks.
- Identifying specific kinase-substrate interactions and responsible kinases for phosphorylation events remains challenging.
- High-throughput methods for systematic kinase-substrate pair identification are needed.
Purpose of the Study:
- To develop and validate a novel proteomic strategy, fluorescence complementation mass spectrometry (FCMS), for high-throughput identification of kinase-substrate pairs.
- To apply FCMS for identifying kinases of the transcription factor cAMP response element-binding protein (CREB).
Main Methods:
- Developed fluorescence complementation mass spectrometry (FCMS) using fluorescence-fused substrates and kinase libraries.
- Stabilized transient kinase-substrate interactions in living cells via fluorescence protein fragment association.
- Isolated, identified, and quantified kinase-substrate pairs using Liquid Chromatography-Mass Spectrometry (LC-MS).
Main Results:
- Successfully applied FCMS to identify known and novel kinases of cAMP response element-binding protein (CREB).
- Validated novel CREB kinases using in vitro kinase assays.
- Unambiguously located phosphorylation sites on CREB, revealing potential new signaling pathway roles.
Conclusions:
- FCMS is a powerful and specific proteomic strategy for high-throughput kinase-substrate pair identification.
- The study uncovered novel kinases for CREB, expanding understanding of its regulatory networks.
- FCMS demonstrates significant potential for dissecting complex cellular signaling pathways.
