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Real time characterization of the MAPK pathway using native mass spectrometry
Elena Scott1, Sangho D Yun1, Zahra Moghadamchargari1
1Department of Chemistry, Texas A&M University, College Station, TX, 77843, USA.
Communications Biology
|April 16, 2025
Summary
Native mass spectrometry (MS) offers new insights into the Mitogen-Activated Protein Kinase (MAPK) pathway. This research details protein interactions and nucleotide binding, advancing our understanding of cancer-related signaling.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- The Mitogen-Activated Protein Kinase (MAPK) pathway is vital for cell signaling, connecting external stimuli to internal cellular activities.
- Dysregulation of the MAPK pathway, often due to activating mutations, is implicated in the development of various cancers.
- Existing methods for studying the MAPK pathway are extensive, but native mass spectrometry (MS) offers a novel approach for detailed molecular characterization.
Purpose of the Study:
- To employ native mass spectrometry (MS) for a comprehensive characterization of the MAPK pathway.
- To investigate nucleotide, drug, and protein interactions within the MAPK pathway using native MS.
- To monitor the MAPK phosphorylation cascade in real-time.
Main Methods:
- Utilized native mass spectrometry (MS) to analyze protein complexes and interactions within the MAPK pathway.
- Characterized nucleotide and drug binding to BRAF complexes, assessing the influence of MEK1.
- Examined the complex formation between different CRAF segments and KRAS, including the Ras binding domain (RBD) and cysteine-rich domain (CRD).
Main Results:
- Native MS provided detailed insights into nucleotide and drug interactions with BRAF, showing modulation by MEK1.
- Different CRAF segments exhibited varied complex formation with KRAS; the CRD significantly enhanced binding compared to RBD alone.
- KRAS displayed notably increased GTPase activity when interacting with specific CRAF fragments, indicating functional consequences of these interactions.
- ERK2 was used as a downstream reporter to track the MAPK phosphorylation cascade.
Conclusions:
- Native MS is a powerful tool for detailed characterization of individual MAPK pathway components.
- This technique allows for real-time monitoring of the complex MAPK phosphorylation cascade.
- The findings enhance our understanding of MAPK pathway regulation and its role in diseases like cancer.
