Proteoform-Resolved Phosphorylation Dynamics in Kinase Complexes by Hybrid Precision Mass Spectrometry

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Protein phosphorylation generates diverse proteoforms, but analyzing their temporal dynamics and combinatorial patterns is challenging.
• Understanding phosphorylation is crucial for kinase regulation and cellular signaling pathways.

Purpose of the Study:

• To develop and apply an integrated mass spectrometry (MS) strategy for resolving phosphorylation dynamics within intact kinase complexes.
• To characterize the proteoform landscape and kinetic hierarchies of AMP-activated protein kinase (AMPK) activation.

Main Methods:

• A hybrid precision mass spectrometry (MS) strategy combining intact mass measurements, bottom-up MS, and top-down MS sequencing.
• Analysis of AMP-activated protein kinase (AMPK) as a model system to study phosphorylation cascades.
• Investigating phosphatase competition effects on specific phosphorylation sites.

Main Results:

• Uncovered coordinated autophosphorylation cascades in AMPK with kinetic hierarchies, identifying α1-S496 as highly efficient.
• Demonstrated allosteric activation bypassing canonical phosphorylation sites, enabling autophosphorylation in mutants.
• Identified a predominant β1 proteoform with specific double phosphorylation linked to distribution and responsiveness.
• Showed selective removal of activation-loop phosphorylation by PP1A while autophosphorylation sites were protected.

Conclusions:

• The integrated MS strategy effectively resolves complex phosphorylation dynamics in intact kinase complexes.
• Revealed novel insights into AMPK activation mechanisms, including kinetic hierarchies and allosteric regulation.
• Provides a versatile framework for studying phosphorylation-based regulation in various kinase systems.