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Updated: Oct 8, 2025

Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Cellular ATP Levels Determine the Stability of a Nucleotide Kinase
Oliver Brylski1,2,3, Puja Shrestha1,2, Patricia Gnutt3
1Institute of Physical and Theoretical Chemistry, TU Braunschweig, Braunschweig, Germany.
Abstract:
The energy currency of the cell ATP, is used by kinases to drive key cellular processes. However, the connection of cellular ATP abundance and protein stability is still under investigation. Using Fast Relaxation Imaging paired with alanine scanning and ATP depletion experiments, we study the nucleotide kinase (APSK) domain of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) synthase, a marginally stable protein. Here, we show that the in-cell stability of the APSK is determined by ligand binding and directly connected to cellular ATP levels. The observed protein stability change for different ligand-bound states or under ATP-depleted conditions ranges from ΔGf 0 = -10.7 to +13.8 kJ/mol, which is remarkable since it exceeds changes measured previously, for example upon osmotic pressure, cellular stress or differentiation. The results have implications for protein stability during the catalytic cycle of APS kinase and suggest that the cellular ATP level functions as a global regulator of kinase activity.
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