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Updated: Aug 9, 2025
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Burst kinetics and CNNM binding are evolutionarily conserved properties of phosphatases of regenerating liver
Rayan Fakih1, Robert H Goldstein1, Guennadi Kozlov1
1Department of Biochemistry, Centre for Structural Biology, McGill University, Montreal, Quebec, Canada.
Abstract:
Phosphatases of regenerating liver (PRL or PTP4A) are a family of enigmatic protein phosphatases implicated in cell growth and metabolism. Despite their relevance in metastatic cancer, much remains unknown about the PRL family. They act as pseudophosphatases to regulate the CNNM family of magnesium transporters yet also have enzymatic activity on unknown substrates. In mammals, PRLs are mostly found trapped in an intermediate state that regulates their pseudophosphatase activity. Phosphocysteine, which is formed as an intermediate in the phosphatase catalytic cycle, is inefficiently hydrolyzed leading to burst enzyme kinetics and turnover numbers of less than one per hour. In flies, PRLs have recently been shown to have neuroprotective and neurodevelopmental roles raising the question whether they act as phosphatases, pseudophosphatases, or both. Here, we characterize the evolutionary development of PRLs and ask whether their unique structural and functional properties are conserved. We purified recombinant PRL proteins from 15 phylogenetically diverse organisms and characterized their catalytic activities and ability to bind CNNM proteins. We observed PRLs from humans to amoebae form a stable phosphocysteine intermediate and exhibit burst kinetics. Isothermal titration calorimetry experiments confirmed that the PRL-CNNM interaction is broadly conserved with nanomolar affinity in vertebrates. Lastly, we determined the crystal structure of the Drosophila melanogaster PRL-CNNM complex and identified mutants that specifically impair either phosphatase activity or CNNM binding. Our results reveal the unique properties of PRLs are conserved throughout the animal kingdom and open the door to using model organisms to dissect PRL function in cell signaling.
Insights
Phosphatases of regenerating liver (PRL) proteins are conserved across animals, acting as both enzymes and regulators of magnesium transporters. Their unique burst kinetics and pseudophosphatase activity are maintained from humans to amoebae.
Area of Science:
- Biochemistry
- Molecular Biology
- Evolutionary Biology
Background:
- Phosphatases of regenerating liver (PRL) are protein phosphatases involved in cell growth and metabolism.
- Their roles in metastatic cancer and as pseudophosphatases regulating CNNM magnesium transporters are known, but their precise functions remain unclear.
- PRLs in mammals exist in an intermediate state, leading to inefficient phosphocysteine hydrolysis, burst enzyme kinetics, and low turnover rates.
Purpose of the Study:
- To investigate the evolutionary conservation of PRL protein structure and function.
- To determine if the unique properties of PRLs, including their catalytic activity and interaction with CNNM proteins, are conserved across diverse species.
- To explore the dual role of PRLs as phosphatases and pseudophosphatases.
Main Methods:
- Purification of recombinant PRL proteins from 15 phylogenetically diverse organisms.
- Characterization of PRL catalytic activities and binding affinity to CNNM proteins using isothermal titration calorimetry.
- Determination of the crystal structure of the Drosophila melanogaster PRL-CNNM complex.
- Identification of specific mutants affecting phosphatase activity or CNNM binding.
Main Results:
- PRL proteins from humans to amoebae consistently form a stable phosphocysteine intermediate, exhibiting burst kinetics.
- The interaction between PRL and CNNM proteins is broadly conserved, with nanomolar affinity observed in vertebrates.
- Crystal structure analysis revealed specific mutations impacting either PRL's phosphatase activity or its binding to CNNM.
Conclusions:
- The unique structural and functional properties of PRLs, including their burst kinetics and conserved interaction with CNNM proteins, are highly conserved throughout the animal kingdom.
- These findings support the use of model organisms to further elucidate the complex roles of PRLs in cellular signaling.
- PRLs function as both enzymes and pseudophosphatases, with these dual roles being evolutionarily conserved.
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