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Phosphorylated WNK kinase networks in recoded bacteria recapitulate physiological function.

Paula Schiapparelli1, Natasha L Pirman2, Kyle Mohler2

  • 1Department of Neurologic Surgery, Mayo Clinic, Jacksonville, FL 32224, USA.

Cell Reports
|July 21, 2021
PubMed
Summary

Researchers engineered bacteria to produce human phosphoproteins, synthetically activating kinase networks. This protein engineering platform advances research into phosphorylated protein networks and kinase inhibitors for diseases like glioblastoma.

Keywords:
SPAKWNK1glioblastoma cell migrationkinasesmall-molecule kinase inhibitorsynthetic biology

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • Genetic code expansion allows site-specific protein post-translational modifications.
  • Human kinases, including WNK and SPAK/OSR, play critical roles in cellular signaling pathways.

Purpose of the Study:

  • To engineer a bacterial system for producing site-specifically phosphorylated human kinases.
  • To synthetically activate the WNK-SPAK/OSR kinase network in bacteria.
  • To utilize this platform for identifying kinase properties, substrates, and inhibitors.

Main Methods:

  • Utilized a recoded bacterial strain with an expanded genetic code to incorporate phosphoserine.
  • Directly encoded phosphoserine into WNK1 and WNK4 kinases at multiple sites.
  • Investigated the biochemical properties and kinase activities within the engineered bacterial system.

Main Results:

  • Successfully produced activated, phosphorylated WNK kinases in bacteria.
  • Demonstrated synthetic activation of the WNK-SPAK/OSR kinase network.
  • Identified a SPAK substrate motif and developed small-molecule inhibitors for phosphorylated SPAK.
  • Showcased inhibitor efficacy in modulating SPAK substrates, cell volume, and glioblastoma cell migration.

Conclusions:

  • Established a novel protein-engineering platform for studying phosphorylated protein networks.
  • Demonstrated the potential of synthetically active kinase networks to model cellular systems.
  • Highlighted the utility of this platform for discovering kinase inhibitors and advancing cancer research.