Phosphorylated WNK kinase networks in recoded bacteria recapitulate physiological function

• Published in: Cell reports (Cambridge) 2021-07, Vol.36 (3), p.109416-109416, Article 109416
• Main Authors: Schiapparelli, Paula, Pirman, Natasha L., Mohler, Kyle, Miranda-Herrera, Pierre A., Zarco, Natanael, Kilic, Onur, Miller, Chad, Shah, Sagar R., Rogulina, Svetlana, Hungerford, William, Abriola, Laura, Hoyer, Denton, Turk, Benjamin E., Guerrero-Cázares, Hugo, Isaacs, Farren J., Quiñones-Hinojosa, Alfredo, Levchenko, Andre, Rinehart, Jesse
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cell Line, Tumor; Cell Movement - drug effects; Cell Proliferation - drug effects; Escherichia coli - metabolism; Female; Glioblastoma - pathology; glioblastoma cell migration; HEK293 Cells; Humans; kinase; Male; Mice; Mice, Nude; Middle Aged; Phosphorylation - drug effects; Phosphoserine - metabolism; Protein Serine-Threonine Kinases - chemistry; Protein Serine-Threonine Kinases - metabolism; Recombinant Proteins - metabolism; Signal Transduction - drug effects; Small Molecule Libraries - pharmacology; small-molecule kinase inhibitor; SPAK; Substrate Specificity; synthetic biology; WNK Lysine-Deficient Protein Kinase 1 - metabolism; WNK1
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2021.109416

Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria. We used this approach to identify biochemical properties of WNK kinases, a motif for SPAK substrates, and small-molecule kinase inhibitors for phosphorylated SPAK. We show that the kinase inhibitors modulate SPAK substrates in cells, alter cell volume, and reduce migration of glioblastoma cells. Our work establishes a protein-engineering platform technology that demonstrates that synthetically active WNK kinase networks can accurately model cellular systems and can be used more broadly to target networks of phosphorylated proteins for research and discovery. [Display omitted] •Bacterial strain with expanded genetic code incorporates phosphoserine into WNK1•Synthetically phosphorylated WNK1 activates SPAK and enables substrate discovery•Active WNK1/SPAK pathway yields a screen for small-molecule kinase inhibitors•Inhibitors modulate WNK/SPAK control of cell volume and reduce GBM migration Schiapparelli et al. describe a protein-engineering platform technology to synthetically activate the WNK/SPAK/OSR1 kinase network. Using this approach, they identify biochemical properties of WNK and SPAK kinases along with small-molecule inhibitors for SPAK. Cellular systems, both in vitro and in vivo, translate findings from the engineered kinase network.