TY - JOUR
T1 - Phosphorylated WNK kinase networks in recoded bacteria recapitulate physiological function
AU - Schiapparelli, Paula
AU - Pirman, Natasha L.
AU - Mohler, Kyle
AU - Miranda-Herrera, Pierre A.
AU - Zarco, Natanael
AU - Kilic, Onur
AU - Miller, Chad
AU - Shah, Sagar R.
AU - Rogulina, Svetlana
AU - Hungerford, William
AU - Abriola, Laura
AU - Hoyer, Denton
AU - Turk, Benjamin E.
AU - Guerrero-Cázares, Hugo
AU - Isaacs, Farren J.
AU - Quiñones-Hinojosa, Alfredo
AU - Levchenko, Andre
AU - Rinehart, Jesse
N1 - Publisher Copyright:
© 2021 The Author(s)
PY - 2021/7/20
Y1 - 2021/7/20
N2 - 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.
AB - 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.
KW - SPAK
KW - WNK1
KW - glioblastoma cell migration
KW - kinase
KW - small-molecule kinase inhibitor
KW - synthetic biology
UR - http://www.scopus.com/inward/record.url?scp=85110556665&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85110556665&partnerID=8YFLogxK
U2 - 10.1016/j.celrep.2021.109416
DO - 10.1016/j.celrep.2021.109416
M3 - Article
C2 - 34289367
AN - SCOPUS:85110556665
SN - 2211-1247
VL - 36
JO - Cell reports
JF - Cell reports
IS - 3
M1 - 109416
ER -