Publications

• Protein AMPylation by an Evolutionarily Conserved Pseudokinase.

  Sreelatha A., Yee S.S., Lopez V.A., Park B.C., Kinch L.N., Pilch S., Servage K.A., Zhang J., Jiou J., Karasiewicz-Urbanska M., Lobocka M., Grishin N.V., Orth K., Kucharczyk R., Pawlowski K., Tomchick D.R., Tagliabracci V.S.

  Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates ( ... >> More

  Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities. << Less

  Cell 175:809-821(2018) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Kinetic and structural insights into the mechanism of AMPylation by VopS Fic domain.

  Luong P., Kinch L.N., Brautigam C.A., Grishin N.V., Tomchick D.R., Orth K.

  The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylation of a conserved threonine residue in the switch 1 region of R ... >> More

  The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylation of a conserved threonine residue in the switch 1 region of Rho GTPases. The modified GTPases are no longer able to interact with downstream effectors due to steric hindrance by the covalently linked AMP moiety. Herein we analyze the structure of VopS and its evolutionarily conserved catalytic residues. Steady-state analysis of VopS mutants provides kinetic understanding on the functional role of each residue for AMPylation activity by the Fic domain. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. Discovery of a ternary reaction mechanism along with structural insight provides critical groundwork for future studies for the family of AMPylators that modify hydroxyl-containing residues with AMP. << Less

  J. Biol. Chem. 285:20155-20163(2010) [PubMed] [EuropePMC]

• AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling.

  Yarbrough M.L., Li Y., Kinch L.N., Grishin N.V., Ball H.L., Orth K.

  The Vibrio parahaemolyticus type III effector VopS is implicated in cell rounding and the collapse of the actin cytoskeleton by inhibiting Rho guanosine triphosphatases (GTPases). We found that VopS could act to covalently modify a conserved threonine residue on Rho, Rac, and Cdc42 with adenosine ... >> More

  The Vibrio parahaemolyticus type III effector VopS is implicated in cell rounding and the collapse of the actin cytoskeleton by inhibiting Rho guanosine triphosphatases (GTPases). We found that VopS could act to covalently modify a conserved threonine residue on Rho, Rac, and Cdc42 with adenosine 5'-monophosphate (AMP). The resulting AMPylation prevented the interaction of Rho GTPases with downstream effectors, thereby inhibiting actin assembly in the infected cell. Eukaryotic proteins were also directly modified with AMP, potentially expanding the repertoire of posttranslational modifications for molecular signaling. << Less

  Science 323:269-272(2009) [PubMed] [EuropePMC]