Glutaredoxin GrxC2 catalyzes the glutathionylation and inactivation of Arabidopsis BRI1-ASSOCIATED RECEPTOR-LIKE KINASE 1 (BAK1) in vitro

Authors: BENDER, KYLE - University Of Illinois; WANG, XUEJUN - University Of Illinois; CHENG, GEO BIING-WEN - University Of Illinois; KIM, HYOUNG SEOK - University Of Illinois; ZIELINSKI, RAYMOND - University Of Illinois; Huber, Steven

Submitted to: Biochemical Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/1/2015
Publication Date: 5/1/2015
Citation: Bender, K.W., Wang, X., Cheng, G., Kim, H., Zielinski, R.E., Huber, S.C. 2015. Glutaredoxin GrxC2 catalyzes the glutathionylation and inactivation of Arabidopsis BRI1-ASSOCIATED RECEPTOR-LIKE KINASE 1 (BAK1) in vitro. Biochemical Journal. 467(3):399-413.

Interpretive Summary: Plants contain a large family of receptor kinases that regulate many aspects of plant growth and development and response to stress and the environment. Receptor kinases are proteins that are embedded in the plasma membrane and transmit signals from the outside of the cell to the interior. The signals are specific molecules referred to ligands that bind to the extracellular portion of the receptor kinase. In plants, binding of the ligand induces the receptor kinase to interact with a specific co-receptor kinase, which then results in their mutual activation by a process involving protein phosphorylation and perhaps other post-translational modifications. Additional proteins can bind to the receptor kinase:co-receptor kinase to generate a signaling complex. Thus, identifying proteins that can physically interact with receptor kinases is an important approach to determine which proteins may be part of the signaling complex. The classic experimental approach known as the yeast two-hybrid system was used to identify proteins that interact with the cytoplasmic domain of the BAK1 receptor kinase. A number of potential interacting proteins were identified, including several that suggested that BAK1 might be regulated by reduction-oxidation of cysteine residues. One putative interacting protein, known as glutaredoxin C2, was studied in detail. Glutaredoxin C2 was confirmed as an interacting protein in vitro, and was shown to catalyze the covalent addition of a glutathione molecule to specific cysteine residues on BAK1. These results are the first demonstration of S-glutathionylation of a plant protein kinase and suggest for the first time that redox regulation may play a role in control of BAK1 activity. Moreover, glutaredoxin proteins usually catalyze disulfide reduction (i.e., glutathione removal from a protein), not the covalent addition of glutathionylation as described in the present study. Consequently, it appears that certain members of the glutaredoxin family may have unexpected functions. The function of glutathionylation of BAK1 in vivo remains to be determined, but conceivably could provide another strategy to engineer the properties of this important receptor kinase.

Technical Abstract: Reversible protein phosphorylation, catalyzed by protein kinases, is the most widely studied post-translational modification (PTM) both in terms of its occurrence and the regulatory consequences of phosphorylation events on phosphorylated proteins. In addition to reversible phosphorylation, many proteins in plant and animal systems undergo other types of PTM including acetylation, methylation, and S-thiolation, however, the global analysis of these other modifications in plants is in its relative infancy when compared to phosphorylation. In a yeast-2-hybrid screen, we identified a number of novel putative BAK1 (BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1) interacting proteins including several proteins related to redox regulation, which prompted us to explore the possibility that BAK1 is redox regulated. Using recombinant proteins, we confirmed the interaction between BAK1 and glutaredoxin C2 (AtCRXC2) in vitro. We show that BAK1 peptide kinase activity is sensitive to the oxidizing agents H2O2 and diamide in vitro, suggesting that Cys oxidation inhbits BAK1 kinase activity. Furthermore, BAK1 could be glutathionylated non-enzymatically ia a thiolate-dependent reaction with GSSG or a H2O2-dependent reaction with GSH, with both resulting in inhibition of kinase activity. Unexpectedly, both reactions could be catalyzed by AtGRXC2 such that glutathionylation and inhibition of BAK1 activity occurred at lower concentrations of GSSG or GSH. Using MALDI-TOF mass spectrometry, we identified Cys353, Cys374, and Cys408 as sites of glutathionylation on the BAK1 cytoplasmic domain. Collectively, these results highlight the potential for redox control of BAK1. Importantly, we describe the ability of AtGRXC2 to catalyze protein glutathionylation, a function not previously identified for any plant glutaredoxin. This work presents a foundation for future studies of glutathionylation of plant receptor-like protein kinases as well as for the analysis of activities of plant glutaredoxins.