Abstract
Reversible protein phosphorylation, catalysed by protein kinases, is the most widely studied post-translational modification (PTM), whereas the analysis of other modifications such as S-thiolation is in its relative infancy. In a yeast-two-hybrid (Y2H) screen, we identified a number of novel putative brassinosteroid insensitive 1 (BR1)-associated receptor-like kinase 1 (BAK1) interacting proteins including several proteins related to redox regulation. Glutaredoxin (GRX) C2 (AtGRXC2) was among candidate proteins identified in the Y2H screen and its interaction with recombinant Flag-BAK1 cytoplasmic domain was confirmed using an in vitro pull-down approach. We show that BAK1 peptide kinase activity is sensitive to the oxidizing agents H2O2 and diamide in vitro, suggesting that cysteine oxidation might contribute to control of BAK1 activity. Furthermore, BAK1 was glutathionylated and this reaction could occur via a thiolatedependent reactionwith GSSG or aH2O2-dependent reactionwith GSH and inhibited kinase activity. Surprisingly, both reactions were catalysed by AtGRXC2 at lower concentrations of GSSG or GSH than reacted non-enzymatically. Using MALDI-TOF MS, we identified Cys353, Cys374 and Cys408 as potential sites of glutathionylation on the BAK1 cytoplasmic domain and directed mutagenesis suggests that Cys353 and Cys408 are major sites of GRXC2-mediated glutathionylation. Collectively, these results highlight the potential for redox control of BAK1 and demonstrate the ability of AtGRXC2 to catalyse protein glutathionylation, a function not previously described for any plant GRX. The present work presents a foundation for future studies of glutathionylation of plant receptor-like protein kinases (RLKs) as well as for the analysis of activities of plant GRXs.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 399-413 |
| Number of pages | 15 |
| Journal | Biochemical Journal |
| Volume | 467 |
| Issue number | 3 |
| DOIs | |
| State | Published - May 1 2015 |
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Keywords
- Brassinosteroid insensitive 1-associated receptor-like kinase 1 (BAK1)
- Glutaredoxin
- Glutathionylation
- Receptor-like kinase
- Redox regulation
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
Glutaredoxin AtGRXC2 catalyses inhibitory glutathionylation of arabidopsis BRI1-associated receptor-like kinase 1 (BAK1) in vitro. / Bender, Kyle W.; Wang, Xuejun; Cheng, George B.; Kim, Hyoung Seok; Zielinski, Raymond E; Huber, Steven C.
In: Biochemical Journal, Vol. 467, No. 3, 01.05.2015, p. 399-413.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Glutaredoxin AtGRXC2 catalyses inhibitory glutathionylation of arabidopsis BRI1-associated receptor-like kinase 1 (BAK1) in vitro
AU - Bender, Kyle W.
AU - Wang, Xuejun
AU - Cheng, George B.
AU - Kim, Hyoung Seok
AU - Zielinski, Raymond E
AU - Huber, Steven C
PY - 2015/5/1
Y1 - 2015/5/1
N2 - Reversible protein phosphorylation, catalysed by protein kinases, is the most widely studied post-translational modification (PTM), whereas the analysis of other modifications such as S-thiolation is in its relative infancy. In a yeast-two-hybrid (Y2H) screen, we identified a number of novel putative brassinosteroid insensitive 1 (BR1)-associated receptor-like kinase 1 (BAK1) interacting proteins including several proteins related to redox regulation. Glutaredoxin (GRX) C2 (AtGRXC2) was among candidate proteins identified in the Y2H screen and its interaction with recombinant Flag-BAK1 cytoplasmic domain was confirmed using an in vitro pull-down approach. We show that BAK1 peptide kinase activity is sensitive to the oxidizing agents H2O2 and diamide in vitro, suggesting that cysteine oxidation might contribute to control of BAK1 activity. Furthermore, BAK1 was glutathionylated and this reaction could occur via a thiolatedependent reactionwith GSSG or aH2O2-dependent reactionwith GSH and inhibited kinase activity. Surprisingly, both reactions were catalysed by AtGRXC2 at lower concentrations of GSSG or GSH than reacted non-enzymatically. Using MALDI-TOF MS, we identified Cys353, Cys374 and Cys408 as potential sites of glutathionylation on the BAK1 cytoplasmic domain and directed mutagenesis suggests that Cys353 and Cys408 are major sites of GRXC2-mediated glutathionylation. Collectively, these results highlight the potential for redox control of BAK1 and demonstrate the ability of AtGRXC2 to catalyse protein glutathionylation, a function not previously described for any plant GRX. The present work presents a foundation for future studies of glutathionylation of plant receptor-like protein kinases (RLKs) as well as for the analysis of activities of plant GRXs.
AB - Reversible protein phosphorylation, catalysed by protein kinases, is the most widely studied post-translational modification (PTM), whereas the analysis of other modifications such as S-thiolation is in its relative infancy. In a yeast-two-hybrid (Y2H) screen, we identified a number of novel putative brassinosteroid insensitive 1 (BR1)-associated receptor-like kinase 1 (BAK1) interacting proteins including several proteins related to redox regulation. Glutaredoxin (GRX) C2 (AtGRXC2) was among candidate proteins identified in the Y2H screen and its interaction with recombinant Flag-BAK1 cytoplasmic domain was confirmed using an in vitro pull-down approach. We show that BAK1 peptide kinase activity is sensitive to the oxidizing agents H2O2 and diamide in vitro, suggesting that cysteine oxidation might contribute to control of BAK1 activity. Furthermore, BAK1 was glutathionylated and this reaction could occur via a thiolatedependent reactionwith GSSG or aH2O2-dependent reactionwith GSH and inhibited kinase activity. Surprisingly, both reactions were catalysed by AtGRXC2 at lower concentrations of GSSG or GSH than reacted non-enzymatically. Using MALDI-TOF MS, we identified Cys353, Cys374 and Cys408 as potential sites of glutathionylation on the BAK1 cytoplasmic domain and directed mutagenesis suggests that Cys353 and Cys408 are major sites of GRXC2-mediated glutathionylation. Collectively, these results highlight the potential for redox control of BAK1 and demonstrate the ability of AtGRXC2 to catalyse protein glutathionylation, a function not previously described for any plant GRX. The present work presents a foundation for future studies of glutathionylation of plant receptor-like protein kinases (RLKs) as well as for the analysis of activities of plant GRXs.
KW - Brassinosteroid insensitive 1-associated receptor-like kinase 1 (BAK1)
KW - Glutaredoxin
KW - Glutathionylation
KW - Receptor-like kinase
KW - Redox regulation
UR - http://www.scopus.com/inward/record.url?scp=84932165836&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84932165836&partnerID=8YFLogxK
U2 - 10.1042/BJ20141403
DO - 10.1042/BJ20141403
M3 - Article
C2 - 25678081
AN - SCOPUS:84932165836
VL - 467
SP - 399
EP - 413
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 3
ER -