S-Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)

Jian-Zhong Liu,Jicheng Duan,Min Ni,Zhen Liu,Wen-Li Qiu,Steven A Whitham,Wei-Jun Qian

doi:10.1074/jbc.m117.803882

Abstract

It is well known that the reactive oxygen species NO can trigger cell death in plants and other organisms, but the underlying molecular mechanisms are not well understood. Here we provide evidence that NO may trigger cell death in tomato (Solanum lycopersicum) by inhibiting the activity of phosphoinositide-dependent kinase 1 (SlPDK1), a conserved negative regulator of cell death in yeasts, mammals, and plants, via S-nitrosylation. Biotin-switch assays indicated that SlPDK1 is a target of S-nitrosylation. Moreover, the kinase activity of SlPDK1 was inhibited by S-nitrosoglutathione in a concentration-dependent manner, indicating that SlPDK1 activity is abrogated by S-nitrosylation. The S-nitrosoglutathione-induced inhibition was reversible in the presence of a reducing agent but additively enhanced by hydrogen peroxide (H2O2). Our LC-MS/MS analyses further indicated that SlPDK1 is primarily S-nitrosylated on a cysteine residue at position 128 (Cys128), and substitution of Cys128 with serine completely abolished SlPDK1 kinase activity, suggesting that S-nitrosylation of Cys128 is responsible for SlPDK1 inhibition. In summary, our results establish a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1.

Highlights

It is well known that the reactive oxygen species NO can trigger cell death in plants and other organisms, but the underlying molecular mechanisms are not well understood
To test whether the SlPDK1 is S-nitrosylated in planta, the FLAG–SlPDK1 fusion protein was transiently expressed in NbGSNOR1-silenced Nicotiana benthamiana leaves via Agrobacterium infiltration
It has been reported that loss of GSNOR1 results in a substantial increase in the level of protein S-nitrosylation and increased cell death in response to pathogen infections both in mammals and plants [7, 19, 42]

Summary

Edited by Joseph Jez

It is well known that the reactive oxygen species NO can trigger cell death in plants and other organisms, but the underlying molecular mechanisms are not well understood. We provide evidence that NO may trigger cell death in tomato (Solanum lycopersicum) by inhibiting the activity of phosphoinositide-dependent kinase 1 (SlPDK1), a conserved negative regulator of cell death in yeasts, mammals, and plants, via S-nitrosylation. Our results establish a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1. The PKB/Akt homolog Adi (AvrPto-dependent Pto-interacting protein 3) physically interacts with and is phosphorylated by SlPDK1 [39] Silencing both SlPDK1 and Adi or treatment with a PDK1 inhibitor results in MAPKK kinase ␣– dependent cell death, indicating that Adi functions analogously to the mammalian PKB/Akt by negatively regulating cell death via PDK1 phosphorylation [39]. Our results suggest that inhibition of SlPDK1 activity by S-nitrosylation at Cys128 might be a key molecular event underpinning NO-triggered cell death

Results

Discussion

Plant materials

Kinase assays