Regulation of Nicotiana tabacum osmotic stress-activated protein kinase and its cellular partner GAPDH by nitric oxide in response to salinity

Abstract

Several studies focusing on elucidating the mechanism of NO (nitric oxide) signalling in plant cells have highlighted that its biological effects are partly mediated by protein kinases. The identity of these kinases and details of how NO modulates their activities, however, remain poorly investigated. In the present study, we have attempted to clarify the mechanisms underlying NO action in the regulation of NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SNF1 (sucrose non-fermenting 1)-related protein kinase 2 family. We found that in tobacco BY-2 (bright-yellow 2) cells exposed to salt stress, NtOSAK is rapidly activated, partly through a NO-dependent process. This activation, as well as the one observed following treatment of BY-2 cells with the NO donor DEA/NO (diethylamine-NONOate), involved the phosphorylation of two residues located in the kinase activation loop, one being identified as Ser158. Our results indicate that NtOSAK does not undergo the direct chemical modifications of its cysteine residues by S-nitrosylation. Using a co-immunoprecipitation-based strategy, we identified several proteins present in immunocomplex with NtOSAK in salt-treated cells including the glycolytic enzyme GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Our results indicate that NtOSAK directly interacts with GAPDH in planta. Furthermore, in response to salt, GAPDH showed a transient increase in its S-nitrosylation level which was correlated with the time course of NtOSAK activation. However, GADPH S-nitrosylation did not influence its interaction with NtOSAK and did not have an impact on the activity of the protein kinase. Taken together, the results support the hypothesis that NtOSAK and GAPDH form a cellular complex and that both proteins are regulated directly or indirectly by NO.

Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Cadmium-Stressed Arabidopsis Roots

“…Although complex formation in the nucleus was not unaffected by stress conditions or NO donors, the interaction was abolished when both active-site cysteines of GAPDH were mutated into serines (Wawer et al, 2010). These studies were performed with Arabidopsis protoplasts expressing GAPDH under the control of the p35S promoter (Holtgrefe et al, 2008;Wawer et al, 2010), and in similar experiments, we could nicely confirm the nuclear localization of GAPC1-YFP expressed under the control of p35S (data not shown). However, replacing this constitutive strong promoter with the GAPC1 endogenous promoter largely prevented, in control conditions, the nuclear accumulation of GAPC1 (Supplemental Fig.…”

“…In animal cells, mechanisms independent on S-nitrosylation of catalytic Cys were also proposed for nuclear localization of GAPDH under several conditions (Sirover, 2011). Interestingly, Wawer et al (2010) also found that nuclear localization of the GAPDH-NtOSAK complex was not stimulated by NO donors, though complex formation did depend, somehow, on GAPDH catalytic Cys. Interestingly, in mammalian cells, several nonmetabolic functions have been reported for GAPDH in the nucleus, including posttranscriptional regulation and apoptosis (Sirover, 2011;Tristan et al, 2011).…”

“…Holtgrefe et al (2008) have proposed that, in Arabidopsis protoplasts, both GAPC1 and GAPC2 could localize also in the nucleus, in addition to the cytosol, and bind DNA. In the same Arabidopsis system, tobacco (Nicotiana tabacum) cytosolic GAPDH was shown to interact, both in the cytosol and in the nucleus, with an osmotic stress-activated protein kinase (NtOSAK) from tobacco too (Wawer et al, 2010). Although complex formation in the nucleus was not unaffected by stress conditions or NO donors, the interaction was abolished when both active-site cysteines of GAPDH were mutated into serines (Wawer et al, 2010).…”

“…In the same Arabidopsis system, tobacco (Nicotiana tabacum) cytosolic GAPDH was shown to interact, both in the cytosol and in the nucleus, with an osmotic stress-activated protein kinase (NtOSAK) from tobacco too (Wawer et al, 2010). Although complex formation in the nucleus was not unaffected by stress conditions or NO donors, the interaction was abolished when both active-site cysteines of GAPDH were mutated into serines (Wawer et al, 2010). These studies were performed with Arabidopsis protoplasts expressing GAPDH under the control of the p35S promoter (Holtgrefe et al, 2008;Wawer et al, 2010), and in similar experiments, we could nicely confirm the nuclear localization of GAPC1-YFP expressed under the control of p35S (data not shown).…”

Le monoxyde d’azote

“…À ce jour, une cinquantaine de protéines S-nitrosylées a ainsi été identifiée, dont une dizaine ont fait l'objet d'études fonctionnelles approfondies (Tableau I). Il ressort de ces études que la S-nitrosylation module l'activité de ces protéines en ciblant directement des résidus Cys de sites actifs [24][25][26][27][28], en promouvant la formation de ponts disulfures [29,30], et en bloquant la fixation de cofacteurs (FAD ou ATP) par encombrement stérique [31,32]. Les données acquises pour les protéines NPR1 (nonexpressor of pathogenesis-related gene 1) et RBOHD (respiratory burst oxidase homologue D) sont détaillées ci-dessous à titre d'exemple (Figure 2).…”