Biochemical Characterization of the Tobacco 42-kD Protein Kinase Activated by Osmotic Stress

Abstract

In tobacco (Nicotiana tabacum), hyperosmotic stress induces rapid activation of a 42-kD protein kinase, referred to as Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK). cDNA encoding the kinase was cloned and, based on the predicted amino acid sequence, the enzyme was assigned to the SNF1-related protein kinase type 2 (SnRK2) family. The identity of the enzyme was confirmed by immunoprecipitation of the active kinase from tobacco cells subjected to osmotic stress using antibodies raised against a peptide corresponding to the C-terminal sequence of the kinase predicted from the cloned cDNA. A detailed biochemical characterization of NtOSAK purified from stressed tobacco cells was performed. Our results show that NtOSAK is a calcium-independent Ser/Thr protein kinase. The sequence of putative phosphorylation sites recognized by NtOSAK, predicted by the computer program PREDIKIN, resembled the substrate consensus sequence defined for animal and yeast (Saccharomyces cerevisiae) AMPK/SNF1 kinases. Our experimental data confirmed these results, as various targets for AMPK/SNF1 kinases were also efficiently phosphorylated by NtOSAK. A range of protein kinase inhibitors was tested as potential modulators of NtOSAK, but only staurosporine, a rather nonspecific protein kinase inhibitor, was found to abolish the enzyme activity. In phosphorylation reactions, NtOSAK exhibited a preference for Mg 21 over Mn 21 ions and an inability to use GTP instead of ATP as a phosphate donor. The enzyme activity was not modulated by 5#-AMP. To our knowledge, these results represent the first detailed biochemical characterization of a kinase of the SnRK2 family.

Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes

“…Several members of the SnRK3 subfamily have been extensively characterized. One of best studied kinases in the SnRK3 family is SOS2, which is required for sodium and K ϩ homeostasis and abiotic stress tolerance (15). The activity of SnRK3 kinases are regulated in a Ca 2ϩ -dependent manner, and their activity is induced by interactions with a calcineurin-B like Ca 2ϩ binding protein in response to abiotic stress (16)(17)(18).…”

Nitric oxide production and its functional link with OIPK in tobacco defense response elicited by chitooligosaccharide

“…Published papers report that MAPK cascades play an important role in regulating NO signal (She and Song 2008;Asai et al 2008). Others report that protein kinase may play a role downstream of NO signal in plants under normal conditions or environmental stress (Kelner et al 2004;Lamotte et al 2004;Sokolovski et al 2005). …”

Sucrose non-fermenting 1-related protein kinase 2 (SnRK2): a family of protein kinases involved in hyperosmotic stress signaling

“…At least four members of this family were found activated in vivo during hyperosmotic stress, while in a transient expression system nine of them were activated by hyperosmotic stress using mannitol and NaCl (Boudsocq et al, 2004). Based on the amino acid sequence, a 42-kDa protein kinase (NtOSAK) activated by osmotic stress in tobacco (Nicotiana tabacum) has been assigned to the SnRK2 family (Kelner et al, 2004). A maize SnRK2b homologue (ZmSPK1) was shown to be expressed in roots, mature leaves and vassels but most abundantly in the reproductive organs (Zou et al, 2006).…”

“…In vitro treatment with phosphatases has shown that kinase phosphorylation is important for activation: Tobacco homolog of Arabidopsis SnRK2 (ASK1/ SnRK2.4) purified from BY-2 cells was inactivated by protein phosphatase 2A (PP2A) (Miklajczyk et al, 2000;Kelner et al, 2004); alkaline phosphatase abolished salt activation of two rice SnRK2s, SAPK1 and SAPK2 (Kobayashi et al, 2004). Suppression of OST1 kinase activation in response to ABA in the dominant abi1-1 mutant (Mustilli et al, 2002) provided the evidence for negative regulation of ABA signal transduction upstream of OST1 by protein phosphatase 2C (PP2C) ABI1 (Koornneef et al, 1984;Leung et al, 1994).…”

“…bZIP transcription factors, such as TaAbF from wheat (Johnson et al, 2002) and TRAB1 from rice (Kagaya et al, 2002), were shown to be substrates for SnRK2. Prediction for a sequence motif for a substrate recognized by NtOSAK (Kelner et al, 2004) came about using the computer program PREDIKIN (Brinkworth et al, 2003)…”