Osmotic Stress Induces Rapid Activation of a Salicylic Acid-Induced Protein Kinase and a Homolog of Protein Kinase ASK1 in Tobacco Cells

Mikołajczyk, Monika; Awotunde, Olubunmi S.; Muszyńska, Grażyna; Klessig, Daniel F.; Dobrowolska, Grażyna

doi:10.2307/3871037

Cited by 70 publications

(109 citation statements)

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“…Our previous results concerning NtOSAK of the SnRK2 subfamily (25), the results obtained by the Hattori group on two rice SnRK2s (14), and the very recent ones by Belin et al (26) on Arabidopsis OST1 kinase suggested that these enzymes were activated by phosphorylation. Contrary to this, Hoyos and Zhang (27) claimed that high osmolarity stress-activated kinase from tobacco, whose properties resemble those of NtOSAK, was activated by dephosphorylation.…”

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confidence: 75%

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Nicotiana tabacum Osmotic Stress-activated Kinase Is Regulated by Phosphorylation on Ser-154 and Ser-158 in the Kinase Activation Loop

Burza

Pekala

Sikora

et al. 2006

Journal of Biological Chemistry

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NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SnRK2 subfamily, is activated rapidly in response to hyperosmotic stress. Our previous results as well as data presented by others indicate that phosphorylation is involved in activation of SnRK2 kinases. Here, we have mapped the regulatory phosphorylation sites of NtOSAK by mass spectrometry with collision-induced peptide fragmentation. We show that active NtOSAK, isolated from NaCl-treated tobacco BY-2 cells, is phosphorylated on Ser-154 and Ser-158 in the kinase activation loop. Prediction of the NtOSAK three-dimensional structure indicates that phosphorylation of Ser-154 and Ser-158 triggers changes in enzyme conformation resulting in its activation. The involvement of Ser-154 and Ser-158 phosphorylation in regulation of NtOSAK activity was confirmed by site-directed mutagenesis of NtOSAK expressed in bacteria and in maize protoplasts. Our data reveal that phosphorylation of Ser-158 is essential for NtOSAK activation, whereas phosphorylation of Ser-154 most probably facilitates Ser-158 phosphorylation. The time course of NtOSAK phosphorylation on Ser-154 and Ser-158 in BY-2 cells subjected to osmotic stress correlates with NtOSAK activity, indicating that NtOSAK is regulated by reversible phosphorylation of these residues in vivo. Importantly, Ser-154 and Ser-158 are conserved in all SnRK2 subfamily members, suggesting that phosphorylation at these sites may be a general mechanism for SnRK2 activation.Plants growing in natural conditions are challenged by various environmental stresses (e.g. drought, extreme temperatures, and high salinity), all of which cause osmotic stress in cells. To survive, plants induce various defense mechanisms. Perception of environmental stimuli and proper signal transduction are crucial for plant acclimatization and development. Protein kinases and phosphoprotein phosphatases play a pivotal role in these processes. Therefore, the activity of these enzymes has to be regulated very strictly inside the cell. Besides the enzymes belonging to the MAPK cascades and several other protein kinases conserved in all eukaryotic organisms ranging from yeast to human, plant genomes encode also plant-specific protein kinases, involved in the responses to harsh environmental conditions. Representative of such enzymes are some of the SNF1-related protein kinases (SnRKs). Plant SnRKs are classified into three subfamilies: SnRK1, SnRK2, and SnRK3 (for reviews, see Refs. 4 -6). Enzymes belonging to the SnRK1 group structurally and functionally resemble the yeast and animal SNF1/AMPK kinases, which play a role in protecting cells against nutritional and environmental stresses (for reviews, see Refs. 4,7,and 8). The SnRK2 and SnRK3 subfamilies are specific to plants (for reviews, see Refs. 4 and 5). Amassing data suggest that these enzymes participate in environmental stress signaling. Members of the SnRK3 subfamily, especially those that interact with calcineurin B-like proteins, are relatively well characterized. The...

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confidence: 75%

“…Purification of NtOSAK-NtOSAK was purified from BY-2 cells treated for 5 min with 250 mM NaCl according to the method described previously (25).…”

Section: Methodsmentioning

confidence: 99%

Nicotiana tabacum Osmotic Stress-activated Kinase Is Regulated by Phosphorylation on Ser-154 and Ser-158 in the Kinase Activation Loop

Burza

Pekala

Sikora

et al. 2006

Journal of Biological Chemistry

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“…Arabidopsis suspension cultures were grown in Gamborg B5 medium as described by Yamada et al (24); BY-2 cells were grown under conditions described elsewhere (4,25). Cells were subcultured every 7 days.…”

Section: Methodsmentioning

confidence: 99%

“…Purification of NtOSAK from Tobacco BY-2 Cells-NtOSAK was purified from BY-2 cells treated for 5 min with 250 mM NaCl according to the method described previously (4,5).…”

Section: Methodsmentioning

confidence: 99%

SNF1-related Protein Kinases 2 Are Negatively Regulated by a Plant-specific Calcium Sensor

Bucholc

Ciesielski

Goch

et al. 2011

Journal of Biological Chemistry

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SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb 3؉ as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ؎ 0.9 ؋ 10 5 M ؊1 . The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.Plants respond to environmental stresses by induction of various defense mechanisms. Stress signals are recognized and transmitted to different cellular compartments by specialized signaling pathways in which protein kinases and phosphatases are key components. The SnRK2 family members are plant-specific kinases considered as important regulators of plant response to abiotic stresses. Ten members of the SnRK2 family have been identified in both Arabidopsis thaliana and Oryza sativa (1, 2). All of them, except SnRK2.9 from A. thaliana, were shown by transient expression in protoplasts to be rapidly activated by treatment with different osmolytes, such as sucrose, mannitol, sorbitol, or NaCl and some of them also by abscisic acid (ABA), 3 suggesting that these kinases are involved in a general response to osmotic stress (1-3). It was also reported that in tobacco BY-2 cells Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 subfamily, is activated rapidly in response to hyperosmotic stress (4 -6).Ample data indicate that SnRK2s are positive regulators of plant response to drought. ABA-activated protein kinase (AAPK) is activated by ABA in guard cells of fava bean (Vicia faba) in response to drought and is involved in the regula...

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“…Among three kinases, MAPK has been best characterized in plants, as exemplified by AtMPK3, AtMPK4, AtMPK6 from Arabidopsis, SAMK and SIMK from alfalfa, and WIPK and SIPK from tobacco. [3][4][5][6][7] WIPK (wound induced protein kinase) was originally identified as a wound and pathogen responsive kinase, of which transcripts were induced as early as 10 min after mechanical wounding in both wounded and adjacent unwounded leaves, and considered to play a critical role in stress response in tobacco plants. 8 One of its direct upstream MEKs was shown to be NtMEK2, as its constitutive activation induced WIPK activity together with hypersensitive responsive cell death.…”

Section: Map Kinase Cascadementioning

confidence: 99%

Finding a missing link in MAP kinase cascade

Chung¹,

Sano

2008

Plant Signaling & Behavior

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Mitogen-activated protein kinase (MAPK) cascade is one of the major signaling systems in eukaryotes. External signals are tranduced through three protein kinases, which successively relay phosphorylation to finally activate target genes/proteins. However, few information on targets of MAPK have so far been available. In this study, we identified a novel transcription factor, NtWIF, which is directly phosphorylated by a wound-induced protein kinase (WIPK), a typical MAPK from tobacco plants. Phosphorylated NtWIF recognizes the auxin responsive element (ARE), and transcriptionally activates ARE-driven Luciferase-reporter genes. Transgenic tobacco plants, in which NtWIF was overexpressed or suppressed, showed distinct features not only in pathogen resistance, but also in seed development and root growth. Micro-array assay using transgenic lines identified 178 differentially expressed genes, among which nearly half was related to defense and development. Screening of the available promoter regions revealed that multiple genes encoding such as pathogenesis-related protein Q (PR-Q), β-1,3-glucanase, aminocyclopropane carboxylic acid synthase 2, P-450 and WIPK itself possess the ARE motif. Upon coexpression in cultured cells, NtWIF transcriptionally activated the Luciferase-reporter gene driven by intact promoters of PR-Q and WIPK. Since ARE is commonly found in auxin-responsive genes, NtWIF possibly targets diverse genes for defense and development by sharing processes that involve auxins. Transcriptional activation of WIPK by NtWIF suggests that WIPK is produced through a feed-back controlling system. It was thus concluded that NtWIF is a missing link between WIPK and its down-stream proteins, and that WIPK cascade is auto-regulated through a self-amplifying circuit.

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Osmotic Stress Induces Rapid Activation of a Salicylic Acid-Induced Protein Kinase and a Homolog of Protein Kinase ASK1 in Tobacco Cells

Cited by 70 publications

References 0 publications

Nicotiana tabacum Osmotic Stress-activated Kinase Is Regulated by Phosphorylation on Ser-154 and Ser-158 in the Kinase Activation Loop

Nicotiana tabacum Osmotic Stress-activated Kinase Is Regulated by Phosphorylation on Ser-154 and Ser-158 in the Kinase Activation Loop

SNF1-related Protein Kinases 2 Are Negatively Regulated by a Plant-specific Calcium Sensor

Finding a missing link in MAP kinase cascade

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