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

Maria Bucholc,Anna Maria Burza,Pawel Malagocki,Izabela Pękala,Grażyna Dobrowolska,Luiza Koper,Pawel Siedlecki,Michal Dadlez,Piotr Zielenkiewicz,Jacek Sikora

doi:10.1074/jbc.m601977200

Abstract

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.

Highlights

Enzymes has to be regulated very strictly inside the cell
To understand the mechanism of NtOSAK activation in response to hyperosmotic stress, we analyzed the effect of osmotic stress on NtOSAK expression at the mRNA and protein levels and on NtOSAK phosphorylation in BY-2 tobacco cells subjected to osmotic stress
NtOSAK Expression Is Transiently Up-regulated by Osmotic Stress—The level of NtOSAK transcript was analyzed by reverse transcription-PCR in BY-2 cells untreated and treated with 250 mM NaCl or 500 mM sorbitol for various times

Summary

Mechanism of NtOSAK Activation

Bidopsis thaliana and in Oryza sativa, there are 10 members of the SnRK2 family [14, 15]. Except SnRK2.9 from Arabidopsis, are activated by different osmolytes, such as sucrose, mannitol, sorbitol, and NaCl, and some of them by abscisic acid, suggesting that these kinases are involved in a general response to osmotic stress [14, 15]. Expression of genes encoding the SPK-3 and SPK-4 kinases from soybean, both members of the SnRK2 subfamily, is induced by dehydration and high salinity [23]. 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. The identified sites are conserved in all members of the SnRK2 family, which suggests that this can be a general mechanism of regulation of the activity of these kinases

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION