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...