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

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. 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. Several protein kinases involved in osmotic stress signal transduction in plants have been identified (for a review, see Ref. 1Boudsocq M. Lauriere C. Plant Physiol. 2005; 138: 1185-1194Crossref PubMed Scopus (208) Google Scholar). Most of the available information concerns mitogen-activated protein kinase (MAPK) 3The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; ABA, abscisic acid; SnRK, Snf1-related protein kinase; MBP, myelin basic protein; GST, glutathione S-transferase; GFP, green fluorescent protein; LC, liquid chromatography; MS, mass spectrometry. 3The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; ABA, abscisic acid; SnRK, Snf1-related protein kinase; MBP, myelin basic protein; GST, glutathione S-transferase; GFP, green fluorescent protein; LC, liquid chromatography; MS, mass spectrometry. cascades, which in all eukaryotic organisms play a central role in stress signal transduction. All MAPKs, as well as MAPKKs, including plant enzymes, are activated by phosphorylation in their activation loops by specific upstream kinases, MAPKKs and MAPKK kinases, respectively (for a review, see Ref. 2Tibbles L.A. Woodgett J.R. Cell Mol. Life Sci. 1999; 55: 230-254Crossref Scopus (553) Google Scholar), and become inactivated by phosphoprotein phosphatases (for a review, see Ref. 3Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (705) Google Scholar). 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. 4Halford N.G. Hardie D.G. Plant Mol. Biol. 1998; 37: 735-748Crossref PubMed Scopus (303) Google Scholar, 5Hrabak E.M. Chan C.W.M. Gribskov M. Harper J.F. Choi J.H. Halford N. Kudla M. Plant Physiol. PubMed Scopus Google Scholar, Biol. Google Scholar). belonging to the and the yeast and kinases, which play a role in cells and environmental stresses (for reviews, see Refs. 4Halford N.G. Hardie D.G. Plant Mol. Biol. 1998; 37: 735-748Crossref PubMed Scopus (303) Google Scholar, D.G. 1999; Google Scholar, and D.G. PubMed Scopus Google Scholar). The SnRK2 and SnRK3 are specific to plants (for reviews, see Refs. 4Halford N.G. Hardie D.G. Plant Mol. Biol. 1998; 37: 735-748Crossref PubMed Scopus (303) Google and 5Hrabak E.M. Chan C.W.M. Gribskov M. Harper J.F. Choi J.H. Halford N. Kudla M. Plant Physiol. PubMed Scopus Google Scholar). data that these enzymes in environmental stress of the SnRK3 subfamily, that with are well in the of plant cells stresses M. Lauriere C. Plant Physiol. 2005; 138: 1185-1194Crossref PubMed Scopus (208) Google Scholar, Kudla M. Plant PubMed Scopus Google Scholar, 55: PubMed Scopus Google Scholar, Plant Physiol. PubMed Scopus Google Scholar). kinases are activated by with and most probably by in the of several enzymes of subfamily, of a conserved in the kinase activation with the results in active enzymes M. Plant PubMed Scopus Google Scholar, C. Biol. PubMed Scopus Google Scholar). of information enzymes belonging to the SnRK2 subfamily also indicate their role in stress in has been that in and in are of the SnRK2 Plant PubMed Scopus Google Scholar, M. Lauriere C. Biol. PubMed Scopus Google Scholar). All of from are activated by such as and and some of also by abscisic suggesting that these kinases are involved in a general response to osmotic stress Plant PubMed Scopus Google Scholar, M. Lauriere C. Biol. PubMed Scopus Google Scholar). The of the SnRK2 are kinase by abscisic the of which is activated by as well as by and Sci. PubMed Scopus Google Scholar, Plant Physiol. PubMed Scopus Google protein kinase), activated by in cells of in response to drought, involved in the regulation of and S.M. Plant PubMed Google Scholar, S.M. 2000; PubMed Scopus Google and protein enzyme to the regulation of by and upstream of Plant PubMed Scopus Google Scholar). The with a in and has a J.R. Plant Cell Physiol. PubMed Scopus Google Scholar). has been that kinase is involved in stress Sci. PubMed Scopus Google Scholar). The enzyme the of plants by involved in stress signal transduction have been of the and kinases from of the SnRK2 subfamily, is by and high C. Mol. PubMed Scopus Google Scholar). other protein kinases, and from the are to of plant to hyperosmotic stress Plant PubMed Scopus Google Scholar). is that in stress in plants and are activated very rapidly in response to osmotic is very information the mechanism of their activation. Our previous results NtOSAK of the SnRK2 subfamily M. Plant 2000; 12: Google Scholar), the results by the on Plant PubMed Scopus Google Scholar), and the very by C. C. Plant Physiol. PubMed Scopus Google on kinase that these enzymes activated by phosphorylation. to and M. Plant Physiol. 2000; PubMed Scopus Google that high stress-activated kinase from of NtOSAK, was activated by to and to the mechanism of SnRK2 activation osmotic stress on NtOSAK activation The results presented show that phosphorylation of and in the NtOSAK activation is involved in the kinase activation in response to hyperosmotic stress. The identified sites are conserved in all of the SnRK2 which that be a general mechanism of regulation of the activity of these kinases. Plant tobacco cells, by as by Scopus Google Scholar). The cells in and with and The cells The cells with for the by in liquid and at of Protein as M. Plant 2000; 12: Google Scholar, Hardie D.G. Plant Physiol. PubMed Scopus Google Scholar). was isolated from and BY-2 to the by the The was from of the was for at in of of of the was used for in of and a of the conditions as and The on and by as on NtOSAK on NtOSAK a of the and of the of was the and was as Hardie D.G. Plant Physiol. PubMed Scopus Google the peptide of the kinase in the and by The as the of with in to the with and in and at and for in the with the at at with the results by with of was from BY-2 cells for with to the M. Plant 2000; 12: Google Scholar). of in of the of and of the protein by Hardie D.G. Plant Physiol. PubMed Scopus Google Scholar). was for at and at and of NtOSAK was used as the for site-directed mutagenesis with the site-directed mutagenesis with the and All to the and of The into and the expressed and to the as for Hardie D.G. Plant Physiol. PubMed Scopus Google Scholar). was as M. Plant 2000; 12: Google Scholar, Hardie D.G. Plant Physiol. PubMed Scopus Google with some from with in at for on a of protein was and the was for by and three with and with the the into in a of The was used for kinase activity whereas the was used for of phosphorylation sites by mass spectrometry by with of was to the and the was at for was by kinase activity Protein as well as kinase activity as M. 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Our results are in with the data presented by C. C. Plant Physiol. PubMed Scopus Google Scholar), that in kinase belonging to of the SnRK2 kinases to the of and Lauriere M. Lauriere C. Plant Physiol. 2005; 138: 1185-1194Crossref PubMed Scopus (208) Google Scholar), to Ser-158 of and to of is for the kinase activity and the enzyme in with and the NtOSAK the conserved the The of as the NtOSAK in the of kinases PubMed Scopus Google Scholar, Mol. PubMed Scopus Google Scholar). 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The maize was several by and has been used to several signal transduction PubMed Scopus Google Scholar, Plant Physiol. PubMed Scopus Google Scholar). NtOSAK is expressed in all plant and its are in plant in we that is for with the its with Ser-154 Ser-158 with with activity of the expressed of NtOSAK was by kinase as a subjected to osmotic we a kinase activity of of the expressed The kinase activity was at a in with we to kinase activity of the expressed confirmed mutagenesis in the indicating that phosphorylation of Ser-158 is crucial for the kinase in to the results the of Ser-154 to to the NtOSAK activity, the subjected to hyperosmotic stress results indicate role of identified in NtOSAK activation in the plant response to osmotic stress. NtOSAK, a member of of the SnRK2 to the of and Lauriere M. Lauriere C. Plant Physiol. 2005; 138: 1185-1194Crossref PubMed Scopus (208) Google Scholar), was identified as a kinase rapidly activated in tobacco cells subjected to osmotic stress M. Plant 2000; 12: Google Scholar). Our to the mechanism of regulation of NtOSAK activity in response to osmotic stress. we changes in NtOSAK at the and protein and its phosphorylation in response to stress. NtOSAK is at the in cells, cause in the NtOSAK protein The of the NtOSAK protein is the in and NaCl-treated cells, which that NtOSAK activation is to its Our previous that NtOSAK activity was by with protein M. Plant 2000; 12: Google Scholar), indicating that NtOSAK phosphorylation is for its activity and suggesting that be the mechanism of its activation osmotic stress. was confirmed by Plant PubMed Scopus Google Scholar), that all of the SnRK2 subfamily are activated by and very by C. C. Plant Physiol. PubMed Scopus Google Scholar), that phosphorylation is for protein kinase the mass spectrometry we mapped phosphorylation sites in active NtOSAK to from tobacco cells. the active Ser-154 and Ser-158 in the kinase activation To that NtOSAK phosphorylated on the residues in BY-2 cells in response to osmotic we the time course of NtOSAK phosphorylation on Ser-154 and Ser-158 and the enzyme activation in tobacco cells to We a of NtOSAK phosphorylation on the identified and its The kinase was active and phosphorylated in BY-2 cells to osmotic stress as well as a to the stress. that phosphorylation is for NtOSAK activation and that is for its Prediction of the NtOSAK three-dimensional structure that phosphorylation of Ser-154 and Ser-158 has essential on NtOSAK conformation and its mutagenesis on NtOSAK expressed in bacteria as well as in plant cells confirmed expressed in has a activity, which be by of with whereas with a phosphorylated active of activity the enzyme isolated from plant cells. mutagenesis on the expressed in that phosphorylation of Ser-158 is essential for the enzyme activity, whereas phosphorylation of Ser-154 most probably facilitates NtOSAK on The results of of NtOSAK and its in maize also role of Ser-154 and Ser-158 in NtOSAK activation. of to to the kinase activity, that in plants for phosphorylated Ser-158 of The activity was in as well as with Ser-154 was to to the expressed activity in to osmotic stress as well as stress results role of Ser-158 also of Ser-154 in NtOSAK activation in response to osmotic stress. 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The role of Ser-154 phosphorylation is in of the of was to tobacco BY-2 and plants active NtOSAK in to NtOSAK The plant that the of the identified residues with is to a active kinase in of the expressed of NtOSAK a kinase activity in plants to osmotic stress. results by the Plant PubMed Scopus Google Scholar). their to active of several residues in the kinase activation also with their of these in a active of Plant PubMed Scopus Google Scholar). also C. C. Plant Physiol. PubMed Scopus Google that of to to the kinase activity of and its to the The to a active SnRK2 by of the crucial for the kinase activity with be by the very activity of the of the kinases. 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PubMed Google Scholar). on to a on in the kinase activation loop. of the regulatory with on the regulatory a active of the to the kinase that was to be activated by MAPKK, whereas to the kinase activated to a specific activity of of the specific activity of activated kinase Biol. PubMed Google Scholar). are several other that of the in the kinase activation by in a active the of the kinase of the in the kinase activation to to the kinase activity Sci. PubMed Scopus Google Scholar). The of for kinase to to a of the kinase activity in cells Biol. PubMed Scopus Google Scholar). to the their results indicate that phosphorylation of in the activation of is for kinase activity Biol. PubMed Scopus Google Scholar). is also that phosphorylation of the identified residues of NtOSAK is is for NtOSAK activation in We the that are other phosphorylated residues that are for the kinase activation that in We some phosphorylated is to the of the protein by we of NtOSAK isolated from tobacco cells. to some phosphorylated and are very to by was several and identified at the and of the several residues of identified by C. C. Plant Physiol. PubMed Scopus Google Scholar). we see phosphorylation of residues in NtOSAK isolated from NaCl-treated tobacco BY-2 cells in the that such phosphorylation we these Besides other with some specific have a on NtOSAK was that NtOSAK activity in tobacco cells and be a in NtOSAK activation in response to hyperosmotic stress C. Biol. PubMed Scopus Google Scholar). that of NtOSAK may be involved in the regulation of its are also that the regulatory of is involved in their activation Plant PubMed Scopus Google Scholar, C. C. Plant Physiol. 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