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.
Burza
Anna Maria
AM
Department of Plant Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawińskiego 5a, 02-106 Warsaw, Poland.
Pekala
Izabela
I
Sikora
Jacek
J
Siedlecki
Pawel
P
Małagocki
Paweł
P
Bucholc
Maria
M
Koper
Luiza
L
Zielenkiewicz
Piotr
P
Dadlez
Michał
M
Dobrowolska
Grazyna
G
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004789:Enzyme Activation; D018506:Gene Expression Regulation, Plant; D015151:Immunoblotting; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009997:Osmotic Pressure; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism*; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000737:chemistry / Q000201:enzymology*; D003313:Zea mays / Q000254:growth & development / Q000378:metabolism
D010940:Plant Proteins; D012694:Serine; D017346:Protein Serine-Threonine Kinases