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J. Biol. Chem., Vol. 281, Issue 45, 34299-34311, November 10, 2006

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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^*

Anna Maria Burza¹, Izabela Pkala¹, Jacek Sikora, Pawel Siedlecki^¶, Pawel Malagocki, Maria Bucholc, Luiza Koper, Piotr Zielenkiewicz^¶||, Michal Dadlez^**, and Grayna Dobrowolska²

From the Department of Plant Biochemistry, ^¶Department of Bioinformatics, and Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawiskiego 5a, 02-106 Warsaw, Poland, the ^**Faculty of Biology, Warsaw University, Miecznikowa 1, 02-096 Warszawa, Poland, and the ^||Laboratory of Plant Molecular Biology, Warsaw University, ul. Pawiskiego 5a, 02-106 Warsaw, Poland

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.

Received for publication, March 1, 2006 , and in revised form, August 22, 2006.

^* This work was supported by Ministry of Education and Science Grants PBZ-KBN-088/P04/2003 (to M. D.) and PBZ-KBN-110/PO4/2004 (to G. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These two authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 48-22-5925717; Fax: 48-22-6584636; E-mail: dobrowol{at}ibb.waw.pl.

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