Abstract
In tobacco (Nicotiana tabacum), hyperosmotic stress induces rapid activation of a 42-kD protein kinase, referred to as Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK). cDNA encoding the kinase was cloned and, based on the predicted amino acid sequence, the enzyme was assigned to the SNF1-related protein kinase type 2 (SnRK2) family. The identity of the enzyme was confirmed by immunoprecipitation of the active kinase from tobacco cells subjected to osmotic stress using antibodies raised against a peptide corresponding to the C-terminal sequence of the kinase predicted from the cloned cDNA. A detailed biochemical characterization of NtOSAK purified from stressed tobacco cells was performed. Our results show that NtOSAK is a calcium-independent Ser/Thr protein kinase. The sequence of putative phosphorylation sites recognized by NtOSAK, predicted by the computer program PREDIKIN, resembled the substrate consensus sequence defined for animal and yeast (Saccharomyces cerevisiae) AMPK/SNF1 kinases. Our experimental data confirmed these results, as various targets for AMPK/SNF1 kinases were also efficiently phosphorylated by NtOSAK. A range of protein kinase inhibitors was tested as potential modulators of NtOSAK, but only staurosporine, a rather nonspecific protein kinase inhibitor, was found to abolish the enzyme activity. In phosphorylation reactions, NtOSAK exhibited a preference for Mg(2+) over Mn(2+) ions and an inability to use GTP instead of ATP as a phosphate donor. The enzyme activity was not modulated by 5'-AMP. To our knowledge, these results represent the first detailed biochemical characterization of a kinase of the SnRK2 family.
Highlights
In tobacco (Nicotiana tabacum), hyperosmotic stress induces rapid activation of a 42-kD protein kinase, referred to as Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK). cDNA encoding the kinase was cloned and, based on the predicted amino acid sequence, the enzyme was assigned to the SNF1-related protein kinase type 2 (SnRK2) family
We identified in tobacco cells a 42-kD protein kinase that is rapidly activated by osmotic stress (Miko1ajczyk et al, 2000)
Analysis of the deduced amino acid sequence by a standard National Center for Biotechnology Information (NCBI)-BLAST homology search shows the greatest similarity to protein kinases assigned to the SnRK2b subfamily according to Halford and Hardie (1998), i.e. CPPK1 from Craterostigma plantagineum, SPK3 and SPK4 from soybean, and ASK1 and ASK2 from Arabidopsis (Fig. 1A), suggesting that NtOSAK is a member of this subfamily
Summary
NtOSAK, purified to near-homogeneity, was subjected to microsequencing. Partial amino acid sequences of five internal tryptic peptides were obtained (Miko1ajczyk et al, 2000). The antibodies immunoprecipitated the 42-kD protein kinase activated by osmotic stress, confirming that the cloned cDNA encodes the studied enzyme (Fig. 2B). To verify that the p42-kD kinase rapidly activated by osmotic stress is encoded by the cloned cDNA, specific anti-NtOSAK antibodies were generated. Protein extracts prepared from BY-2 cells (untreated and treated for 5 min with 250 mM NaCl) were subjected to western-blot analysis In both extracts, the anti-NtOSAK antibodies recognized a single protein band with a molecular mass of 42 kD (Fig. 2A). NtOSAK was isolated from BY-2 cells treated for 5 min with 250 mM NaCl. The enzyme was purified to near-homogeneity (about 12,000-fold) by four-step chromatography on Source 15Q, phenyl-Sepharose, heparin-Sepharose, and, MonoQ according to a previously described procedure (Miko1ajczyk et al, 2000). Parameters were estimated by fitting of data directly to the MichaelisMenten equation using GraphPad Prism 3.0, and are quoted 6SE
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