Báo cáo Y học: A Ca2+/CaM-dependent kinase from pea is stress regulated and in vitro phosphorylates a protein that binds to AtCaM5 promoter ppt

Eur J Biochem 269, 3193–3204 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02994.x A Ca2+/CaM-dependent kinase from pea is stress regulated and in vitro phosphorylates a protein that binds to AtCaM5 promoter Sona Pandey*, Shiv B Tiwari†, Wricha Tyagi, Mali K Reddy, Kailash C Upadhyaya and Sudhir K Sopory School of Life Sciences, Jawaharlal Nehru University, New Delhi, India and International Center for Genetic Engineering and Biotechnology, New Delhi, India An immuno-homologue of maize Ca2+/calmodulin (CaM)dependent protein kinase with a molecular mass of 72 kDa was identified in pea The pea kinase (PsCCaMK) was upregulated in roots in response to low temperature and increased salinity Exogenous Ca2+ application increased the kinase level and the response was faster than that obtained following stress application Low temperaturemediated, but not salinity-mediated stress kinase increase was inhibited by the application of EGTA and W7, a CaM inhibitor The purification of PsCCaMK using immunoaffinity chromatography resulted in coelution of the kinase with another polypeptide of molecular mass 40 kDa (p40) Western blot revealed the presence of PsCCaMK in nuclear protein extracts and was found to phosphorylate p40 in vitro Gel mobility shift and South-Western analysis showed that p40 is a DNA-binding protein and it interacted specifically with one of the cis acting elements of the Arabidopsis CaM5 gene (AtCaM5) promoter The binding of p40 to the specific elements in the AtCaM5 promoter was dependent of its dephosphorylated state Our results suggest that p40 could be an upstream signal component of the stress responses Plants perceive a variety of signals from the external environment as well as from the internal cellular milieu generated during various developmental processes Signals, such as light, nutrients and various environmental stresses, etc are perceived by specific receptors Following perception, a number of second messengers are generated that regulate the activity of other proteins such as kinases and phosphatases to transduce the signal downstream Towards the end of the signal transduction pathway, these second messengers and/or other accessory protein(s) affected by them modulate the activity of the transcription factors, which regulate the expression of specific gene(s) leading to the final response The events related to perception of a signal and corresponding changes in the activity and concentration of various second messengers have been studied in great detail However, the downstream pathways leading to the final control of gene expression have been elucidated in few cases only [1–3] Calcium ions are the most important second messengers, controlling a variety of cellular and physiological responses [4,5] Cytosolic concentration of calcium ([Ca2+]cyt) changes in response to a number of external stimuli and internal physiological developments [6,7] Besides, a number of other second messenger such as cyclic ADP-ribose, inositol-3-phosphate and various proteins such as calmodulin (CaM) and calcium-dependent protein kinases (CDPKs) are also affected by changes in ([Ca2+]cyt) [6,8] The information through ([Ca2+]cyt) is transduced by two main pathways, one involving CaM and CaM-related proteins, and the other involving CDPKs Both of these pathways cross-talk at various points in the signaling cascade [9] The presence of CaM has been detected in different plant cell compartments especially in the nuclei [10] In animal systems, it has been shown conclusively that nuclear CaM, in combination with nuclear Ca2+ changes, regulates the expression of various genes either by interacting with the transcription factors directly [11] or via specific calmodulin kinases (CaMKs) [12–14] In plant systems too, recent studies have shown that CaM is involved in the regulation of gene expression Szymanski et al [15] have shown that CaM affects the binding of TGA3 to the Arabidopsis CaM3 promoter A study by van der Luit et al [16] showed that distinct calcium signaling pathways that operate during cold (predominantly cytoplasmic) and wind (predominantly nucleus) signaling are regulated via CaM gene expression They have identified two different CaM isoforms, having different nucleotide sequences, but coding for the same polypeptide, and only one of these (NpCaM-1) is affected by cold and wind signaling CaMKs are important junctions in signal transduction where Ca2+-dependent and CaM-dependent signaling pathways converge and these kinases are potential candidates in regulating gene expression Reports on the existence Correspondence to S Pandey, 208 Mueller Laboratory, Biology Department, Pennsylvania State University, University Park, PA 16802, USA Fax: + 814 865 9131, E-mail: sxp49@psu.edu Abbreviations: CaM, calmodulin; CaMK, calmodulin kinase; CDPK, calcium-dependent protein kinase; GMSA, gel mobility shift analysis; W7, N-(6-aminohexyl)-5-chloro-1-napthalenesulphonamide hydrochloride; HMG, high mobility group Note: S Pandey and S B Tiwari contributed equally to this work *Present address: 208 Mueller Laboratory, Biology Department, The Pennsylvania State University, University Park, PA 16802, USA  Present address: Biochemistry Department, 117 Schweitzer Hall, University of Missouri, Columbia, MO 65211, USA (Received 15 February 2002, revised May 2002, accepted 13 May 2002) Keywords: calmodulin; DNA–protein interaction; plant protein kinase; protein phosphorylation; stress signaling Ó FEBS 2002 3194 S Pandey et al (Eur J Biochem 269) of this class of kinase are limited in plants [17–21] The Ca2+/CaM kinase isolated from maize roots has been shown to be involved in gravitropism [19] and the Ca2+/ CaM kinase isolated from lily anthers binds with the translational elongation factor II [22] We reported previously the purification and characterization of a novel Ca2+/CaM-dependent protein kinase, ZmCCaMK from etiolated maize coleoptiles [21,23] We have now identified an immuno-homologue of this kinase in pea (PsCCaMK) and in this study we show that PsCCaMK is involved in Ca2+/CaM-regulated stress signaling in plants PsCCaMK is tightly associated with its substrate, a 40-kDa protein (p40) that binds to specific sequence elements in the promoter of the Arabidopsis CaM5 gene (AtCaM5) gene in a phosphorylation/dephosphorylationdependent manner EXPERIMENTAL PROCEDURES Plant material, growth conditions and stress treatments Pea (Pisum sativum) plants were grown in moist vermiculite (16-h light/8-h dark) at 25 °C for days For salinity stress, 5-day-old plants were treated in 50, 100, 200, 300 or 500 mM NaCl solution for specified time periods For osmotic stress, plants were similarly treated with 300 mM mannitol for 24, 48 or 96 h For temperature stress, 5-day-old plants were transferred either to °C (cold stress) or 40 °C (heat stress) for specified time periods Calcium treatment (10, 25, 50 or 100 mM) was given to the 5-day-old plants for 3, 6, 12 or 24 h Plants grown under normal conditions for the same time period served as controls For pharmacological experiments, 5-day-old plants were treated with EGTA (10 mM), lanthanum (10 mM) or W7 (60 lM) for 12 h These plants were further subjected to different stresses as described earlier Plants treated with these compounds but without stress conditions served as control for these experiments After the treatments roots and shoots of the treated as well as untreated (control) plants were harvested separately, frozen in liquid N2 and stored at )80 °C until use (Sigma Chemical Co.) was used as secondary antibody and the antigen–antibody complex was visualized by the reaction of 5-bromo-4-chloro-3-indocyl phosphate/nitroblue tetrazolium (Sigma Chemical Co.) as described by Harlow & Lane [26] Protein purification using immuno-affinity chromatography For purification of the pea immuno-homologue of ZmCCaMK, 5-day-old pea plants were treated overnight with CaCl2 (100 mM); roots were harvested and washed extensively with water Further steps were performed at °C unless stated otherwise Tissue was ground in presence of liquid N2, extracted with vol extraction buffer as described earlier and centrifuged at 15000 g for 45 The crude protein extract obtained was precipitated with 0–40%, 40–50% and 50–80% ammonium sulfate and dialyzed extensively against 50 vols extraction buffer To detect the presence of the ZmCCaMK homologue, all the three fractions were immunoblotted with anti-ZmCCaMK Ig The 40–50% ammonium sulfate precipitated proteins containing the kinase homologue were utilized for immunoaffinity chromatography Protein A sepharose purified anti-ZmCCaMK Ig were linked to CNBr-activated Sepharose 6B (Pharmacia Biotech) according to the manufacturer’s instructions and the matrix was packed in a 4-mL column The column was equilibrated with buffer containing 50 mM Tris pH 7.5, 10 mM MgSO4, mM phenylmethanesulfonyl fluoride, mM dithiothreitol, 0.1% Triton-X 100, 10% glycerol (v/v) The proteins were bound to the column by recirculating twice through it The bound proteins were washed extensively with the same buffer containing 50 mM NaCl until the A280 of the column flow-through reached zero Specifically bound proteins were eluted either with a salt gradient of 0.05–1 M in the presence of 50 mM Tris pH 7.5, 10 mM MgSO4, mM phenylmethanesulfonyl fluoride, mM dithiothreitol, 10 mM 2-mercaptoethanol, 0.5% Triton-X 100 and 10% (v/v) glycerol or with 2.9 pH glycine buffer [26] When low pH glycine buffer was used for elution, the samples were neutralized immediately using a calibrated amount of Tris pH 7.5 Protein extraction, SDS/PAGE and immunoblotting Frozen tissue was ground to a fine powder in liquid N2 and extracted with vols extraction buffer [20 mM Hepes pH 7.5, mM EDTA, mM EGTA, mM phenylmethanesulfonyl fluoride, mM dithiothreitol and 10% glycerol (v/v)] on ice The slurry obtained was centrifuged at 12 000 g for 30 and the supernatant was used for SDS/ PAGE and immunoblotting studies Protein estimation was carried out according to Bradford [24] and equal amounts of proteins were resolved on SDS/PAGE according to Laemmli [25] Gels were run in duplicate; one part was used for staining with Coomassie brilliant blue R250 to ascertain equal loading of proteins, and the second part was electrophoretically transferred to nitrocellulose membrane Equal amount of the proteins on blots was further confirmed by staining with Ponceau S (Sigma Chemical Co.) The blots were probed with antibodies raised in rabbit against purified ZmCCaMK [21] at : 25000 dilution Goat anti-(rabbit IgG) Ig conjugated with alkaline phosphatase In vitro phosphorylation assays In vitro phosphorylation assays were performed as described earlier [21] Briefly, protein eluted from the affinity column (1 lg) was incubated in phosphorylation buffer (30 mM Hepes pH 7.5, mM MgCl2, 0.5 mM dithiothreitol, 25 mM NaCl) in the absence (control) or presence of 100 lM calcium and 110 nM CaM The reaction was started by the addition of 100 lM [c-32P]ATP (Amersham Biosciences Corp.) to the reaction mix in a total volume of 50 lL and incubated at 30 °C for An equal volume of SDS sample buffer was added to stop the reaction The reaction mix was boiled at 100 °C for and resolved by SDS/ PAGE The gel was dried and exposed for autoradiography To test the effect of KN-62, 50 lM of the compound was also included in the reaction mix To determine if antiZmCCaMK Ig blocked the phosphorylation reaction, the protein fraction was first incubated with lg of antibodies at room temperature for h with shaking The antigen– Ó FEBS 2002 Role of PsCCaMK in stress signaling (Eur J Biochem 269) 3195 antibody complex was then precipitated out using protein A sepharose beads and the resulting supernatant was used for phosphorylation assay RT-PCR Approximately 0.5 g plant tissue (control and stress treated) was ground in liquid nitrogen and total RNA was extracted with TRIzol reagent (Life Technologies) according to the manufacturer’s protocol Total RNA concentration was determined by UV absorbance at 260 nm For each sample lg total RNA was reverse transcribed with an oligo dT primer and Superscript II (Life Technologies) according to manufacturer’s instructions One ll of reaction product was used as template in PCR reaction with AtCaM5 primers (forward primer: 5¢-GATGTTGATGGTGATGGTCA-3¢; reverse primer: 5¢-AAACCAGCCATGAATGAAAT-3¢) and with actin primers (forward primer: 5¢-GTTGGGAT GAACCAGAAGGA-3¢; reverse primer: 5¢-GAACCA CCGATCCAGACACT-3¢) as a control Reactions with no DNA added served as a negative control The PCR cycling profile was: denaturation at 92 °C for 30 s, annealing at 58 °C for and extension at 72 °C for 1.5 for 25 cycles PCR products were analyzed on 1% agarose gels Preparation of nuclear protein extract, heparin–agarose chromatography and gel mobility shift analysis Pea nuclei were isolated as described previously [27] and purified on a discontinuous percoll gradient For preparation of nuclear protein extract, the nuclei were washed with buffer containing 50 mM Tris pH 7.8, mM MgCl2, mM dithiothreitol, 20% glycerol (v/v) and collected by gentle centrifugation Nuclei were resuspended in washing buffer containing 110 mM KCl, 10 mM phenylmethanesulfonyl fluoride, and lgỈmL)1 each of antipain and leupeptin and the suspension was brought to 40% ammonium sulfate saturation The suspension was centrifuged at 100 000 g for h in a Beckman Ti 75 rotor The supernatant obtained was brought to 70% saturation and the nuclear proteins were obtained by centrifugation at 100 000 g for h The proteins were finally resuspended in the same buffer without MgCl2 and stored frozen in small aliquots at )80 °C A pre-packed 2.5-mL heparin–agarose column (Sigma Chemical Co.) was equilibrated with 10 column vols of binding buffer (50 mM Tris pH 7.2, 10 mM MgSO4, mM dithiothreitol, mM phenylmethanesulfonyl fluoride and 10% glycerol, v/v) Ten to 20 mg proteins were loaded on to the column and washed with binding buffer containing 50 mM NaCl until the A260 of the flow through reached zero The proteins specifically bound to the column were eluted with a 0.05 to M NaCl gradient in binding buffer and eluted proteins were tested for binding with the AtCaM5 promoter fragment Active fractions were pooled, dialyzed and stored in small aliquots at )80 °C Gel mobility shift analysis (GMSA) were performed according to Ausubel et al [28] either with the labeled AtCaM5 promoter fragment ()588 to )339) or with specific oligonucleotides designed from the same promoter fragment The sequences of the oligonucleotides used for these experiments are: Oligo I, 5¢-CAAGGACGTTCGATGCA CTTCCAAAAAACATATAAT-3¢; Oligo II, 5¢-CAAT GTAGTATTAAAAAGTAGTAGTTAAAAGC-3¢; Oligo III, 5¢-GTTTTTATCCGATGCAAATTTTTGCTTTGT GATTG-3¢ The reaction was performed in 20 lL DNA-binding buffer containing (50 mM Tris pH 7.4, 50 mM KCl, mM dithiothreitol, 6% glycerol, v/v) supplemented with lg sonicated calf thymus DNA Labeled probe ( 10 000 c.p.m.) was incubated with the required amount of protein at room temperature for 10 DNA–protein complexes formed were fractionated by 5% nondenaturing PAGE and autoradiographed To abolish any protein– protein interaction, 0.5% deoxycholate (Sigma Chemical Co) was incubated in the reaction mixture For supershift analysis, the required dilution of anti-ZmCCaMK Ig was included in the reaction mixture with or without deoxycholate Competition analyses were performed by including 1000 · concentration of self or nonself oligonucleotides in the reaction mix To test the affect of phosphorylation on binding of p40 with DNA, p40 was phosphorylated using cold ATP as described above and used for assays An identical experiment performed with labeled ATP was run on a gel to verify the phosphorylation and to confirm the integrity of protein (data not shown) For dephosphorylation 10 lg of phosphorylated protein was incubated in buffer containing 50 mM Hepes pH 7.5, mM MgCl2, 0.5 mM dithiothreitol and calf intestinal phosphatase (10 U) in a total volume of 50 lL, at 30 °C for 10 Reactions were stopped by the addition lL 100 mM sodium pyrophosphate Protein was precipitated using ice-cold acetone and resuspended in DNA-binding buffer to study DNA–protein interaction For South-Western analysis, proteins separated on 10% SDS/PAGE were electro-blotted on nitrocellulose membrane The membrane was blocked with the binding buffer (described above) containing 3% BSA at room temperature with gentle shaking The membrane was washed twice with same buffer containing 0.25% BSA Hybridization was carried out in the presence of 50 lgỈmL)1 sonicated calf thymus DNA and labeled probe at room temperature for h The membrane was washed, dried and exposed for autoradiography RESULTS Immuno-homologue of maize ZmCCaMK is upregulated by low temperature and salinity stresses in pea roots Western blot analysis using anti-ZmCCaMK Ig of total protein extracts from pea shoots and roots showed the presence immuno-homologue of maize kinase in pea (PsCCaMK) PsCCaMK showed a development-dependent and tissue-specific expression (S Pandey & S K Sopory, unpublished data) The level of PsCCaMK was very low in roots as compared to the shoots (Fig 1, control lanes) As some protein kinases are involved in stress signaling pathways [29] and recent work points towards the involvement of Ca2+/CaM-dependent protein kinases in stress signaling [16], changes in the level of PsCCaMK was evaluated under various stress conditions Five-day-old pea seedlings were subjected to temperature, salinity and osmotic stress (as described in Experimental procedures) and the level of PsCCaMK was monitored by Western blotting in both roots and shoots using antiZmCCaMK Ig It was seen that PsCCaMK level remained 3196 S Pandey et al (Eur J Biochem 269) Fig Western blot analysis of level of PsCCaMK in response to different stresses in roots and shoots of pea Five-day-old pea plants were given heat stress (42 °C), low temperature stress (4 °C), salt stress (0.3 M NaCl) and osmotic stress (0.3 M mannitol) for 24 h and roots and shoots were harvested separately Extracted proteins were separated by SDS/PAGE (25 lg per lane) and probed with anti-ZmCCaMK Ig Roots and shoots from normal vermiculite-grown plants served as controls Numbers on the left indicate molecular weight markers in kDa unchanged under all the conditions tested in shoots (Fig 1) However, a strong upregulation of the protein level was observed in roots when salt (0.3 M NaCl) or low temperature (4 °C) treatment was given to the plants Osmotic stress or heat shock had no effect on the level of PsCCaMK suggesting that this kinase is not a general stress-regulated kinase but may specifically be involved in a signaling pathway associated with salinity and low temperature stress As both NaCl and low temperature upregulated the kinase level, time-kinetics experiments were performed for these stress treatments The optimum concentration of NaCl required to upregulate the kinase level was also determined As shown in Fig 2, the level of PsCCaMK started increasing in response to 50 mM NaCl, reached maxima at 300 mM and then remained constant up to 500 mM NaCl The kinase level started increasing after h of treatment of plants with NaCl as well as low temperature and the maximum level was observed following 24 h of treatment Calcium upregulates the PsCCaMK level in a timeand concentration-dependent manner Because PsCCaMK showed a strong upregulation in response to low temperature and salinity stress in pea roots under in vivo conditions, and the signaling pathways for both of these stresses are often mediated by Ca2+, the effect of exogenous Ca2+ was analyzed on the protein level of PsCCaMK As shown in Fig 3, the level of the kinase was strongly upregulated by Ca2+ The kinase level started Ó FEBS 2002 Fig Western blots showing the kinetics of induction of PsCCaMK following NaCl and cold treatment Five-day-old pea plants were given NaCl or cold treatment for indicated time periods and concentrations Roots were harvested and immediately frozen Twenty-five lg total protein extracts were separated by SDS/PAGE, and Western blotting was performed using anti-ZmCCaMK Ig Equal loading of proteins per lane was confirmed by Ponceau S staining of the Western blot Lane C denotes protein isolated from control plants that were not given any stress treatment Fig Effect of exogenous calcium on the level of PsCCaMK Calcium treatment was given to the 5-day-old pea plants for the indicated time periods and concentration and roots were harvested Total protein extracted (25 lg per lane) was Western blotted and probed with antiZmCCaMK Ig Mg denotes plants treated with 50 mM MgCl2 instead of CaCl2 Lane C denotes protein isolated from control plants not given any calcium treatment increasing at 10–25 mM exogenous Ca2+ and the maximum level was observed at 100 mM The time-kinetics data showed that the appearance of PsCCaMK after Ca2+ Ó FEBS 2002 Role of PsCCaMK in stress signaling (Eur J Biochem 269) 3197 treatment was earlier (at h) than that obtained following NaCl and low temperature stress treatment (at h) To exclude the possibility of this upregulation being mediated via a divalent cation in general, the effect of Mg2+ was also tested Plants treated with 50 mM Mg2+ for 24 h did not show any upregulation of the PsCCaMK level Low temperature, but not salinity-stimulated kinase level is mediated via a Ca2+/CaM pathway To further confirm that the upregulation of PsCCaMK by NaCl and low temperature is mediated via a Ca2+/CaM signaling pathway, the plants were pretreated with EGTA (10 mM), lanthanum (10 mM) or W7 (60 lM), before exposure to low temperature, NaCl or Ca2+ Plants treated with these compounds but not given any further stress treatments served as controls along with normal vermiculite-grown plants The Western blot analysis showed that EGTA as well as W7 almost completely blocked low temperature- and Ca2+-induced upregulation of the kinase but that the NaCl-stimulated kinase level was unaffected by these treatments (Fig 4) These results suggest that the low temperature-induced response may be mediated by Ca2+/CaM whereas the salt-induced upregulation might be mediated via some other pathway Lanthanum had no effect on the expression level of the kinase Purification of PsCCaMK by immuno-affinity chromatography: a 40-kDa protein always coelutes To purify PsCCaMK, 5-day-old pea plants were treated overnight with 100 mM Ca2+ and then roots were excised for purification of protein The total soluble protein extract was fractionated with 40–50% ammonium sulfate and loaded on to an immuno-affinity column prepared using anti-ZmCCaMK Ig The protein was bound in the presence of 50 mM NaCl and eluted either using a salt gradient of 0.05–1 M containing 0.5% TritonX-100 and 10 mM 2-mercaptoethanol or with low pH glycine buffer (pH 2.9) Under both of these sets of conditions a 40-kDa protein eluted first from the column followed by elution of the 72-kDa protein corresponding to the PsCCaMK (Fig 5) Under all elution conditions tested, the 72-kDa protein could not be eluted independently of the 40-kDa protein Western blot analysis of the eluted fractions with the same antibodies that were used for making the immuno-affinity column showed cross-reactivity with the 72-kDa PsCCaMK protein only This suggests that the 40-kDa protein does not bind to the antibodies on the column directly but possibly it is very tightly associated with PsCCaMK PsCCaMK is present in nuclear protein extracts and possibly interacts with DNA A number of CaM kinases from animal systems have been reported to be present in nuclei and to regulate gene expression [30] Our studies with the AtCaM5 promoter, which is induced under different stress conditions (S B Tiwari & K C Upadhyaya, unpublished data), gave indications that the anti-ZmCCaMK Ig affected binding of the AtCaM5 promoter with specific proteins eluted from Fig Effect of various pharmacological compounds on the expression level of PsCCaMK Five-day-old pea plants were pretreated with EGTA (10 mM), lanthanum (La, 10 mM) or W7 (60 lM) Pre-treated plants were given low temperature (4 °C), NaCl (0.3 M) or calcium (100 mM) treatment for 24 h Pre-treated plants, not given further treatment, as well as normal vermiculite-grown plants (C) served as different controls Twenty-five lg total proteins extracted from roots were separated by SDS/PAGE, Western blotted and probed with antiZmCCaMK Ig Fig Purification of PsCCaMK from immuno-affinity columns Roots of 5-day-old plants treated with calcium (100 mM) for 24 h were used for the purification of kinase using the immuno-affinity column prepared using anti-ZmCCaMK Ig Elution was with a 0.05–1 M NaCl gradient The left panel shows the SDS/PAGE profile of proteins from fractions 7, and 11 after silver staining The right panel shows the Western blot of the same fractions using anti-ZmCCaMK Ig 3198 S Pandey et al (Eur J Biochem 269) Ó FEBS 2002 Fig RT-PCR, Western blot and supershift analyses (A) RT-PCR of AtCaM5 gene RNA isolated from roots of control, low temperature (4 °C) and salinity (0.3 M NaCl) stressed plants was reverse transcribed and amplified with AtCaM5 gene-specific primers PCR with actin primers is included as control (B) Western blot analysis of pea nuclear extracts Five lg pea nuclear proteins (NP) and lg pea nuclear proteins fractionated on heparin–agarose column (HAFr) were separated by SDS/PAGE in duplicate One set was silver stained while the other set was Western blotted and probed with anti-ZmCCaMK Ig (C) Interaction of pea nuclear proteins with AtCaM5 promoter and supershift analysis The nuclear proteins were used in GMSAs to analyze their interaction with AtCaM5 promoter fragment Analysis was also performed in the presence or absence of 0.5% deoxycholate and anti-ZmCCaMK Ig to determine supershift Antibodies (Abs) alone were included as control the heparin–agarose column Moreover, expression of AtCaM5 gene is strongly upregulated in response to identical conditions of low temperature and salinity stress as analyzed by RT-PCR (Fig 6A) Taking clues from these observations we tested for the presence of PsCCaMK in nuclear protein fractions as well as in the proteins fractionated on the heparin–agarose column As shown in Fig 6B, the antibodies cross-reacted with a 72-kDa protein in the total nuclear protein fractions indicating its possible nuclear localization The antibodies also cross-reacted with the nuclear proteins fractionated on the heparin–agarose column The 72-kDa kinase band could be detected specifically in the 0.2–0.4 M salt-eluted protein fractions The same fractions also showed binding to the AtCaM5 promoter under different physiological conditions (unpublished data) As the salt concentration used was high enough not to let nonspecific proteins interact with the heparin column, it was predicted that this protein might be interacting with DNA directly To confirm this observation, we performed GMSA of total pea nuclear protein extract with a 249-bp labeled AtCaM5 promoter region ()588 to )339) in the presence and absence of antiZmCCaMK Ig This particular region was selected as it showed maximum protection and structural changes when foot-printing analysis was performed (S B Tiwari & K C Upadhyaya, unpublished data) As shown in Fig 6B, a strong DNA–protein complex was formed with the AtCaM5 promoter fragment and total pea nuclear protein extract, which showed a supershift with the anti-ZmCCaMK Ig Addition of 0.5% deoxycholate to abolish any ionic protein–protein interactions showed the presence of two loose complexes Addition of antibodies along with deoxycholate also showed a supershift, giving further indications that the kinase interacts with DNA either directly or it is strongly associated with some DNA binding protein Antibodies alone showed no interaction with DNA We could not determine the direct binding of the purified kinase with DNA as we could not obtain the kinase preparation without p40 under any conditions and attempts to purify the kinase by gel elution gave a very low yield and the eluted protein was highly labile The 40-kDa protein is phosphorylated by PsCCaMK and binds directly with the AtCaM5 promoter As p40 remains tightly bound to the PsCCaMK during the purification process, the possibility of it being a substrate for PsCCaMK was tested Further it was also examined if p40 binds with the specific regions of AtCaM5 promoter To ascertain these facts, proteins eluted from the immuno-affinity column were pooled in two different fractions, one containing pure p40 (fraction A) and other containing both the p72 and p40 (fraction B) Both of these fractions were used for in vitro phosphorylation experiments As shown in Fig 7A, no phosphorylation could be detected in fraction A under any of the Ó FEBS 2002 Role of PsCCaMK in stress signaling (Eur J Biochem 269) 3199 Fig In vitro protein phosphorylation, South-Western and DNA GMSA with pea nuclear proteins fractionated by immuno-affinity chromatography (A) Protein fractions eluted from kinase antibody affinity column containing lg purified p40 (fraction A) and those containing lg of both p40 and p72 PsCCaMK kinase (fraction B) were used for in vitro phosphorylation in the absence or presence of Ca2+, CaM, kinase antibodies and KN62 In one set 50 ng fraction B was added to fraction A (B) Proteins from fraction A and B were separated by PAGE and probed with labeled AtCaM5 promoter fragment in South-Western analysis (C) GMSA was performed in the absence (control) or presence of fraction B proteins with labeled AtCaM5 promoter fragment conditions tested, but a Ca2+-dependent, CaM-stimulated phosphorylation of p40 could be seen in fraction B This phosphorylation could be specifically blocked by KN-62, a Ca2+/CaM kinase inhibitor as well as with anti-ZmCCaMK Ig Addition of 50 ng fraction B proteins into fraction A led to phosphorylation of p40 in fraction A also On longer exposure of the blots a faint signal could be detected at the 72-kDa position, corresponding to the autophosphorylated PsCCaMK in fraction B, but no such signal was observed with fraction A (data not shown), confirming that p40 has no phosphorylation activity of its own These results clearly established that the PsCCaMK has Ca2+/CaM kinase activity and uses p40 as its in vitro substrate To ensure the DNA-binding property of p40, both fractions A and B were used for South-Western analysis with the AtCaM5 promoter fragment ()588 to )339) that was used earlier for GMSA A strong signal was observed at the 40-kDa position with both the fractions, showing that p40 binds to this promoter fragment (Fig 7B) To confirm this observation GMSA was performed with the AtCaM5 promoter fragment and fraction B, in the presence of excess of calf thymus DNA As shown in Fig 7C two specific DNA–protein complexes were formed These data showed that p40 binds directly to the AtCaM5 promoter, but whether PsCCaMK binds with DNA directly or through p40 remains inconclusive p40 binds to the specific cis-elements in the AtCaM5 promoter region and the binding is affected by phosphorylation To further analyze the specific binding of p40 to DNA, GMSAs were performed with fraction A (pure p40) and three specific oligonucleotides (see Experimental procedures) These oligonucleotides were designed based on the protected regions of the AtCaM5 promoter fragment ()588 to )339) in the foot-printing experiments (data not shown) p40 showed binding with two of the sequences, Oligo I and Oligo III but not with Oligo II (data for Oligos I and II are shown in Fig 8) The specificity of the binding was further confirmed by competition assays where excess concentrations (1000 ·) of the self-oligonucleotides as well as nonself oligonucleotides were used As shown in the autoradiogram (Fig 8), the DNA–protein complex could not be detected when the self-oligonucleotides were used at higher concentration, whereas no such effect could be seen with the nonself oligonucletides As p40 was found to be an in vitro substrate for the PsCCaMK, the effect of phosphorylation on its DNAbinding property was tested The protein was in vitro phosphorylated using cold ATP and used for DNA binding studies No binding was detected when prephosphorylated protein was used for the DNA binding reaction To confirm the reversibility of the phosphorylation reaction and the associated DNA-binding activity, phosphorylated p40 was 3200 S Pandey et al (Eur J Biochem 269) Ó FEBS 2002 Fig GMSA with p40 with specific cis elements (oligonucleotides) of AtCaM5 promoter Oligonucleotides I and II representing specific cis-regions of the AtCaM5 promoter were used to study the specific interaction of p40 Proteins from fraction A (see Fig 7) interacted specifically with Oligo I (A) but not with Oligo II (B) For competition a 1000 · excess of self or nonself oligonucleotides were included in the reaction mix Addition of ATP abolished the DNA–protein complex formation with Oligo I The binding with Oligo I was also studied following phosphorylation/dephosphorylation of p40 (C) treated with calf intestinal phosphatase A DNA–protein complex could be detected using the dephosphorylated protein, confirming that p40 could bind to the AtCaM5 promoter only in the dephosphorylated form DISCUSSION Role of PsCCaMK in stress signaling Protein kinases and phosphatases are important components of signaling cascades, which by changing the phosphorylation status of the target proteins transduce the signal to elicit the final response [31] A number of studies in recent years have linked the stress signaling with changes in the calcium level and the upregulation of various protein kinase transcripts [1–3,32–36] A receptor protein kinase RPK1 is regulated in response to multiple stresses and probably has a role at the very beginning of multiple stress signaling pathways [37] Involvement of CDPKs in stress signaling has also been shown by using chimeric gene constructs containing abscisic-acidresponsive elements and the GFP reporter gene [29] In this system, transient expression of CDPKs could be observed in response to various stresses as well as exogenous calcium A number of kinases of the mitogen activated protein signaling cascade have also been reported to be involved in stress signaling pathways [38] Upregulation of protein kinase transcripts is a complex process, as multiple signals (stress, light as well as phytohormones) affect the level of same kinase and in turn different kinases are modulated by the same signal, depending on the specificity of calcium signal and position of the kinase in the signaling cascade Though the role of some protein kinases is established in the stress signaling pathways, the exact sequence of events that leads to the final gene expression in response to a particular signal is not very well elucidated We have reported earlier the purification and characterization of ZmCCaMK and the presence of its immunohomologues in a variety of other plants [21] including Arabidopsis (data not shown) We now show that the kinase homologue from pea is involved in stress signaling Conditions such as 0.3 M NaCl and °C are widely reported to cause severe stress to plants Under these conditions, pea plants showed visible effect of stress in both roots and shoots However, the upregulation of kinase level was observed in roots only Shoots had higher kinase levels compared to roots to begin with, and it was not altered by stress treatment This is similar to the Nicotiana tobacum CDPKl transcript that could not be detected in leaves of the normal plants, but shows a strong upregulation in response to different stresses [39] The kinase level also increases in response to exogenous Ca2+ and even though a maximum increase in the kinase Ó FEBS 2002 Role of PsCCaMK in stress signaling (Eur J Biochem 269) 3201 level was obtained with 100 mM Ca2+, stimulatory effects are seen at much lower concentrations (10–25 mM) as well As these treatments were given to whole plants (not to isolated protoplasts or proteins) for very short duration, the actual Ca2+ uptake would be much lower Moreover, the low temperature- and Ca2+-induced increase in kinase level could be blocked by EGTA and W7 (a CaM inhibitor) and the high Mg2+ treatment given for 24 h has no effect on the kinase level; it is therefore very likely that Ca2+ is acting as a signal molecule However, the possibility that at such high concentrations Ca2+ might be causing some pleiotropic effects to cell physiology cannot be ruled out totally It has been shown earlier that both salt- and low temperature stress-mediated signaling pathways are modulated via Ca2+ [2,40–42] In most of the cases the same protein is affected in response to both of these stresses, but proteins affected by low temperature but not by salt or dehydration and vice versa are also known [34,43,44] Recently, the role of a novel kinase SOS2 in salt tolerance has been suggested [36,45] Specific sequence elements have been identified (e.g DRE) that are important for cold and/ or dehydration responses [46] In some cases different proteins bind to the same sequence under different physiological conditions to give specific responses [43,47,48] PsCCaMK does not appear to be a general stressresponsive kinase, as its expression does not change in response to mannitol or heat shock As dehydration or osmotic shock are known to affect the level of proteins regulated by salt stress, this kinase falls into a different category The response of PsCCaMK to NaCl is similar to that of the SOS3 gene [42] and Ca2+ appears to be involved in its regulation by upregulating the protein level; also the response is faster than that to either NaCl or to low temperature treatment The differential effects of EGTA and W7 on low temperature- and salinity-mediated expression of PsCCaMK indicate that their responses are mediated by different signaling pathways and that Ca2+/CaM signaling is involved only in the upregulation of PsCCaMK in response to low temperature To ascertain whether this kinase acts as a junction point of two different signaling pathways needs further work Purification of PsCCaMK and its substrate protein and their functional characterization PsCCaMK is present in the nuclear protein fractions as well as in the protein fractions eluted from the heparin–agarose column and possibly interacts with the AtCaM5 promoter In animal systems, a number of CaM kinases have been identified in the nuclei that are shown to affect the expression of specific genes by changing the phosphorylation status of transcription factors [30,49] It has been shown that plant nuclear extracts could be phosphorylated [50], showing the presence of kinase(s) in the nuclei In addition, different signaling pathways point towards the possibility of Ca2+ and CaM acting through the nucleus via specific CaM kinases [16] However, our study provides strong evidence of the presence of a CaM kinase and its possible function in plant nuclei It has been observed that during purification of PsCCaMK using an immuno-affinity column, p40 always coelutes with the PsCCaMK, even under very stringent conditions Moreover, p40 does not cross-react with the kinase antibodies used to make the column, showing that it is not interacting directly with the antibodies linked with the column but is possibly very tightly associated with the PsCCaMK There are other examples where substrate or interacting protein has been eluted from the affinity column along with the relevant protein [45] In vitro phosphorylation experiments show that p40 could not be phosphorylated on its own, but only in the presence of PsCCaMK, in a Ca2+-dependent, CaM-stimulated manner The phosphorylation of p40 could be blocked by KN-62 (a specific CaM kinase inhibitor) as well as the anti-ZmCCaMK Ig (Fig 7) This shows that PsCCaMK properties are similar to those of ZmCCaMK and p40 is one of its in vitro substrates To check the interaction of these proteins with DNA, we selected the promoter of the Arabidopsis CaM5 gene (AtCaM5) The reason to use this promoter was that the AtCaM5 gene was strongly upregulated in response to identical conditions of salinity and low temperature stress Besides, PsCCaMK was specifically present in the same fractions of heparin–agarose eluted proteins that show binding with the AtCaM5 gene promoter We also had preliminary data for this promoter, using GMSA and DNase I foot-printing, about the regions of the promoter showing structural changes under different physiological conditions Based on these studies we used the )588 to )399 fragment of this promoter (that showed maximum protection and changes in footprinting analysis) for our studies Detection of DNA–protein complexes by South-Western analysis, using the labeled fragment and the affinity purified protein fraction, shows the binding of the promoter with the p40 protein and not with PsCCaMK (Fig 7) The absence of binding with PsCCaMK could be due either to the fact that it does not bind to DNA at all, to a weak interaction, or to a very much lower amount of protein that could not be detected However, GMSAs with the nuclear protein extract and anti-ZmCCaMK Ig showed a supershift though weak, even in presence of high concentration of deoxycholate, indicating the possible interaction of this kinase directly with the DNA (Fig 6B) On the other hand, pure p40 protein showed direct binding with the AtCaM5 gene promoter fragment by GMSA Binding experiments with specific oligonucleotides confirm that the p40 interacts with defined sequence elements of the promoter, and that the binding is highly specific The same oligonucleotides, which not show binding with p40 when tested for binding with total pea nuclear extract, show a strong binding (data not shown) which further confirms that p40 binds to some specific cis sequences only The question of whether PsCCaMK binds directly or via its association with p40 has not been fully resolved During purification of the protein the association of the protein kinase and its substrate was not affected even though stringent conditions were used It is possible therefore, that these two proteins physically interact and even in the presence of 0.5% deoxycholate this interaction remains intact In this case the supershift could be a result of antibody interacting with the PsCCaMK bound to p40, which in turn interacts directly with DNA Of the several strategies that modulate the binding of a protein with its target DNA, to effect transcription, phosphorylation is regarded as one of the major mechanisms [51,52] In plants too, a number of studies have shown that phosphorylating the transcription factors affects their Ó FEBS 2002 3202 S Pandey et al (Eur J Biochem 269) binding with DNA [27,53–55] We have found that the binding of p40 is dependent on the dephosphorylation status of the protein and the binding is fully abolished once the protein is phosphorylated Analysis of the specific sequence elements, with which the p40 interacts, shows that these elements are not yet reported in any of the stress responsive genes On computational analysis, both of the sequences with which p40 interacts show the presence of a binding site for high mobility group proteins (HMG boxes) HMG box proteins have been identified from many plant species and it has been shown that their binding with DNA is affected by the phosphorylation status of the proteins [56– 58] In animal systems it has been shown conclusively that these proteins usually interact with the AT-rich sequences that show high structural changes [59–62], which is the case with the oligonucleotides that we used It could therefore be speculated that p40 is a protein similar to HMG box proteins; this, however, requires further characterization Proposed mechanism of action Based on all these results we propose that p40 is a negative regulator of the CaM5 gene promoter that may act in a similar way to the DREAM protein, a negative transcriptional regulator that acts in a calcium-dependent manner [63] During normal growth and development, p40 is bound to the promoter When the plant is under salinity or low temperature stress, the calcium level inside the cell increases This elevated calcium could then affect the level and activity of the Ca2+/CaM-dependent protein kinase homologue, which is either always present in the nuclei or it gets translocated to the nuclei [55,64] Once activated, the kinase phosphorylates p40 and as a result its binding to DNA is abolished and the protein is released from the DNA We also have preliminary data (not shown) which show that some other protein of unknown identity binds to the same sequence elements, after p40 dissociates from it following phosphorylation There are earlier reports which show that different proteins, though unrelated, bind to the same sequence elements, as in case of DREB1A and DREB2A [48] The present study thus proposes that protein kinases that are upregulated in response to specific stress function in the nuclei, might use DNA binding proteins as substrate(s) and affect their binding property thereby regulating the expression of stress-induced genes To be able to apply this statement in a broader perspective, it would be essential to look 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specificity J Biol Chem 274, 20116–20122 63 Carrion, A.M., Link, W.A., Ledo, F., Mellstrom, B & Naranjo, J.R (1999) DREAM is a Ca2+-regulated transcriptional repressor Nature 398, 80–84 64 Jensen, A.B., Goday, A., Figueras, M., Jessop, A.C & Pages, M (1998) Phosphorylation mediates the nuclear targeting of the maize Rab 17 protein Plant J 13, 691–697  ... 5¢-CAAGGACGTTCGATGCA CTTCCAAAAAACATATAAT-3¢; Oligo II, 5¢-CAAT GTAGTATTAAAAAGTAGTAGTTAAAAGC-3¢; Oligo III, 5¢-GTTTTTATCCGATGCAAATTTTTGCTTTGT GATTG-3¢ The reaction was performed in 20 lL DNA-binding... PsCCaMK suggesting that this kinase is not a general stress- regulated kinase but may specifically be involved in a signaling pathway associated with salinity and low temperature stress As both NaCl... lanes) As some protein kinases are involved in stress signaling pathways [29] and recent work points towards the involvement of Ca2+/CaM-dependent protein kinases in stress signaling [16], changes