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Ion Binding Properties of the Dehydrin ERD14 Are Dependent upon Phosphorylation

2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês

10.1074/jbc.m307151200

1083-351X

Muath Alsheikh, Bruce J. Heyen, Stephen K. Randall,

Plant Molecular Biology Research

The ERD14 protein (early response to dehydration) is a member of the dehydrin family of proteins which accumulate in response to dehydration-related environmental stresses. Here we show the Arabidopsis dehydrin, ERD14, possesses ion binding properties. ERD14 is an in vitro substrate of casein kinase II; the phosphorylation resulting both in a shift in apparent molecular mass on SDS-PAGE gels and increased calcium binding activity. The phosphorylated protein bound significantly more calcium than the nonphosphorylated protein, with a dissociation constant of 120 μm and 2.86 mol of calcium bound per mol of protein. ERD14 is phosphorylated by extracts of cold-treated tissues, suggesting that the phosphorylation status of this protein might be modulated by cold-regulated kinases or phosphatases. Calcium binding properties of ERD14 purified from Arabidopsis extracts were comparable with phosphorylated Escherichia coli-expressed ERD14. Approximately 2 mol of phosphate were incorporated per mol of ERD14, indicating a minimum of two phosphorylation sites. Western blot analyses confirmed that threonine and serine are possible phosphorylation sites on ERD14. Utilizing matrix assisted laser desorption ionization-time of flight/mass spectrometry we identified five phosphorylated peptides that were present in both in vivo and in vitro phosphorylated ERD14. Our results suggest that the polyserine (S) domain is most likely the site of phosphorylation in ERD14 responsible for the activation of calcium binding. The ERD14 protein (early response to dehydration) is a member of the dehydrin family of proteins which accumulate in response to dehydration-related environmental stresses. Here we show the Arabidopsis dehydrin, ERD14, possesses ion binding properties. ERD14 is an in vitro substrate of casein kinase II; the phosphorylation resulting both in a shift in apparent molecular mass on SDS-PAGE gels and increased calcium binding activity. The phosphorylated protein bound significantly more calcium than the nonphosphorylated protein, with a dissociation constant of 120 μm and 2.86 mol of calcium bound per mol of protein. ERD14 is phosphorylated by extracts of cold-treated tissues, suggesting that the phosphorylation status of this protein might be modulated by cold-regulated kinases or phosphatases. Calcium binding properties of ERD14 purified from Arabidopsis extracts were comparable with phosphorylated Escherichia coli-expressed ERD14. Approximately 2 mol of phosphate were incorporated per mol of ERD14, indicating a minimum of two phosphorylation sites. Western blot analyses confirmed that threonine and serine are possible phosphorylation sites on ERD14. Utilizing matrix assisted laser desorption ionization-time of flight/mass spectrometry we identified five phosphorylated peptides that were present in both in vivo and in vitro phosphorylated ERD14. Our results suggest that the polyserine (S) domain is most likely the site of phosphorylation in ERD14 responsible for the activation of calcium binding. Environmental stresses such as cold and drought have significant impact on plant growth and development; hence, agricultural productivity. Plants have evolved a wide variety of molecular responses to enable them to survive severe abiotic stresses (1Palva E.T. Tahtiharju S.T. Tamminen I. Puhakainen T. Laitinen R. Svensson J. Helenius E. Heino P. JIRCAS Work. Rep. 2002; 23: 9-15Google Scholar, 2Kim K.N. Cheong Y.H. Grant J.J. Pandey G.K. Luan S. Plant Cell. 2003; 15: 411-423Crossref PubMed Scopus (323) Google Scholar). Among these responses is the alteration of expression of a family of genes that encodes dehydrins, a subfamily of the group II late embryogenesis abundant mRNAs (LEAs). Levels of dehydrins are increased in response to low temperature, drought, or osmotic stress or are abscissic acid-induced (3Close T.J. Fenton R.D. Moonan F. Plant Mol. Biol. 1993; 23: 279-286Crossref PubMed Scopus (243) Google Scholar, 4Close T.J. Fenton R.D. Yang A. Asghar R. DeMason D.A. Crone D.E. Meyer N.C. Moonan F. Curr. Top. Plant Physiol. Am. Soc. Plant Physiol. Ser. 1993; 10: 104-118Google Scholar, 5Welin B.V. Olson A. Nylander M. Palva E.T. Plant Mol. Biol. 1994; 26: 131-144Crossref PubMed Scopus (167) Google Scholar, 6Shinozaki K. Yamaguchi-Shinozaki K. Curr. Opin. Biotechnol. 1996; 7: 161-167Crossref PubMed Scopus (393) Google Scholar, 7Close T.J. Physiol. Plant. 1997; 100: 291-296Crossref Google Scholar, 8Nylander M. Svensson J. Palva E.T. Welin B.V. Plant Mol. Biol. 2001; 45: 263-279Crossref PubMed Scopus (322) Google Scholar). In Arabidopsis, dehydrins can be characterized as the acidic dehydrins, pI 4.6–6.4 (COR47, ERD10, ERD14, 1The abbreviations used are: ERD14, early response to dehydration; CKII, casein kinase II; MALDI-TOF/MS, matrix-assisted laser desorption ionization-time of flight/mass spectrometry; SAP, shrimp alkaline phosphatase. NP_195554, NP_195624, and X91920), which are generally highly enriched in glutamic acid, and the neutral/basic dehydrins, pI 7.6–9.8 (RAB18, XERO1, and XERO2) which are enriched in glycine. Dehydrin proteins remain soluble after boiling, are extremely hydrophilic, and can be defined by the presence of at least one lysine-rich consensus sequence, the K domain (EKKGIMDKIKEKLPG), which is similar to class A2 amphipathic α-helical domains found in lipid-binding proteins (7Close T.J. Physiol. Plant. 1997; 100: 291-296Crossref Google Scholar, 9Koag M.C. Fenton R.D. Wilkens S. Close T.J. Plant Physiol. 2003; 131: 309-316Crossref PubMed Scopus (274) Google Scholar). Some dehydrins meet the criteria for hydrophillins (10Garay-Arroyo A. Colmenero-Flores J.M. Garciarrubio A. Covarrubias A.A. J. Biol. Chem. 2000; 275: 5668-5674Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Additionally, many dehydrins contain the S-domain, a Ser-rich sequence (3Close T.J. Fenton R.D. Moonan F. Plant Mol. Biol. 1993; 23: 279-286Crossref PubMed Scopus (243) Google Scholar, 11Vilardell J. Goday A. Freire M.A. Torrent M. Martinez M.C. Torne J.M. Pages M. Plant Mol. Biol. 1990; 14: 423-432Crossref PubMed Scopus (159) Google Scholar, 12Plana M. Itarte E. Eritja R. Goday A. Pages M. Martinez M.C. J. Biol. Chem. 1991; 266: 22510-22514Abstract Full Text PDF PubMed Google Scholar, 13Jensen A.B. Goday A. Figueras M. Jessop A.C. Pages M. Plant J. 1998; 13: 691-697Crossref PubMed Scopus (106) Google Scholar). Although their biochemical and physiological roles are still unclear, it has been suggested that dehydrins may play a role in stabilizing proteins or membrane structures under environmental stresses through interactions with an amphipathic α-helix (9Koag M.C. Fenton R.D. Wilkens S. Close T.J. Plant Physiol. 2003; 131: 309-316Crossref PubMed Scopus (274) Google Scholar). Calcium levels control a variety of plant developmental and signal transduction processes (14Bush D.S. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 95-122Crossref Scopus (911) Google Scholar, 15Sanders D. Brownlee C. Harper J.F. Plant Cell. 1999; 11: 691-706Crossref PubMed Scopus (780) Google Scholar), and in particular, calcium signaling is requisite for plant responses to environmental signals (16Knight H. Trewavas A.J. Knight M.R. Plant Cell. 1996; 8: 489-503Crossref PubMed Scopus (726) Google Scholar, 17Polisensky D.H. Braam J. 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Plant Physiol. 1997; 114: 29-37Crossref PubMed Scopus (76) Google Scholar). Calcium levels in the cytosol increases by several orders of magnitude upon signaling; however, sustained elevation of calcium can be toxic (31Hepler P.K. Wayne R.O. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1985; 36: 397-439Crossref Google Scholar). It has been suggested that cold-induced [Ca2+]cyt increases are caused by calcium leakage across membranes initiated at the plasma membrane and perpetuated by calcium release mainly from vacuole and endoplasmic reticulum (16Knight H. Trewavas A.J. Knight M.R. Plant Cell. 1996; 8: 489-503Crossref PubMed Scopus (726) Google Scholar, 17Polisensky D.H. Braam J. Plant Physiol. 1996; 111: 1271-1279Crossref PubMed Scopus (141) Google Scholar, 32Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2268) Google Scholar). The connection between stress-induced increases in cytosolic calcium and the accumulation of dehydrins has suggested to us that a potential physiological role for dehydrins in cold stress management could be ion binding. It was recently determined by our laboratory that a celery protein, similar in sequence to dehydrins (VCaB45), binds calcium (33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar), and it has been reported that another dehydrin-like protein found in castor bean has metal binding capability (34Kruger C. Berkowitz O. Stephan U.W. Hell R. J. Biol. Chem. 2002; 277: 25062-25069Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The present study is a characterization of a known Arabidopsis dehydrin, ERD14, its ion binding activity, and its regulation by phosphorylation. Plant Materials and Extractions—Arabidopsis thaliana (Columbia ecotype) seedlings were grown with 16/8 h light/dark period at 20 °C. Low temperature treatment was performed on 3–4-week-old plants for 2 days 4.7 °C. Total protein extracts of Arabidopsis plants were obtained from cold-treated and control tissues (33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar). In some cases low density membranes were isolated from cold-treated A. thaliana in the presence of protease and phosphatase inhibitors as described in Heyen et al. (33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar). Triton X-100 (0.2% w/w) was used to release the contents of the membrane vesicles (33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar). Supernatants derived from the permeabilized membranes were heat-treated in an 85 °C water bath for 20 min and quickly cooled in an ethanol bath (–50 °C) for 5 min. The soluble fraction was then obtained after ultracentrifugation for 1 h at 100,000 × g at 4 °C. Anion exchange chromatography was performed on a Mono Q column (Amersham Biosciences) in 20 mm Tris-HCl, pH 8.2 at 4 °C. ERD14 Cloning and Expression—A λ-PRL2 expression library (obtained from Ohio State Plant Resource Center) was subcloned into the pHEP2 vector (50Biermann B.J. Morehead T.A. Tate S.E. Price J.R. Randall S.K. Crowell D.N. J. Biol. Chem. 1994; 269: 25251-25254Abstract Full Text PDF PubMed Google Scholar) using the PstI and XbaI sites. Bacterial colonies were screened on replicate lifts after induction with isopropyl-1-thio-β-d-galactopyranoside as reported previously (50Biermann B.J. Morehead T.A. Tate S.E. Price J.R. Randall S.K. Crowell D.N. J. Biol. Chem. 1994; 269: 25251-25254Abstract Full Text PDF PubMed Google Scholar), with the antibody raised against the celery dehydrin-like protein, VCaB45 (33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar), using a secondary antibody of anti-mouse/alkaline phosphatase. Blots were developed with the colorimetric substrate nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. The fusion protein was expected to be ∼5 kDa greater than ERD14 due to the inclusion of v-Ras sequences (50Biermann B.J. Morehead T.A. Tate S.E. Price J.R. Randall S.K. Crowell D.N. J. Biol. Chem. 1994; 269: 25251-25254Abstract Full Text PDF PubMed Google Scholar). However, the induced ERD14 was present as two bands. A minor band (of the expected size for the fusion protein) and an ∼5-kDa smaller band (major band) were obtained. The major band was purified and sequenced by Edman degradation from the amino terminus. This revealed that the v-Ras portion of the fusion was missing, and additionally, the two amino-terminal amino acids (methionine-alanine) of ERD14 had been removed by proteolysis. The presence of the predicted amino-terminal tryptic peptide (lacking Met-Ala) was also confirmed by matrix assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS). This product, ERD14 lacking the amino-terminal two residues, was used for all the studies described here. Purification of ERD14 —Induced cells (1 mm isopropyl-1-thio-β-d-galactopyranoside) were harvested and resuspended in 2× homogenizing buffer in the presence of protease inhibitors (33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar). Samples were sonicated for 5 min and heat-treated as described before. Lysates were then centrifuged at 4,700 × g for 15 min at 4 °C. Supernatants were treated with 0.2% protamine sulfate to precipitate DNA and ultracentrifuged for 1 h at 100,000 × g at 4 °C. The high speed supernatant was fractionated by anion exchange chromatography on a 40-ml packed bed of diethyl aminoethyl (DEAE)-Sepharose (Amersham Biosciences) (1 ml/min flow rate). ERD14 fractions were further concentrated on a Mono Q column (Amersham Biosciences). Western Blotting—Antibodies raised against phosphoserine and phosphothreonine were obtained from Zymed Laboratories Inc. and Cell Signaling Technology, respectively. Anti-dehydrin antiserum was kindly supplied by Dr. Timothy J. Close (3Close T.J. Fenton R.D. Moonan F. Plant Mol. Biol. 1993; 23: 279-286Crossref PubMed Scopus (243) Google Scholar). Samples were separated by 10% SDS-PAGE and electroblotted onto nitrocellulose membranes (Protran, Mid-West Scientific). For anti-dehydrin antibody, membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline. When anti-phosphoserine and anti-phosphothreonine were used, membranes were blocked with 5% Tween 20 and 5% nonfat dry milk, 0.1% Tween 20, respectively, in Tris-buffered saline. Anti-rabbit and anti-mouse immunoglobulin G (goat) conjugated to horseradish peroxidase (Sigma) were used as secondary antibodies. Antibody detection procedures were with SuperSignal West Pico reagent (Pierce). ERD14 in Vitro Phosphorylation and Dephosphorylation—Purified E. coli-expressed ERD14 (∼7.5 μg) was incubated with 10 units of casein kinase II in 1× kinase buffer (BIOMOL Research Laboratories, Inc.) and 1 mm ATP at 30 °C in a total of 10 μl. In some cases, 1 mm Staurosporine (BIOMOL Research Laboratories, Inc.) was added after 30 min to inhibit protein kinase activity. Dephosphorylation was performed with shrimp alkaline phosphatase (Roche Applied Science) (33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar). Reactions were stopped by the addition of SDS-PAGE sample buffer and were boiled for 5 min. For the 32P incorporation experiments, purified ERD14 was phosphorylated in the presence of 0.8 mm ATP (0.17 μCi of [γ-32P]ATP). 32P incorporation was visualized with a PhosphorImager (STORM 840, Molecular Dynamics). In some cases, reactions were terminated with 25% trichloroacetic acid, and precipitates were collected on GFA glass fiber filters (Mid-West Scientific) and washed with 5% trichloroacetic acid and then 100% acetone. In such cases, the incorporated 32P was determined by liquid scintillation counting. Phosphorylation of ERD14 by A. thaliana Protein Extract—Extracts from whole A. thaliana plants treated for 2 days at 4 or 20 °C were incubated with or without the bacterial-purified ERD14 (final concentration of 95 ng/μl), 0.1 mm ATP containing [γ-32P]ATP (1 μCi) in 50 mm Hepes, pH 7.2, 0.5 mm CaCl2, 10 mm MgCl2, and 10 mm NaF at 30 °C. Reactions were stopped by adding SDS-PAGE loading buffer and boiling for 5 min. Calcium Binding Activity Assays—Calcium binding activity was determined by ligand blotting and equilibrium dialysis (33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar). Equilibrium dialysis assays were performed with Teflon Micro Dialyzer cells (QuizSep, Mid-West Scientific) and Spectra/Por MWCO 6–8,000 membranes (Spectrum Laboratories, Inc.). MALDI-TOF Mass Spectrometry—Protein samples (purified from E. coli) were mixed with an equal volume of 8 m urea and incubated for 30 min at 55 °. To reduce the urea concentration to 1 m, 10 mm ammonium bicarbonate buffer, pH 8.5, was added. Protein was then digested overnight with shaking at 37 °C with 1 μg of freshly prepared sequencing grade trypsin (Promega, V511A) dissolved in 50 mm acetic acid. A C18 Zip-Tip (Millipore) was used to remove urea and desalt the digestion solution. ERD14 purified from Arabidopsis was separated on 10% SDS-PAGE gels and Coomassie-stained. Protein bands corresponding to ERD14 were excised and manually processed. Samples were destained with 50% acetonitrile in water for 15 min at 20 °C, 50% acetonitrile in 50 mm ammonium bicarbonate for 15 min at 20 °C and 100% acetonitrile for 10 min at 20 °C. After removing the destain solution, 10 mm dithiothreitol in 100 mm ammonium bicarbonate was added to cover the gel pieces and incubated for 45 min at 50 °C. For alkylation, dithiothreitol was replaced with 55 mm iodoacetamide in 50 mm ammonium bicarbonate, and the samples were incubated for 30 min in the dark at 20 °C. Gel pieces were rinsed twice with 50 mm ammonium bicarbonate for 15 min at 20 °C. Protein bands were digested overnight at 37 °C with 150 ng of freshly prepared sequencing grade trypsin. Equal volumes of water and 100 mm ammonium bicarbonate were added to the digestion solution to give a final trypsin solution of 1 ng/μl. After a C18 Zip-Tip (Millipore) purification, the protein solution was mixed with an equal volume of α-cyano-4-hydroxycinnamic acid matrix (Sigma) and analyzed by MALDI-TOF/MS (MALDI LR™, Micromass, UK, located at the Indiana University, Purdue University Indianapolis Proteomic Core Facility). The mass of peptides obtained from each band were compared with the computer-generated list of deduced tryptic fragments to identify the 80-Da shift corresponding to phosphorylation using the FindMod site (ca.expasy.org/tools/findmod) for single phosphorylation sites and a Command Prompt program written to find multiple phosphorylations on single peptide fragments. Genomic Solutions™ (65.219.84.5/index.html) web site was used to search for protein identification (ProFound™) and to identify the theoretical phosphorylation sites on ERD14 (Protein Association Work Station, PAWS™). In Vitro Phosphorylation and Calcium Binding of ERD14 — ERD14 was cloned from an Arabidopsis expression library using antibody raised against celery VCaB45, a dehydrin-like protein (33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar). The apparent molecular mass of the purified E. coli-expressed ERD14, as estimated by 10% SDS-PAGE, was 37 kDa (See Figs. 1, 2, and 3B). The molecular mass of ERD14, deduced from the amino acid sequence, is 20.9 kDa. The mass of the protein used in these studies is predicted to be 20.6 kDa (lacks the amino-terminal two amino acids). The anomalous migration resulting in the overestimation of mass by SDS-PAGE is commonly found in other dehydrins (5Welin B.V. Olson A. Nylander M. Palva E.T. Plant Mol. Biol. 1994; 26: 131-144Crossref PubMed Scopus (167) Google Scholar, 33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar, 35Gilmour S.J. Artus N.N. Thomashow M.F. Plant Mol. Biol. 1992; 18: 13-21Crossref PubMed Scopus (255) Google Scholar, 36Ceccardi T.L. Meyer N.C. Close T.J. Protein Expression Purif. 1994; 5: 266-269Crossref PubMed Scopus (56) Google Scholar, 37Welin B.V. Olson A. Palva E.T. Plant Mol. Biol. 1995; 29: 391-395Crossref PubMed Scopus (60) Google Scholar, 38Svensson J. Palva E.T. Welin B. Protein Expression Purif. 2000; 20: 169-178Crossref PubMed Scopus (81) Google Scholar).Fig. 2Phosphorylation and apparent mass shift of ERD14 by CKII is reversible. Purified E. coli-expressed ERD14 was treated for 3 h at 30 °C with or without CKII. One portion of the phosphorylated ERD14 was then incubated for 20 min at 37 °C in the presence or absence of 5 units of SAP. After SDS-PAGE analyses, gels were stained with Coomassie Brilliant Blue.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Panel A, purified E. coli-expressed ERD14 is phosphorylated on at least two sites. ERD14 was in vitro phosphorylated at 30 °C with casein kinase II in the presence of [γ-32P]ATP. At the indicated times reactions were terminated with 25% trichloroacetic acid. Precipitates were collected on GFA glass fiber filters and washed with 5% trichloroacetic acid and 100% acetone. 32P incorporation was determined by liquid scintillation counting. Data are the average of three experiments. S.D. are shown as error bars. Panel B, purified E. coli expressed ERD14 is phosphorylated by CKII in threonine and serine sites. After SDS-PAGE, blots were probed with anti-phosphothreonine (top panel) and anti-phosphoserine (bottom panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our previous work with a dehydrin-like protein (33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar) and the presence of potential casein kinase II phosphorylation sites on ERD14 and other dehydrins suggested that ERD14 might be a substrate for CKII. Indeed, treatment of the purified E. coli-expressed ERD14 with CKII resulted in a shift in the apparent molecular mass on SDS-PAGE gels (Figs. 1A and 2), with an apparent increase of ∼5 kDa. The shifted polypeptides were confirmed to be ERD14 with a dehydrin-specific antibody, raised against the conserved K-domain of dehydrins (Fig. 1A). The ERD14 gel shift was nearly completed after a 30-min phosphorylation (Fig. 1A). Dephosphorylation of CKII-phosphorylated ERD14 with shrimp alkaline phosphatase (SAP) resulted in a quantitative return to the smaller molecular mass (Fig. 2). We have utilized this gel shifting phenomena to routinely assess the phosphorylation status of ERD14, as demonstrated for other phosphorylated proteins (33Heyen B.J. Alsheikh M.K. Smith E.A. Torvik C.F. Seals D.F. Randall S.K. Plant Physiol. 2002; 130: 675-687Crossref PubMed Scopus (128) Google Scholar, 39Kitta K. Clement S.A. Remeika J. Blumberg J.B. Suzuki Y.J. Biochem. J. 2001; 359: 375-380Crossref PubMed Scopus (53) Google Scholar, 40Rivedal E. Opsahl H. Carcinogenesis. 2001; 22: 1543-1550Crossref PubMed Scopus (121) Google Scholar). To quantitate ERD14 phosphorylation, ERD14 was phosphorylated with CKII in the presence of [γ-32P]ATP. A rapid increase in phosphorylation of ERD14 was observed in the first 30 min of CKII treatment and approached a plateau after 1 h. Phosphorylation of ERD14 correlated temporally with the gel shifting of ERD14 on SDS-PAGE gels (Fig. 1B). To determine whether the E. coli-expressed ERD14 bound calcium and whether this activity was regulated by phosphorylation, ERD14 was in vitro phosphorylated with CKII and separated on SDS-PAGE. Calcium binding activity was then assessed by calcium (45CaCl2) ligand blots and visualized by phosphorimaging. Fig. 1C demonstrates that E. coli-expressed ERD14 binds calcium only when phosphorylated. Quantitative analysis of the calcium ligand blot showed an increase in ERD14 calcium binding with increased phosphorylation and that ERD14 calcium binding activity approached saturation after 1 h (Fig. 1C). Calcium binding, as measured by a calcium-ligand blot (Fig. 1C), correlated well with the gel shifting of ERD14 on SDS-PAGE gels (Fig. 1A) and with the extent of phosphorylation (Fig. 1B). To analyze the extent of phosphorylation of the E. coli-expressed ERD14, CKII-phosphorylated ERD14 (with [γ-32P] ATP) was precipitated with trichloroacetic acid and collected on glass fiber filters. Approximately two mol of phosphate were incorporated per mol of ERD14 after 3 h of phosphorylation (Fig. 3A), indicating at least 2 possible phosphorylation sites on ERD14. No further 32P was incorporated into ERD14 for up to 5 h of phosphorylation. In some experiments fresh CKII was added after 3 h, 2M. K. Alsheikh and S. K. Randall, unpublished data. with no further incorporation observed. Western blots probed with antibodies raised against phosphoserine and phosphothreonine indicated that both threonine and serine were possible phosphorylation sites on ERD14 (Fig. 3B). Because phosphothreonine was detected only after 3 h of phosphorylation it is unlikely the phosphorylation requisite for the gel shifting and calcium binding activation was due to phosphorylation on a threonine. Western blots probed with anti-phosphoserine showed a subtle increase in serine phosphorylation over time (15–120 min). However, the anti-phosphoserine antibodies used were not specific for serine phosphorylation (Sigma, catalog number P3430, and Zymed Laboratories Inc., catalog number 61-8100) because they reacted with non-phosphorylated ERD14. Because dehydrins seem to lack significant secondary structure and have been considered to be intrinsically unstructured proteins (41Tompa P. Trends Biochem. Sci. 2002; 27: 527-533Abstract Full Text Full Text PDF PubMed Scopus (1677) Google Scholar), we thought it was possible that activation of calcium binding could be due to a rapid phosphorylation but require a slower induced structural change. To test this hypothesis ERD14 was treated for 30 min with CKII, further phosphorylation was prevented by adding staurosporine, and the incubation was continued. No further increase in calcium binding occurred with further incubation in the presence of staurosporine (Fig. 4). These results suggest that gel shifting requires an enzymatically active kinase and that calcium binding activity of the purified ERD14 does not require slow phosphorylation-dependent conformational changes. The quantitative effect of the phosphorylation on ERD14 calcium binding activity is illustrated by equilibrium dialysis experiments, which show saturable binding of calcium to native ERD14 (see Fig. 5). Scatchard plot analysis (Fig. 5B) indicated that CKII-phosphorylated ERD14 binds ∼3 mol of calcium per mol of ERD14 with relatively low affinity, having an apparent Kd for calcium of 120 μm. W