Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase

Spinach (Spinacea oleracea L.) nitrate reductase (NR) is inactivated by phosphorylation on serine-543, followed by binding of the phosphorylated enzyme to 14-3-3 proteins. We purified one of several chromatographically distinct NR_serine-543 kinases from spinach leaf extracts, and established by Edman sequencing of 80 amino acid residues that it is a calcium-dependent (calmodulin-domain) protein kinase (CDPK), with peptide sequences very similar to Arabidopsis CDPK6 (accession no. U20623; also known as CPK3). The spinach CDPK was recognized by antibodies raised against Arabidopsis CDPK. Nitrate reductase was phosphorylated at serine-543 by bacterially expressed His-tagged CDPK6, and the phosphorylated NR was inhibited by 14-3-3 proteins. However, the bacterially expressed CDPK6 had a specific activity approx. 200-fold lower than that of the purified spinach enzyme. The physiological control of NR by CDPK is discussed, and the regulatory properties of the purified CDPK are considered with reference to current models for reversible intramolecular binding of the calmodulin-like domain to the autoinhibitory junction of CDPKs. Received: 12 February 1998 / Accepted: 28 May 1998     

Ca2+-dependent protein kinase from alfalfa (Medicago varia): Partial purification and autophosphorylation

A calcium-dependent protein kinase (CDPK) was purified to 1400-fold from the soluble fraction of alfalfa (Medicago varia) cells by ammonium sulfate fractionation, Sephacryl-300, DEAE-Sephacel, Phenyl-Sepharose and Hydroxylapatite column chromatography. The enzyme is mainly monomeric. During the course of the purification steps a 50 kDa phosphoprotein doublet and a 56 kDa phosphoprotein copurified with the CDPK activity. Mobility shift of these proteins have been shown by SDS PAGE in Ca²⁺ free conditions. Tests on enzyme activity after separation by native gel electrophoresis revealed two protein kinase activities in our enzyme preparation and the phosphorylation of the 50 kDa and 56 kDa proteins. We suggest that these proteins are the autophosphorylated forms of calcium dependent protein kinases. Preincubation of the CDPK in ATP resulted in a marked increase in enzyme activity, but did not alter the Ca²⁺ sensitivity of the protein kinase.     

A rice membrane calcium-dependent protein kinase is induced by gibberellin.

A rice (Oryza sativa) seed plasma-membrane calcium-dependent serine/threonine protein kinase (CDPK) has been partially purified. Comparing results in seeds that were treated with and without the plant hormone gibberellin (GA) for 10 min showed that rice CDPK was highly induced by GA. After separating solubilized membrane proteins by sodium dodecyl sulfate-gel electrophoresis, followed by renaturation, a radiolabeled phosphoprotein band of approximately 58 kD was detected, and it was apparently produced by autophosphorylation. There are five aspects of the rice CDPK that show similarity to mammalian protein kinase C (PKC) and to other plant CDPKs: (a) Histone IIIS and PKC peptide-ser25 (19-31) are phosphorylated by rice CDPK. (b) The phosphorylation reaction is strictly dependent on calcium. (c) The activity of the rice CDPK is inhibited by either staurosporine or the PKC inhibitory peptide (19-36). (d) Addition of calmodulin has no effect on the activity of the enzyme; however, the CDPK is inhibited by the calmodulin antagonists trifluoperazine and W-7. (e) The rice CDPK reacts with a mammalian anti-PKC antibody in immunoblotting analysis. However, there is one major difference between the rice CDPK and other CDPKs: the rice CDPK is induced by GA, whereas no mammalian PKC or other plant CDPKs are known to be induced by any hormone.     

The plasma membrane H+‐ATPase from maize roots is phosphorylated in the C‐terminal domain by a calcium‐dependent protein kinase

A protein kinase activity associated with maize root plasma membranes was partially purified and characterized. Biochemical properties, such as calcium dependence, inhibition by calmodulin antagonists, and absence of calmodulin stimulation, indicated that the enzyme belongs to the calcium‐dependent protein kinase (CDPK) family. By means of an in‐gel phosphorylation assay the molecular mass of active polypeptides was determined: two bands of 55 and 51 kDa became labelled. The same proteins were also immunodecorated by monoclonal antibodies against soybean CDPK. Results from in vitro assays demonstrated that maize H⁺‐ATPase was a suitable substrate for this protein kinase and that the phosphorylation site was located at the C‐terminal domain of the enzyme. This result was confirmed by using as substrate in phosphorylation assays the isolated C‐terminal domain of the H⁺‐ATPase expressed in Escherichia coli as a glutathione‐transferase fusion protein.     

Biochemical evidence for a calcium-dependent protein kinase from Pharbitis nil and its involvement in photoperiodic flower induction

A soluble Ca(2+)-dependent protein kinase (CDPK) was isolated from seedlings of the short-day plant Pharbitis nil and purified to homogeneity. Activity of Pharbitis nil CDPK (PnCDPK) was strictly dependent on the presence of Ca(2+) (K(0,5)=4,9 microM). The enzyme was autophosphorylated on serine and threonine residues and phosphorylated a wide diversity of substrates only on serine residues. Histone III-S and syntide-2 were the best phosphate acceptors (K(m) for histone III-S=0,178 mg ml(-1)). Polyclonal antibodies directed to a regulatory region of the soybean CDPK recognized 54 and 62 kDa polypeptides from Pharbitis nil. However, only 54 kDa protein was able to catalyse autophosphorylation and phosphorylation of substrates in a Ca(2+)-dependent manner. CDPK autophosphorylation was high in 5-day-old Pharbitis nil seedlings grown under non-inductive continuous white light and was reduced to one-half of its original when plants were grown in the long inductive night. Also, the pattern of proteins phosphorylation has changed. After 16-h-long inductive night phosphorylation of endogenous target (specific band of 82 kDa) increased in the presence of calcium ions. It may suggest that Ca(2+)-dependent protein kinase is involved in this process and it is dependent on light/dark conditions.     

Phosphorylation of style S-RNases by Ca2+-dependent protein kinases from pollen tubes

Solanaceous plants with gametophytic self-incompatibility produce ribonucleases in the transmitting tract of the style that interact with self-pollen and inhibit its growth. These ribonucleases are a series of allelic products of the S-locus, which controls self-incompatibility. Little is known about the pollen components involved in this interaction or whether a signal transduction pathway is activated during the self-incompatibility response. We have partially purified a soluble protein kinase from pollen tubes of Nicotiana alata that phosphorylates the self-incompatibility RNases (S-RNases) from N. alata but not Lycopersicon peruvianum. The soluble protein kinase (Nak-1) has several features shared by the calcium-dependent protein kinase (CDPK) class of plant protein kinases, including substrate specificity, calcium dependence, inhibition by the calmodulin antagonist calmidazolium, and cross-reaction with monoclonal antibodies raised to a CDPK from soybean. Phosphorylation of S ₂-RNase by Nak-1 is restricted to serine residues, but the site(s) of phosphorylation has not been determined and there is no evidence for allele-specific phosphorylation. The microsomal fraction from pollen tubes also phosphorylates S-RNases and this activity may be associated with proteins of M_r60 K and 69 K that cross-react with the monoclonal antibody to the soybean CDPK. These results are discussed in the context of the involvement of phosphorylation in other self-incompatibility systems.     

The motor complex of Plasmodium falciparum: phosphorylation by a calcium-dependent protein kinase

Calcium-dependent protein kinases (CDPKs) of Apicomplexan parasites are crucial for the survival of the parasite throughout its life cycle. CDPK1 is expressed in the asexual blood stages of the parasite, particularly late stage schizonts. We have identified two substrates of Plasmodium falciparum CDPK1: myosin A tail domain-interacting protein (MTIP) and glideosome-associated protein 45 (GAP45), both of which are components of the motor complex that generates the force required by the parasite to actively invade host cells. Indirect immunofluorescence shows that CDPK1 localizes to the periphery of P. falciparum merozoites and is therefore suitably located to act on MTIP and GAP45 at the inner membrane complex. A proportion of both GAP45 and MTIP is phosphorylated in schizonts, and we demonstrate that both proteins can be efficiently phosphorylated by CDPK1 in vitro. A primary phosphorylation of MTIP occurs at serine 47, whereas GAP45 is phosphorylated at two sites, one of which could also be detected in phosphopeptides purified from parasite lysates. Both CDPK1 activity and host cell invasion can be inhibited by the kinase inhibitor K252a, suggesting that CDPK1 is a suitable target for antimalarial drug development.     

Purification and characterization of wheat and pine small basic protein substrates for plant calcium-dependent protein kinase.

A wheat basic protein (WBP) was purified to homogeneity from wheat germ by a protocol involving extraction, centrifugation, batchwise elution from carboxymethylcellulose (CM-52), acidification with trifluoroacetic acid, neutralization and HPLC on a SP5PW cation exchange column. WBP is a 10 kDa protein and is phosphorylated on serine residues by wheat germ Ca(2+)-dependent protein kinase (CDPK). [32P]phosphoWBP exactly comigrates with WBP on SDS-PAGE. WBP does not inhibit either wheat germ CDPK or calmodulin-dependent myosin light chain kinase. Apart from histone H1, WBP is the best endogenous substrate yet found for wheat embryo CDPK. A 12 kDa pine basic protein (PBP) was purified to homogeneity from seeds of stone pine (Pinus pinea L.) by a simple procedure involving batchwise elution from carboxymethylcellulose and cation exchange HPLC. PBP is also a good substrate for CDPK and is phosphorylated on Ser residues. N-terminal sequencing of WBP and PBP revealed that these proteins are homologous to a family of small basic plant proteins having a phospholipid transfer function.     

A calcium- and phospholipid-dependent protein kinase from rice (Oryza sativa) leaves

Protein kinases in plants have not been examined in detail, but protein phosphorylation has been shown to be essential for regulating plant growth via the signal transduction system. A Ca²⁺- and phospholipid-dependent protein kinase, possibly involved in the intracellular signal transduction system from rice leaves, was partially purified by sequential chromatography on DE52, Phenyl Superose and Superose 12. This protein kinase phosphorylated the substrate, histone III-S, in the presence of Ca²⁺ and phosphatidylserine. The apparent molecular mass of the Ca²⁺- and phosphatidylserine-dependent protein kinase (Ca²⁺/PS PK), determined by phosphorylation in SDS-polyacrylamide gel containing histone III-S, was 50 kDa. The protein kinase differed from Ca²⁺-dependent protein kinase (CDPK) in rice leaves in that Ca²⁺/PS PK showed phospholipid dependency and the molecular mass of Ca²⁺/PS PK exceeded that of CDPK. Investigations were carried out on changes in Ca²⁺/PS PK and CDPK activity in the cytosolic and membrane fractions during germination. The maximum activity of Ca²⁺/PS PK in the cytosolic fraction was observed before imbibition and that of CDPK in the membrane fraction was noted at 6 days following imbibition. Protein kinases are likely to regulate plant growth through protein phosphorylation.     

Involvement of calcium-dependent protein kinase in rice (Oryza sativa L.) lamina inclination caused by brassinolide

Promotive effect of brassinolide (BL) on green lamina inclination was concentration-dependent when excised rice (Oryza sativa L.) lamina was floated on BL solution under continuous light conditions. Protein kinase inhibitor staurosporine and Ca2+ channel blocker LaCl3 could completely, while Ca2+ chelator EGTA could partially inhibit the lamina inclination caused by BL. Two protein kinases with apparent molecular masses of 45 and 54 kDa were detected using an in-gel kinase assay with histone III-S as a substrate. In particular, the changes in 45 kDa protein kinase activity correlated with lamina inclination caused by BL. The 45 kDa kinase activity was inhibited by Ca2+ chelator EGTA, protein kinase inhibitor, staurosporine and calmodulin antagonist W-7. Therefore, this 45 kDa protein kinase was identified as a Ca2+ -dependent protein kinase (CDPK). Patterns of 2-dimensional PAGE after in vitro phosphorylation of crude extracts showed that the phosphorylation of 56 and 41 kDa proteins, which was Ca2+ -dependent, was strongly increased by BL treatment. These results suggested that CDPK and Ca2+ -dependent protein phosphorylation are involved in BL-induced rice lamina inclination.     

Phosphorylation of a putative myosin light chain inChara by calcium-dependent protein kinase

Summary Ca²⁺-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca²⁺ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-³²P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10^–4M free Ca²⁺ compared to <10^–9M free Ca²⁺. The Ca²⁺-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca²⁺-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK calcium-dependent protein kinase - mAb monoclonal antibody     

Determination of the site of phosphorylation of nodulin 26 by the calcium-dependent protein kinase from soybean nodules.

Nodulin 26 is a nodule-specific protein that is associated with the symbiosome membrane of soybean root nodules. Nodulin 26 is an endogenous substrate for a novel calcium-dependent protein kinase (CDPK) of soybean root nodules. By phosphopeptide mapping of endoproteinase Lys-C-digested nodulin 26 and automated and manual peptide sequence analyses, we have identified the site on nodulin 26 phosphorylated by CDPK. We have also established that the phosphorylation site of nodulin 26 is identical to the phosphorylation site of CK-15, a synthetic peptide with the carboxyl-terminal sequence of nodulin 26. The phosphorylation of nodulin 26 occurs at position Ser262, and the phosphorylation of CK-15 occurs at the analogous position, Ser,6 in vitro. Thus, the CK-15 sequence apparently contains sufficient structural features of the phosphorylation site of nodulin 26 to be recognized by CDPK. On the basis of peptide mapping analysis of nodulin 26 from nodules that are metabolically labeled with [32P]phosphate, it appears that the site of nodulin 26 that is phosphorylated in vitro is also labeled in vivo. The data indicate that the carboxyl terminus of nodulin 26 is phosphorylated by CDPK and provide initial sequence data for the phosphorylation site of an endogenous substrate for a plant CDPK.     

Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose

Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.     

组蛋白激活拟南芥CDPK催化CaMBP10的体外磷酸化反应

植物转脂蛋白 (plant lipid transfer proteins, LTPs) 是高等植物中广泛存在的多基因编码的小分子碱性蛋白. 本研究室已经证明白菜和豌豆LTPs可分别被内源胞浆可溶性和膜结合钙依赖性蛋白激酶 (calcium-dependent protein kinase, CDPK) 磷酸化. 为深入研究CDPK对白菜钙调素结合蛋白10 (calmodulin-binding protein-10, CaMBP10) 的磷酸化性质及特征, 本文从拟南芥可溶性蛋白粗提物中检测到1个分子量约为54 kD的CDPK对CaMBP10有磷酸化作用. 研究表明, 组蛋白可增强 CDPK对CaMBP10的磷酸化活性, 促进磷酸化进程. 而且组蛋白和Ca2+对CDPK具有协同调节效应, 二者共同作用时比Ca2+单独作用时, 激酶的活力增强约12倍. 此外, 不同组蛋白对CDPK的激活能力不同, 组蛋白1对该激酶活性的激活能力要比组蛋白3高约8倍.     

Changes in Calcium-Dependent Protein Kinase Activity during in Vitro Tuberization in Potato

A soluble Ca2+-dependent protein kinase (CDPK) was purified to homogeneity in potato (Solanum tuberosum L.) plants. Potato CDPK was strictly dependent on Ca2+ (one-half maximal activation 0.6 [mu]M) and phosphorylated a wide diversity of substrates, in which Syntide 2 was the best phosphate acceptor (Michaelis constant = 30 [mu]M). The kinase was inhibited by Ca2+-chelating agents, phenotiazine derivatives, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (one-half maximal inhibition = 0.25 mM). Polyclonal antibodies directed against the regulatory region of the soybean CDPK recognized a 53-kD polypeptide. In an autophosphorylation assay, this same band was strongly labeled with [[gamma]-32P]ATP in the presence of Ca2+. CDPK activity was high in nontuberized plants, but increased 2.5-fold at the onset of tuber development and was reduced to one-half of its original activity when the tuber had completed formation. In the early stages of tuberization, Ca2+-dependent phosphorylation of endogenous targets (specific bands of 68, 51, and 46 kD) was observed. These polypeptides were not labeled in nontuberizing plants or in completely formed tubers, indicating that this phosphorylation is a stage-specific event. In addition, dephosphorylation of specific polypeptides was detected in tuberizing plants, suggesting the involvement of a phosphatase. Preincubation of crude extracts with phosphatase inhibitors rendered a 100% increase in CDPK activity.     

Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response

In the Cf-9/Avr9 gene-for-gene interaction, the Cf-9 resistance gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum, which expresses the corresponding pathogen-derived avirulence product Avr9. To understand R gene function and dissect the signaling mechanisms involved in the induction of plant defenses, we studied Cf-9/Avr9-dependent activation of protein kinases in transgenic Cf9 tobacco cell cultures. Using a modified in-gel kinase assay with histone as substrate, we identified a membrane-bound, calcium-dependent protein kinase (CDPK) that showed a shift in electrophoretic mobility from 68 to 70 kD within 5 min after Avr9 elicitor was added. This transition from the nonelicited to the elicited CDPK form was caused by a phosphorylation event and was verified when antibodies to CDPK were used for protein gel blot analysis. In addition, the interconversion of the corresponding CDPK forms could be induced in vitro in both directions by treatment with either phosphatase or ATP. In vitro protein kinase activity toward syntide-2 or histone with membrane extracts or gel-purified enzyme was dependent on Ca(2)+ content and was compromised by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) but not by its inactive isoform N-(6-aminohexyl)-1-naphthalenesulfonamide. In these assays, the CDPK activity in elicited samples, reflecting predominantly the phosphorylated 70-kD CDPK form, was greater than in nonelicited samples. Thus, Avr9/Cf-9-dependent phosphorylation and subsequent transition from the nonelicited to the elicited form correlate with the activation of a CDPK isoform after in vivo stimulation. Because that transition was not inhibited by W-7, the in vivo CDPK activation probably is not the result of autophosphorylation. Studies with pharmacological inhibitors indicated that the identified CDPK is independent of or is located upstream from a signaling pathway that is required for the Avr9-induced active oxygen species.     

Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin

Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3zeta, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3zeta binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.     

The phosphorylation site of Ca2+-dependent protein kinase from alfalfa

A 50 kDa, calcium-dependent protein kinase (CDPK) was purified about 1000-fold from cultured cells of alfalfa (Medicago varia) on the basis of its histone H1 phosphorylation activity. The major polypeptide from bovine histone H1 phosphorylated by either animal protein kinase C (PK-C) or by the alfalfa CDPK gave an identical phosphopeptide pattern. The phosphoamino acid determination showed phosphorylation of serine residues in histone H1 by the plant enzyme. Histone-related oligopeptides known to be substrates for animal histone kinases also served as substrates for the alfalfa kinase. Both of the studied peptides (GKKRKRSRKA; AAASFKAKK) inhibited phosphorylation of H1 histones by bovine and alfalfa kinases. The results of competition studies with the nonapeptide (AAASFKAKK), which is a PK-C specific substrate, suggest common features in target recognition between the plant Ca²⁺-dependent kinase and animal protein kinase C. We also propose that synthetic peptides like AAASFKAKK can be used as a tool to study substrates of plant kinases in crude cell extracts.