Polypeptides from maize seedlings with protein kinase functions

Low molecular weight protein kinase from maize seedlings was isolated. The molecular weight of the enzyme was below 10 000. The enzyme preferentially utilized ATP and casein as donor and acceptor of phosphorus, respectively. Casein kinase was a cyclic nucleotide-independent, heparin-sensitive enzyme. The low molecular weight casein kinase was resolved into basic, neutral and slightly acidic peaks of activity by chromatofocussing.     

Calcium-dependent protein kinase from maize seedlings activated by phospholipids.

A calcium- and phospholipid-dependent protein kinase of apparent molecular mass 54 kDa (designated ZmCPKp54) was partially purified from etiolated maize seedlings. Activity of ZmCPKp54 is stimulated by phosphatidylserine and phosphatidylinositol, but is not essentially affected by diolein and phorbol esters. The enzyme cross-reacts with polyclonal antibodies against the calmodulin like-domain of the calcium-dependent protein kinase, but not with antibodies against catalytic or regulatory domains of protein kinase C. ZmCPKp54 is not able to phosphorylate the specific substrates of protein kinase C (MARCKS peptide and protein kinase C substrate peptide derived from pseudosubstrate sequence) and its activity is not inhibited by specific PKC inhibitors (bisindolylmaleimide, protein kinase C pseudosubstrate inhibitory peptide). The substrate specificity and sensitivity to the inhibitors of the maize enzyme resembles calcium-dependent protein kinase. The biochemical and immunological properties indicate that ZmCPKp54 belongs to the calcium-dependent protein kinase family.     

Peptide phosphorylation by calcium-dependent protein kinase from maize seedlings.

Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA.     

Dual positional specificity of wound-responsive lipoxygenase from maize seedlings

Wound-responsive lipoxygenase full-length cDNA from Zea mays was used to heterologously express the lipoxygenase enzyme and positional specificity of the lipoxygenase reaction was determined. The purified lipoxygenase catalyzed the conversion of α-linolenic acid into both 13-hydroperoxylinolenic acid and 9-hydroperoxylinolenic acid with a ratio of 6 to 4. The phylogenetic tree analysis indicated that the lipoxygenase is a type 1-lipoxygenase and belongs to 9-lipoxygenase subfamily with exceptional positional specificity. Dual positional specificity of the wound-responsive lipoxygenase indicates the versatile utilization of a singular lipoxygenase species as both 13-lipoxygenase and 9-lipoxygenase.     

Characterization of a CK II protein kinase from etiolated oat seedlings

Protein kinases play a central role in controlling the cellular metabolism of living organisms. A protein kinase was purified from etiolated oat seedlings by several steps of ion-exchange and affinity chromatographies. The kinase was a 150-kDa tetrameric protein and composed of three subunits of 34, 37, and 40 kDa proteins. The 34 and 40 kDa proteins had ATP binding sites, suggesting that they are catalytic subunits and that the 37-kDa protein is a regulatory subunit. In the in vitro phosphorylation of a crude oat cell extract, it intensively phosphorylated a serine residue of a 110-kDa protein. The 110-kDa protein was tentatively identified as a DNA topoisomerase I, based on an amino acid sequence homology. Phosphorylation of the 110-kDa protein by the kinase required ATP or GTP as a phosphoryl group donor. The kinase activity was inhibited by 50% at a concentration of 0.05 microg/ml heparin. These results, therefore, indicate that the purified kinase is a CK II protein kinase and may be involved in the regulation of DNA topoisomerase I activity.     

Purification of a basic phospholipid transfer protein from maize seedlings

A phospholipid transfer protein has been purified 125-fold from maize seedlings. The successive steps of purification comprised gel filtration on Sephadex G-75, DEAE- and CM-chromatography and chromatofocusing. The homogeneity of the protein was determined by polyacrylamide gel electrophoresis with and without SDS and by isoelectric focusing. The protein has an apparent molecular weight of 20000, as estimated from SDS electrophoresis, and an isoelectric point of 8.8 ± 0.2. The amino acid composition of the protein is characterized by a high content of alanine, glycine, cysteine and serine and a small amount of lysine. A molecular weight of 14058 was calculated from this amino acid composition. The protein only loses 25% of its activity after 5 min heating at 95°C. The purified protein is able to transfer phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine between liposomes and mitochondria at the rates of, respectively, 100, 56 and 1.6. After incubation of the purified protein with [³H]phosphatidylcholine, a labelled phosphatidylcholine-protein complex was obtained after chromatofocusing. This suggests that the protein acts by carrying phosphatidylcholine from a membrane toward another one.     

Phospholipid transfer protein from maize seedlings is partly membrane-bound

Specific antibodies raised against phospholipid transfer protein from maize seeds, react with mitochondria or microsomes solubilized by sodium deoxycholate. A single precipitin line was observed with both types of solubilized membranes when the double immunodiffusion technique was used. When the solubilized membranes were separated by fast-protein liquid chromatography (FPLC) on a reverse phase column, prior to the immunodiffusion, only fractions co-migrating with the pure phospholipid transfer protein, reacted with the antibody. Alternatively, solubilized membranes were submitted to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting; the detection of antigen-antibody complexes by a peroxydase reagent revealed the presence of a band co-migrating with the pure protein. All these observations strongly suggest that phospholipid transfer proteins are membrane-bound. The physiological significance of this finding will be discussed.     

Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [³⁵S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 M KN-93, but binding is not affected by 5 M KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 M KN-93, but not by 5 M KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.Abbreviations CaM calmodulin - CaMK (II) Ca²⁺/calmodulin-dependent protein kinase (II) - CBP CaM-binding protein - CDPK Ca²⁺-dependent protein kinase - MCK1 maize homolog of mamalian CaMK This work is supported by the National Aeronautics and Space Administration grant No: NAGW 238.     

Mutational analysis of stress-responsive peanut dual specificity protein kinase. Identification of tyrosine residues involved in regulation of protein kinase activity

We recently reported that Arachis hypogaea serine/threonine/tyrosine (STY) protein kinase is developmentally regulated and is induced by abiotic stresses (Rudrabhatla, P., and Rajasekharan, R. (2002) Plant Physiol. 130, 380-390). Other than MAPKs, the site of tyrosine phosphorylation has not been documented for any plant kinases. To study the role of tyrosines in the phosphorylation of STY protein kinase, four conserved tyrosine residues were sequentially substituted with phenylalanine and expressed as histidine fusion proteins. Mass spectrometry experiments showed that STY protein kinase autophosphorylated within the predicted kinase ATP-binding motif, activation loop, and an additional site in the C terminus. The protein kinase activity was abolished by substitution of Tyr(297) with Phe in the activation loop between subdomains VII and VIII. In addition, replacing Tyr(148) in the ATP-binding motif and Tyr(317) in the C-terminal domain with Phe not only obliterated the ability of the STY protein kinase protein to be phosphorylated, but also inhibited histone phosphorylation, suggesting that STY protein kinase is phosphorylated at multiple sites. Replacing Tyr(213) in the Thr-Glu-Tyr sequence motif with Phe resulted in a 4-fold increase in autophosphorylation and 2.8-fold increase in substrate phosphorylation activities. Mutants Y148F, Y297F, and Y317F displayed dramatically lower phosphorylation efficiency (k(cat)/K(m)) with ATP and histone, whereas mutant Y213F showed increased phosphorylation. Our results suggest that autophosphorylation of Tyr(148), Tyr(213), Tyr(297), and Tyr(317) is important for the regulation of STY protein kinase activity. Our study reveals the first example of Thr-Glu-Tyr domain-mediated autoinhibition of kinases.     

Characterisation of two protein phosphatase 2A holoenzymes from maize seedlings

Two holoenzymes of protein phosphatase 2A (PP2A), designated PP2AI and PP2AII, were purified from maize seedlings. The subunit composition of maize holoenzymes generally resembled those of animal PP2A. Using SDS/PAGE and Western blots with antibodies generated against peptides derived from animal PP2A, we established the subunit composition of plant protein phosphatase 2A. In both maize holoenzymes, a 38000 catalytic (PP2Ac) and a 66000 constant regulatory subunit (A) constituting the core dimer of PP2A were present. In addition, PP2AI (180000-200000) contained a protein of 57000 which reacted with antibodies generated against the peptide (EFDYLKSLEIEE) conserved in all eukaryotic Balpha regulatory subunits. In contrast, none of the proteins visualised in PP2AII (140000-160000) by double staining reacted with these antibodies. The activity of PP2AI measured with (32)P-labelled phosphorylase a in the presence of protamine and ammonium sulfate is about two times higher than that of PP2AII. PP2AI and PP2AII displayed different patterns of activation by protamine, polylysine and histone H1 and exhibit high sensitivity toward inhibition by okadaic acid. The data obtained provide direct biochemical evidence for the existence in plants of PP2A holoenzymes composed of a catalytic subunit complexed with one or two regulatory subunits.     

The dual specificity protein kinase CLK3 is abundantly expressed in mature mouse spermatozoa

CLK3, a member of the LAMMER family of dual-specificity protein kinases, is abundantly expressed in the reproductive system of male mice. Specifically, high levels of CLK3 protein expression are found in mature spermatozoa in the testis and epididymis. The majority of the CLK3 protein in the testis is a full-length kinase-containing form, and only a small amount of a catalytically inactive N-terminally truncated splice variant protein product is observed. Within the mature spermatozoa CLK3 is localized to the acrosome and tail. CLK3 is expelled from the sperm following the acrosome reaction and inactivated, likely by degradation by the proteases released by the sperm during the acrosome reaction. The CLK family of kinases has previously been implicated in mRNA splicing; however, the bulk of the CLK3 protein in these cells is located in the cytoplasm, suggesting that CLK3 may have additional roles in the cell.     

Characterization of an HMG-CoA reductase from Listeria monocytogenes that exhibits dual coenzyme specificity

HMG-CoA reductase (HMGR) is an enzyme critical for cellular cholesterol synthesis in mammals and isoprenoid synthesis in certain eubacteria, catalyzing the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have isolated the gene encoding HMG-CoA reductase from Listeria monocytogenes and expressed the recombinant 6x-His-tagged form in Escherichia coli. Using NAD(P)(H), the enzyme catalyzes HMG-CoA reduction approximately 200-fold more efficiently than mevalonate oxidation in vitro. The purified enzyme exhibits dual coenzyme specificity, utilizing both NAD(H) and NADP(H) in catalysis; however, catalytic efficiency using NADP(H) is approximately 200 times greater than when using NAD(H). The statins mevinolin and mevastatin are weak inhibitors of L. monocytogenes HMG-CoA reductase, requiring micromolar concentrations for inhibition. Three-dimensional modeling reveals that the overall structure of L. monocytogenes HMG-CoA reductase is likely similar to the known structure of the class II enzyme from Pseudomonas mevalonii. It appears that the enzyme has catalytic amino acids in analogous positions that likely play similar roles and also has a flap domain that brings a catalytic histidine into the active site. However, in L. monocytogenes HMG-CoA reductase histidine 143 and methionine 186 are present in the putative NAD(P)(H)-selective site, possibly interacting with the 2' phosphate of NADP(H) or 2' hydroxyl of NAD(H) and providing the active site architecture necessary for dual coenzyme specificity.     

Mitogen-activated protein kinase phosphatase 1/dual specificity phosphatase 1 mediates glucocorticoid inhibition of osteoblast proliferation

Steroid-induced osteoporosis is a common side effect of long-term treatment with glucocorticoid (GC) drugs. GCs have multiple systemic effects that may influence bone metabolism but also directly affect osteoblasts by decreasing proliferation. This may be beneficial at low concentrations, enhancing differentiation. However, high-dose treatment produces a severe deficit in the proliferative osteoblastic compartment. We provide causal evidence that this effect of GC is mediated by induction of the dual-specificity MAPK phosphatase, MKP-1/DUSP1. Excessive MKP-1 production is both necessary and sufficient to account for the impaired osteoblastic response to mitogens. Overexpression of MKP-1 after either GC treatment or transfection ablates the mitogenic response in osteoblasts. Knockdown of MKP-1 using either immunodepletion of MKP-1 before in vitro dephosphorylation assay or short interference RNA transfection prevents inactivation of ERK by GCs. Neither c-jun N-terminal kinase nor p38 MAPK is activated by the mitogenic cocktail in 20% fetal calf serum, but their activation by a DNA-damaging agent (UV irradiation) was inhibited by either GC treatment or overexpression of MKP-1, indicating regulation of all three MAPKs by MKP-1 in osteoblasts. However, an inhibitor of the MAPK/ERK kinase-ERK pathway inhibited osteoblast proliferation whereas inhibitors of c-jun N-terminal kinase or p38 MAPK had no effect, suggesting that ERK is the MAPK that controls osteoblast proliferation. Regulation of ERK by MKP-1 provides a novel mechanism for control of osteoblast proliferation by GCs.     

Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase.

The MPS1 gene has been previously identified by a mutant allele that shows defects in spindle pole body (SPB) duplication and cell cycle control. The SPB is the centrosome-equivalent organelle in the yeast Saccharomyces cerevisiae, and it nucleates all the microtubules in the cell. We report the isolation of the MPS1 gene, which encodes an essential protein kinase homolog. The MPS1 open reading frame has been fused to those that encode the LexA protein or the GST protein and both of these constructs function in yeast. The fusion proteins have been affinity-purified from yeast extracts and the GST chimeric protein has been found to be a phosphoprotein. Both proteins have been used to demonstrate intrinsic in vitro protein kinase activity of Mps1p against exogenous substrates and itself (autophosphorylation). A mutation predicted to abolish kinase function not only eliminates in vitro protein kinase activity, but also behaves like a null mutation in vivo, suggesting that kinase activity contributes to the essential function of the protein. Phosphoamino acid analysis of substrates phosphorylated by Mps1p indicates that this kinase can phosphorylate serine, threonine and tyrosine residues, identifying Mps1p as a dual specificity protein kinase.     

Identification of the dual specificity and the functional domains of the cardiac-specific protein kinase TNNI3K

Molecular cloning of cardiac troponin I-interacting kinase (TNNI3K), a novel cardiac-specific protein kinase containing seven N-terminal ankyrin (ANK) repeats followed by a protein kinase domain and a C-terminal Ser-rich domain, has previously been reported. In the present study, we show that the C-terminal functional region of TNNI3K negatively regulates the kinase activity, and the N-terminal ANK domain is necessary for autophosphorylation. An in vitro kinase assay shows that TNNI3K exhibits dual-specific kinase activity and forms dimers or oligomers that may be necessary for its activation.     

Crystallization and preliminary X-ray crystallographic analysis of phospholipid transfer protein from maize seedlings

Phospholipid transfer protein from maize seedlings has been crystallized using trisodium citrate as precipitant. The crystal belongs to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 24.46 Å, b = 49.97 Å, and c = 69.99 Å. The presence of one molecule in the asymmetric unit gives a crystal volume per protein mass (V_m) of 2.36 Å ³/Da and a solvent content of 48% by volume. The X-ray diffraction pattern extends at least to 1.6 Å Bragg spacing when exposed to both CuK_α and synchrotron X-rays. A set of X-ray data to approximately 1.9 Å Bragg spacing has been collected from a native crystal. © 1994 Wiley-Liss, Inc.     

Hydroxamic Acid glucosyltransferases from maize seedlings

Hydroxamic acids occur in several forms in maize (Zea mays L.) with 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) being the predominant form and others including 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) being found at lower concentrations. Two enzymes capable of glucosylating hydroxamic acids were identified in maize protein extracts and partially purified and characterized. The total enzyme activity per seedling increased during the first 4 days of germination and was concurrent with the accumulation of DIMBOA. Purification of the enzymes by ammonium sulfate precipitation followed by Sephadex G-200 and Q-Sepharose gel chromatography resulted in a 13-fold increase in specific activity. The enzymes are initially separated into two peaks (peak 1 and peak 2) of activity by Q-Sepharose gel chromatography. The peak 1 glucosyltransferase had 3.6% of the DIMBOA glucosylating activity when DIBOA was used as substrate, whereas this percentage increased to 57% for the peak 2 enzyme. The enzyme in peak 2 has a K_m of 174 micromolar for DIMBOA and a K_m of 638 micromolar for DIBOA; the enzyme in peak 1 has a K_m of 217 micromolar for DIMBOA and its activity on DIBOA was too low to determine a K_m. The identification of two glucosyltransferases capable of glucosylating hydroxamic acids in vitro serves as an initial step in the characterization of the enzymes involved in production of hydroxamic acids in maize.     

Cloning and characterization of a novel CBL-interacting protein kinase from maize

A novel CBL-interacting protein kinase (CIPK) gene, ZmCIPK16, was isolated from maize (Zea mays), which has been certified to have two copies in the genome. The ZmCIPK16 is strongly induced in maize seedlings by PEG, NaCl, ABA, dehydration, heat and drought, but not by cold. A yeast two-hybrid assay demonstrated that ZmCIPK16 interacted with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8. Bimolecular fluorescence complementation (BiFC) assays prove that ZmCIPK16 can interact with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8 in vivo. Subcellular localization showed that ZmCIPK16 is distributed in the nucleus, plasma membrane and cytoplasm; this is different from the specific localization of ZmCBL3, ZmCBL4, and ZmCBL5, which are found in the plasma membrane. The results also showed that overexpression of ZmCIPK16 in the Arabidopsis sos2 mutant induced the expression of the SOS1 gene and enhanced salt tolerance. These findings indicate that ZmCIPK16 may be involved in the CBL-CIPK signaling network in maize responses to salt stress. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jinfeng Zhao and Zhenfei Sun are contributed equally to this work.     

Calcium-activated protein kinase from soluble and membrane fractions of maize coleoptiles

This paper describes the results of experiments in which phenyl Sepharose was used to partially purify Ca2(+)-activated protein kinase (CPK) from maize soluble and membrane-solubilized proteins. It is shown that CPK has very similar properties to Ca2(+)-activated, calmodulin independent protein kinase from other plant tissues, and that chromatography on phenyl Sepharose resolves two closely related forms of CPK from both soluble and membrane-solubilized proteins. The amount of each of these forms differs in the two fractions, and it is suggested that the kinase requiring EGTA for elution from phenyl Sepharose at high pH may be either a non-proteolitically digested form or an acylated form of CPK.