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1.
M. M. K. KHAN S. YANG Y. IWASAKI Y. FUJISAWA H. FUKUDA & S. KOMATSU 《Plant, cell & environment》2005,28(5):679-687
The GA-signal transduction pathways downstream to the Gα protein in rice seedling root were investigated using in-gel kinase assay and in vitro protein phosphorylation techniques with a Gα protein defective mutant, d1. A 50-kDa protein kinase was detected downstream to Gα protein in the membrane fraction of rice seedling roots using an in-gel kinase assay with histone III-S as a substrate. The activity of a 50-kDa protein kinase increased in the wild-type rice by gibberellin (GA3) treatment, but did not change in the d1 mutant. This protein kinase activity was inhibited by the Ca2+ chelator ethyleneglycol-bis-(beta-aminoethylether)-N,N,N 1,N 1-tetraacetic acid (EGTA), protein kinase inhibitors, staurosporine and H7, and calmodulin antagonist, trifluoperazine, suggesting that the 50-kDa protein kinase is a putative plant Ca2+-dependent protein kinase (CDPK). The activity of the 50-kDa putative CDPK reached its highest level at 3 h after GA3 treatment and then gradually declined with time. In order to identify the endogenous substrate for 50-kDa putative CDPK, two-dimensional polyacrylamide gel electrophoresis followed by in vitro protein phosphorylation was carried out. The phosphorylation activity of an endogenous protein PP30, identified as an unknown protein having molecular weight 30 kDa and isoelectric point 5.8 was increased in the wild-type rice by GA3 treatment, compared with the d1 mutant. The addition of GA3 treated membrane fraction, which predominantly represent a 50-kDa putative CDPK further increased the phosphorylation of PP30. Almost similar to GA3 treatment, phosphorylation activity of PP30 was also increased by the treatment with cholera toxin in the wild-type rice but not in d1 mutant. These results suggest that the 50-kDa putative CDPK and an unknown protein, PP30 promoted by GA3 treatment are G-protein mediated in rice seedling roots. 相似文献
2.
A Ca2+-dependent protein kinase (CDPK) that has been partiallypurified and characterized previously [Yuasa and Muto (1992)Arch. Biochem. Biophys. 296: 175] was further purified to about20,000-fold from the soluble fraction of Dunaliella tertiolecta.The enzyme preparation contained 60- and 52-kDa polypeptidesboth of which phosphorylated casein as a substrate. Both polypeptidesshowed a Ca2+-dependent increase in mobility during SDS-PAGEand 45Ca2+-binding activity after SDS-PAGE and electroblottingonto a nitrocellulose membrane, suggesting that both the 60-and 52-kDa CDPKs directly bind Ca2+. The protein kinase inhibitors,K-252a and staurosporine, inhibited the CDPK competitively withrespect to ATP. An antibody raised against the 60-kDa CDPK crossreactedwith both the 60- and 52-kDa polypeptides. Both molecular specieswere autophosphorylated in the presence of Ca2+, and a highlyphosphorylated 80-kDa band appeared in addition to these phosphorylatedbands at 60 and 52 kDa in SDS-PAGE. However, the specific activityof CDPK was not changed by prior autophosphorylation when theautophosphorylated enzyme was assayed as a mixture of thesephosphorylated molecular species. Only the 60-kDa polypeptidewas immunodetected in subcellular fractions of Dunaliella cells.The 52-kDa polypeptide increased during storage of the enzyme.These results suggest that the 52-kDa polypeptide is a proteolyticartifact produced during purification. Immunoreactive bandsof 60-kDa were detected in extracts of several green algae butnot in extracts of higher plants or a brown alga.
1This research was partly supported by Grants-in-Aid from theMinistry of Education, Science and Culture, Japan (No. 06454013and 06304023) and Research Fellowship of the Japan Society forthe Promotion of Science for Young Sciencists.
2Research Fellow (PD) of the Japan Society for the Promotionof Science. 相似文献
3.
Localization, solubilization and characterization of plant membrane-associated calcium-dependent protein kinases 总被引:5,自引:4,他引:1
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Membrane fractions from mature silver beet (Beta vulgaris) deveined leaf and leaf stem homogenates have associated Ca2+ -dependent protein kinase. The Ca2+ -dependent protein kinase activity is associated with plasma membranes (density 1.14-1.18 grams per cubic centimeter) as determined from copurification on isopycnic centrifugation with plasma membrane markers such as β-glucan synthetase, eosin-5-maleimidelabeling, and specific naphthylphthalamic acid-binding. The Ca2+ -dependent protein kinase is not specifically associated with chloroplasts or mitochondria. The membrane-bound Ca2+ -dependent protein kinases were solubilized with 0.8% (volume/volume) Nonidet P40. The solubilized enzymes were extensively purified by a protocol involving binding to diethylaminoethyl-cellulose (Whatman DE-52), Ca2+ -dependent binding to phenyl-Sepharose CL-4B, gradient elution from diethylaminoethyl-Sephacel (resolving two distinct Ca2+ -dependent protein kinases), and gel filtration on Ultrogel AcA 44. These two membrane-derived enzymes have similar molecular weights but differ in protein substrate specificity, in Km values for ATP, and in Ca2+ -independent activation by unsaturated fatty acids. The membrane-bound enzymes correspond closely in these properties to two Ca2+ -dependent protein kinases present in the soluble phase. 相似文献
4.
《Plant science》1988,58(2):135-144
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. 相似文献
5.
《Plant Physiology and Biochemistry》2003,41(4):369-374
Ca2+-dependent protein kinases (CDPKs) (EC 2.7.1.37) are the predominant Ca2+-regulated serine/threonine protein kinase in plants and their genes are encoded by a multigene family. CDPKs are important components in signal transduction, but the precise role of each individual CDPK is still largely unknown. A CDPK gene designated as OsCDPK13 was cloned from rice seedlings and it showed a high level of sequence similarities to rice and other plant CDPK genes. OsCDPK13 contains all conserved regions found in CDPKs. It was a single copy gene and was highly expressed in root and leaf sheath tissues of rice seedlings. OsCDPK13 expression was increased in leaf sheath segments treated with gibberellin or subjected to cold stress. The results in this investigation, together with our previous studies, suggest that OsCDPK13 may be an important signaling component in rice seedlings under cold stress condition and in response to gibberellin. 相似文献
6.
7.
Several plasma-membrane proteins from beet root (Beta vulgaris L.) have been functionally incorporated into reconstituted proteoliposomes. These showed H+-ATPase activity, measured both as ATP hydrolysis and H+ transport. The proton-transport specific activity was 10 times higher than in plasma membranes, and was greatly stimulated
by potassium and valinomycin. These proteoliposomes also showed calcium-regulated protein kinase activity. This kinase activity
is probably due to a calmodulin-like domain protein kinase (CDPK), since two protein bands were recognized by antibodies against
soybean and Arabidopsis CDPK. This kinase phosphorylated histone and syntide-2 in a Ca2+-dependent manner. Among the plasma-membrane proteins phosphorylated by this kinase, was the H+-ATPase. When the H+-ATPase was either prephosphorylated or assayed in the presence of Ca2+, both the ATP-hydrolysis and the proton-transport activities were slower. This inhibition was reversed by an alkaline-phosphatase
treatment. A trypsin treatment (that has been reported to remove the C-terminal autoinhibitory domain from the H+-ATPase) also reversed the inhibition caused by phosphorylation. These results indicate that a Ca2+-dependent phosphorylation, probably caused by a CDPK, inhibits the H+-ATPase activities. The substrate of this regulatory phosphorylation could be the H+-ATPase itself, or a different protein influencing the ATPase activities.
Received: 1 May 1997 / Accepted: 25 June 1997 相似文献
8.
Several calcium-dependent protein kinases (CDPKs) are located in plant plasma membranes where they phosphorylate enzymes and transporters, like the H+-ATPase and water channels, thereby regulating their activities. In order to determine which kinases phosphorylate the H+-ATPase, a calcium-dependent kinase was purified from beetroot (Beta vulgaris L.) plasma membranes by anion-exchange chromatography, centrifugation in glycerol gradients and hydrophobic interaction chromatography. The kinetic parameters of this kinase were determined (V
max: 3.5 μmol mg−1 min−1, K
m
for ATP: 67 μM, K
m
for syntide 2: 15 μM). The kinase showed an optimum pH of 6.8 and a marked dependence on low-micromolar Ca2+ concentrations (K
d
: 0.77 μM). During the purification procedure, a 63-kDa protein with an isoelectric point of 4.7 was enriched. However, this protein was shown not to be a kinase by mass spectrometry. Kinase activity gels showed that a 50-kDa protein could be responsible for most of the activity in purified kinase preparations. This protein was confirmed to be a CDPK by mass spectrometry, possibly the red beet ortholog of rice CDPK2 and Arabidopsis thaliana CPK9, both found associated with membranes. This kinase was able to phosphorylate purified H+-ATPase in a Ca2+-dependent manner.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . 相似文献
9.
Involvement of calcium-dependent protein kinase in rice (Oryza sativa L.) lamina inclination caused by brassinolide 总被引:12,自引:0,他引:12
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. 相似文献
10.
11.
Ogawa Noriyuki; Yabuta Naohiro; Ueno Yoshihisa; Izui Katsura 《Plant & cell physiology》1998,39(10):1010-1019
Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31
[EC]
] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca2+-requiring protein kinase from Ca2+-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa2+-dependent but calmodulin-independent protein kinase (CDPK).Although the Mr, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an Mr of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed.
1Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan.
2Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan.
4N.O. and N.Y. contributed equally to this work. 相似文献
12.
Claudia M. Ribaudo José A. Curá María L. Cantore 《World journal of microbiology & biotechnology》2017,33(2):22
Rice seedlings (Oryza sativa) inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense FT326 showed an enhanced development of the root system 3 days after inoculation. Later on, a remarkable enlargement of shoots was also evident. An increase in the Ca2+-dependent histone kinase activity was also detected as a result of inoculation. The biochemical characterization and Western-blot analysis of the kinase strongly supports the hypothesis that it belongs to a member of the rice CDPK family. The fact that the amount of the protein did not change upon inoculation seems to indicate that a posttranslational activation is responsible for the change in the enzymatic activity. An in-gel kinase experiment identified a 46 kDa CDPK like protein kinase as a putative component of the signal transduction pathway triggered by Azospirillum inoculation. To our knowledge, this is the first report on the possible involvement of a Ca2+-dependent protein kinase in promotion of rice plants growth by A. brasilense. 相似文献
13.
Cytoplasmic streaming in the characean algae is inhibited by micromolar rises in the level of cytosolic free Ca2+, but both the mechanism of action and the molecular components involved in this process are unknown. We have used monoclonal antibodies against soybean Ca2+-dependent protein kinase (CDPK), a kinase that is activated by micromolar Ca2+ and co-localizes with actin filaments in higher-plant cells (Putnam-Evans et al., 1989, Cell Motil. Cytoskel.12, 12–22) to identify and localize its characean homologue. Immunoblot analysis revealed that CDPK inChara corralina Klein ex. Wild shares the same relative molecular mass (51–55 kDa) as the kinase purified from soybean, and after electrophoresis in denaturing gels is capable of phosphorylating histone III-S in a Ca2+-dependent manner. Immunofluorescence microscopy localized CDPK inChara to the subcortical actin bundles and the surface of small organelles and other membrane components of the streaming endoplasm. The endoplasmic sites carrying CDPK were extracted from internodal cells by vacuolar perfusion with 1 mM ATP or 10–4 M Ca2+. Both the localization of CDPK and its extraction from internodal cells by perfusion with ATP or high Ca2+ are properties similar to that reported for the heavy chain of myosin inChara (Grolig et al., 1988, Eur. J. Cell Biol.47, 22–31). Based on its endoplasmic location and inferred enzymatic properties, we suggest that CDPK may be a putative element of the signal-transduction pathway that mediates the rapid Ca2+-induced inhibition of streaming that occurs in the characean algae.Abbreviations CDPK
calcium-dependent protein kinase
- kDa
kilodalton
- mAb
monoclonal antibody
- Mr
relative molecular mass
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
We thank Dr. Richard Williamson (Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University) for valuable discussions during the course of this research. This work was supported by funds from a Queen Elizabeth II Fellowship awarded to D.W.McC. and U.S. Department of Agriculture (88-37261-4199) and National Research Inititive Competitive Grants Program (91-37304-6654) grants to A.C.H. 相似文献
14.
Koh-ichi Sugiyama Izumi C. Mori Koji Takahashi Shoshi Muto Ikuko Shihira-Ishikawa 《Journal of phycology》2000,36(6):1145-1152
The cytoplasm around a wound made in the multinucleate unicellular green alga Ventricaria ventricosa ( J. Agardh) Olsen et West formed an aggregation-ring surrounding the wound immediately after injury. A contraction of the ring then brought about wound healing in culture medium containing Ca2 + . Involvement of a calcium-dependent protein kinase (CDPK) as a regulator of wound healing was examined using an anti- Dunaliella tertiolecta CDPK antibody. A 52-kDa protein cross-reacting with the antibody was detected by Western blotting. Protein kinases of 60 kDa and 52 kDa, which were markedly activated by Ca2 + , and a 40-kDa Ca2 + -independent protein kinase were detected by an in-gel protein kinase assay using myelin basic protein as the substrate. A 52-kDa band with Ca2 + -dependent protein kinase activity was immunoprecipitated from the cytoplasmic extract, indicating that these 52-kDa proteins are identical and possess CDPK activity. Microscopic observation showed that the contraction of the aggregation ring was suppressed by application of the anti-CDPK to the culture medium. A protein kinase inhibitor, K-252a, and the calmodulin inhibitors, calmidazolium and compound 48 / 80, which inhibit CDPK activity, also suppressed the contraction of the aggregation-ring. Immunofluorescence microscopy showed a similar distribution of 52-kDa CDPK to the distribution of f-actin, which was randomly distributed in an intact cell and formed a bundle during wound healing. Further, f-actin was not recruited after injury in the presence of the antibody to CDPK. These results suggest that the 52-kDa CDPK functions as a Ca2 + receptor in wound healing and simultaneously participates in the organization and contraction of f-actin to heal the wound. 相似文献
15.
Involvement of a Calcium-Dependent Protein Kinase in Hypoosmotic Turgor Regulation in a Brackish Water Characeae Lamprothamnium succinctum 总被引:1,自引:0,他引:1
Yuasa Takashi; Okazaki Yoshiji; Iwasaki Naohiko; Muto Shoshi 《Plant & cell physiology》1997,38(5):586-596
Calcium ion is a key messenger in turgor regulation of internodalcells of Lamprothamnium succinctum in response to hypoosmotictreatment. An increase in the concentration of cytosolic freecalcium ion ([Ca2+]c) is prerequisite for the turgor regulation[Okazaki and Tazawa (1990) J. Membr. Biol. 114: 189], We examinedwhether or not a calcium-dependent protein kinase (CDPK) isinvolved in the Ca2+-mediated turgor regulation of Lamprothamniumcells. A 53-kDa CDPK which phosphorylated preferentially histoneH1 but poorly myelin basic protein or casein, was detected inthe cell extract of Lamprothamnium by an in-gel protein kinaseassay. This protein kinase was detected by Western blottingand was immunoprecipitated using an anti-Dunaliella tertiolectaCDPK antibody which can neutralize the Dunaliella CDPK activity[Yuasa et al. (1995) Plant Cell Physiol. 36: 699]. The 53-kDaCDPK was partially purified from Lamprothamnium and its activitywas shown to be inhibited by the antibody and K-252a, a proteinkinase inhibitor. Microinjection of the antibody into the cytosblof Lamprothamnium cells inhibited the decrease in turgor pressurein response to hypoosmotic treatment. However, a transient increasein [Ca2+]c, which was suggested by a transient reduction ofthe velocity of cytoplasmic streaming, was induced in antibody-injectedcells after hypoosmotic treatment. Turgor regulation upon hypoosmotictreatment was inhibited when the cells were treated with K-252a.These results imply that CDPK of Lamprothamnium functions ata down-stream position of Ca2+-mobilization in processing turgorregulation in response to hypoosmotic treatment.
2 These authors contributed equally to the work. 相似文献
16.
17.
Guard Cells Possess a Calcium-Dependent Protein Kinase That
Phosphorylates the KAT1 Potassium Channel 总被引:25,自引:2,他引:23
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Increasing evidence suggests that changes in cytosolic Ca2+ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca2+ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca2+-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca2+ dependent. CDPK exhibits a Ca2+-induced electrophoretic mobility shift and its Ca2+-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca2+ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca2+-dependent protein phosphatase calcineurin enhances the Ca2+-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K+ channel KAT1 protein in a Ca2+-dependent manner. These results suggest that CDPK may be an important component of Ca2+ signaling in guard cells. 相似文献
18.
19.
Parvathi K. Gayathri J. Maralihalli G.B. Bhagwat A.S. Raghavendra A.S. 《Photosynthetica》2000,38(1):23-28
PEP carboxylase (PEPC) in leaves of C4 plants is activated by phosphorylation of enzyme by a PEPC-protein kinase (PEPC-PK). We reevaluated the pattern of PEPC phosphorylation in leaf extracts of Amaranthus hypochondriacus. It was dependent on Ca2+, the optimum concentration of which for stimulation was 10 mM. The extent of stimulation was inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), a Ca2+ chelator. The inhibition by BAPTA was relieved by the addition of Ca2+ but not by the addition of Mg2+. The stimulation by Ca2+ of PEPC phosphorylation was marginally enhanced by calmodulin (CaM), but not by diacylglycerol (DAG). Phosphorylation was strongly restricted by Ca2+ or Ca2+-CaM-dependent protein kinase inhibitors. Thus phosphorylation of PEPC is Ca2+-dependent in leaves of A. hypochondriacus and a calcium-dependent protein kinase (CDPK) may modulate PEPC-PK and subsequently the phosphorylation status of PEPC. 相似文献
20.
Reinhard Pinontoan Takashi Yuasa† ‡ Marinela I. Anderca Takashi Matsuoka Nobuyuki Uozumi Hitoshi Mori Shoshi Muto 《Journal of phycology》2000,36(3):545-552
A cDNA clone encoding a Ca2 +-dependent protein kinase (DtCPK1) with a calculated molecular mass of 65,746 Da was isolated by sequential immuno- and hybridization-screening from a cDNA library of the halotolerant green alga, Dunaliella tertiolecta Butcher (Chlorophyceae). Primary structure analysis of DtCPK1 revealed a long variable domain preceding a catalytic domain, an autoinhibitory junction domain, and a C-terminal calmodulin-like domain containing 4 EF-hand motifs. Database searches showed that DtCPK1 has a high similarity to CCK1 , a CDPK from the green alga, Chlamydomonas eugamentos Moewus . The N-terminal long variable domain of DtCPK1 contains neither the N-myristoylation motif, which is found in many CDPKs, nor the PEST motif, which is associated with rapid protein turnover and found in one CDPK subfamily. However, a putative Ca2 +-dependent lipid binding domain that might be responsible for the association of cytosolic DtCPK1 with the cell membrane was identified in the variable domain. Three CDPKs, with molecular masses of 62, 54, and 47 kDa respectively, were observed in an in-gel protein kinase assay of D. tertiolecta cells extract. No change in the activities of these CDPKs were observed for up to 30 min after D. tertiolecta cells had been subjected to a hypoosmotic shock. An antibody raised against a CDPK purified from D. tertiolecta and used to isolate the DtCPK1 cDNA clone cross-reacted strongly with the 62-kDa CDPK but weakly with the 54-kDa CDPK in a Western blot, indicating that the 62-kDa CDPK is identical to DtCPK1. There was no change in the intensity of these bands after hypoosmotic shock, implying that the cellular level of the enzyme protein is not associated with hypoosmotic shock. These results indicate that CDPK is activated only by the increase in cytosolic-free Ca2 + concentration in vivo . 相似文献