CaMKII knockdown attenuates H2O2-induced phosphorylation of ERK1/2, PKB/Akt, and IGF-1R in vascular smooth muscle cells

We have shown earlier a requirement for Ca²⁺ and calmodulin (CaM) in the H₂O₂-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key mediators of growth-promoting, proliferative, and hypertrophic responses in vascular smooth muscle cells (VSMC). Because the effect of CaM is mediated through CaM-dependent protein kinase II (CaMKII), we have investigated here the potential role of CaMKII in H₂O₂-induced ERK1/2 and PKB phosphorylation by using pharmacological inhibitors of CaM and CaMKII, a CaMKII inhibitor peptide, and siRNA knockdown strategies for CaMKIIα. Calmidazolium and W-7, antagonists of CaM, as well as KN-93, a specific inhibitor of CaMKII, attenuated H₂O₂-induced responses of ERK1/2 and PKB phosphorylation in a dose-dependent fashion. Similar to H₂O₂, calmidazolium and KN-93 also exhibited an inhibitory effect on glucose/glucose oxidase-induced phosphorylation of ERK1/2 and PKB in these cells. Transfection of VSMC with CaMKII autoinhibitory peptide corresponding to the autoinhibitory domain (aa 281–309) of CaMKII and with siRNA of CaMKIIα attenuated the H₂O₂-induced phosphorylation of ERK1/2 and PKB. In addition, calmidazolium and KN-93 blocked H₂O₂-induced Pyk2 and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation. Moreover, treatment of VSMC with CaMKIIα siRNA abolished the H₂O₂-induced IGF-1R phosphorylation. H₂O₂ treatment also induced Thr²⁸⁶ phosphorylation of CaMKII, which was inhibited by both calmidazolium and KN-93. These results demonstrate that CaMKII plays a critical upstream role in mediating the effects of H₂O₂ on ERK1/2, PKB, and IGF-1R phosphorylation.     

Sphingosine 1-phosphate attenuates H2O2-induced apoptosis in endothelial cells

Reactive oxygen species including H₂O₂ lead vascular endothelial cells (EC) to undergo apoptosis. Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid mediator that elicits various EC responses. We aimed to explore whether and how S1P modulates EC apoptosis induced by H₂O₂. Treatment of cultured bovine aortic EC (BAEC) with H₂O₂ (750 μM for 6 h) led to DNA fragmentation (ELISA), DNA nick formation (TUNEL staining), and cleavage of caspase-3, key features of EC apoptosis. These responses elicited by H₂O₂ were alike markedly attenuated by pretreatment with S1P (1 μM, 30 min). H₂O₂ induced robust phosphorylation of both p38 and JNK MAP kinases. However, pretreatment with S1P decreased phosphorylation of only p38 MAP kinase, but not that of JNK; conversely, an inhibitor of p38 MAP kinase, but not that of JNK, attenuated H₂O₂-induced caspase-3 activation. Thus S1P attenuates H₂O₂-induced apoptosis of cultured BAEC, involving p38 MAP kinase.     

Avian Reovirus activates a novel proapoptotic signal by linking Src to p53

We have previously shown that avian reovirus (ARV) S1133 and its structural protein σC cause apoptosis in cultured Vero cells through an unknown intracellular signaling pathway. This work investigates how ARV S1133 induces proapoptotic signals. Upon ARV S1133 infection and subsequent apoptosis, levels of p53 mRNA and protein, and p53 serine-46 and serine-392 phosphorylation increased. In addition, p53-driven reporter activity and levels of the p53-induced apoptotic protein bax were increased, and Src tyrosine-418 phosphorylation was elevated. UV-inactivated virus failed to activate Src, p53 or induce apoptosis. Over-expression of dominant negative p53, or treatment with tyrosine kinase inhibitor genistein protected cells from ARV S1133-induced apoptosis. Inhibition of Src by over-expression of C-terminal Src kinase (Csk) or treatment with Src family tyrosine kinase inhibitor SU-6656 diminished the ARV S1133-induced p53 expression, activation, and apoptosis. Over-expression of σC resulted in the upregulation of p53, p53 serine-46 phosphorylation, p53-driven reporter activity and accumulation of bax. σC expression during ARV S1133 infection was concomitant with the onset of apoptosis. These studies provide strong evidence that the viral gene expression is required for ARV S1133 to initiate a proapoptotic signal via Src to p53. In addition, σC was able to utilize a p53-dependent pathway to elicit apoptosis.     

The involvement of a P38-like MAP kinase in ABA-induced and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-mediated stomatal closure in <Emphasis Type="Italic">Vicia faba</Emphasis> L.

SB203580 is a specific inhibitor of p38 mitogen-activated protein (MAP) kinase and has been widely used to investigate the physiological roles of p38 in animal and yeast cells. Here by using an epidermal strip bioassay, laser-scanning confocal microscopy and whole-cell patch clamp analysis, we assess the effects of pyridinyl imidazoles-like SB203580 on the H₂O₂ signaling in guard cells of Vicia faba L. The results indicated that SB203580 blocks H₂O₂- or ABA-induced stomatal closure, ABA-induced H₂O₂ generation, and decrease in K⁺ fluxing across plasma membrane of Vicia guard cells by application of ABA and H₂O₂, whereas its analog SB202474 had no effect on these events. Thus, these results suggest that activation of p38-like MAP kinase modulates guard cell ROS signaling in response to stress.     

Competitive inhibition of hydrogen peroxide-induced aggregation of calf platelets by prostaglandin H2/thromboxane A2 receptor ligands

Hydrogen peroxide (H₂O₂)-induced aggregation of calf platelets and its modification by agents with specific properties were characterized employing a spectrophotometric assay. An Arrhenius activation energy of 20 ± 1 kcal/mol was found in the temperature range of 25‡-36‡C. Rate inhibition occurred on either side of this temperature range, and under anaerobic conditions. Exogenous Ca²⁺ ions were not required but Ca²⁺ ions, at 1 mM-concentration, optimally increased rates and extent of aggregation at suboptimal H₂O₂ concentrations but only extent of aggregation at optimal H₂O₂ concentrations. Ba²⁺, Sr²⁺, Cd²⁺, Mn²⁺ and Ni²⁺ ions (1 mM) and Zn²⁺, Pb²⁺ and Hg²⁺ ions (10 mM) were inhibitory. The cyclo-oxygenase inhibitor, indomethacin (10-30 mM) exerted only mild inhibition by a competitive mechanism. Another cyclo-oxygenase inhibitor, aspirin, functioned to increase aggregation. Ligands acting directly at the prostaglandin H₂/thromboxane A, receptor (5Z. 9, 11, 13E, 15(S) 15-hydroxy 9(11) epoxy methano prosta 5, 13-dien-1-oic acid, pinane thromboxane A₂, arachidonic acid, eicosapentaenoic acid, and N-ethylmaleimide) functioned as competitive inhibitors. Another platelet-activating sulphydryl reagent, thimerosal, also inhibited competitively while the protein kinase C inhibitor, sphingosine, and the protein kinase C modulator, Zn²⁺ ions, inhibited by different mechanisms. The results indicate direct action of H₂O₂ at the prostaglandin H₂/thromboxane A₂ receptor, possibly its sulphydryls, to activate the protein kinase C pathway, independently of cyclo-oxygenase products. The results underscored the power of the kinetic approach for investigating mechanisms of platelet activation.     

Role of the JAK-STAT pathway in protection of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress in PC12 cells

The aim of this study was to investigate the role of JAK-STAT pathway in the cytoprotection afforded by preconditioning with H₂O₂. It was shown that (1) Preconditioning with 100 μmol/L H₂O₂ can markedly protect PC12 cells against apoptosis and cytotoxicity induced by 300 μmol/L H₂O₂; (2) The expression and tyrosine phosphorylation of JAK2, not JAK1 were rapidly increased at 5 min after H₂O₂ preconditioning; (3) The expression of STAT1 and STAT3 were significantly increased at 15 min after H₂O₂ preconditioning, and the pTyr-STAT1 and pTyr-STAT3 were markedly increased at 60 min after H₂O₂ preconditioning; (4) Pretreatment with the JAK inhibitor AG-490 (10 μmol/L) 20 min before H₂O₂ preconditioning blocked not only the activation of JAK2, STAT1 and STAT3, but also the cytoprotection of H₂O₂ preconditioning against apoptosis and cytotoxicity induced by oxidative stress. These findings suggested that preconditioning with H₂O₂ activated the JAK-STAT pathway that played an important role in the cytoprotection induced by H₂O₂ preconditioning.     

Role of ERK in Hydrogen Peroxide-Induced Cell Death of Human Glioma Cells

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. Activation of extracellular signal-regulated kinase (ERK) in oxidative stress remains controversial. In some cellular systems, the ERK activation is associated with protection against oxidative stress, while in other system, the ERK activation is involved in apoptotic cell death. The present study was undertaken to examine the role of ERK activation in H₂O₂-induced cell death of human glioma (A172) cells. H₂O₂ resulted in a time- and dose-dependent cell death, which was largely attributed to apoptosis. H₂O₂ treatment caused marked sustained activation of ERK. The ERK activation and cell death induced by H₂O₂ was prevented by catalase, the hydrogen peroxide scavenger, and U0126, an inhibitor of ERK upstream kinase MEK1/2. Transient transfection with constitutive active MEK1, an upstream activator of ERK1/2, increased H₂O₂-induced cell death, whereas transfection with dominant-negative mutants of MEK1 decreased the cell death. The ERK activation and cell death caused by H₂O₂ was inhibited by antioxidants (N-acetylcysteine and trolox), Ras inhibitor, and suramin. H₂O₂ produced depolarization of mitochondrial membrane potential and its effect was prevented by catalase and U0126. Taken together, these findings suggest that growth factor receptor/Ras/MEK/ERK signaling pathway plays an active role in mediating H₂O₂-induced apoptosis of human glioma cells and functions upstream of mitochondria-dependent pathway to initiate the apoptotic signal.     

Hydrogen peroxide primes heart regeneration with a derepression mechanism

While the adult human heart has very limited regenerative potential, the adult zebrafish heart can fully regenerate after 20% ventricular resection. Although previous reports suggest that developmental signaling pathways such as FGF and PDGF are reused in adult heart regeneration, the underlying intracellular mechanisms remain largely unknown. Here we show that H₂O₂ acts as a novel epicardial and myocardial signal to prime the heart for regeneration in adult zebrafish. Live imaging of intact hearts revealed highly localized H₂O₂ (∼30 μM) production in the epicardium and adjacent compact myocardium at the resection site. Decreasing H₂O₂ formation with the Duox inhibitors diphenyleneiodonium (DPI) or apocynin, or scavenging H₂O₂ by catalase overexpression markedly impaired cardiac regeneration while exogenous H₂O₂ rescued the inhibitory effects of DPI on cardiac regeneration, indicating that H₂O₂ is an essential and sufficient signal in this process. Mechanistically, elevated H₂O₂ destabilized the redox-sensitive phosphatase Dusp6 and hence increased the phosphorylation of Erk1/2. The Dusp6 inhibitor BCI achieved similar pro-regenerative effects while transgenic overexpression of dusp6 impaired cardiac regeneration. H₂O₂ plays a dual role in recruiting immune cells and promoting heart regeneration through two relatively independent pathways. We conclude that H₂O₂ potentially generated from Duox/Nox2 promotes heart regeneration in zebrafish by unleashing MAP kinase signaling through a derepression mechanism involving Dusp6.     

Modulation of Erythrocyte Acetylcholinesterase Activity and Its Association with G Protein-Band 3 Interactions

Circulating acetylcholine, substrate of membrane acetylcholinesterase (AChE), is known to enhance the band 3 protein degree of phosphorylation. The purpose of this study was to verify whether the band 3 phosphorylation status is associated with a G protein and whether it is an influent factor on AChE enzyme activity. From blood samples of healthy donors, erythrocyte suspensions were prepared and incubated with AChE substrate (acetylcholine) and inhibitor (velnacrine), along with protein tyrosine kinase (PTK) and tyrosine phosphatase (PTP) inhibitors. AChE activity was determined by spectrophotometry and extract samples were analyzed by western blotting using primary antibodies to different G protein subunits. Our results with phosphorylated band 3 (PTP inhibitor) show an increase in erythrocyte AChE (p < 0.0001). A dephosphorylated band 3 state (PTK inhibitor) shows a significant decrease. We identified a potential linkage of protein subunits Gα_i1/2 and G_β with band 3 protein. Gα_i1/2 and G_β may be linked to the band 3 C-terminal site. Gα_i1/2 is associated with the band 3 N-terminal domain, except for the control and ACh aliquots. G_β is associated with both phosphorylated and dephosphorylated band 3 in the presence of velnacrine. We conclude that an erythrocyte G protein with subunits Gα_i1/2 and G_β is associated with band 3. AChE depends on the degree of band 3 phosphorylation and its association with Gα_i1/2 and G_β.     

Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210

Abstract

Background. Insulin protects cardiomyocytes from reactive oxygen species (ROS)-induced apoptosis after ischemic/reperfusion injury, but the mechanism is not clear. This study investigated the protective mechanism of insulin in preventing cardiomyocyte apoptosis from ROS injury. Methods. Rat cardiomyoblast H9c2 cells were treated with hydrogen peroxide (H₂O₂) or insulin at various concentrations for various periods of time, or with insulin and H₂O₂ for various periods of time. Cell viability was measured by the methylthiazolydiphenyl-tetrazolium bromide method. Cellular miR-210 levels were quantified using real-time RT-PCR. MiR-210 expression was also manipulated through lentivirus-mediated transfection. LY294002 was used to investigate involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Results. The percentage of viable cells was significantly and inversely associated with H₂O₂ concentration, an effect that was seemingly attenuated by insulin pretreatment. Treatments with H₂O₂ or insulin were associated with a significant increase in miR-210 levels. Manipulation of miR-210 expression by gene transfection showed that miR-210 could attenuate H₂O₂-induced cellular injury. Inhibition of the PI3K/Akt pathway by the Akt inhibitor LY294002 was associated with a decrease in miR-210 expression. Conclusion. Insulin stimulated the expression of miR-210 through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injury that had been induced by H₂O₂/oxygen species. Our results provide novel evidence regarding the mechanism underlying the protective effect of insulin.     

UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation

Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′-triphosphate (UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the most potent agonist among several nucleotides tested. Adenosine 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also potent agonists, characteristic of the P2Y₂ or P2Y₄ receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2 phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is mediated by a P2Y₂ or P2Y₄ receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR.     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

Human cord blood stem cell paracrine factors activate the survival protein kinase Akt and inhibit death protein kinases JNK and p38 in injured cardiomyocytes

Background aimsWe hypothesized that paracrine factors from human umbilical cord blood mononuclear cells (hUCBC) activate in injured cardiomyocytes the survival protein kinase Akt and limit activation of death protein kinases JNK and p38.MethodsWe treated hUCBC with H₂O₂ and measured growth factors and cytokines secreted by hUCBC. We then treated cardiomyocytes with H₂O₂ for 24 h and measured Akt, JNK and p38 activation by means of Western blots. We also measured myocyte viability and apoptosis with the use of fluorescence-activated cell-sorting cytometry. We then investigated myocytes treated for 24 h with H₂O₂ plus hUCBC and myocytes without hUCBC or H₂O₂. Four million hUCBC were placed in transwells permeable only to hUCBC paracrine factors, and the transwells were placed in flasks with H₂O₂+Dulbecco's modified Eagle's medium or in flasks with myocytes plus H₂O₂+Dulbecco's modified Eagle's medium.ResultshUCBC increased secretion during H₂O₂ of hepatocyte growth factor by 338%, insulin-like growth factor by 200%, interleukin-4 by 200%, vascular endothelial cell growth factor by 192%, placental growth factor by 150%, interleukin-10 by 150% and angiogenin by 121%. H₂O₂ increased myocyte JNK activation by 237% and p38 activation by 60%, decreased myocyte viability by 38% and increased necrosis by 34% (all P < 0.01). hUCBC paracrine factors increased in myocytes with H₂O₂ Akt activation by ≥25%, decreased JNK and p38 activation by >35%, increased viability by >22% and decreased apoptosis by >33% (all P < 0.05). Akt inhibitor API-1 prevented the effects of hUCBC and enhanced H₂O₂ decrease of myocyte viability. Addition of JNK inhibitor SP600125 or p38 inhibitor SB203580 to myocytes plus H₂O₂ prevented H₂O₂ decrease in viability and increased hUCBC beneficial effects.ConclusionsDuring free radical stress, hUCBC paracrine factors activate myocyte Akt, which increases myocyte viability by decreasing activation of death-promoting protein kinases JNK and p38.     

Defective nuclear translocation of stress-activated signaling in senescent diploid human fibroblasts: a possible explanation for aging-associated apoptosis resistance

In order to study the nature of aging-dependent apoptosis resistance, we compared the activation pattern of mitogen-activated protein kinases (MAPK) in response to three different stress modalities: hydrogen peroxide (H₂O₂), staurosporine, and thapsigargin. We observed the agonist-specific activation pattern of MAP kinases in human diploid fibroblasts (HDFs). When young HDFs were treated with PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK), H₂O₂-induced apoptosis was blocked, whereas staurosporine-induced apoptosis was inhibited by treatment with SB203580, a specific inhibitor of p38. In addition, the levels of anti-apoptotic protein Bcl-2 (B-cell lymphoma protein-2) were restored by PD98059 or SB239063 in cells treated with H₂O₂ or staurosporine, respectively. We also found that inhibition of the nuclear import of p-Erk and p-p38 using wheat germ agglutinin induced apoptosis resistance in young HDF cells in response to H₂O₂ or staurosporine. These data indicate a potential role of the nuclear translocation of apoptotic signals in the induction of apoptosis. Moreover, the nuclear translocation of activated ERK1/2 and p38 in response to H₂O₂ or staurosporine was significantly compromised in senescent HDFs, compared with young cells. Taken together, we propose that the apoptosis resistance of senescent HDFs might be related to the defective nuclear translocation of stress-activated signals in an agonist-specific manner, which would imply the operation of an aging-dependent functional nucleo-cytoplasmic trafficking barrier.     

Purification and characterization of an NADH oxidase from extremely thermophilic anaerobic bacterium Thermotoga hypogea

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It was found to be able to grow in the presence of micromolar molecular oxygen (O₂). Activity of NADH oxidase was detected in the cell-free extract of T. hypogea, from which an NADH oxidase was purified to homogeneity. The purified enzyme was a homodimeric flavoprotein with a subunit of 50 kDa, revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It catalyzed the reduction of O₂ to hydrogen peroxide (H₂O₂), specifically using NADH as electron donor. Its catalytic properties showed that the NADH oxidase had an apparent V_max value of 37 mol NADH oxidized min^–1 mg^–1 protein. Apparent K_m values for NADH and O₂ were determined to be 7.5 M and 85 M, respectively. The enzyme exhibited a pH optimum of 7.0 and temperature optimum above 85°C. The NADH-dependent peroxidase activity was also present in the cell-free extract, which could reduce H₂O₂ produced by the NADH oxidase to H₂O. It seems possible that O₂ can be reduced to H₂O by the oxidase and peroxidase, but further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic T. hypogea from accidental exposure to O₂.     

Role of receptor and nonreceptor protein tyrosine kinases in H2O2-induced PKB and ERK1/2 signaling

Excessive generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of many diseases, including atherosclerosis, hypertension, and vascular complications of diabetes. However, the precise mechanisms by which ROS contribute to the development of these diseases are not fully characterized. Hydrogen peroxide (H₂O₂), a ROS, has been shown to activate several signaling protein kinases, such as extracellular signal-regulated kinase (ERK)1/2 and protein kinase B (PKB) in different cell types, notably in vascular smooth muscle cells. Because these pathways regulate cellular mitogenesis, migration, proliferation, survival, and death responses, their aberrant activtion has been suggested to be a potential mechanism of ROS-induced pathologies. The upstream elements responsible for H₂O₂-induced ERK1/2 and PKB activation remain poorly characterized, but a potential role of receptor and nonreceptor protein tyrosine kinases (PTKs) as triggers that initiate such events has been postulated. Therefore, the aim of this review is to highlight the involvement of receptor and nonreceptor PTKs in modulating H₂O₂-induced ERK1/2 and PKB signaling.     

Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes

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 .