Redox regulation of the calcium/calmodulin-dependent protein kinases

Reactive oxygen intermediates (ROI) have been viewed traditionally as damaging to the cell. However, a predominance of evidence has shown that ROI can also function as important activators of key cellular processes, and ROI have been shown to play a vital role in cell signaling networks. The calcium/calmodulin-dependent protein kinases (CaM kinases) are a family of related kinases that are activated in response to increased intracellular calcium concentrations. In this report we demonstrate that hydrogen peroxide treatment results in the activation of both CaM kinase II and IV in Jurkat T lymphocytes. Surprisingly, this activation occurs in the absence of any detectable calcium flux, suggesting a novel means for the activation of these kinases. Treatment of Jurkat cells with phorbol 12-myristate 13-acetate (PMA), which does not cause a calcium flux, also activated the CaM kinases. The addition of catalase to the cultures inhibited PMA-induced activation of the CaM kinases, suggesting that similar to hydrogen peroxide, PMA also activates the CaM kinases via the production of ROI. One mechanism by which this likely occurs is through oxidation and consequential inactivation of cellular phosphatases. In support of this concept, okadaic acid and microcystin-LR, which are inhibitors of protein phosphatase 2A (PP2A), induced CaM kinase II and IV activity in these cells. Overall, these results demonstrate a novel mechanism by which ROI can induce CaM kinase activation in T lymphocytes.     

Activation of the Ikappa B kinases by RIP via IKKgamma /NEMO-mediated oligomerization

To understand the mechanism of activation of the IkappaB kinase (IKK) complex in the tumor necrosis factor (TNF) receptor 1 pathway, we examined the possibility that oligomerization of the IKK complex triggered by ligand-induced trimerization of the TNF receptor 1 complex is responsible for activation of the IKKs. Gel filtration analysis of the IKK complex revealed that TNFalpha stimulation induces a large increase in the size of this complex, suggesting oligomerization. Substitution of the C-terminal region of IKKgamma, which interacts with RIP, with a truncated DR4 lacking its cytoplasmic death domain, produced a molecule that could induce IKK and NF-kappaB activation in cells in response to TRAIL. Enforced oligomerization of the N terminus of IKKgamma or truncated IKKalpha or IKKbeta lacking their serine-cluster domains can also induce IKK and NF-kappaB activation. These data suggest that IKKgamma functions as a signaling adaptor between the upstream regulators such as RIP and the IKKs and that oligomerization of the IKK complex by upstream regulators is a critical step in activation of this complex.     

Specificity of calcium/calmodulin-dependent protein kinases in mouse egg activation

CaMKIIγ, the predominant CaMKII isoform in mouse eggs, controls egg activation by regulating cell cycle resumption. In this study we further characterize the involvement and specificity of CaMKIIγ in mouse egg activation. Using exogenous expression of different cRNAs in Camk2g^−/− eggs, we show that the other multifunctional CaM kinases, CaMKI, and CaMKIV, are not capable of substituting CaMKIIγ to initiate cell cycle resumption in response to a rise in intracellular Ca²⁺. Exogenous expression of Camk2g or Camk2d results in activation of nearly 80% of Camk2g^−/− MII eggs after stimulation with SrCl₂, which does not differ from the incidence of activation of wild-type eggs expressing exogenous Egfp. In contrast, none of the Camk2g^−/− MII eggs expressing Camk1 or Camk4 activate in response to SrCl₂ treatment. Expression of a constitutively active form of Camk4 (ca-Camk4), but not Camk1, triggers egg activation. EMI2, an APC/C repressor, is a key component in regulating egg activation downstream of CaMKII in both Xenopus laevis and mouse. We show that exogenous expression of either Camk2g, Camk2d, or ca-Camk4, but not Camk1, Camk4, or a catalytically inactive mutant form of CaMKIIγ (kinase-dead) in Camk2g^−/− mouse eggs leads to almost complete degradation (~90%) of exogenously expressed EMI2 followed by cell cycle resumption. Thus, degradation of EMI2 following its phosphorylation specifically by CaMKII is mechanistically linked to and promotes cell cycle resumption in MII eggs.     

Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation

AMP-activated protein kinase (AMPK) responds to oxidative stress. Previous work has shown that ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMPK and reduced the amount of AMPK protein. Ethanol generates oxidative stress in the liver. Since AMPK is activated by reactive oxygen species, it seems paradoxical that ethanol would inhibit AMPK in the hepatoma cells. In an attempt to understand the mechanism whereby ethanol inhibits AMPK, we studied the effect of ethanol on AMPK activation by exogenous hydrogen peroxide. The effects of ethanol, hydrogen peroxide, and inhibitors of protein phosphatase 2A (PP2A) [either okadaic acid or PP2A small interference RNA (siRNA)] on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3) and HeLa cells. In H4IIEC3 cells, hydrogen peroxide (H(2)O(2), 1 mM) transiently increased the level of phospho-AMPK to 1.5-fold over control (P < 0.05). Similar findings were observed in HeLa cells, which do not express the upstream AMPK kinase, LKB1. H(2)O(2) markedly increased the phosphorylation of LKB1 in H4IIEC3 cells. Ethanol significantly inhibited the phosphorylation of PKC-zeta, LKB1, and AMPK caused by exposure to H(2)O(2). This inhibitory effect of ethanol required its metabolism. More importantly, the inhibitory effects of ethanol on H(2)O(2)-induced AMPK phosphorylation were attenuated by the presence of the PP2A inhibitor, okadaic acid, or PP2A siRNA. The inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of PKC-zeta and LKB1 phosphorylation and the activation of PP2A.     

Studies on the phosphorylation of protein kinase B by Ca(2+)/calmodulin-dependent protein kinases

Protein kinase B (PKB) was recently reported to be activated on the phosphorylation of Thr(308) by Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaM-kinase kinase alpha), suggesting that PKB was regulated through not only the phosphoinositide 3-kinase pathway but also the Ca(2+)/calmodulin protein kinase pathway. The activation of PKB by CaM-kinase kinase alpha was as high as 300-fold after incubation for 30 min under the phosphorylation conditions, and still increased thereafter, suggesting that the maximal activation of PKB on phosphorylation of the Thr(308) residue is several hundred fold. On the other hand, the V(max) value of CaM-kinase kinase alpha for the phosphorylation of PKB was more than two orders of magnitude lower than that for CaM-kinase IV, although the K(m) values for PKB and CaM-kinase IV were not significantly different, raising the question of whether or not PKB is a physiological substrate of CaM-kinase kinase alpha. Besides CaM-kinase kinase alpha, CaM-kinase II also remarkably activated PKB. However, the specific activities of CaM-kinase kinase alpha and CaM-kinase II as to the activation of PKB were more than three orders of magnitude lower than that of 3-phosphoinositide-dependent protein kinase 1 (PDK1).     

Characterization of distinct tyrosine-specific protein kinases in B and T lymphocytes

Lymphocyte membrane fractions from both normal and neoplastic sources exhibit tyrosine-specific protein kinase activity. The molecular weights of the endogenous substrates phosphorylated on tyrosine residues differ in B and T cells. To further characterize membrane tyrosine phosphorylation in the two major classes of lymphocytes, the tryptic phosphopeptides of their endogenous substrates were compared and the sensitivity of the kinases to inhibition by N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) was determined. The two major B cell substrates (61,000 and 55,000 daltons, p61 and p55) were gel purified after phosphorylation and exhaustively digested with trypsin. Separation by reverse phase high pressure liquid chromatography demonstrated that these two substrates had two identical phosphotyrosine containing tryptic phosphopeptides. p61 had an additional phosphotyrosine site. Parallel analysis of the two T cell substrates (64,000 and 58,000 daltons, p64 and p58) showed that they also contained two phosphotyrosine sites that were identical. However, the tryptic phosphopeptides from the B and T cell substrate pairs were clearly distinct suggesting that they arise from different gene products. When B and T cell membrane fractions were preincubated with TLCK (21 degrees C, 30 min) a dose-dependent decrease in p64 and p58 phosphorylation resulted. p61 and p55 phosphorylation was not affected at concentrations up to 10 mM TLCK. Tyrosine-specific kinase activity was also assessed by measuring phosphorylation of a tyrosine containing synthetic peptide. The kinase activity of T cell plasma membrane fractions was inhibited by TLCK; the B cell activity was unaffected. The results suggest that membrane fractions from normal and some neoplastic B and T cells have at least two different tyrosine-specific kinases.     

Prolactin induces calcium influx and release from intracellular pools in human T lymphocytes by activation of tyrosine kinases

The early events related to intracellular signals after prolactin (PRL) activation in T lymphocytes are not clearly established. The aim of this work was to study the effect of PRL in cytosolic calcium levels in human T lymphocytes. By using the dye FURA-2 AM, the variations in cytosolic Ca(2+) were studied in peripheral human T lymphocytes isolated from extracted blood from healthy donors. Fifty nanograms per milliliter PRL induces a small increase in cytosolic calcium. When the cells are preincubated overnight (16-20 h) in the presence of PRL, the increase in calcium is higher. This high increase is due to the release from intracellular pools and to the influx from the extracellular media. That is, after overnight incubation with PRL, calcium influx in T cells follows the capacitative model. Since PRL receptor (PRL-R) activation involves the tyrosine kinase pathway, we check calcium effect in the presence of genistein, a known inhibitor of tyrosine kinases. When cells are preincubated in the presence of 10 microM genistein, and PRL is immediately added, no increase in cytosolic calcium is observed. The presence of genistein also completely blocks the increase in cytosolic calcium stimulated by PRL after overnight incubation with PRL. In the presence of PRL and N,N-dimethyl-D-erythro-sphingosine (DMS), a stimulus that increases cytosolic calcium in T cells by tyrosine kinase stimulation, a high, even insignificant, calcium influx is induced. However, when the cells are incubated overnight in the presence of PRL, and then DMS is added, a significant increase in cytosolic calcium levels takes place. This increase is associated with an increase in calcium release from intracellular pools and an increase in calcium uptake. Genistein reduces the influx of external calcium induced by DMS after short incubation with PRL and significantly inhibits both, calcium pools empty, and calcium influx is induced by DMS after overnight incubation with PRL. In summary, PRL induces calcium influx in normal T lymphocytes. The influx is magnified after long PRL exposures, intracellular Ca(2+) pool-dependent, and activated through tyrosine kinases.     

Effects of resveratrol and 4-hexylresorcinol on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

The protective effects of resveratrol and 4-hexylresorcinol against oxidative DNA damage in human lymphocytes induced by hydrogen peroxide were investigated. Resveratrol and 4-hexylresorcinol showed no cytotoxicity to human lymphocytes at the tested concentration (10-100 μM). In addition, DNA damage in human lymphocytes induced by H 2 O 2 was inhibited by resveratrol and 4-hexylresorcinol. Resveratrol and 4-hexylresorcinol at concentrations of 10-100 μM induced an increase in glutathione (GSH) levels in a concentration-dependent manner. Moreover, these two compounds also induced activity of glutathione peroxidase (GPX) and glutathione reductase (GR). The activity of glutathione-S-transferase (GST) in human lymphocytes was induced by resveratrol. Resveratrol and 4-hexylresorcinol inhibited the activity of catalase (CAT). These data indicate that the inhibition of resveratrol and 4-hexylresorcinol on oxidative DNA damage in human lymphocytes induced by H 2 O 2 might be attributed to increase levels of GSH and modulation of antioxidant enzymes (GPX, GR and GST).     

Mechanisms of hydrogen peroxide-induced calcium dysregulation in PC12 cells

The mechanisms of H(2)O(2)-induced elevated calcium baselines in PC12 cells were investigated in the present study by using fura-2-fluorescent image analysis. The results showed that the calcium comes from both intracellular and extracellular sources. Although the major intracellular source was mitochondria, only the extracellular calcium influx was responsible for the sustained post-H(2)O(2)-exposure increases. This calcium influx was partially blocked by calcium channel antagonists [verapamil (L-type) or mibefradil (nonselective)] and was more effectively blocked by the sodium channel antagonist, tetrodotoxin (TTX). Membrane depolarization following H(2)O(2) exposure contributed to the opening of the ion channels. The H(2)O(2)-induced calcium influx was blocked by TTX even in a sodium-free buffer, indicating that calcium directly fluxed through sodium channels. Sodium-calcium exchangers (NCX) on the plasma membrane did not play a role, because use of a specific reverse mode NCX inhibitor, No. 7943, was ineffective in blocking the influx. The H(2)O(2)-induced calcium influx was mimicked by using a thiol-selective oxidizing reagent, 2', 2'-dithiodipyridine, and in both situations, the calcium levels were completely reversed by a thiol-selective reducing reagent, dithiothreitol. Our results indicated that mechanisms of oxidant-induced elevated calcium baselines in PC12 cells involved calcium influx through sodium and calcium channels that may be directly or indirectly attributed to thiol oxidation.     

Changes of synaptotagmin interaction with t-SNARE proteins in vitro after calcium/calmodulin-dependent phosphorylation

The regulation of multiple phases of the life cycle of synaptic vesicles is carried out by a complex series of protein-protein interactions. According to the SNARE hypothesis the core of these interactions is a heterotrimeric complex formed by syntaxin, SNAP-25, and VAMP-synaptobrevin. Other proteins interacting with the core of the SNARE complex, such as voltage-activated calcium channels and synaptotagmin (a putative calcium sensor), are considered crucial for the calcium dependence of release and also molecular mediators of synaptic plasticity. Here the interaction of synaptotagmin with SNARE proteins was studied in immunoprecipitated native complexes, and the effects of previous phosphorylation-dephosphorylation on this interaction were analyzed. It is surprising that the interaction of synaptotagmin with syntaxin and SNAP-25 in native complexes was not found to be calcium-dependent. However, previous incubation under dephosphorylating conditions decreased the synaptotagmin-syntaxin interaction. Stimulation of Ca2+/calmodulin-dependent protein kinase II, which endogenously phosphorylates synaptotagmin in synaptic vesicles, increased the interaction of syntaxin and SNAP-25 with synaptotagmin (particularly when measured in the presence of calcium), as well as increasing the binding of the kinase itself. These results suggest that calcium decreases synaptotagmin-t-SNARE interactions after dephosphorylation and increases them after phosphorylation. Overall, these results imply a phosphorylation-dephosphorylation balance in regulation of the synaptotagmin-t-SNARE interaction and suggest a role for protein phosphorylation in the modulation of calcium sensitivity in transmitter release.     

The roles of calcium/calmodulin-dependent and Ras/mitogen-activated protein kinases in the development of psychostimulant-induced behavioral sensitization

Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium-stimulated second messengers, including the calcium/calmodulin-dependent protein kinases and the Ras/mitogen-activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium-stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long-term expression of psychostimulant-induced behavioral sensitization.     

Modulation of the phosphorylation and activity of calcium/calmodulin-dependent protein kinase II by zinc

Calcium/calmodulin-dependent protein kinase II (CaMPK-II) is a key regulatory enzyme in living cells. Modulation of its activity, therefore, could have a major impact on many cellular processes. We found that Zn(2+) has multiple functional effects on CaMPK-II. Zn(2+) generated a Ca(2+)/CaM-independent activity that correlated with the autophosphorylation of Thr(286), inhibited Ca(2+)/CaM binding that correlated with the autophosphorylation of Thr(306), and inhibited CaMPK-II activity at high concentrations that correlated with the autophosphorylation of Ser(279). The relative level of autophosphorylation of these three sites was dependent on the concentration of zinc used. The autophosphorylation of at least these three sites, together with Zn(2+) binding, generated an increased mobility form of CaMPK-II on sodium dodecyl sulfate gels. Overall, autophosphorylation induced by Zn(2+) converts CaMPK-II into a different form than the binding of Ca(2+)/CaM. In certain nerve terminals, where Zn(2+) has been shown to play a neuromodulatory role and is present in high concentrations, Zn(2+) may turn CaMPK-II into a form that would be unable to respond to calcium signals.     

Regulation of Ikappa B kinase (IKK)gamma /NEMO function by IKKbeta -mediated phosphorylation

The IkappaB kinase (IKK) complex includes the catalytic components IKKalpha and IKKbeta in addition to the scaffold protein IKKgamma/NEMO. Increases in the activity of the IKK complex result in the phosphorylation and subsequent degradation of IkappaB and the activation of the NF-kappaB pathway. Recent data indicate that the constitutive activation of the NF-kappaB pathway by the human T-cell lymphotrophic virus, type I, Tax protein leads to enhanced phosphorylation of IKKgamma/NEMO by IKKbeta. To address further the significance of IKKbeta-mediated phosphorylation of IKKgamma/NEMO, we determined the sites in IKKgamma/NEMO that were phosphorylated by IKKbeta, and we assayed whether IKKgamma/NEMO phosphorylation was involved in modulating IKKbeta activity. IKKgamma/NEMO is rapidly phosphorylated following treatment of cells with stimuli such as tumor necrosis factor-alpha and interleukin-1 that activate the NF-kappaB pathway. By using both in vitro and in vivo assays, IKKbeta was found to phosphorylate IKKgamma/NEMO predominantly in its carboxyl terminus on serine residue 369 in addition to sites in the central region of this protein. Surprisingly, mutation of these carboxyl-terminal serine residues increased the ability of IKKgamma/NEMO to stimulate IKKbeta kinase activity. These results indicate that the differential phosphorylation of IKKgamma/NEMO by IKKbeta and perhaps other kinases may be important in regulating IKK activity.     

Stimulus-induced phosphorylation of PKC theta at the C-terminal hydrophobic-motif in human T lymphocytes

Protein kinase C (PKC) is a family of serine/threonine kinases whose activity is controlled, in part, by phosphorylation on three conserved residues that are located on the catalytic domain of the enzyme, known as the activation-loop, the turn-motif, and the C-terminal hydrophobic-motif sites. Using a panel of phospho-specific antibodies, we have determined that PKC beta(I) and delta are constitutively phosphorylated on all three sites in unstimulated and activated T cells. Although PKC theta is constitutively phosphorylated at the activation-loop and turn-motif sites in T cells, PMA or anti-CD3/CD28 stimulation results in an increase in phosphorylation at the hydrophobic-motif (Ser695), an event that coincides with translocation of the enzyme from the cytosol/cytoskeleton to the membrane. Studies on the stimulus-induced phosphorylation of PKC theta demonstrate that an upstream kinase activity involving a conventional PKC isoform(s) and the PI3-kinase pathway, rather than autophosphorylation or the rapamycin-sensitive mTOR pathway, regulates this site in T lymphocytes. However, hydrophobic-motif phosphorylation does not appear to control membrane translocation, suggesting that this site may control other aspects of PKC theta signalling.     

Active calcium transport and calcium-dependent membrane phosphorylation in human peripheral blood lymphocytes

The characteristics of the calcium pump were investigated in intact human peripheral blood lymphocytes /PBL/ and in inside-out vesicles prepared from their plasma membranes. Intact PBL were loaded with calcium by a short exposure to A23187 ionophore. After the elimination of the ionophore, calcium-loaded PBL produced an ATP-dependent, external lanthanum sensitive, uphill calcium extrusion. Calcium pump in intact PBL was insensitive to ouabain and /until cellular ATP was provided/ to oligomycin and dinitrophenol. Maximum calcium extrusion rate and the alkali cation sensitivity of the process were similar to those in human red cells. Calcium was partially sequestered by PBL, and this calcium could be released by A23187 ionophore only.Inside-out plasma membrane vesicles prepared from hypotonically lysed PBL showed and ATP + Mg²⁺-dependent uphill calcium uptake. This calcium transport was insensitive to ouabain, oligomycin, or dinitrophenol, while blocked by lanthanum and quercetin. Calmodulin significantly stimulated calcium pumping in EDTA-washed vesicles. ATP-dependent and -independent calcium uptake rates, respectively, showed different calcium concentration dependences.When PBL membrane vesicles were phosphorylated by γ ³²P-ATP, a calcium-induced, hydroxylamine-sensitive incorporation of ³²P was found in 120–150 000 molecular weight proteins. Depending on the way of membrane preparation, the molecular weight of the phosphoprotein was shifted. Similarly to that found in red cell membranes, sensitivity to calmodulin stimulation and partial proteolysis of the calcium pump molecule showed an inverse relationship.     

Tyrosine and calcium/calmodulin kinases are common signaling components in the generation of reactive oxygen species in human lymphocytes

This study examined the signaling mechanism involved in the generation of reactive oxygen species (ROS) in human lymphocytes activated by formyl-Met-Leu-Phenylalanine (fMLP; 200 nmol/L) or phorbol-myristate-acetate (PMA; 100 nmol/L). ROS were monitored spectrophotometrically using dichlorofluorescin diacetate. fMLP and PMA significantly increased ROS above the control levels (p<0.05 and 0.001, respectively). These increases were significantly inhibited by catalase, sodium azide, and dimethylsulfoxide but not by superoxide dismutase, suggesting that the ROS apparently included hydrogen peroxide, singlet oxygen and hydroxyl ion but not superoxide anion. PMA-induced responses were reduced by tyrphostin (p<0.01), ST-638 (p<0.05), KN-62 (p<0.001), bisindolylmaleimide (p<0.001), RO-31-8220 (p<0.001), and by LY-83583 (p<0.001), suggesting significant involvement of tyrosine kinase, calcium/calmodulin kinase II, protein kinase C and guanylyl cyclase. fMLP-induced responses were significantly reduced by only tyrphostin (p<0.001), ST-638 (p<0.05), and KN-62 (p<0.01). The results show that tyrosine kinase and calcium/calmodulin kinase II are common signalling components in the production of reactive oxygen species in activated lymphocytes.     

Chromosomal proteins in human B and T lymphocytes.

Histones and non-histone chromosomal proteins were characterized in B and T human lymphocytes by means of polyacrylamide disc gel electrophoresis. It was found that while histones do not present appreciable differences in the two examined populations, non-histone chromosomal proteins exhibit distinct electrophoretic profiles. Low molecular weight proteins predominate in B lymphocytes whereas high and intermediate proteins are largely represented in T lymphocytes. The latter proteins may be related to the capability of these resting cells to proliferate under appropriate antigenic stimuli.     

Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes

Two src family kinases, lck and fyn, participate in the activation of T lymphocytes. Both of these protein tyrosine kinases are thought to function via their interaction with cell surface receptors. Thus, lck is associated with CD4, CD8, and Thy-1, whereas fyn is associated with the T cell antigen receptor and Thy-1. In this study, the intracellular localization of these two protein tyrosine kinases in T cells was analyzed by immunofluorescence and confocal microscopy. Lck was present at the plasma membrane, consistent with its proposed role in transmembrane signalling, and was also associated with pericentrosomal vesicles which co-localized with the cation-independent mannose 6- phosphate receptor. Surprisingly, fyn was not detected at the plasma membrane in either Jurkat T cells or T lymphoblasts but was closely associated with the centrosome and to microtubule bundles radiating from the centrosome. In mitotic cells, fyn co-localized with the mitotic spindle and poles. The essentially non-overlapping intracellular distributions of lck and fyn suggest that these kinases may be accessible to distinct regulatory proteins and substrates and, therefore, may regulate different aspects of T cell activation. Anti- phosphotyrosine antibody staining at the plasma membrane increases dramatically after CD3 cross-linking of Jurkat T cells. The localization of lck to the plasma membrane suggests that it may participate in mediating this increase in tyrosine phosphorylation, rather than fyn. Furthermore, the distribution of fyn in mitotic cells raises the possibility that it functions at the M phase of the cell cycle.