Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

EGF, a single-chain polypeptide growth factor important for many cellular functions including glycolysis and protein phosphorylation, is known to modulate calcium metabolism in several cell systems. EGF causes an increase in Ca2+ influx and accumulation of inositol triphosphate and probably exhibits many, if not all, of its effects via the calcium messenger system. Lead is known to interact with and perturb normal calcium signaling pathways; therefore, the purpose of this work was to determine if lead perturbs EGF modulation of calcium metabolism in ROS 17/2.8 cells and if lead impairs collagen synthesis, which is controlled by EGF. To characterize 45Ca kinetics, cells were labelled with 45Ca (1.87 mM Ca) for 20 hr in the presence of 5 microM Pb, 50 ng/ml EGF, or 5 microM Pb and 50 ng/ml EGF. Kinetic parameters were determined from 45Ca efflux curves. Three kinetic compartments described the intracellular metabolism of 45Ca; 5 microM Pb significantly altered the effect of EGF on intracellular calcium metabolism. Calcium distribution was shifted from the fast-exchanging, quantitatively small calcium pools S1 and S2 to the slow-exchanging, quantitatively large S3. There was also a 50% increase in total cell calcium in cells treated with 5 microM Pb and 50 ng/ml EGF over calcium in cells treated with 50 ng/ml EGF alone. Because EGF and phorbol 12-myristate 13-acetate (PMA) have similar effects on protein kinase C (PKC) and collagen metabolism, the transient effects of EGF and PMA on 45Ca and 210Pb were also characterized. EGF caused a rapid transient increase in efflux of both isotopes, which was further increased by the addition of PMA. In contrast, PMA pretreatment, which depletes PKC, significantly attenuated the latter effect of EGF, suggesting that downregulation by PKC of EGF-induced increases in 45Ca and 210Pb efflux. Moreover, collagen synthesis was decreased by lead, EGF, and PMA in a similar manner, further suggesting PKC as the common modulator of these effects. These data show that Pb impairs the normal modulation of intracellular calcium homeostasis and collagen synthesis by EGF. Furthermore, these results provide additional support to the postulate that an early and discrete effect of lead involves perturbation of the calcium messenger system at one or several loci.
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PMID:Lead perturbs epidermal growth factor (EGF) modulation of intracellular calcium metabolism and collagen synthesis in clonal rat osteoblastic (ROS 17/2.8) cells. 158 73

In the present report, we demonstrate that Tb3+ binds to protein kinase C and serves as a luminescent reporter of certain cationic metal-binding sites. Tb3+ titration of 50 nM protein kinase C results in a 20-fold enhancement of Tb3+ luminescence which is half-maximal at 12 microM Tb3+. A Kd of approximately 145 nM was determined for Tb3+ binding to the enzyme. The excitation spectrum of bound Tb3+ exhibits a peak at 280 nm characteristic of energy transfer from protein tryptophan or tyrosine residues. The luminescence of this complex can be markedly decreased by other metals, including Pb2+ (IC50 = 25 microM), La3+ (IC50 = 50 microM), Hg2+ (IC50 = 300 microM), Ca2+ (IC50 = 6 mM), and Zn2+ (IC50 greater than 10 mM), and chelation of Tb3+ by 2 mM EGTA. Tb3+ binding to protein kinase C is correlated with its inhibition of protein kinase activity (IC50 = 8 microM), r = 0.99) and phorbol ester binding (IC50 = 15 microM, r = 0.98). Tb3+ inhibition of protein kinase C activity cannot be overcome by excess Ca2+, but can be partially overcome with excess phosphatidylserine or by chelation of Tb3+ with EGTA. Tb3+ noncompetitively inhibits phorbol ester binding by decreasing the maximal extent of binding without significantly altering binding affinity. The results suggest that the Tb3(+)-binding site is at or allosterically related to the enzyme's phosphatidylserine-binding site, but is distinct from the phorbol ester-binding domain and the Ca2(+)-binding site that regulates enzyme activity.
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PMID:Terbium as a luminescent probe of metal-binding sites in protein kinase C. 210 17

We investigated membrane currents activated by intracellular divalent cations in two types of molluscan pacemaker neurons. A fast and quantitative pressure injection technique was used to apply Ca2+ and other divalent cations. Ca2+ was most effective in activating a nonspecific cation current and two types of K+ currents found in these cells. One type of outward current was quickly activated following injections with increasing effectiveness for divalent cations of ionic radii that were closer to the radius of Ca2+ (Ca2+ greater than Cd2+ greater than Hg2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than Pb2+ greater than Sr2+ greater than Mg2+ greater than Ba2+). The other type of outward current was activated with a delay by Ca2+ greater than Sr2+ greater than Hg2+ greater than Pb2+. Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, and Ni2+ were ineffective in concentrations up to 5 mM. Comparison with properties of Ca2(+)-sensitive proteins related to the binding of divalent cations suggests that a Ca2(+)-binding protein of the calmodulin/troponin C type is involved in Ca2(+)-dependent activation of the fast-activated type of K+ current. Th sequence obtained for the slowly activated type is compatible with the effectiveness of different divalent cations in activating protein kinase C. The nonspecific cation current was activated by Ca2+ greater than Hg2+ greater than Ba2+ greater than Pb2+ greater than Sr2+, a sequence unlike sequences for known Ca2(+)-binding proteins.
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PMID:Activation of three types of membrane currents by various divalent cations in identified molluscan pacemaker neurons. 255 42

The interaction of some cations with the enzymatic activity of soluble protein kinase C was determined in order to elucidate whether protein kinase C can be activated by other metal cations besides Ca2+. Protein kinase C was activated by Ca2+ and Sr2+ having EC50 values of nearly 10 microM and 200 microM, respectively. Ba2+ likewise activated protein kinase C but was less potent. Co2+, Ni2+ and Mn2+ had no activating effects on the activity in the absence of Ca2+, but was slightly reduced in the presence of Ca2+ (0.5 mM). Cations with ionic radii close to Ca2+ (0.99 A) inhibited the activity irrespective of the absence or presence of Ca2+. The order of potency is as follows: Hg2+ greater than Cd2+ approximately Cu2+ much greater than Sm3+ greater than Tb3+ greater than La3+. Pb2+ and Zn2+, which showed a high affinity to SH-groups, as well as Hg2+, Cd2+ and Cu2+, which also inhibited the activity. Thus, among the ions investigated, the alkaline-earth ions Sr2+ and Ba2+ could be substituted for Ca2+, irrespective of ionic radii. The serious environmental pollutants such as Hg2+, Cd2+ or Pb2+ impaired the activity of protein kinase C probably due to SH-blocking.
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PMID:The interaction of cations with activity of soluble protein kinase C from mouse brain. 319 43

Specific cellular sites of action of the environmental pollutant, lead, have not been completely defined. The present investigations were conducted to test the hypothesis that lead exposure perturbs glucocorticoid-mediated effects in hormonal target tissues. The cell culture model chosen for these investigations was the effects of lead on glucocorticoid-regulated tyrosine aminotransferase (TAT) specific activity in the H4-II-C3 hepatoma cells. Cells were treated with 300 nM-10 microM lead acetate for 24 or 48 h in absence or presence of the inducing agent, dexamethasone. Lead dose-dependently inhibited TAT specific activity up to 52% and 61% following 24 and 48 h lead treatments, respectively. These treatment times and concentrations of lead acetate did not significantly alter total cell numbers, [3H]thymidine incorporation or trypan blue exclusion. Glucocorticoid receptor-binding studies yielded a Kd = 8.3 nM and a Bmax = 290 fmol/mg protein in untreated cells versus a Kd = 9.2 nM and Bmax = 262 fmol/mg protein in cells exposed to 10 microM lead acetate for 48 h. Treatment with lead did not significantly perturb uptake of the inducing glucocorticoids or initial cytosolic receptor-binding events. To sustain induced levels of TAT, glucocorticoid must be continuously present. Following steroid withdrawal, enzyme de-induction was significantly altered in lead-treated cells. At 6 h following dexamethasone withdrawal, TAT levels had decreased to 51% of maximum in sodium acetate-treated cells. This was significantly reduced to 33% of maximum in lead acetate-treated cells. Lead treatment of HTC cells was also shown to ameliorate PMA amplification of dexamethasone-induced TAT activity. Taken together, these results suggest that acute exposure of cells to lead may inhibit processes involved in glucocorticoid-mediated enzyme induction within the hormonal target cell. Results suggest that lead may be acting to increase the turnover of TAT by actions at the transcription, translation and/or posttranslational level. Lead may also be affecting PKC-mediated phosphorylations in the glucocorticoid-TAT signal transduction system.
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PMID:The acute effect of lead acetate on glucocorticoid regulation of tyrosine aminotransferase in hepatoma cells. 762 83

Inorganic lead inhibits neurite initiation in cultured rat hippocampal neurons at concentrations as low as 100 nM. Conflicting reports suggest that Pb2+ may stimulate or inhibit protein kinase C, adenylyl cyclase, phosphodiesterase, and calmodulin, or increase intracellular free Ca2+ concentrations. Therefore, Pb2+ may alter the activities of Ca2+/calmodulin-dependent protein kinase (CaM kinase) or protein kinases C or A. We cultured rat hippocampal neurons in 100 nM PbCI2 alone or in combination with kinase or calmodulin inhibitors. Inhibiting protein kinase C with calphostin C exacerbated the inhibition of neurite initiation caused by PbCI2, but inhibiting protein kinase A with KT5720, CaM kinase with KN62, or calmodulin with calmidazolium completely reversed the effects of PbCI2. These results indicate that Pb2+ may inhibit neurite initiation by inappropriately stimulating protein phosphorylation by CaM kinase or cyclic AMP-dependent protein kinase (PKA), possibly by stimulating calmodulin. This hypothesis is supported by findings that other treatments that should increase protein phosphorylation (okadaic acid, a protein phosphatase inhibitor, and Sp-cAMPS, a PKA activator) also reduced neurite initiation. Whole-cell intracellular free Ca2+ ion concentrations were not significantly altered by 100 nM PbCI2 at 4, 12, 24, or 48 hr. Therefore, the hypothesized stimulatory effects of Pb2+ exposure on calmodulin, CaM kinase, or PKA are probably not caused by increases in whole-cell intracellular free Ca2+, but may be attributable either to intracellular Pb2+ or to localized increases in [Ca2+]in that are not reflected in whole-cell measurements.
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PMID:Inorganic lead may inhibit neurite development in cultured rat hippocampal neurons through hyperphosphorylation. 767 45

We investigated the interaction between Pb2+ and protein kinase C (PKC) in the Pb(2+)-induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb2+ interacts with the adrenal enzyme at multiple sites. Pb2+ activates the enzyme through high-affinity (KA(Pb) = 2.4 x 10(-12) M) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar-(Ki = 7.1 x 10(-9) M) and micromolar-(Ki = 2.8 x 10(-7) M) affinity sites, respectively. Activation of PKC by 12-O-tetradecanoyl-phorbol 13-acetate (TPA) in Ca(2+)-deficient, Pb(2+)-containing medium, enhances the Pb(2+)-induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb2+ required for half-maximal activation of the secretory response from 7.5 x 10(-10) to 5.7 x 10(-11) M. The PKC inhibitors staurosporine and pseudosubstrate PKC (19-36) abolish the effect of TPA without affecting the Pb(2+)-induced secretion in the absence of TPA. These results indicate that (a) Pb2+ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb2+ results in increased sensitivity of exocytosis to Pb2+ but is not obligatory for Pb(2+)-triggered secretion.
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PMID:Multisite interactions between Pb2+ and protein kinase C and its role in norepinephrine release from bovine adrenal chromaffin cells. 776 46

The interaction of Pb and Ca with cellular sites depends upon the concentration of free ions present (Pb2+, Ca2+). The ability of Pb2+ to form complexes with simple anions such as Cl- and OH-, the formation of precipitates such as Pb(OH)2 and Pb3(PO4)2, and the ubiquity of Pb as a contaminant in laboratory reagents implies that particular care is needed in order to define the Pb2+ concentration of a solution. The free Pb2+ concentration may be controlled with Pb2+ buffers, and measured with a Pb2+ selective electrode, a fluorescent dye, fura-2, or an NMR indicator, 19F-BAPTA. Pb(2+)-Ca2+ interactions occur in three main situations at the cellular level. Pb2+ and Ca2+ compete at the plasma membrane for transport systems which effect their entry or exit, such as Ca2+ channels, and the Ca2+ pump. Intracellular Ca2+ is buffered to around 10(-7) M by proteins, endoplasmic reticulum and mitochondria. Pb2+ disturbs intracellular Ca2+ homeostasis. Ca(2+)-Pb2+ interactions at mitochondria have been described, but other mechanisms have not yet been explored. Increases in intracellular [Ca2+] act as a signal (or second messenger). Pb2+ interacts with a number of Ca(2+)-dependent effector mechanisms, such as calmodulin (a Ca2+ receptor protein which couples to several enzymes e.g., phosphodiesterase, protein kinases), protein kinase C, Ca(2+)-dependent K+ channels in the plasma membrane and neurotransmitter release. The actions of Pb2+ on neurotransmission may be relevant to Pb(2+)-induced human neuropathy and encephalopathy.
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PMID:Lead-calcium interactions in cellular lead toxicity. 824 14

The intracellular mechanism of Pb(2+)-induced release of norepinephrine (NE) was investigated in comparison with Ca2+ in bovine chromaffin cells permeabilized with staphylococcal alpha-toxin. Pb2+ activated NE release at considerably lower concentrations [concentration of free metal giving half maximal metal-dependent release (K0.5) 4.6 nM] than Ca2+ (K0.5 2.4 microM). The release of NE was associated with the release of dopamine-beta-hydroxylase but not lactate dehydrogenase. The maximal secretory responses produced by Pb2+ and Ca2+ were similar and nonadditive. Pb(2+)- and Ca(2+)-dependent releases showed a similar requirement for MgATP and were equally enhanced by protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) but not by kinase A activator 8-bromoadenosine 3',5'-cyclic monophosphate free base. The protein kinase C inhibitor staurosporine blocked the TPA-stimulated component of secretion but had no effect on the NE release in the absence of TPA. Calmidazolium, an inhibitor of calmodulin, inhibited the secretion evoked by both metals to similar extent. Agents interacting with microtubules (colchicine and vinblastine) or microfilaments (cytochalasin B and phalloidin) had no effect on secretion induced by either metal cation. These observations indicate that both Pb2+ and Ca2+ act at a common site and activate the exocytotic release of NE by an analogous mechanism.
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PMID:Intracellular mechanism of Pb(2+)-induced norepinephrine release from bovine chromaffin cells. 827 23

Lead (Pb2+) has been reported to activate calcium/phospholipid-dependent protein kinase C (PKC) at subnanomolar concentrations (Markovac, J., and Goldstein, G. W. (1988) Nature 334, 732-734); however, others have failed to find any Pb(2+)-induced activation of PKC (Murakami, K., Feng, G., and Chen, S. G. (1993) J. Pharmacol. Exp. Ther. 264, 757-761). In neither of these studies was the actual free Pb2+ or Ca2+ concentration measured. In this study, 1,2-bis(2-amino-5-fluorophenoxy)ethane N,N,N',N'-tetraacetic acid (5F-BAPTA) was used to buffer Pb2+ and Ca2+ concentrations in the PKC reaction mixture. The specific free ion concentrations of Pb2+ and Ca2+, as well as Zn2+ and other divalent cations contained in the PKC reaction mixtures, were determined by 19F NMR spectroscopy. Using this approach to set and confirm the free Pb2+ and Ca2+ concentrations, we measured the Pb(2+)-dependent and the Ca(2+)-dependent activation of phosphotydylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous acid precipitable proteins in the 100,000 x g supernatant from homogenized rat brain cortex. We found that free Pb2+ activates PKC in the range from 10(-11) to 10(-8) M, Kact = 5.5 x 10(-11) M, while Ca2+ activates PKC in the range from 10(-8) to 10(-5) M, Kact = 2.56 x 10(-7) M. These findings clearly resolve the activation of PKC by subnanomolar concentrations of free Pb2+ from activation induced by Ca2+ or other divalent cations. Furthermore, it documents the utility of 5F-BAPTA as buffer and indicator when resolving the contributions of multiple divalent cations in biochemical processes.
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PMID:Lead activation of protein kinase C from rat brain. Determination of free calcium, lead, and zinc by 19F NMR. 828 36


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