Novel compounds, ‘1,3-selenazine derivatives’ as specific inhibitors of eukaryotic elongation factor-2 kinase

The inhibitory activities of 5,6-dihydro-4H-1,3-selenazine derivatives on protein kinases were investigated. In a multiple protein kinase assay using a postnuclear fraction of v-src-transformed NIH3T3 cells, 4-ethyl-4-hydroxy-2-p-tolyl-5,6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-6-isopropyl-4-methyl-2-p-tolyl-5,6-dihydro-4H-1,3-selenazine (TS-4) exhibited selective inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K) over protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). In further experiments using purified kinases, TS-2 (IC₅₀=0.36 μM) and TS-4 (IC₅₀=0.31 μM) inhibited eEF-2K about 25-fold more effectively than calmodulin-dependent protein kinase-I (CaMK-I), and about 6-fold (TS-2) or 33-fold (TS-4) more effectively than calmodulin-dependent protein kinase-II (CaMK-II), respectively. TS-2 and TS-4 showed much weaker inhibitory activity toward PKA and PKC, while TS-4, but not TS-2, moderately inhibited immunoprecipitated v-src kinase. TS-2 (10.7-fold) and TS-4 (12.5-fold) demonstrated more potent and more specific eEF-2K inhibitory activity than rottlerin, a previously identified eEF-2K inhibitor. TS-2 inhibited ATP or eEF-2 binding to eEF-2K in a competitive or non-competitive manner, respectively. In cultured v-src-transformed NIH3T3 cells, TS-2 also decreased phospho-eEF-2 protein level (IC₅₀=4.7 μM) without changing the total eEF-2 protein level. Taken together, these results suggest that TS-2 and TS-4 are the first identified selective eEF-2K inhibitors and should be useful tools for studying the function of eEF-2K.     

Inhibition of calcium-independent luminal uptake of L-dopa by calmodulin antagonists in immortalized rat capillary cerebral endothelial cells

The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca(2+)/calmodulin mediated pathways on the luminal uptake of L-DOPA through the L-type amino acid transporter in immortalized rat capillary cerebral endothelial (REB-4) cells. Non-linear analysis of the saturation curve for L-DOPA revealed a K(m)value (in microM) of 71+/-9 and a V(max)value of 17+/-1 (in nmol mg protein/6 min). L-DOPA uptake at the luminal cell border was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 m m), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC(50)=140 microM). The Ca(2+)/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC(50)s of 23 and 33 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutyrate, staurosporine and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in RBE-4 cells is promoted through the L-type amino acid transporter and appears to be under the control of calmodulin mediated pathways.     

Design, synthesis and biological evaluation of pyrazolyl-thiazolinone derivatives as potential EGFR and HER-2 kinase inhibitors

A series of pyrazolyl-thiazolinone derivatives (E1-E36) have been designed and synthesized and their biological activities were also evaluated as potential EGFR and HER-2 kinase inhibitors. Thirty-four of the 36 compounds were reported for the first time. Among them, compound 2-(5-(4-bromophenyl)-3-p-tolyl-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (E28) displayed the most potent inhibitory activity (IC(50)=0.24μM for EGFR and IC(50)=1.07μM for HER-2). Antiproliferative assay results indicated that compound E28 owned high antiproliferative activity against MCF-7, B16-F10 and HCT-116 in vitro, with IC(50) value of 0.30, 0.54, and 0.70μM, respectively. Docking simulation was further performed to position compound E28 into the EGFR active site to determine the probable binding model. Based on the preliminary results, compound E28 with potent inhibitory activity in tumor growth would be a potential anticancer agent.     

Inhibitory effect of H-7, H-8 and polymyxin B on liver protein kinase C-induced phosphorylation of endogenous substrates

Inhibitory actions of 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), N-[2-(methylamine)ethyl]-5-isoquinolinesulfonamide [H-8] and polymyxin B on the calcium-activated, phospholipid-dependent protein kinase (protein kinase C) of rat liver were compared. Using a partially purified liver protein kinase C and an exogenous substrate histone-III S, polymyxin B showed maximum inhibition (IC50, 9.5 microM) followed by H-7 (IC50, 25 microM) and H-8 (IC50, 36 microM). These inhibitors also inhibited protein kinase C-induced phosphorylation of endogenous cytosolic and particulate proteins in a dose-dependent manner though polymyxin B was relatively less effective with the particulate fraction. With the aid of protein kinase-C activators and these inhibitors, seven proteins in cytosolic (Mr 170K, 150K, 43K, 34K, 30K, 25K and 19K daltons) and six proteins in particulate (Mr 150K, 43K, 34K, 25K, 19K and 16K daltons) fractions were identified as probable substrates for protein kinase C in liver. The identity of these proteins remains to be determined.     

Synthesis, molecular docking and evaluation of thiazolyl-pyrazoline derivatives as EGFR TK inhibitors and potential anticancer agents

Fourty-two thiazolyl-pyrazoline derivatives were synthesized to screen for their EGFR kinase inhibitory activity. Compound 4-(4-chlorophenyl)-2-(3-(3,4-dimethylphenyl)-5-p-tolyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole (11) displayed the most potent EGFR TK inhibitory activity with IC(50) of 0.06 μM, which was comparable to the positive control. Molecular docking results indicated that compound 11 was nicely bound to the EGFR kinase. Compound 11 also showed significant antiproliferative activity against MCF-7 with IC(50) of 0.07 μM, which would be a potential anticancer agent.     

Direct inhibition of a PKA inhibitor, H-89 on KV channels in rabbit coronary arterial smooth muscle cells

We examined the effects of the protein kinase A (PKA) inhibitor H-89 on voltage-dependent K(+) (K(V)) currents in freshly isolated rabbit coronary arterial smooth muscle cells, using a whole-cell patch clamp technique. H-89 inhibited the K(V) current in a concentration-dependent manner, with a K(d) value of 1.02 microM. However, the PKA inhibitors KT 5720 and Rp-8-CPT-cAMPS did not significantly alter the K(V) current or the inhibitory effects of H-89 on the K(V) current. Moreover, H-85, a structurally similar but inactive analog of H-89, showed similar inhibitory effects on the K(V) channel. H-89 had no effect on the voltage-dependency of activation or inactivation, or on recovery kinetics. These results suggest that in rabbit coronary arterial smooth muscle cells, H-89 inhibits the K(V) current directly by blocking the pore cavity, an effect independent of PKA inhibition.     

Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-949: mechanism of inhibitory activity

CI-949 [5-methyl-3-(1-methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H- indole-2- carboxamide, L-arginine salt] inhibits human neutrophil activation in response to stimuli which promote calcium mobilization or calcium influx. This report further examines the effect of CI-949 on phosphoinositide-dependent stimulus-response coupling. At 100 microM, CI-949 had no inhibitory effect on human neutrophil phospholipase C or protein kinase C. In contrast, CI-949 inhibited FMLP-stimulated intracellular calcium mobilization with an IC50 of 8.4 microM. The compound was also a potent calmodulin antagonist, inhibiting calmodulin-dependent phosphodiesterase activity with an IC50 of 31.0 microM. The calmodulin antagonist activity of CI-949 was confirmed by fluorescence spectroscopy. These results demonstrate that CI-949 may function through inhibition of calcium- and calmodulin-dependent signal transduction processes.     

Kinetic characterisation of the enzymatic activity of the eEF-2-specific Ca2(+)-and calmodulin-dependent protein kinase III purified from rabbit reticulocytes.

The Ca2(+)-and calmodulin-dependent protein kinase III, which specifically phosphorylates the eukaryotic elongation factor 2 (eEF-2), has been purified to apparent homogeneity from the post-ribosomal fraction of rabbit reticulocytes by an efficient four-step method. The method results in a more than 4000-fold purification of the enzyme. SDS-gel electrophoresis showed that the purified kinase contained only one polypeptide with the apparent molecular mass of 90 kDa. The kinase activity was associated with the 90-kDa protein as shown by analyzing the phosphorylating activity of SDS gel electrophoretically purified protein electroblotted to nitrocellulose membranes. The purified kinase was dependent on Ca2+, Mg2+ and calmodulin for activity. Kinetic analysis of the phosphorylation reaction indicates that the turnover number of the kinase was approximately 1 s-1. The Km for the two substrates ATP and eEF-2 was calculated to be approximately 100 microM and 10 microM, respectively. The activity of the kinase was competitively inhibited by cAMP. The inhibition constant Ki (0.5 mM) was found to be in the same order of magnitude as that calculated for the competitive product inhibition caused by ADP. GTP was ten-times less efficient as competitor, indicating that the kinase had a preference for adenosine nucleotides. Phosphorylation of eEF-2 did not interfere with the diphtheria-toxin-catalysed ADP-ribosylation of the factor nor did ADP-ribosylation inhibit phosphorylation.     

Synthesis and evaluation of 2',4',6'-trihydroxychalcones as a new class of tyrosinase inhibitors

In this study, we synthesized a series of hydroxychalcones and examined their tyrosinase inhibitory activity. The results showed that 2',4',6'-trihydroxychalcone (1), 2,2',3,4',6'-pentahydroxychalcone (4), 2',3,4,4',5,6'-hexahydroxychalcone (5), 2',4',6'-trihydroxy- 3,4-dimethoxychalcone (9) and 2,2',4,4',6'-pentahydroxychalcone (15) exhibited high inhibitory effects on tyrosinase with respect to l-tyrosine as a substrate. By the structure-activity relationship study, it was suggested that the 2',4',6'-trihydroxyl substructure in the chalcone skeleton were efficacious for the inhibition of tyrosinase activity. And also, the catechol structure on B-ring of chalcones was not advantageous for the inhibitory potency. Furthermore, 15 (IC(50)=1microM) was found to show the highest activity out of a set of 15 hydroxychalcones, even better than both 2,2',4,4'-tetrahydroxychalcone (13, IC(50)=5microM) and kojic acid (16, IC(50)=12microM), which were known as potent tyrosinase inhibitors. Kinetic study revealed that 15 acts as a competitive inhibitor of tyrosinase with K(i) value of 3.1microM.     

Regulation of elongation factor-2 kinase by pH

Elongation factor-2 kinase (eEF-2K) is a Ca(2+)/calmodulin-dependent protein kinase that phosphorylates and inactivates eEF-2 and that can regulate the rate of protein synthesis at the elongation stage. Here we report that a slight decrease in pH, within the range observed in vivo, leads to a dramatic activation of eEF-2K. The activity of eEF-2K in mouse liver extracts, as well as the activity of purified recombinant human eEF-2K, is low at pH 7.2-7.4 and is increased by severalfold when the pH drops to 6.6-6.8. eEF-2K requires calmodulin for activity at neutral as well as acidic pH. Kinetic studies demonstrate that the pH does not affect the K(M) for ATP or eEF-2 and activation of eEF-2K at acidic pH is due to an increase in V(max). To analyze the potential role of eEF-2K in regulating protein synthesis by pH, we constructed a mouse fibroblast cell line that expresses eEF-2K in a tetracycline-regulated manner. Overexpression of eEF-2K led to a decreased rate of protein synthesis at acidic pH, but not at neutral pH. Our results suggest that pH-dependent activation of eEF-2K may play a role in the global inhibition of protein synthesis during tissue acidosis, which accompanies such processes as hypoxia and ischemia.     

N(omega)-nitro-L-arginine inhibits inducible HSP-70 via Ca(2+), PKC, and PKA in human intestinal epithelial T84 cells

The nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) inhibits heat stress (HS)-induced NO production and the inducible 70-kDa heat shock protein (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of L-NNA on HS-induced HSP-70i expression. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2, and protein kinase C (PKC), and PKA activities were determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45 degrees C for 10 min and allowed to recover for 6 h. L-NNA treatment for 1 h before HS inhibited the induction of HSP-70i mRNA and protein, with an IC(50) of 0.0471 +/- 0.0007 microM. Because the HS-induced increase in HSP-70i mRNA and protein is Ca(2+) dependent, we measured [Ca(2+)](i) after treating cells with L-NNA. L-NNA at 100 microM significantly decreased resting [Ca(2+)](i). Likewise, treatment with 1 microM GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca(2+)](i) and inhibited HS-induced increase in HSP-70i. GF-109203X- or H-89-treated cells failed to respond to L-NNA by further decreasing [Ca(2+)](i) and HSP-70i. L-NNA effectively blocked heat shock factor-1 (HSF1) translocation from the cytosol to the nucleus, a process requiring PKC phosphorylation. These results suggest that L-NNA inhibits HSP-70i by reducing [Ca(2+)](i) and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus.     

Membrane interactions of amphiphilic polypeptides mastoparan, melittin, polymyxin B, and cardiotoxin. Differential inhibition of protein kinase C, Ca2+/calmodulin-dependent protein kinase II and synaptosomal membrane Na,K-ATPase, and Na+ pump and differentiation of HL60 cells.

Interactions of certain naturally occurring, amphiphilic polypeptides with membranes were investigated. Mastoparan (wasp venom toxin), melittin (bee venom toxin), cardiotoxin (cobra venom toxin), and polymyxin B (antibacterial antibiotic) inhibited protein kinase C stimulated by phosphatidylserine bilayer or arachidonate monomer and blocked binding of [3H] phorbol 12,13-dibutyrate to protein kinase C in the presence of phosphatidylserine bilayer, with IC50 values (concentrations causing 50% inhibition) of 1-8 microM. Mastoparan and polymyxin B were much less inhibitory (IC50, 10-20 microM), whereas melittin and cardiotoxin were similarly inhibitory (IC50, 1-4 microM), when protein kinase C was activated instead by synaptosomal membrane. Kinetic analysis indicate that mastoparan inhibited protein kinase C, assayed using phosphatidylserine or synaptosomal membrane as the phospholipid cofactor, competitively with the phospholipid cofactor, in a mixed manner with CaCl2 or diacylglycerol, noncompetitively with histone, and uncompetitively with ATP, with apparent Ki values of 1.6-18.7 microM. Inhibition of Na,K-ATPase in the membrane by these polypeptides had relative potencies different from those for their inhibition of protein kinase C activated by the same membrane preparation; mastoparan and melittin inhibited the two activities with comparable potencies, but polymyxin B and cardiotoxin were far less effective in inhibiting Na,K-ATPase. The same relative inhibitory potencies of the polypeptides (melittin greater than mastoparan greater than polymyxin B) for inhibition of Na,K-ATPase were also noted for their inhibition of Ca2+/calmodulin-dependent protein kinase II, 86Rb uptake (Na+ pump) by HL60 cells and the phorbol ester-induced differentiation of the leukemia cells. These findings were consistent with discrete interactions of the polypeptides with functionally distinct sites on the membrane, leading to differential inhibition of biological activities associated with the membrane. Actions of certain polypeptides appeared to be more specific compared to those of lipid second messengers such as lyso-phosphatidylcholine and sphingosine, and the antineoplastic ether lipid analogs such as 1-O-octadecyl-2-methyl-rac-glycero-3-ophosphocholine.     

Geraniol inhibits prostate cancer growth by targeting cell cycle and apoptosis pathways

Elongation factor-2 kinase (eEF-2 kinase, also known as calmodulin-dependent protein kinase III), is a unique calcium/calmodulin-dependent enzyme that inhibits protein synthesis by phosphorylating and inactivating elongation factor-2 (eEF-2). We previously reported that expression/activity of eEF-2 kinase was up-regulated in several types of malignancies including Gliomas, and was associated with response of tumor cells to certain therapeutic stress. In the current study, we sought to determine whether eEF-2 kinase expression affected sensitivity of glioma cells to treatment with tumor the necrosis factor-related apoptosis-inducing ligand (TRAIL), a targeted therapy able to induce apoptosis in cancer cells but causes no toxicity in most normal cells. We found that inhibition of eEF-2 kinase by RNA interference (RNAi) or by a pharmacological inhibitor (NH125) enhanced TRAIL-induced apoptosis in the human glioma cells, as evidenced by an increase in apoptosis in the tumor cells treated with eEF-2 kinase siRNA or the eEF-2 kinase inhibitor. We further demonstrated that sensitization of tumor cells to TRAIL was accompanied by a down-regulation of the anti-apoptotic protein, Bcl-xL, and that overexpression of Bcl-xL could abrogate the sensitizing effect of inhibiting eEF-2 kinase on TRAIL. The results of this study may help devise a new therapeutic strategy for enhancing the efficacy of TRAIL against malignant glioma by targeting eEF-2 kinase.     

eEF-2 kinase,another meddler in the “Yin and Yang” of Akt–mediated cell fate?

Eukaryotic elongation factor-2 (eEF-2) kinase, also known as calmodulin-dependent protein kinase III, is a unique calcium/calmodulin-dependent enzyme. eEF-2 kinase can act as a negative regulator of protein synthesis and a positive regulator of autophagy under environmental or metabolic stresses. Akt, a key downstream effector of the PI3K signaling pathway that regulates cell survival and proliferation, is an attractive therapeutic target for anticancer treatment. Akt inhibition leads to activation of both apoptosis, type I programmed cell death and autophagy, a cellular degradation process via lysosomal machinery (also termed type II programmed cell death). However, the underlying mechanisms that dictate functional relationship between autophagy and apoptosis in response to Akt inhibition remain to be delineated. Our recent study demonstrated that inhibition of eEF-2 kinase can suppress autophagy but promote apoptosis in tumor cells subjected to Akt inhibition, indicating a role of eEF-2 kinase as a controller in the crosstalk between autophagy and apoptosis. Furthermore, inhibition of eEF-2 kinase can reinforce the efficacy of a novel Akt inhibitor, MK-2206, against human glioma. These findings may help optimize the use of Akt inhibitors in the treatment of cancer and other diseases.     

eEF-2 kinase: Another meddler in the “yin and yang” of Akt-mediated cell fate?

Eukaryotic elongation factor-2 (eEF-2) kinase, also known as calmodulin-dependent protein kinase III, is a unique calcium/calmodulin-dependent enzyme. eEF-2 kinase can act as a negative regulator of protein synthesis and a positive regulator of autophagy under environmental or metabolic stresses. Akt, a key downstream effector of the PI3K signaling pathway that regulates cell survival and proliferation, is an attractive therapeutic target for anticancer treatment. Akt inhibition leads to activation of both apoptosis, type I programmed cell death and autophagy, a cellular degradation process via lysosomal machinery (also termed type II programmed cell death). However, the underlying mechanisms that dictate functional relationship between autophagy and apoptosis in response to Akt inhibition remain to be delineated. Our recent study demonstrated that inhibition of eEF-2 kinase can suppress autophagy but promote apoptosis in tumor cells subjected to Akt inhibition, indicating a role of eEF-2 kinase as a controller in the crosstalk between autophagy and apoptosis. Furthermore, inhibition of eEF-2 kinase can reinforce the efficacy of a novel Akt inhibitor, MK-2206, against human glioma. These findings may help optimize the use of Akt inhibitors in the treatment of cancer and other diseases.     

PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle

Regulation of adenylyl cyclase type V/VI and cAMP-specific, cGMP-inhibited phosphodiesterase (PDE) 3 and cAMP-specific PDE4 by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) was examined in gastric smooth muscle cells. Expression of PDE3A but not PDE3B was demonstrated by RT-PCR and Western blot. Basal PDE3 and PDE4 activities were present in a ratio of 2:1. Forskolin, isoproterenol, and the PKA activator 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole 3',5'-cyclic monophosphate, SP-isomer, stimulated PDE3A phosphorylation and both PDE3A and PDE4 activities. Phosphorylation of PDE3A and activation of PDE3A and PDE4 were blocked by the PKA inhibitors [protein kinase inhibitor (PKI) and H-89] but not by the PKG inhibitor (KT-5823). Sodium nitroprusside inhibited PDE3 activity and augmented forskolin- and isoproterenol-stimulated cAMP levels; PDE3 inhibition was reversed by blockade of cGMP synthesis. Forskolin stimulated adenylyl cyclase phosphorylation and activity; PKI blocked phosphorylation and enhanced activity. Stimulation of cAMP and inhibition of inositol 1,4,5-trisphosphate-induced Ca(2+) release and muscle contraction by isoproterenol were augmented additively by PDE3 and PDE4 inhibitors. The results indicate that PKA regulates cAMP levels in smooth muscle via stimulatory phosphorylation of PDE3A and PDE4 and inhibitory phosphorylation of adenylyl cyclase type V/VI. Concurrent generation of cGMP inhibits PDE3 activity and augments cAMP levels.     

Discovery of cyclin-dependent kinase inhibitor, CR229, using structurebased drug screening

To generate new scaffold candidates as highly selective and potent cyclin-dependent kinase (CDK) inhibitors, structure-based drug screening was performed utilizing 3D pharmacophore conformations of known potent inhibitors. As a result, CR229 (6-bromo-2,3,4,9-tetrahydro-carbolin-1-one) was generated as the hit-compound. A computational docking study using the X-ray crystallographic structure of CDK2 in complex with CR229 was evaluated. This predicted binding mode study of CR229 with CDK2 demonstrated that CR229 interacted effectively with the Leu83 and Glu81 residues in the ATP-binding pocket of CDK2 for the possible hydrogen bond formation. Furthermore, biochemical studies on inhibitory effects of CR229 on various kinases in the human cervical cancer HeLa cells demonstrated that CR229 was a potent inhibitor of CDK2 (IC50: 3 microM), CDK1 (IC50: 4.9 microM), and CDK4 (IC50: 3 microM), yet had much less inhibitory effect (IC50: >20 microM) on other kinases, such as casein kinase 2-1 (CK2- alpha1), protein kinase A (PKA), and protein kinase C (PKC). Accordingly, these data demonstrate that CR229 is a potent CDK inhibitor with anticancer efficacy.     

Ca2+/calmodulin mediated pathways regulate the uptake of L-DOPA in mouse neuroblastoma neuro 2A cells

The present study examined the involvement of protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), protein tyrosine kinase (PTK) and Ca2+/calmodulin mediated pathways on the uptake of L-DOPA through the L-type amino acid transporter in Neuro 2A cells, an in vitro model of neuronal cells. Non-linear analysis of the saturation curve for L-DOPA revealed a Km value (in microM) of 54+/-2 and a Vmax value (in nmol mg protein/6 min) of 34+/-1. L-DOPA uptake was a sodium-independent process and insensitive to N-(methylamino)-isobutyric acid (MeAIB, 1 mM), but sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC, IC50=82 microM). The Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA (2.5 microM) uptake with IC50's of 33 and 105 microM, respectively. The inhibitory effect of BHC on the accumulation of L-DOPA was of the competitive type, whereas that of calmidazolium and trifluoperazine was of the non-competitive type. Modulators of PKA (cyclic AMP, forskolin, isobutylmethylxanthine and cholera toxin), PKG (cyclic GMP, zaprinast, LY 83583 and sodium nitroprusside), PKC (phorbol 12,13-dibutirate, phorbol 12-myristate 13-acetate and chelerythrine) and PTK (genistein and tyrphostin 25) failed to affect the accumulation of a non-saturating (2.5 microM) concentration of L-DOPA. It is concluded that L-DOPA uptake in Neuro 2A cells is promoted through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin mediated pathways.     

Detection of anti-elongation factor 2 kinase (calmodulin-dependent protein kinase III) antibodies in patients with systemic lupus erythematosus

Elongation factor 2 kinase (eEF-2K), also known as calmodulin-dependent protein kinase III, is a member of the calmodulin-mediated signaling pathway that links activation of cell surface receptors to cell division. The activity of eEF-2K is increased in many human cancers and may be a valid target for anti-cancer treatment. It is one of the unconventional eukaryotic protein kinases with respect to its structural domains in comparison to other members of the serine/threonine protein kinase superfamily. eEF-2K is highly conserved in nature. For example, the amino acid sequence of human eEF-2K is 90% identical to mouse and rat eEF-2Ks and 40% identical to that of the C. elegans enzyme. Therefore it has been difficult to generate high-titer and high-specificity antibodies to the human enzyme by traditional techniques. Patients with systemic lupus erythematosus (SLE) produce auto-antibodies to a variety of cellular proteins, including members of the protein translation apparatus. Hence, we developed an ELISA assay that could detect anti-eEF2K antibodies from sera of SLE patients using purified eEF-2K as an antigen. We screened 117 sera from SLE patients. High-titer anti-eEF-2K antibodies were detected in 72 subjects. One of the high-titer sera was used for further characterization. The auto-antibody recognized eEF-2K on immunoblots and immunoprecipitated the kinase with intact enzyme activity. In conclusion, anti-eEF-2K antibodies are found in sera of SLE patients and are useful tools to study the role of this highly conserved enzyme.     

Lysophosphatidylcholine metabolism and lipoprotein secretion by cultured rat hepatocytes deficient in choline.

A protein kinase activity was identified in pig brain that co-purified with microtubules through repeated cycles of temperature-dependent assembly and disassembly. The microtubule-associated protein kinase (MTAK) phosphorylated histone H1; this activity was not stimulated by cyclic nucleotides. Ca2+ plus calmodulin, phospholipids or polyamines. MTAK did not phosphorylate synthetic peptides which are substrates for cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase. Ca2+/calmodulin-dependent protein kinase II, protein kinase C or casein kinase II. MTAK activity was inhibited by trifluoperazine [IC50 (median inhibitory concn.) = 600 microM] in a Ca2+-independent fashion. Ca2+ alone was inhibitory [IC50 = 4 mM). MTAK was not inhibited by heparin, a potent inhibitor of casein kinase II, nor a synthetic peptide inhibitor of cyclic AMP-dependent protein kinase. MTAK demonstrated a broad pH maximum (7.5-8.5) and an apparent Km for ATP of 45 microM. Mg2+ was required for enzyme activity and could not be replaced by Mn2+. MTAK phosphorylated serine and threonine residues on histone H1. MTAK is a unique cofactor-independent protein kinase that binds to microtubule structures.