Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Productive human immunodeficiency virus type 1 (HIV-1) infection causes sustained NF-kappaB DNA-binding activity in chronically infected monocytic cells. A direct temporal correlation exists between HIV infection and the appearance of NF-kappaB DNA-binding activity in myelomonoblastic PLB-985 cells. To examine the molecular basis of constitutive NF-kappaB DNA-binding activity in HIV1 -infected cells, we analyzed the phosphorylation and turnover of IkappaBalpha protein, the activity of the double-stranded RNA-dependent protein kinase (PKR) and the intracellular levels of NF-kappaB subunits in the PLB-985 and U937 myeloid cell models. HIV-1 infection resulted in constitutive, low-level expression of type 1 interferon (IFN) at the mRNA level. Constitutive PKR activity was also detected in HIV-1-infected cells as a result of low-level IFN production, since the addition of anti-IFN-alpha/beta antibody to the cells decreased PKR expression. Furthermore, the analysis of IkappaBalpha turnover demonstrated an increased degradation of IkappaBalpha in HIV-1-infected cells that may account for the constitutive DNA binding activity. A dramatic increase in the intracellular levels of NF-kappaB subunits c-Rel and NF-kappaB2 p100 and a moderate increase in NF-kappaB2 p52 and RelA(p65) were detected in HIV-1-infected cells, whereas NF-kappaB1 p105/p50 levels were not altered relative to the levels in uninfected cells. We suggest that HIV-1 infection of myeloid cells induces IFN production and PKR activity, which in turn contribute to enhanced IkappaBalpha phosphorylation and subsequent degradation. Nuclear translocation of NF-kappaB subunits may ultimately increase the intracellular pool of NF-kappaB/IkappaBalpha by an autoregulatory mechanism. Enhanced turnover of IkappaBalpha and the accumulation of NF-kappaB/Rel proteins may contribute to the chronically activated state of HIV-1-infected cells.
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PMID:Chronic human immunodeficiency virus type 1 infection of myeloid cells disrupts the autoregulatory control of the NF-kappaB/Rel pathway via enhanced IkappaBalpha degradation. 876 27

NF-kappa B/Rel transcription factors participate in the activation of numerous genes involved in immune regulation/inflammation including cytokines, cell surface receptors, adhesion molecules, and acute phase proteins. NF-kappa B activity is controlled by inhibitory proteins, I kappa Bs, that maintain the DNA-binding forms of NF-kappa B in an inactive state in the cytoplasm. Many viruses, including the human retroviruses HIV-1 and HTLV-1, also utilize the NF-kappa B/I kappa B pathway to their transcriptional advantage during viral infection. Our recent studies have focused on the I kappa B alpha inhibitor and have characterized several protein interactions that modulate the functional activity of I kappa B alpha during human retrovirus infection. In this article, we summarise recent studies demonstrating that (1) chronic HIV-1 infection of human myelomonoblastic PLB-985 cells leads to constitutive NF-kappa B activity, activated in part due to enhanced I kappa B alpha turnover and increased NF-kappa B/Rel production; (2) HTLV-1 Tax protein physically associates with the I kappa B alpha protein in vivo and in vitro and also mediates a 20- to 40-fold stimulation of NF-kappa B DNA binding activity mediated via an enhancement of NF-kappa B dimer formation; (3) casein kinase II phosphorylates I kappa B alpha at multiple sites in the C-terminal PEST domains and regulates I kappa B alpha function; (4) transdominant forms of I kappa B alpha, mutated in critical Ser or Thr residues required for inducer-mediated (S32A,S36A) and/or constitutive phosphorylation block HIV LTR trans-activation and also effectively inhibit HIV-1 multiplication in a single cycle infection model; and (5) the amino-terminal 55aa of I kappa B alpha (NIK) interacts with the human homologue of dynein light chain 1, a small 9-kDa human homologue of the dynein light chain protein involved in microtubule and cytoskeletal dynamics. Together, our results highlight a number of intriguing molecular interactions between I kappa B alpha and cellular or viral proteins that modulate transcription factor activity and nuclear-cytoplasmic flow of host proteins.
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PMID:Cellular and viral protein interactions regulating I kappa B alpha activity during human retrovirus infection. 922 98

Quantitative immunoassays to discriminate and quantitate phospholamban and its phosphorylation states in heart homogenates were developed using known amounts of protein determined by amino acid analysis. Synthetic 1-52 phospholamban, the hydrophilic 1-25 peptide, and 1-25 phosphopeptides containing P-Ser(16), P-Thr(17), and dually phosphorylated (P-Ser(16), P-Thr(17)) were used to calibrate immunoblot systems. In addition, synthetic 1-52 peptide was phosphorylated using cAMP-dependent protein kinase (P-Ser(16)) or Ca(2+)-calmodulin protein kinase (P-Thr(17)) and then separated from unphosphorylated 1-52 by HPLC prior to quantitation. Further, canine cardiac sarcoplasmic reticulum was phosphorylated in vitro using [gamma-(32)P]-ATP with cAMP-dependent protein kinase and/or Ca(2+)-calmodulin-dependent protein kinase as well as sequential phosphorylation in both orders to assess the veracity of antibody recognition of phosphorylated forms. Western blots proved useful in characterizing the reactivity of the different antibodies to phospholamban and phosphorylated phospholamban, but were inefficient for accurate quantitation and problems with antibody recognition of dually phosphorylated phospholamban were found. mAb 1D11 recognized all forms of phospholamban, polyclonal antibodies 285 and PS-16 were highly selective for P-Ser(16) phospholamban but had diminished reactivity to diphosphorylated (P-Ser(16), P-Thr(17)) phospholamban, and polyclonal antibody PT-17, although selective for P-Thr(17) phospholamban, generated very weak signals on Western blots and reacted poorly with diphosphorylated phospholamban. Results in quantitative immunodot blot experiments were even more compelling. None of the phosphorylation specific antibodies reacted with the diphospho 1-25 phospholamban peptide. Transgenic mouse hearts expressing varying levels of PLB and ferret heart biopsy samples taken before and after isoproterenol perfusion were analyzed. In all samples containing phospholamban, a basal level of Ser(16) phosphorylation (about 4% of the total PLB population) and a lesser amount of Thr(17) phosphorylation was observed. Upon isoproterenol perfusion, Ser(16) phosphorylation increased only to 17% of the total phospholamban population with a similar change in Thr(17) phosphorylation. This suggests that phospholamban phosphorylation may serve as an electrostatic switch that dissociates inactive calcium pump complexes into catalytically active units. Thus, direct correlations between phospholamban phosphorylation state and contractile parameters may not be valid.
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PMID:Characterization and quantitation of phospholamban and its phosphorylation state using antibodies. 1062 71

In order to improve transient gene transfer into PLB-985 cells, we treated cells with 12-tetradecanoylphorbol 13-acetate (TPA) 4 h before transfection, and increased by 540-fold the reporter activity of the firefly luciferase gene transfected by TFL-01, a cationic liposome. Dioctanolyglycerol added before TPA addition inhibited the TPA-dependent increase in activity, suggesting it to be a TPA competitor for PKC binding. H7, staurosporine, GO 6976, and H8, but not GF 109203X and forskolin, inhibited the TPA-dependent increase in reporter activity when added 8 h after TFL-01/gene addition. Forskolin and GF 109203X also inhibited the increase when added before TPA. Therefore, for the potentiation of transfection by the TPA/TFL-01 method, conventional PKC activity with significant but low protein kinase A (PKA) activity are first required, and then a novel PKC activity with significant PKA activity. TPA enhanced the uptake of FITC-labeled phosphorothioated oligonucleotides and their prolonged maintenance by cells, suggesting increased TFL-01-assisted plasmid uptake and its stabilization in TPA-treated PLB-985 cells. This method was used successfully for the sensitive analysis of the promoter function of the gp91(phox) gene, implying the method to be generally useful for promoter analyses of various genes expressed in differentiated human monocytic cells.
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PMID:Phorbol ester-potentiated liposomal transfection to monocytic PLB-985 cells. 1109 42

The regulation of protein phosphatase (PP) activity by cardiac beta-adrenergic receptor stimulation with isoproterenol (ISO) was studied in four groups of guinea pigs consisting of seven animals each. Group 1 received the vehicle solution only intraperitoneally; group 2, 6 microg/kg of ISO; group 3, 60 microg/kg of ISO; and group 4, 600 microg/kg of ISO. Total PP activity (consisting of both type 1 and type 2A PP), activity of each PP subtype, the cAMP-dependent protein kinase activity ratio (-cAMP/+cAMP), the phosphorylation of PP inhibitor 1, and the phosphorylation of phospholamban were measured in ventricular tissue. PP activity was also studied in ventricular cardiomyocytes isolated from guinea pigs treated with and without 1 microM ISO or 1 microM ISO plus 10 microM propranolol, an antagonist of the beta-adrenoceptor. PP activity decreased significantly in membrane vesicles, but not in cytosolic fractions, of guinea pigs treated with 60 and 600 microg/kg of ISO compared with untreated animals. The PKA activity ratio, PLB phosphorylation, and PP inhibitor 1 phosphorylation increased in ventricles of guinea pigs treated with 60 and 600 microg/kg of ISO compared with vehicle-treated animals. The decrease in overall PP activity was due primarily to a reduction in type 1 but not type 2A PP activity. In isolated ventricular cardiomyocytes, PP activity was decreased significantly after treatment with 1 microM ISO, and this inhibition was reversed by treatment with 10 microM propranolol. The membrane vesicles of group 1 animals did not release any catalytic subunit of type 1 PP upon phosphorylation by exogenous PKA. These results indicate that activation of cardiac beta-adrenoceptors inhibits type 1 PP activity via phosphorylation of PP inhibitor 1 in the ventricles. This effect is associated with the well-known effect of ISO on increases in the PKA activity ratio and PLB phosphorylation. Inhibition of type 1 PP activity could be one possible mechanism, in addition to activation of adenylate cyclase, by which ISO mediates enhanced contractility of the heart.
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PMID:Inhibition of type 1 protein phosphatase activity by activation of beta-adrenoceptors in ventricular myocardium. 1193 39

Frequency-dependent acceleration of relaxation (FDAR) is an intrinsic physiological mechanism, which allows more rapid ventricular diastolic filling at higher heart rates. FDAR is also observed in isolated myocardial trabeculae and cardiac myocytes, but its mechanism is still poorly understood. We tested the hypothesis that FDAR results mainly from Ca/calmodulin-dependent protein kinase II (CaMKII) dependent stimulation of sarcoplasmic reticulum (SR) Ca transport, but does not require phospholamban. Experiments were performed at 23 or 35 degrees C in isolated ventricular muscle and single myocytes from wild-type (WT) and phospholamban knockout (PLB-KO) mice and rat ventricular myocytes. Isometric twitch force of muscles and unloaded shortening and Ca transients in myocytes were measured ([Ca](o)=1mM) in the absence and presence of CaMKII inhibitors (1 microM KN-93 or 20 microM autocamtide-2 related inhibitory peptide, AIP). Stimulation frequency was altered over a wide range (0.2-8Hz) and post-rest vs steady state twitches were also compared. In both WT and PLB-KO mouse muscles FDAR of twitch force was prominent, but was largely suppressed by KN-93. FDAR of twitch contractions was associated with FDAR of Ca transients in PLB-KO myocytes, and both were inhibited by KN-93. Similarly, a different CaMKII inhibitor (AIP) inhibited FDAR of contraction and Ca transients in rat ventricular myocytes. We conclude that FDAR results mainly from CaMKII-dependent stimulation of SR Ca transport, but does not require phospholamban.
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PMID:Frequency-dependent acceleration of relaxation in the heart depends on CaMKII, but not phospholamban. 1223 67

We have previously described a cardiomyopathy induced by culturing ventricular myocytes from normal adult rats in a medium containing high concentrations of glucose, which recapitulates cellular changes associated with early onset diabetic cardiomyopathy. This investigation was designed to evaluate cellular mechanisms that could contribute to slowed cytosolic Ca(2+) removal and myocyte relaxation in glucose-induced cardiomyopathy. Isolated ventricular myocytes were cultured overnight in medium containing normal glucose (n=5.5mM) or high glucose (HG=25.5mM). Cytosolic Ca(2+) removal was monitored with fluo-3 and myocyte mechanics with video-edge detection. Electrically stimulated Ca(2+) transients were prolonged in HG cells (A(T/PK)=215+/-7ms, n=41) compared to N myocytes (A(T/PK)=173+/-5ms, n=34). By pharmacological and ionic manipulations, Ca(2+) removal attributable to SERCA was slower in the HG group (A(D/PK)=290+/-17ms,n =41) compared to N (A(D/PK)=219+/-10, n=34), whereas NCX function was similar in both groups of cells. Total PKA activity was depressed in HG myocytes by 56% compared to N cells. beta-adrenergic receptor stimulation with ISO (10(-7)M) normalized myocyte relaxation, Ca(2+) transients and PKA activity in HG myocytes. Furthermore, inhibition of PKA with H89 (10(-5)M) depressed peak fractional shortening (PS) and slowed relengthening (A(R/PK)) to a greater extent in N (-50% for PS and 92% for A(R/PK)) than in HG cells (-25% for PS and 48% A(R/PK)). Depressed cytosolic Ca(2+) removal was not, however, associated with changes in basal levels of phosphorylated PLB, nor levels of SERCA, NCX or PLB proteins. We conclude that cellular mechanisms associated with the early onset glucose-induced cardiomyocyte dysfunction involves alterations in Ca(2+) regulation, which may be a common manifestation of other forms of cardiomyopathies.
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PMID:Depressed PKA activity contributes to impaired SERCA function and is linked to the pathogenesis of glucose-induced cardiomyopathy. 1223 68

Phospholamban is an integral membrane protein that regulates the contractility of cardiac muscle by maintaining cardiomyocyte calcium homeostasis. Abnormalities in association of protein kinase A with PLB have recently been linked to human heart failure, where a single mutation is responsible for dilated cardiomyopathy. To date, a high-resolution structure of phospholamban in a lipid environment has been elusive. Here, we describe the first structure of recombinant, monomeric, biologically active phospholamban in lipid-mimicking dodecylphosphocholine micelles as determined by multidimensional NMR experiments. The overall structure of phospholamban is "L-shaped" with the hydrophobic domain approximately perpendicular to the cytoplasmic portion. This is in agreement with our previously published solid-state NMR data. In addition, there are two striking discrepancies between our structure and those reported previously for synthetic phospholamban in organic solvents: a), in our structure, the orientation of the cytoplasmic helix is consistent with the amphipathic nature of these residues; and b), within the hydrophobic helix, residues are positioned on two discrete faces of the helix as consistent with their functional roles ascribed by mutagenesis. This topology renders the two phosphorylation sites, Ser-16 and Thr-17, more accessible to kinases.
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PMID:NMR solution structure and topological orientation of monomeric phospholamban in dodecylphosphocholine micelles. 1450 21

A classical velocity centrifugation technique was used to study the in vitro uptake of the new ketolide ABT-773 by human polymorphonuclear neutrophils (PMNs) and a myelomonoblastic cell line, PLB-985, which can be differentiated into PMNs under certain culture conditions, compared to that of HMR 3004. ABT-773 was rapidly taken up by PMNs (cellular concentration to extracellular concentration ratio [C/E], about 34 at 30 s and up to 207 at 5 min), and uptake plateaued from 30 to 180 min (C/E, about 300). ABT-773 was accumulated significantly better than HMR 3004 from 5 to 180 min. Nondifferentiated PLB-985 cells (ND-PLB) accumulated significantly less ABT-773 and HMR 3004 than PMNs and PLB-985 cells differentiated into PMNs (D-PLB). Whatever the cell type and in contrast to the results obtained with HMR 3004, ABT-773 was mainly located in the cytosol (about 75%) and was rapidly released from loaded cells (about 40% at 5 min), followed by a plateau, likely owing to avid reuptake. Verapamil and H89, an inhibitor of protein kinase A, increased drug efflux. Uptake was sensitive to external pH, and the activation energy was moderate (about 50 kJ/mol). The existence of an active transport system on the PMN membrane was suggested by the following findings: concentration-dependent and saturable uptake (V(max), about 10,000 ng/2.5 x 10(6) PMNs/5 min; K(m), about 60 microg/ml) the inhibitory effects of PMN activators or inhibitors (phorbol myristate acetate, verapamil, Ni(2+)) and the significantly decreased levels of accumulation by killed cells and cells treated at low temperatures. In addition, various macrolides impaired ABT-773 uptake, contrary to the findings for the quinolone levofloxacin. ND- and D-PLB also presented saturation kinetics that defined an active transport system (V(max) and K(m) values were similar to those obtained with PMNs), but the activation pathway of the carrier system did not seem to be fully functional in ND-PLB. As has been observed with other erythromycin A derivatives, ABT-773 impaired oxidant production by phagocytes in a time- and concentration-dependent manner. These data extend our previous results on the existence of an active transport system common to all macrolides and ketolides, at least in PMNs.
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PMID:Interaction of the new ketolide ABT-773 (cethromycin) with human polymorphonuclear neutrophils and the phagocytic cell line PLB-985 in vitro. 1504 7

We have recently demonstrated that in human heart, beta2-adrenergic receptors (beta2-ARs) are biochemically coupled not only to the classical adenylyl cyclase (AC) pathway but also to the cytosolic phospholipase A2 (cPLA2) pathway (Pavoine, C., Behforouz, N., Gauthier, C., Le Gouvello, S., Roudot-Thoraval, F., Martin, C. R., Pawlak, A., Feral, C., Defer, N., Houel, R., Magne, S., Amadou, A., Loisance, D., Duvaldestin, P., and Pecker, F. (2003) Mol. Pharmacol. 64, 1117-1125). In this study, using Fura-2-loaded cardiomyocytes isolated from adult rats, we showed that stimulation of beta2-ARs triggered an increase in the amplitude of electrically stimulated [Ca2+]i transients and contractions. This effect was abolished with the PKA inhibitor, H89, but greatly enhanced upon addition of the selective cPLA2 inhibitor, AACOCF3. The beta2-AR/cPLA2 inhibitory pathway involved G(i) and MSK1. Potentiation of beta2-AR/AC/PKA-induced Ca2+ responses by AACOCF3 did not rely on the enhancement of AC activity but was associated with eNOS phosphorylation (Ser1177) and L-NAME-sensitive NO production. This was correlated with PKA-dependent phosphorylation of PLB (Ser16). The constraint exerted by the beta2-AR/cPLA2 pathway on the beta2-AR/AC/PKA-induced Ca2+ responses required integrity of caveolar structures and was impaired by Filipin III treatment. Immunoblot analyses demonstrated zinterol-induced translocation of cPLA and its cosedimentation with MSK1, eNOS, PLB, and sarcoplasmic reticulum Ca2+ pump (SERCA) 2a in a low density caveolin-3-enriched membrane fraction. This inferred the gathering of beta2-AR signaling effectors around caveolae/sarcoplasmic reticulum (SR) functional platforms. Taken together, these data highlight cPLA as a cardiac beta2-AR signaling pathway that limits beta2-AR/AC/PKA-induced Ca2+ responses in adult rat cardiomyocytes through the impairment of eNOS activation and PLB phosphorylation.
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PMID:The cytosolic phospholipase A2 pathway, a safeguard of beta2-adrenergic cardiac effects in rat. 1572 87


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