scholarly journals Inhibition of protein kinases C prevents murine cytomegalovirus replication

2005 ◽  
Vol 86 (8) ◽  
pp. 2153-2161 ◽  
Author(s):  
Natalia Kučić ◽  
Hana Mahmutefendić ◽  
Pero Lučin
Keyword(s):  
Protein Phosphorylation ◽  
Protein Kinases ◽  
Phorbol Esters ◽  
Early Stage ◽  
Specific Inhibitor ◽  
Cellular Protein ◽  
Protein Kinase Inhibitors ◽  
Murine Cytomegalovirus ◽  
Mcmv Infection

For successful establishment of infection and initiation of the replication cycle, murine cytomegalovirus (MCMV) utilizes cellular structures and functions, including cell-membrane penetration, capsid dismantling and cytosolic transport of viral DNA into the nucleus. These early events of MCMV infections are dependent on cellular regulatory mechanisms, primarily protein phosphorylation. In the present study, protein kinase inhibitors were used to explore the role of protein phosphorylation mediated by protein kinases C (PKCs) in the very early events of MCMV infection. Inhibitory effects were determined by immunofluorescence and Western blot analysis of MCMV IE1 and E1 protein expression and by production of infectious virions in cell culture. It was found that H-7, a broadly specific inhibitor of cellular protein kinases, prevented virus replication in a dose-dependent and reversible manner, and that the block in replication occurred very early in infection. More specific PKC inhibitors (sangivamycin, calphostin C and bisindolylmaleimide II), Ca2+/calmodulin inhibitors (EDTA and W7) and phorbol esters (PMA) were used to dissect PKC-subclass contribution in the very early events of MCMV replication. The results indicate that the role of diacylglycerol/phorbol ester-dependent but calcium-independent PKCs is essential for establishment of MCMV infection in the host cell, starting at a very early stage of infection.

Protein kinases in seeds

1998 ◽  
Vol 8 (2) ◽  
pp. 193-200 ◽  
Author(s):  
M. K. Walker-Simmons
Keyword(s):  
Amino Acid ◽  
Environmental Stress ◽  
Protein Phosphorylation ◽  
Protein Kinases ◽  
Protein Interaction ◽  
Stress Responses ◽  
Amino Acid Residues ◽  
Protein Protein Interaction ◽  
Reversible Protein Phosphorylation

AbstractReversible phosphorylation is catalysed by protein kinases that transfer the γ-phosphate from ATP to amino acid residues of proteins. The process can be reversed by protein phosphatases. Phosphorylation can dramatically activate or inhibit enzymes and affect protein-protein interaction. Through phosphorylation protein kinases can amplify and propagate cellular signals. In plants and now seeds, protein kinases involved in hormone, defence and environmental stress responses are being identified. Increasingly, these protein kinases are being cloned and characterized, demonstrating the major role of reversible protein phosphorylation in seeds.

scholarly journals Phosphorylation-dependent and -independent pathways of platelet aggregation

1989 ◽  
Vol 258 (2) ◽  
pp. 479-485 ◽  
Author(s):  
S P Watson ◽  
S Hambleton
Keyword(s):  
Protein Phosphorylation ◽  
Platelet Activating Factor ◽  
Specific Inhibitor ◽  
Human Platelets ◽  
Signalling Pathways ◽  
Ionophore A23187 ◽  
Phorbol Dibutyrate ◽  
Kinase C ◽  
Dependent Pathway

We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.

scholarly journals Identification of SRPK1 and SRPK2 as the Major Cellular Protein Kinases Phosphorylating Hepatitis B Virus Core Protein

2002 ◽  
Vol 76 (16) ◽  
pp. 8124-8137 ◽  
Author(s):  
Henrik Daub ◽  
Stephanie Blencke ◽  
Peter Habenberger ◽  
Alexander Kurtenbach ◽  
Julia Dennenmoser ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Protein Kinase ◽  
Hepatitis B ◽  
Protein Phosphorylation ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Core Protein ◽  
Cellular Protein ◽  
Total Cell ◽  
B Virus

ABSTRACT Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.

Modulation of neutrophil activation by okadaic acid, a protein phosphatase inhibitor

1992 ◽  
Vol 262 (1) ◽  
pp. C39-C49 ◽  
Author(s):  
D. J. Lu ◽  
A. Takai ◽  
T. L. Leto ◽  
S. Grinstein
Keyword(s):  
Nadph Oxidase ◽  
Protein Kinases ◽  
Phorbol Ester ◽  
Okadaic Acid ◽  
Phorbol Esters ◽  
Specific Inhibitor ◽  
Human Neutrophils ◽  
Respiratory Response ◽  
Dependent Inhibition ◽  
Reduced Rate

We determined the effects of okadaic acid (OA), a specific inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), on protein phosphorylation and on the activation of the NADPH oxidase in human neutrophils. In otherwise unstimulated cells, OA induced phosphoprotein accumulation, revealing the presence of constitutively active protein kinases. Pulse-chase experiments in electropermeabilized cells confirmed that this effect was due, at least in part, to inhibition of dephosphorylation. OA potentiated phosphoprotein accumulation induced by phorbol esters and by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In phorbol ester-stimulated cells, OA prolonged the respiratory response after inhibition of protein kinase C (PKC) with staurosporine, consistent with a reduced rate of dephosphorylation of active phosphorylated components. Similarly, OA delayed the inactivation of the burst after displacement of FMLP from its receptor by a competitive antagonist. This suggests that the substrates of the protein kinases activated by FMLP are dephosphorylated by PP1 and/or PP2A. That phosphatases control the intensity and duration of the respiratory response is suggested by the finding that OA magnified and prolonged the oxidative burst elicited by FMLP. In contrast, pretreatment with OA produced a time-dependent inhibition of the phorbol ester-induced respiratory burst. Under conditions where inhibition of the phorbol ester response was nearly complete, activation by the chemoattractant peptide not only persisted but was in fact accentuated. These findings provide strong evidence that receptor-mediated stimulation of the NADPH oxidase can occur by pathways not involving PKC.

Calcium-activated sodium and chloride fluxes modulate platelet volume: role of Ca2+ stores

1994 ◽  
Vol 267 (5) ◽  
pp. C1435-C1441 ◽  
Author(s):  
B. P. Fine ◽  
E. S. Marques ◽  
K. A. Hansen
Keyword(s):  
Protein Kinases ◽  
Volume Change ◽  
Kinase Inhibitors ◽  
Cell Types ◽  
Protein Kinase Inhibitors ◽  
Disulfonic Acid ◽  
Platelet Volume ◽  
Ionic Fluxes ◽  
Dependent Protein

An increase in cytosolic ionized Ca2+ concentration ([Ca2+]i) initiates volume changes in various types of cells. In response to increases in [Ca2+]i most cell types contract by efflux of K+ and Cl-, whereas platelets expand in response to rises in [Ca2+]i. This study examined the importance of the source of Ca2+, the flux of ions responsible for the volume change, and the role of Ca(2+)-dependent protein kinases in regulating these ionic fluxes. The baseline platelet volume was independent of extracellular Ca2+ but when stimulated by the Ca2+ ionophore A-23187 (50 nM) the volume increased in both the presence and absence of extracellular Ca2+ (1.18 +/- 0.08 vs. 0.83 +/- 0.06 fl, respectively). The increased volume was caused by the gain of Na+ and Cl-. Na+ entered through both conductive and nonconductive (Na+/H+ exchange) pathways, whereas the influx of Cl- was conductive and inhibited by the Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The Ca(2+)-induced volume change was blocked by both calmodulin and protein kinase inhibitors. Thus the activation of Ca(2+)-dependent protein kinases promotes platelet swelling by stimulating Na+ and Cl- influx.

scholarly journals Latent Murine Cytomegalovirus Infection Contributes to EAE Pathogenesis / Latentna Infekcija Mišjim Citomegalovirusom Ima Ulogu U Patogenezi Eksperimentalnog Autoimunskog Encefalomijelitisa

2014 ◽  
Vol 15 (4) ◽  
pp. 183-190 ◽  
Author(s):  
Jelena Milovanovic ◽  
Aleksandar Arsenijevic ◽  
Bojana Stojanovic ◽  
Marija Milovanovic ◽  
Stipan Jonjic ◽  
...  
Keyword(s):  
Viral Infection ◽  
Murine Cytomegalovirus ◽  
Autoimmune Encephalomyelitis ◽  
Mcmv Infection ◽  
Th 17 ◽  
Murine Homolog ◽  
Environmental Trigger ◽  
The Central Nervous System ◽  
Ms Pathogenesis

ABSTRACT Viral infection has been identified as the most likely environmental trigger of multiple sclerosis (MS). There are conflicting data regarding the role of cytomegalovirus (CMV) in MS pathogenesis. We utilised experimental autoimmune encephalomyelitis (EAE)-resistant BALB/c mice and murine cytomegalovirus (MCMV), the murine homolog of CMV, to examine the mechanism by which viral infection enhances autoimmune neuroinflammation. Mice subjected to latent neonatal MCMV infection developed the typical characteristics of EAE. Similar to MS, the MCMV-infected EAE-induced mice developed infiltrates in the central nervous system (CNS) composed of similar percentages of CD4+ and CD8+ T cells. The influx of both Th 1 and Th 17 cells into the CNS of MCMV- infected EAE-induced mice was observed. Interestingly, the development of autoimmune neuroinflammation after latent MCMV infection was accompanied by a significant influx of Tc17 cells (CD8+IL-17+ and CD8+RoRγt+) but not Tc1, cells. Our results suggest that latent MCMV infection affects the development of inflammatory lymphocytes that exhibit encephalitogenic potential, thereby mediating increased CNS pathology following EAE induction, and that CMV represents a possible environmental factor in the pathogenesis of MS and other autoimmune diseases

scholarly journals The selectivity of protein kinase inhibitors: a further update

2007 ◽  
Vol 408 (3) ◽  
pp. 297-315 ◽  
Author(s):  
Jenny Bain ◽  
Lorna Plater ◽  
Matt Elliott ◽  
Natalia Shpiro ◽  
C. James Hastie ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Kinase Inhibitor ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Protein Kinase Inhibitors ◽  
Mitogen Activated Protein Kinase ◽  
Casein Kinase 1

The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70–80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)–raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes.

scholarly journals Murine Cytomegalovirus m38.5 Protein Inhibits Bax-Mediated Cell Death

2008 ◽  
Vol 82 (10) ◽  
pp. 4812-4822 ◽  
Author(s):  
Igor Jurak ◽  
Uwe Schumacher ◽  
Hrvoje Simic ◽  
Sebastian Voigt ◽  
Wolfram Brune
Keyword(s):  
Cell Death ◽  
Proteasome Inhibitors ◽  
Early Time ◽  
Murine Cytomegalovirus ◽  
Wild Type ◽  
Mcmv Infection ◽  
Infected Cells ◽  
Increased Sensitivity ◽  
Induced Apoptosis

ABSTRACT Many viruses encode proteins that inhibit the induction of programmed cell death at the mitochondrial checkpoint. Murine cytomegalovirus (MCMV) encodes the m38.5 protein, which localizes to mitochondria and protects human HeLa cells and fibroblasts from apoptosis triggered by proteasome inhibitors but not from Fas-induced apoptosis. However, the ability of this protein to suppress the apoptosis of murine cells and its role during MCMV infection have not been investigated previously. Here we show that m38.5 is expressed at early time points during MCMV infection. Cells infected with MCMVs lacking m38.5 showed increased sensitivity to cell death induced by staurosporine, MG132, or the viral infection itself compared to the sensitivity of cells infected with wild-type MCMV. This defect was eliminated when an m38.5 or Bcl-XL gene was inserted into the genome of a deletion mutant. Using fibroblasts deficient in the proapoptotic Bcl-2 family proteins Bak and/or Bax, we further demonstrated that m38.5 protected from Bax- but not Bak-mediated apoptosis and interacted with Bax in infected cells. These results consolidate the role of m38.5 as a viral mitochondrion-localized inhibitor of apoptosis and its functional similarity to the human cytomegalovirus UL37x1 gene product. Although the m38.5 gene is not homologous to the UL37x1 gene at the sequence level, m38.5 is conserved among rodent cytomegaloviruses. Moreover, the fact that MCMV-infected cells are protected from both Bak- and Bax-mediated cell death suggests that MCMV possesses an additional, as-yet-unidentified mechanism to block Bak-mediated apoptosis.

scholarly journals Interferon γ Regulates Acute and Latent Murine Cytomegalovirus Infection and Chronic Disease of the Great Vessels

1998 ◽  
Vol 188 (3) ◽  
pp. 577-588 ◽  
Author(s):  
Rachel M. Presti ◽  
Jessica L. Pollock ◽  
Albert J. Dal Canto ◽  
Andrew K. O'Guin ◽  
Herbert W. Virgin
Keyword(s):  
Immune System ◽  
Chronic Disease ◽  
Interferon Γ ◽  
Murine Cytomegalovirus ◽  
Mcmv Infection ◽  
Great Vessels ◽  
Ifn Γ

To define immune mechanisms that regulate chronic and latent herpesvirus infection, we analyzed the role of interferon γ (IFN-γ) during murine cytomegalovirus (MCMV) infection. Lethality studies demonstrated a net protective role for IFN-γ, independent of IFN-α/β, during acute MCMV infection. Mice lacking the IFN-γ receptor (IFN-γR−/−) developed and maintained striking chronic aortic inflammation. Arteritis was associated with inclusion bodies and MCMV antigen in the aortic media. To understand how lack of IFN-γ responses could lead to chronic vascular disease, we evaluated the role of IFN-γ in MCMV latency. MCMV-infected IFN-γR−/− mice shed preformed infectious MCMV in spleen, peritoneal exudate cells, and salivary gland for up to 6 mo after infection, whereas the majority of congenic control animals cleared chronic productive infection. However, the IFN-γR was not required for establishment of latency. Using an in vitro explant reactivation model, we showed that IFN-γ reversibly inhibited MCMV reactivation from latency. This was at least partly explained by IFN-γ– mediated blockade of growth of low levels of MCMV in tissue explants. These in vivo and in vitro data suggest that IFN-γ regulation of reactivation from latency contributes to control of chronic vascular disease caused by MCMV. These studies are the first to demonstrate that a component of the immune system (IFN-γ) is necessary to regulate MCMV-associated elastic arteritis and latency in vivo and reactivation of a herpesvirus from latency in vitro. This provides a new model for analysis of the interrelationships among herpesvirus latency, the immune system, and chronic disease of the great vessels.

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