scholarly journals Evidence for an Essential Catalytic Role of the F10 Protein Kinase in Vaccinia Virus Morphogenesis

2004 ◽  
Vol 78 (1) ◽  
pp. 257-265 ◽  
Author(s):  
Patricia Szajner ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Protein Kinase ◽  
Vaccinia Virus ◽  
Early Stage ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Membrane Formation ◽  
Virus Morphogenesis ◽  
Catalytic Role

ABSTRACT Temperature-sensitive mutants of vaccinia virus, with genetic changes that map to the open reading frame encoding the F10 protein kinase, exhibit a defect at an early stage of viral morphogenesis. To further study the role of the enzyme, we constructed recombinant vaccinia virus vF10V5i, which expresses inducible V5 epitope-tagged F10 and is dependent on a chemical inducer for plaque formation and replication. In the absence of inducer, viral membrane formation was delayed and crescents and occasional immature forms were detected only late in infection. When the temperature was raised from 37 to 39°C, the block in membrane formation persisted throughout the infection. The increased stringency may be explained by a mild temperature sensitivity of the wild-type F10 kinase, which reduced the activity of the very small amount expressed in the absence of inducer, or by the thermolability of an unphosphorylated kinase substrate or uncomplexed F10-interacting protein. Further analyses demonstrated that tyrosine and threonine phosphorylation of the A17 membrane component was inhibited in the absence of inducer. The phosphorylation defect could be overcome by transfection of plasmids that express wild-type F10, but not by plasmids that express F10 with single amino acid substitutions that abolished catalytic activity. Although the mutated forms of F10 were stable and concentrated in viral factories, only the wild-type protein complemented the assembly and replication defects of vF10V5i in the absence of inducer. These studies provide evidence for an essential catalytic role of the F10 kinase in vaccinia virus morphogenesis.

scholarly journals Distinct roles of the Drosophila ninaC kinase and myosin domains revealed by systematic mutagenesis

1993 ◽  
Vol 122 (3) ◽  
pp. 601-612 ◽  
Author(s):  
JA Porter ◽  
C Montell
Keyword(s):  
Protein Kinase ◽  
Retinal Degeneration ◽  
Kinase Domain ◽  
Specific Protein ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Multiple Functions

The Drosophila ninaC locus encodes a rhabdomere specific protein (p174) with linked protein kinase and myosin domains, required for a wild-type ERG and to prevent retinal degeneration. To investigate the role for linked kinase and myosin domains, we analyzed mutants generated by site-directed mutagenesis. Mutation of the kinase domain resulted in an ERG phenotype but no retinal degeneration. Deletion of the myosin domain caused a change in the subcellular distribution of p174 and resulted in both ERG and retinal degeneration phenotypes. Temperature-sensitive mutations in the myosin domain resulted in retinal degeneration, but no ERG phenotype. These results indicated that the ERG and retinal degeneration phenotypes were not strictly coupled suggesting that the myosin domain has multiple functions. We propose that the role of the kinase domain is to regulate other rhabdomeric proteins important in phototransduction and that the myosin domain has at least two roles: to traffic the kinase into the rhabdomeres and to maintain the rhabdomeres.

scholarly journals Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

2006 ◽  
Vol 189 (5) ◽  
pp. 1565-1572 ◽  
Author(s):  
Venkata Ramana Vepachedu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Small Molecules ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Ph Optimum ◽  
Inner Membrane ◽  
Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies

2008 ◽  
Vol 294 (5) ◽  
pp. H2352-H2362 ◽  
Author(s):  
Andreas A. Werdich ◽  
Eduardo A. Lima ◽  
Igor Dzhura ◽  
Madhu V. Singh ◽  
Jingdong Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Myocytes ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Frequency Dependent ◽  
Physiological Range ◽  
Dependent Protein ◽  
Isolated Cardiac Myocytes

In cardiac myocytes, the activity of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is hypothesized to regulate Ca2+ release from and Ca2+ uptake into the sarcoplasmic reticulum via the phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of cytosolic Ca2+ concentration ([Ca2+]i) transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37°C). When compared with that of mice lacking PLN only, the combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca2+ release rate by more than 50% at 10 Hz. Although PLN ablation increased the rate of Ca2+ uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca2+]i transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca2+]i transient, indicating that the PLN-mediated feedback on [Ca2+]i removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes, the combined chronic CaMKII inhibition and PLN ablation slowed Ca2+ release at physiological frequencies: the frequency-dependent decay of the amplitude and shortening of the [Ca2+]i transient occurs independent of chronic CaMKII inhibition and PLN ablation, and the PLN-mediated regulation of Ca2+ uptake is diminished at higher stimulation frequencies within the physiological range.

Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast.

Genetics ◽  
1992 ◽  
Vol 132 (1) ◽  
pp. 53-62 ◽  
Author(s):  
R E Hollingsworth ◽  
R M Ostroff ◽  
M B Klein ◽  
L A Niswander ◽  
R A Sclafani
Keyword(s):  
Protein Kinase ◽  
Insertional Mutagenesis ◽  
Molecular Genetic ◽  
Dna Lesions ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Wild Type ◽  
Mutant Proteins ◽  
Repair Enzymes ◽  
Induced Mutagenesis

Abstract The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.

scholarly journals Role of Pseudorabies Virus Us3 Protein Kinase during Neuronal Infection

2006 ◽  
Vol 80 (13) ◽  
pp. 6387-6398 ◽  
Author(s):  
L. M. Olsen ◽  
T. H. Ch'ng ◽  
J. P. Card ◽  
L. W. Enquist
Keyword(s):  
Protein Kinase ◽  
Pseudorabies Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Spread Of Infection ◽  
Viral Components ◽  
Axonal Targeting ◽  
Null Mutants

ABSTRACT The pseudorabies virus (PRV) Us3 gene is conserved among the alphaherpesviruses and encodes a serine/threonine protein kinase that is not required for growth in standard cell lines. In this report, we used a compartmented culture system to investigate the role of PRV Us3 in viral replication in neurons, in spread from neurons to PK15 cells, and in axon-mediated spread of infection. We also examined the role of Us3 in neuroinvasion and virulence in rodents. Us3 null mutants produce about 10-fold less infectious virus from neurons than wild-type virus and have no discernible phenotypes for axonal targeting of viral components in cultured peripheral nervous system neurons. After eye infection in rodents, Us3 null mutants were slightly attenuated for virulence, with a delayed onset of symptoms compared to the wild type or a Us3 null revertant. While initially delayed, the symptoms increased in severity until they approximated those of the wild-type virus. Us3 null mutants were neuroinvasive, spreading in both efferent and afferent circuits innervating eye tissues.

scholarly journals Casein kinase II is required for efficient transcription by RNA polymerase III.

1996 ◽  
Vol 16 (3) ◽  
pp. 892-898 ◽  
Author(s):  
D J Hockman ◽  
M C Schultz
Keyword(s):  
Protein Kinase ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Rna Polymerases ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Polymerase Iii

Casein kinase II (CKII) is a ubiquitous and highly conserved serine/threonine protein kinase found in the nucleus and cytoplasm of most cells. Using a combined biochemical and genetic approach in the yeast Saccharomyces cerevisiae, we assessed the role of CKII in specific transcription by RNA polymerases I, II, and III. CKII is not required for basal transcription by RNA polymerases I and II but is important for polymerase III transcription. Polymerase III transcription is high in extracts with normal CKII activity but low in extracts from a temperature-sensitive mutant that has decreased CKII activity due to a lesion in the enzyme's catalytic alpha' subunit. Polymerase III transcription of 5S rRNA and tRNA templates in the temperature-sensitive extract is rescued by purified, wild-type CKII. An inhibitor of CKII represses polymerase III transcription in wild-type extract, and this repression is partly overcome by supplementing reaction mixtures with active CKII. Finally, we show that polymerase III transcription in vivo is impaired when CKII is inactivated. Our results demonstrate that CKII, an oncogenic protein kinase previously implicated in cell cycle and growth control, is required for high-level transcription by RNA polymerase III.

scholarly journals Mutations in the Vaccinia Virus A33R and B5R Envelope Proteins That Enhance Release of Extracellular Virions and Eliminate Formation of Actin-Containing Microvilli without Preventing Tyrosine Phosphorylation of the A36R Protein

2003 ◽  
Vol 77 (22) ◽  
pp. 12266-12275 ◽  
Author(s):  
Ehud Katz ◽  
Brian M. Ward ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Envelope Protein ◽  
Actin Polymerization ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Virus Release ◽  
Type Virus ◽  
Wild Type ◽  
Extracellular Virus

ABSTRACT The spread of vaccinia virus in cell cultures is mediated by virions that adhere to the tips of specialized actin-containing microvilli and also by virions that are released into the medium. The use of a small plaque-forming A36R gene deletion mutant to select spontaneous second-site mutants exhibiting enhanced virus release was described previously. Two types of mutations were found: C-terminal truncations of the A33R envelope protein and a single amino acid substitution of the B5R envelope protein. In the present study, we transferred each type of mutation into a wild-type virus background in order to study their effects in vitro and in vivo. The two new mutants conserved the enhanced virus release properties of the original isolates; the A33R mutant produced considerably more extracellular virus than the B5R mutant. The extracellular virus particles contained the truncated A33R protein in one case and the mutated B5R protein in the other. Remarkably, both mutants failed to form actin tails and specialized microvilli, despite the presence of an intact A36R gene. The synthesis of the A36R protein as well as its physical association with the mutated or wild-type A33R protein was demonstrated. Moreover, the A36R protein was tyrosine phosphorylated, a step mediated by a membrane-associated Src kinase that regulates the nucleation of actin polymerization. The presence of large numbers of adherent virions on the cell surface argued against rapid dissociation as having a key role in preventing actin tail formation. Thus, the A33R and B5R proteins may be more directly involved in the formation or stabilization of actin tails than had been previously thought. When mice were inoculated intranasally, the A33R mutant was highly attenuated and the B5R mutant was mildly attenuated compared to wild-type virus. Enhanced virus release, therefore, did not compensate for the loss of actin tails and specialized microvilli.

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