PubMed Health⌕ Search

Biomedical subjects

J Galabru

Publications and source records attributed to J Galabru.

At least 19 recordsLinked to original sources
PubMed1990

Differential expression and distinct structure of 69- and 100-kDa forms of 2-5A synthetase in human cells treated with interferon.

Using specific monoclonal and polyclonal antibodies, the induction, synthesis and subcellular localization of 69- and 100-kDa forms of 2',5'-oligoadenylate (2-5A) synthetase (p69 and p100) were investigated in alpha-interferon-treated human HeLa and Daudi cells. Although both p69 and p100 were induced by interferon, there were significant differences in the interferon dose-response and the kinetics of synthesis of each protein in these cell lines. Both proteins are localized mainly in the cytoplasm. However, immunoenzymatic staining analysis of interferon-treated cells indicated that a proportion of p69 is concentrated around the nuclei and the rest is distributed in a specific pattern in the cytoplasm whereas p100 is found in a diffuse state in the cytoplasm. In accord with its association to cell membranes, p69 is myristilated. The [35S]cysteine and [3H]myristate-labeled p69 preparations were resolved by two-dimensional gel isoelectric focusing with pI values in the pH range of 7.0-8.0 and 7.0-7.5, respectively. These observations suggested the existence of two forms of p69 which might be complexed together as a dimer. In favor with a dimeric form of p69, in gel filtration experiments the peak of p69 was routinely found in fractions corresponding to a molecular mass of 160 kDa whereas p100 was recovered as a monomer. Taken together, these results indicate that p69 and p100 have distinct characteristics and that their expression is a specific property of each cell type in response to interferon.

2',5'-Oligoadenylate Synthetase↗
PubMed1990

Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon.

The double-stranded (ds) RNA-activated protein kinase from human cells is a 68 kd protein (p68 kinase) induced by interferon. On activation by dsRNA in the presence of ATP, the kinase becomes autophosphorylated and can catalyze the phosphorylation of the alpha subunit of eIF2, which leads to an inhibition of the initiation of protein synthesis. Here we report the molecular cloning and characterization of several related cDNAs from which can be deduced the full-length p68 kinase sequence. All of the cDNAs identify a 2.5 kb RNA that is strongly induced by interferon. The deduced amino acid sequence of the p68 kinase predicts a protein of 550 amino acids containing all of the conserved domains specific for members of the protein kinase family, including the catalytic domain characteristic of serine/threonine kinases. In vitro translation of a reconstructed full-length p68 kinase cDNA yields a protein of 68 kd that binds dsRNA, is recognized by a monoclonal antibody raised against the native p68 kinase, and is autophosphorylated.

Amino Acid Sequence↗
PubMed1989

Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress.

Previous studies have shown that the antiviral response induced by interferon in murine cells could be degraded after a heat shock. Here we have confirmed that a similar effect occurs also in interferon-treated human HeLa cells subjected to a heat shock. In addition, we have investigated the fate of the interferon-induced, double-stranded RNA-dependent protein kinase in heat-shocked cells. This protein kinase is a Mr 68,000 protein (p68 kinase) which, when autophosphorylated, catalyzes phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. After heat shock of interferon-treated HeLa cells, the double-stranded RNA-dependent autophosphorylation of p68 kinase in cytoplasmic extracts is greatly reduced whereas the phosphorylation of other cellular proteins is not affected. In vivo, autophosphorylation of p68 kinase is also reduced in heat-shocked cells whereas there is no apparent effect on the phosphorylation state of other proteins. In such cells, the interferon-mediated antiviral response becomes modified according to the virus challenge, i.e. these cells remain resistant to vesicular stomatitis virus but become partially sensitive to encephalomyocarditis virus (EMCV) infection. The reduction in the activity of p68 kinase is due to its reduced nonionic detergent solubility occurring during the heat shock period. The resultant reduced detergent extractibility of p68 kinase is dependent on the intensity of the thermal stress. In contrast to the effect after a heat shock, arsenite treatment of interferon-treated HeLa cells induces heat shock proteins, but neither modifies the antiviral response nor affects the extractibility of p68 kinase. These results indicate that the degradation of the anti-EMCV response and reduced p68 kinase activity occur in response to heat treatment independently of the induction of heat shock proteins. The role of p68 kinase in the mechanism of the antiviral response against EMCV and vesicular stomatitis virus is discussed.

2',5'-Oligoadenylate Synthetase↗
PubMed1989

Preparation and characterization of polyclonal antibodies specific for the 69 and 100 k-dalton forms of human 2-5A synthetase.

Recently, the existence of 40-, 46-, 69- and 100- kDa forms of 2',5'-oligoadenylate (2-5A) synthetase have been established in interferon-treated human cells. Using monoclonal antibodies specific for 69- and 100- kDa forms of 2-5A synthetase, we purified these proteins by immunoaffinity chromatography and raised murine polyclonal antibodies. All immunized mice developed antibodies (anti-69 or anti-100 kDa form) which were characterized by their capacity to immunoprecipitate [35S] cysteine labeled proteins from interferon-treated cells or identify these proteins by electrophoretic transfer immunoblot analysis of extracts from control and interferon-treated cells. The 69 and 100 kDa 2-5A synthetases were induced in different types of human cells, such as Daudi, BJAB, HeLa and differentiated HL-60 cells. These enzymes were not detectable nor induced in MRC5 and undifferentiated HL-60 cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1989

A type 1 phosphoprotein phosphatase active with phosphorylated Mr = 68,000 initiation factor 2 kinase.

A type 1 protein phosphatase from reticulocytes is shown to efficiently dephosphorylate the Mr = 68,000 phosphopeptide of the double-stranded RNA-dependent kinase that phosphorylates the alpha subunit of eukaryotic peptide initiation factor 2, eIF-2. The kinase, activated in the presence of double-stranded RNA with concomitant phosphorylation of the Mr = 68,000 peptide, causes inhibition of peptide initiation and thereby effects translational control of protein synthesis. The Mn2+-dependent phosphatase is classified as a type 1 enzyme in that it is inhibited by inhibitor 2 in nanomolar concentrations and appears to have a Mr = 35,000 catalytic subunit. Dephosphorylation of the Mr = 68,000 peptide by the phosphatase is directly associated with a loss in kinase activity which can be restored by incubation with double-stranded RNA in the presence of ATP. The results demonstrate that the eIF-2 alpha kinase can undergo cyclic activation-inactivation that appears to be directly related to the phosphorylation state of the Mr = 68,000 peptide. They strongly support the previous conclusion that double-stranded RNA is required only for activation of the kinase and phosphorylation of the Mr = 68,000 peptide.

Animals↗
PubMed1989

The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase.

The protein kinase from human cells dependent on double-stranded (ds) RNA is a 68-kDa protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by low concentrations of dsRNA, the p68 kinase becomes phosphorylated and thereby catalyzes the phosphorylation of the protein-synthesis initiation factor, eIF2. Here, we have purified the p68 kinase to homogeneity using a specific monoclonal antibody to investigate its capacity to bind dsRNA, poly(I).poly(C). Our study suggest that p68 kinase has high- and low-affinity binding sites: the high-affinity binding site is responsible for the activation and the low-affinity binding site for the inhibition of kinase activity. This is in accord with the fact that autophosphorylation of p68 kinase occurs at low concentrations of dsRNA whereas high concentrations of dsRNA inhibit its autophosphorylation. We have also investigated the binding of adenoviral VAI RNA to the purified p68 kinase and have found that the affinity of this binding is lower than that of poly(I).poly(C). We show that VAI RNA can activate or inhibit autophosphorylation of p68 kinase in a dose-dependent manner, i.e. activation at less than or equal to 1 microgram/ml or inhibition at greater than 1 microgram/ml of VAI RNA. In spite of its lower affinity of binding, VAI RNA cannot be displaced by poly(I).poly(C) or reovirus dsRNA. These data confirm our previous results to illustrate that VAI RNA can bind p68 kinase and cause its inactivation irreversably.

Adenoviruses, Human↗
PubMed1988

Interferon- and sarcolectin-dependent cellular regulatory interactions.

Interferons, via specific membrane-bound receptors, induce various cellular functions of which antiviral protection is the most extensively studied. We have previously reported the existence of interferon antagonists (referred to as sarcolectins) in various tissue extracts from placental blood, cartilage, brain, muscle, or from sarcomas. These sarcolectins have been fully characterized and purified to homogeneity. In interferon-treated cells, they restore virus sensitivity 4-6 h after the establishment of antiviral protection. In the present study we investigate the effect of sarcolectins on the steady state levels of two double-stranded RNA dependent enzymes, 2-5A (p chi (A2'p)nA) synthetase and protein kinase. Several authors have previously emphasized the role of these enzymes in the mechanism of interferon's antiviral action. Interferon promotes a 4-8 fold increase in protein kinase and 2-5A synthetase in cells. Addition of sarcolectin 5 h after interferon results in a dramatic reduction in the steady state levels of both these enzymes, as shown by their decreased activity and yield observed in Western blot assays. The degradation of the antiviral response in sarcolectin-treated cells might therefore be at least partially attributed to a reduced synthesis of protein kinase and 2-5A synthetase. Since there are no direct interactions between sarcolectins and interferon or its receptors, it can be postulated that sarcolectins exert their effect through these interferon-dependent proteins. We postulate that the opposing biological effects of interferon and sarcolectins strike a balance which may, however, be modified in one direction or the other, depending on their respective concentrations.

2',5'-Oligoadenylate Synthetase↗
PubMed1988

Expression of interferon-induced genes in different tissues of mice.

In vivo responses to interferon (IFN) in mice were determined by measuring the steady-state levels of induced mRNAs following injection of IFN and poly(I)-poly(C). With cDNA probes for mouse 2'-5' oligoadenylate synthetase (2-5A synthetase) and 1-8, constitutive expression of the corresponding mRNA was detectable in different organs of normal C3H/He mice. These mRNA levels were increased by as much as 15-fold over control levels in various tissues, including the brain, after IFN and poly(I)-poly(C) treatment, coincident with increases in 2-5A synthetase enzyme activity. The basal activity level of this enzyme could be reduced in normal mice by treatment with anti-mouse IFN (alpha + beta) antibody. This treatment also reduced the levels of 2-5A synthetase and 1-8 mRNAs. Thus, physiological levels of circulating IFN maintain elevated levels of IFN-induced mRNAs in mice. Furthermore, changes in 2-5A synthetase enzyme activity reflect the changes in gene expression in vivo.

2',5'-Oligoadenylate Synthetase↗
PubMed1987

Autophosphorylation of the protein kinase dependent on double-stranded RNA.

The double-stranded RNA (dsRNA)-dependent protein kinase (p68 kinase) from interferon-treated human cell is a Mr 68,000 protein induced by interferon. By the use of a specific monoclonal antibody, we have been able to study the two distinct protein kinase activities characteristic of purified p68 kinase. The first activity is functional for endogenous phosphorylation of the enzyme (p68 kinase), whereas the second one is responsible for the phosphorylation of exogenous substrates such as eukaryotic initiation factor 2 and histone. When activated by dsRNA in the presence of Mn2+ and ATP, p68 kinase is autophosphorylated and is then capable of catalyzing phosphorylation of histone in the absence of dsRNA. Whereas binding of 8-azido-[alpha-32P] ATP (8-N3ATP) to p68 kinase is dependent on both dsRNA and Mn2+, phosphorylated p68 kinase binds 8-N3ATP independent of dsRNA. This is consistent with a dsRNA requirement for the autophosphorylation of p68 kinase, but not for the phosphorylation of exogenous substrates. p68 kinase is mainly associated with the ribosomal pellet. It could be recovered efficiently by a buffer containing both high salt and a nonionic detergent. Synthesis of p68 kinase is induced several-fold by interferon in different types of human cells. Partial proteolysis of [35S]methionine and an 8-N3ATP-labeled p68 kinase preparation by Staphylococcus aureus V8 protease indicated the presence of a major Mr 48,000 polypeptide (p48) with a specific ATP-binding site. p48 probably contains the catalytic unit of p68 kinase and is analogous to a similar protein which we have previously described as a distinct protein present in a complexed form with p68 kinase. We now believe that the presence of p48 in previously purified kinase preparations was due to partial degradation of p68 kinase.

Adenosine Triphosphate↗
PubMed1987

The double-stranded RNA-dependent protein kinase is also activated by heparin.

The double-stranded(ds)-RNA dependent protein kinase from human cells is a Mr 68,000 protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by dsRNA, the p68 kinase becomes autophosphorylated. The phosphorylated p68 kinase then can catalyze the phosphorylation of exogenous substrates, such as eIF2 and histone. The second phosphorylation step can take place in the absence of dsRNA. Here we show that, besides dsRNA other polyanions, especially heparin, can also activate the p68 kinase for the autophosphorylation reaction. Heparin activation of the p68 kinase is reversible since it can be prevented by addition of antithrombin III, heparin-binding protein. However, when antithrombin III is added after autophosphorylation of the p68 kinase then phosphorylation of histone is not affected. The p68 kinase binds to heparin-Sepharose. Further evidence that the p68 kinase can be activated by heparin was provided by photoaffinity labeling with 8-azido-[alpha-32P]ATP. This ATP analog can bind to the p68 kinase only in the presence of heparin or dsRNA. Thus suggesting that the activation of the p68 kinase triggers a conformational modification allowing the binding of ATP. Basic proteins, histone and protamine, prevent the activation process induced by heparin. This is probably due to binding of these basic proteins to heparin and thus sequestering the activator of the protein kinase.

Anions↗
PubMed1987

Four different forms of interferon-induced 2',5'-oligo(A) synthetase identified by immunoblotting in human cells.

Antibodies against synthetic peptides derived from the cDNA sequence of interferon-induced 2',5'-oligo(A) synthetase, and which immunoprecipitate the native enzyme activity, were found to detect multiple enzyme forms in denaturing electrophoretic immunoblots. In some human cell lines, four different interferon-induced proteins of 40, 46, 67, and 100 kDa were found to react with the same peptide antibodies. Each isolated form was shown to have 2',5'-oligo(A) synthetase activity, but the dependence on double-stranded RNA was markedly different for activation of the individual enzymes. The four enzyme forms also differ in their intracellular localization, on microsomes (100 kDa), in nuclei (67, 46, 40 kDa), and on membrane structures (67 kDa). Plasma membranes from interferon-treated Daudi lymphoblastoid cells are highly enriched in the 67-kDa 2',5'-oligo(A) synthetase form. The 2',5'-oligo(A) synthetase activity induced by interferons in human cells appears, therefore, as a complex multienzyme system.

2',5'-Oligoadenylate Synthetase↗
PubMed1987

Identification of 69-kd and 100-kd forms of 2-5A synthetase in interferon-treated human cells by specific monoclonal antibodies.

Recently, the existence of 40-kd and 46-kd 2-5A synthetases in interferon-treated cells has been confirmed by cloning and characterization of cDNA corresponding to these small size enzymes. By the use of specific monoclonal antibodies, we describe here two forms of high mol. wt 2-5A synthetases of 69 and 100 kd in human cells. The monoclonal antibodies immunoprecipitate either a 69- or a 100-kd 2-5A synthetase. These purified 2-5A synthetases in immune complex preparations are active, i.e. addition of poly(I).poly(C) and ATP results in the synthesis of 2-5A. Both 2-5A synthetases are composed of several subspecies with similar isoelectric points in the range of 7-8 but have different subcellular localizations: 100-kd synthetase is recovered from the microsomal pellet whereas 69-kd synthetase is found to be associated with cell membranes as well as with the microsomal pellet. Different types of interferon-treated human cells express both or either forms of these enzymes. The 69- and 100-kd 2-5A synthetases were also identified by electrophoretic transfer immunoblot analysis using rabbit polyclonal antibodies against a synthetic peptide common on both 46- and 40-kd 2-5A synthetases. These results indicate that small and large size isozymes share a common peptide sequence.

2',5'-Oligoadenylate Synthetase↗
PubMed1987

Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity.

We have investigated the interaction of VAI RNA with the interferon-induced, double-stranded (ds) RNA-activated protein kinase, P68, both of which regulate protein synthesis in adenovirus-infected cells. Previous work has shown that during infection by the VAI RNA-negative mutant, dl331, both viral and cellular protein synthesis are inhibited due to phosphorylation of the alpha-subunit of the eukaryotic initiation factor, eIF-2, by the P68 protein kinase. Utilizing monoclonal antibodies specific for P68, we demonstrated that the physical levels of P68 in dl331-infected, wild-type Ad2-infected and uninfected cells were all comparable suggesting that the elevated kinase activity detected during mutant infection was not due to increased P68 synthesis. To examine the basis of the increased activity of P68, the protein kinase was purified from infected-cell extracts using the monoclonal antibody. We found that P68 was heavily autophosphorylated during dl331 infection but not during wild-type or mock infection. The extent of autophosphorylation correlated with elevated P68 activity and the loss of the dsRNA requirements to phosphorylate the exogenous substrates, eIF-1 alpha and histones. We also analyzed VAI RNA function in vitro and present evidence that purified VAI RNA can block the autophosphorylation of P68 in the ribosomal salt wash fraction of interferon-treated cells. Finally we suggest VAI RNA functions through a direct interaction with the P68 protein kinase, since we demonstrated that VAI RNA forms a complex with P68 both in vitro and in vivo.

Adenoviruses, Human↗
PubMed1987

Rapid decrease in the levels of the double-stranded RNA-dependent protein kinase during virus infections.

The double-stranded RNA-dependent protein kinase from human cells is a 68,000 molecular weight protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. With a monoclonal antibody specific for p68 kinase, here we show the phosphorylation and steady-state levels of p68 kinase during virus infection. The p68 kinase is phosphorylated in interferon-treated cells during infection with encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), and vaccinia virus, thus indicating activation of p68 kinase during these virus infections, an essential step required for autophosphorylation of p68 kinase. However, in spite of this activation, the level of p68 kinase is rapidly decreased in virus-infected cells. The half-life of p68 kinase in uninfected cells is 6 to 7 hr, whereas in EMCV-infected cells it is 2 to 3 hr. This decrease in the level of p68 kinase is dependent on the multiplicity of virus infection and it seems to be specific since other cellular proteins as well as the activity of 2'-5'-oligoadenylate synthetase are not modified. Decreased levels of p68 kinase are also observed in cells infected with VSV and vaccinia virus. In the absence of virus infection, decreased levels of p68 kinase occur in cells following incubation with poly(I).poly(C).

2',5'-Oligoadenylate Synthetase↗
PubMed1986

Neutralization of interferon produced early during rabies virus infection in mice.

Inoculation of mice (strain C3H/He) with a purified preparation of fixed rabies virus led to the production of interferon with two different peaks of activity detectable in the plasma: an early peak 24 h after inoculation followed by another peak on the 7th day after infection. The level of splenic 2-5A synthetase was enhanced in parallel with the pattern of interferon activity. Neutralization of the first peak of interferon activity by anti-mouse alpha/beta interferon globulin blocked the induction of splenic 2-5A synthetase and modified the development of disease. Infected mice given anti-interferon globulin had a significantly shorter morbidity period than normally infected mice. These results suggest that interferon produced early after virus inoculation plays a role in the onset of rabies disease.

Animals↗
PubMed1985

Two interferon-induced proteins are involved in the protein kinase complex dependent on double-stranded RNA.

The double-stranded (ds) RNA-dependent protein kinase is a 100,000-110,000 Mr complex of two interferon-induced subunits each having ATP binding sites: a 48,000 Mr protein (p48) which appears to be responsible for the phosphorylation of a 68,000 Mr protein (p68) in the presence of dsRNA. The p68 subunit once phosphorylated is converted to an active protein kinase capable of phosphorylating exogenous substrates such as the alpha subunit of protein synthesis initiation factor eIF2 or calf thymus histone. The phosphorylation of exogenous substrates is highly correlated with the degree of phosphate saturation of p68 and does not require the presence of dsRNA. Both the p68 and the p48 subunits of the protein kinase complex are purified by an immuno-affinity column containing monoclonal antibody specific for the p68 subunit.

Adenosine Triphosphate↗
PubMed1985

Monoclonal antibodies to an interferon-induced Mr 68,000 protein and their use for the detection of double-stranded RNA-dependent protein kinase in human cells.

Extracts from interferon-treated human cells show an enhanced level of a double-stranded RNA-dependent protein kinase activity that is manifested by the phosphorylation of an endogenous Mr 69,000-72,000 protein in its phosphate-saturated state. By using a highly purified protein kinase fraction from interferon-treated human Daudi cells, we can now describe the preparation of murine monoclonal antibodies directed against this phosphoprotein, the Mr of which in its native state is found to be 68,000. These monoclonal antibodies (class IgG1) can identify the electrophoresed protein (p68) in polyacrylamide gels by the electrophoretic transfer blotting technique. Immunoprecipitates formed after incubation of extracts from interferon-treated human cells with the monoclonal antibodies can be conveniently recovered by protein A-Sepharose. Such immune complex preparations have associated protein kinase activity--i.e., addition of [gamma-32P]ATP results in the phosphorylation of p68 and added substrates, calf thymus histone, and eukaryotic initiation factor 2. Immune complex preparations from [35S]methionine-labeled extracts show the specific immunoprecipitation of p68. In addition, several other [35S]methionine-labeled proteins are bound unspecifically in these immune complexes prepared under similar experimental conditions as for the assay of protein kinase activity. These unspecifically bound proteins can be washed out by using a buffer containing detergents or high concentrations of KCl and magnesium acetate. Immune complex preparations washed similarly with these buffers still retain p68 but lose their capacity to phosphorylate p68 or exogenous substrates. These results indicate that p68 by itself has no protein kinase activity. The induction of [35S]methionine-labeled p68 in Daudi cells occurs with as little as 1 unit of human alpha interferon, with maximal synthesis between 6 to 9 hr after the addition of interferon. Actinomycin D blocks this induction.

Adenosine Triphosphate↗