Julien Galabru

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.

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.

Phosphorylation of the α-chain of fibrinogen by a platelet kinase activity enhanced by interferon

Treatment of patients with interferon or inducers of interferon results in an enhanced level of a protein kinase activity found in platelets (1,3). The kinase activity is responsible for the phosphorylation of a 70-72,000 molecular weight protein (72K protein) found in blood plasma. By the means of a technique based on the precipitation of this protein kinase system (the protein kinase and its substrate), we show here that the 72K protein is the alpha-chain of fibrinogen. During the coagulation process induced by thrombin, the 32P-labelled 72K protein is recovered in the clot. After incubation in the presence of thrombin, the 72K protein looses a small polypeptide of 2-3000 in molecular weight resulting a shift in its isoelectric point (pI) from 6.8-7.0 to 7.5. At the end of the coagulation process, the 32P-labelled 72K protein becomes undetectable since it gives rise to a covalently linked alpha-polymer of a high molecular weight. In accord with these results, the 72K protein could be precipitated by antibodies against human fibrinogen.

A protein kinase system from platelet rich plasma

Treatment of platelet rich plasma (PRP) at pH 5 results in the precipitation of a protein kinase system. The protein kinase is associated with the platelet fraction and is capable of phosphorylation of several plasma proteins. Analysis of the 32P-labeled phosphoproteins by two dimensional gel electrophoresis showed the existence of three major phosphoproteins: 72K and 80K proteins with identical isoelectric points (pI) of 6.0 and another 72K protein with a pI of 6.8-7.0. This latter 72K phosphoprotein has recently been identified as the alpha-chain of fibrinogen. The identity of the other 2 proteins remains to be shown. The activity of the protein kinase is markedly enhanced by Mn2+, it phosphorylates calf thymus histone as an exogenous substrate and is independent of cAMP or cGMP. This protein kinase activity is inhibited competitively by ADP.

Plasma protein kinase activity enhanced by interferon is found in platelets

A protein kinase activity analogous to that found in interferon‐treated HeLa cells is detectable in human plasma rich in platelets. This kinase activity is manifested by the phosphorylation of an endogenous M r 72 000 protein which could be conveniently assayed after partial purification on poly(G)—Sepharose. Here, we show that the protein kinase system in the plasma consists of at least 2 components. The protein kinase is found to be localised in the platelet whereas most of the substrate (the M r 72 000 protein) is found free in the plasma and a fraction of it associated with the surface of platelets.

Interferon-induced enzymes in mice and in volunteers in response to glucocorticoids

Summary The induction of 2-5A-synthetase has been reported in lymphoid cells treated with glucocorticoids. However, we have been unable to reproduce these results. On the other hand, in the present work, we show that this enzyme, in conjunction with double-stranded RNA-dependent protein kinase, is enhanced in mice treated with hydrocortisone and in volunteers treated with β-1-24 tetracosatide, a synthetic corticosteroid-stimulating hormone. Dose-response experiments carried out in mice indicated that the enhacement of splenic 2-5A-synthetase was observed 48 h after the injection of hydrocortisone and was independent of the dose (25–300 μg/mouse). Administration of mice with anti-mouse (α+β) inteferon immunoglobulins 6 h after injection of hydrocortisone blocked the enhancement of splenic 2-5A-synthetase. These results suggest that the enhancement of 2-5A-synthetase is due to the production of interferon as a consequence of hydrocortisone treatment. In volunteers, the levels of 2-5A-synthetase and protein kinase in peripheral blood mononuclear cells were enhanced 3 and 2 days after the injection of β-1-24-tetracosatide, respectively. The peak of urinary cortisol was 24 h after the injection of β-1-24-tetracosatide. Circulating interferon was not detectable either in mice or in volunteers with high levels of interferon-induced enzymes. This is probably due to the production of very low levels of interferon and the fact that circulating interferon has a very short half-life.