Interrelationships between protein kinases and spectrin phosphorylation in human erythrocytes.

Casein kinase and histone kinase(s) are solubilized from human erythrocyte membranes by buffered ionic solutions (0.1 mM EDTA and subsequent 0.8 M NaCl, pH 8) containing 0.2% Triton X-100. Casein kinase is separated from histone kinase(s) by submitting the crude extracts directly to chromatography on a phosphocellulose column, eluted with a continuous linear gradient of potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. Under these conditions, the membrane-bound casein kinase activity is almost completely recovered into a quite stable preparation, free of histone kinase activity. In contrast, it undergoes a dramatic loss of activity when the extraction and the subsequent phosphocellulose chromatography are carried out with buffers which do not contain Triton X-100. Isolated spectrin, the most abundant membrane protein, is phosphorylated, in the presence of [gamma-32P]ATP, only by casein kinase while histone kinase is ineffective. Only the smaller subunit (band II) of isolated spectrin (and not the larger one (band I) is involved in such a phosphorylation process, as in the endogenous phosphorylation occurring in intact erythrocytes.

Stimulation of a histone H4 protein kinase in Triton X-100 lysates of rabbit peritoneal neutrophils pretreated with chemotactic factors: effect of leukotriene B4 and cytochalasin B.

The effects of leukotriene B4 on the stimulation of a histone H4 kinase activity in rabbit peritoneal neutrophils have been studied. LTB4 activated the kinase within 10 sec with the increasing activity decaying to near basal level after 20 sec of stimulation. The effect was not inhibited by removal of extracellular calcium or loading the cells with quin-2/AM. Pretreatment of the cells with cytochalasin B greatly enhanced and prolonged the activation of the kinase. Pretreatment of the cells with LTB4 or fMet-Leu-Phe inhibited the effect of the later addition of LTB4. Since similar characteristics of LTB4 and cytochalasin B have also been observed in LTB4-stimulated responses such as secretion and superoxide generation, the results suggest a potential role of H4 kinase in LTB4-stimulated responses.

[Human leukemic-cell protein-kinases. 1 - Non-granulocytic leukemias (author's transl)]. / Protéine-kinases des cellules de leucémies humaines. I. Leucémies non granulocytaires.

The study of human leukemic-cell protein-kinases is justified by two types of arguments: on one hand, abnormal protein-kinases have been found in various malignancies, and on the other the products of virus transforming genes have repeatedly been identified as protein-kinases. Using cytosolic and particulate extracts of normal human lymphocytes we measured protein-kinase activities, then following partial purification by DEAE and phosphocellulose chromatography the isoenzymes of cyclic AMP dependent and independent protein-kinases and casein-kinases were determined in order to establish a profile (12 normal subjects). The same methodology was applied to the lymphocytes of 5 patients with chronic lymphocytic leukemia (CLL) and 3 patients with acute lymphoblastic leukemia (ALL). Compared to normal lymphocytes, the specific activity of cytosolic and particulate extracts from the leukemics was higher for histone and casein-kinases, following elimination of an inhibitory activity present in the crude extracts. Studies of the isoenzymes showed, in some individuals, the presence in both cytosolic and particulate extracts of two important cyclic AMP dependent histone-kinase activities, which were very low or absent in normal lymphocyte extracts. In the particulate extracts we found a constant increase in casein-kinase activities concerning essentially one of the two isoenzymes present. Also, the ratio of the different isoenzymes separated by chromatography was considerably modified with regard to both histone and casein-kinases. These quantitative and qualitative abnormalities were present in some CLL cells and in non-granulocytic acute leukemic cells. They resembled the modification reported during normal lymphocyte stimulation by phytohemagglutinin, and also seemed to reflect the intensity of cellular replication and to some degree the progression of the disease.

Protein kinase activities in mammalian blood fluid.

Two protein kinase activities, one specific for phosvitin and another specific for histone, were detected in serum and plasma of calf as well as of human blood after precipitation with ammonium sulfate (40%) and chromatography on DEAE-Sephacel. The enzymes were separated by chromatography on phosphocellulose. The histone kinase is not related to the cyclic AMP-dependent protein kinase; it may derive at least partly from damaged cells. The phosvitin kinase activity carries characteristics of the so called casein kinase type II similar to that present at the surface of cells including blood cells.

Enzyme markers for the presence of circulating interferon: 2-5A synthetase in blood lymphocytes and protein kinase in platelet-rich plasma.

The level of 2-5A synthetase in extracts of peripheral blood lymphocytes and a specific protein kinase activity in platelet-rich plasma were measured in normal individuals and in patients suffering from viral or bacterial infections. The level of these enzymes was tested at different times during the disease. The level of 2-5A synthetase and the protein kinase activity was enhanced by several-fold during viral and bacterial infections and decreased during the course of the disease in parallel with clinical ameliorations and reversal of clinical symptoms. Among the different types of infections studied, higher levels of these enzymes were observed during viral than bacterial infections. Our results emphasize the use of these enzymes as markers to evaluate the state of the disease and recovery. Furthermore, they provide evidence for the production of interferon during different types of infection.

Mechanism of translational control by hemin in reticulocyte lysates.

The formation of translational inhibitor (active eIF-2 kinase) from proinhibitor (inactive eIF-2 kinase) in reticulocyte lysates, known to be controlled by hemin, can, as we recently reported, be induced by 3':5'-cyclic AMP(cAMP)-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) or its catalytic subunit. We find that in crude preparations from rabbit reticulocyte lysates, hemin inhibits the conversion of proinhibitor to inhibitor catalyzed by endogenous cAMP-dependent protein kinase upon addition of cAMP, but not that caused by the addition of free protein kinase catalytic subunit. Hemin prevents the binding of cAMP to the regulatory subunit of cAMP-dependent protein kinase and blocks the cAMP-induced dissociation of regulatory and catalytic subunits of the enzyme whereby the enzyme is inactivated. The mechanism by which hemin prevents the formation of the inhibitor and maintains protein synthesis in reticulocyte lysates is thus explained.

Characterization of the protein kinase activities of human platelet supernatant and particulate fractions.

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.