Mar 12, 1985 - ation of an endogenous Mr 69,000-72,000 protein in its phos- ... 32P]ATP results in the phosphorylation of p68 and added sub- strates, calf ...
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 4341-4345, July 1985 Biochemistry
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 (eukaryotic initiation factor 2)
ANNE G. LAURENT, BERNARD KRUST, JULIEN GALABRU, JOSETTE SVAB, AND ARA G. HOVANESSIAN Unitd d'Oncologie Virale, Institut Pasteur, 75724 Paris Cddex 15, France
Communicated by Pierre R. Aigrain, March 12, 1985
ABSTRACT 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 IgGl) 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 [Y32P]ATP results in the phosphorylation of p68 and added substrates, calf thymus histone, and eukaryotic initiation factor 2. Immune complex preparations from [35Sjmethionine-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 KCI 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 a interferon, with maximal synthesis between 6 to 9 hr after the addition of interferon. Actinomycin D blocks this induction.
ited by heparin (2). It phosphorylates p65 and p68 by their serine and threonine residues (7, 8). The role of such protein kinase activity is considered to be the phosphorylation of eIF2, thus mediating inhibition of the initiation of protein synthesis in cell-free systems (1, 2, 9-11). The phosphorylation of p65 and p68 has been reported in interferon-treated cells during virus infection (12, 13), but the significance of this and its correlation with the phosphorylation of eIF2 remains to be clarified. It has been suggested that p65 and p68 are substrates of the ds RNA-dependent protein kinase rather than being the kinase themselves (14). Accordingly, we have shown recently that phosphorylation of p65 and p68 in enzyme fractions from control mouse and human cells is enhanced by mixing with extracts from interferon-treated heterologous cells. These proteins, therefore, may serve as suitable substrates for a heterologous kinase (8). Separation of the protein kinase from p65 and p68 has not been achieved by conventional purification methods (3-5, 14-16). This is probably due to the fact that in all of these purification attempts, the presence of p65 and p68 was assayed by the protein kinase activity-i.e., by the phosphorylation of these proteins since their concentration is too low to be detectable even after silver nitrate staining (unpublished results). Here, we describe the preparation of monoclonal antibodies against the phosphoprotein in human cells. The Mr of the [35S]methionine-labeled protein is 68,000, but after phosphorylation it is increased to Mr 70,000. This phosphate-saturated [35S]methionine-labeled protein has an isoelectric point (pl) that is identical with that of the 32P-labeled protein. In view of the molecular weight of this phosphoprotein in its native state, we will refer to it as p68 rather than as p69 or p72, which are currently employed in the literature (2, 8). The monoclonal antibodies against p68 could be used to detect the protein as such by immunoprecipitation of [35S]methionine-labeled extracts or by electrophoretic transfer blot analysis. In addition, they could be used to assay the protein kinase activity after immunoprecipitation of p68.
A protein kinase activity is enhanced in mouse and human cells following treatment with interferon (1, 2). This kinase activity is manifested by the phosphorylation of (i) an endogenous Mr 65,000-67,000 protein (p65) in mouse cells or a Mr 68,000-72,000 protein (p68) in human cells; (it) an endogenous or exogenous Mr 35,000 protein that is the a subunit of protein eukaryotic initiation factor 2 (eIF2); and (iii) added histones (HIIA). In crude cell extracts or partially purified kinase fractions, the phosphorylation of these three proteins (p65 or p68, eIF2, and histones) is enhanced considerably by the presence of double-stranded RNA (ds RNA) (1-6). However, in highly purified enzyme preparations, the protein kinase activity might become independent of ds RNA (3). The protein kinase activity is independent of cyclic AMP or cyclic GMP, is markedly stimulated by Mn2+, and is not inhib-
MATERIALS AND METHODS Materials. [y-32P]ATP and [35S]methionine were supplied by Amersham. Sepharose 4B and protein A-Sepharose CL4B were from Pharmacia. Poly(I)*poly(C)-Sepharose and poly(A)-poly(U)-Sepharose were prepared as described (17). Calf thymus histone HIIA was from Sigma. Human leukocyte a interferon (108 NIH units/mg of protein) was purified in the laboratory (18). Heterologous ds RNA-dependent protein kinase was prepared from interferon-treated mouse L929 cells as described (3, 19). Abbreviations: ds RNA, double-stranded RNA; p68 kinase, ds RNA-dependent protein kinase activity that is manifested by the phosphorylation of an endogenous Mr 68,000 protein in human cells; eIF2, eukaryotic initiation factor 2; NP-40, Nonidet P-40.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Cell Culture and Extracts. Human Daudi cells were grown in suspension in RPMI 1640 medium (GIBCO) containing 0.01 mM 2-mercaptoethanol and 10% fetal calf serum. Treatment of cells with a interferon was at 500 units/ml for 18-22 hr. For the preparation of cell extracts, Daudi cells were washed with phosphate-buffered saline before addition of lysis buffer containing 10 mM Hepes (pH 7.6), 10 mM KCl, 2 mM magnesium acetate, 7 mM 2-mercaptoethanol, 0.5% nonionic detergent Nonidet P-40 (NP-40) (Sigma), and aprotinin at 100 units/ml (Zymofren, Specia). Cell extracts were centrifuged at 12,000 x g and the supernatants were stored at -800C. Immunization of Mice. Five BALB/c mice were injected intraperitoneally, six times at 2-wk intervals, with the protein kinase system (p68 kinase) attached to poly(A)-poly(U)Sepharose (17). Material in each injection was equivalent to that from 2 x 108 Daudi cells treated with interferon. One mouse was found to produce antibodies after five injections. These antibodies were tested by an immunoprecipitation assay in which the p68 kinase is precipitated and then incubated with ATP to phosphorylate p68. Protein Kinase Assay by Immunoprecipitation. The protein kinase activity that is associated in immune complex preparations bound to protein A-Sepharose was assayed by the phosphorylation of endogenous p68 and by the phosphorylation of exogenous substrate, calf thymus histone (20). The immunoprecipitation was carried out in buffer A [10 mM Tris*HCl, pH 7.6/100 mM KCi/2.5 mM magnesium acetate/ aprotinin at 100 units/ml/heparin at 5 units/ml/20% glycerol
(vol/vol)]. Fusion Procedure. Splenocytes from the mouse producing anti-p68 kinase were fused in the presence of 50% polyethylene glycol 1500 (Roth, Karlsruhe, FRG) with x63/Ag8.653 myeloma cells. The fused cells were plated in 24-well plates (Costar) and cultured as described (21). Culture supernatants were assayed for the production of anti-p68 kinase antibodies for the immunoprecipitation assay. Culture supernatants of x63 myeloma cells and anti-p68 kinase polyclonal antibodies (20) were used as negative and positive controls, respectively. Cultures producing specific monoclonal antibodies were immediately subcloned by limiting dilution and injected into pristane-primed mice for production of ascitic fluid. Purification of Immunoglobulins. Immunoglobulins were purified from ascitic fluid as follows. Lipid was partially removed by filtration through a cotton bed and immunoglobulins were precipitated with 50% saturated (NH4)2SO4. The precipitate was dissolved in 10 mM phosphate buffer (pH 8.0) and further purified on a DEAE-cellulose column (DE 52, Whatman) by elution at 40 mM phosphate buffer (pH 8.0). Immunoglobulins purified in this manner were judged to be about 90% pure. Isotyping of monoclonal antibodies was determined by Ouchterlony's immunodiffusion test (21). Electrophoretic Transfer Immunoblot Analysis. Proteins in preparations from control and interferon-treated cells were subjected to electrophoresis in NaDodSO4/polyacrylamide slab gels before being electrophoretically transferred to 0.2I.m nitrocellulose sheets (Schleicher & Schull) in electrode buffer [20 mM Tris base/150 mM glycine/20o methanol (vol/vol)] as described (22). The immobilized antigens were visualized by incubation with the monoclonal antibody and the subsequent binding of 1251-labeled goat anti-mouse immunoglobulins (New England Nuclear, 2-10 ,uCi/pg; 1 Ci = 37 GBq). Radioactive Labeling of Cells. Daudi cells in the absence or presence of a interferon (500 units/ml) were incubated (17 hr, 370C) in RPMI 1640 culture medium containing only 10%o of the normal level of methionine (1.5 pg/ml) and [35S]methionine (12.5 ,uCi/ml; 400-500 Ci/mmol). Cell extracts were prepared by lysis in NP-40 buffer. Cell extracts were immunoprecipitated in the presence of protein A-Sepharose and
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the immune complex preparations were washed either with buffer A (under similar conditions as that for the immune complex protein kinase assay) or with buffer RIPA [10 mM Tris HCl, pH 7.6/150 mM NaCl/1 mM EDTA/1% Triton X100 (vol/vol)/0.1% NaDodSO4 (wt/vol)/10% glycerol (vol/ vol)].
RESULTS AND DISCUSSION It is still not clear whether phosphorylation of p68 in kinase preparations from interferon-treated human cells is mediated by a distinct protein kinase or is due to an autophosphorylation process (2, 8, 14). To rule out any misinterpretation and to facilitate the presentation of our results, we propose to consider that this system is composed of at least two components: a ds RNA-dependent protein kinase (p68 kinase) and its substrate p68. Isolation and Identification of Monoclonal Antibodies Against p68. The purpose of this work was to prepare monoclonal antibodies against the p68 kinase (i.e., the ds RNAdependent protein kinase) and its substrate (p68). The presence of antibodies in the serum of immunized mice and in hybridoma culture supernatants was tested by a technique of immunoprecipitation that we have described previously by using polyclonal antibodies against a preparation of protein kinase (20). Briefly, extracts from interferon-treated Daudi cells were incubated with the antibody preparation and protein A-Sepharose. Immune complexes bound to the solid support were first washed and then incubated with [ 32P]ATP to phosphorylate the endogenous p68. In addition to this, we assayed the phosphorylation of an exogenous substrate, calf thymus histone. By this latter phosphorylation, we expected to detect the protein kinase in the absence of its endogenous substrate. Five mice were injected six times with a purified fraction containing the protein kinase system. Such preparations consisted of poly(A)-poly(U)-Sepharose-bound proteins from interferon-treated Daudi cells (8, 17). After the fifth immunization, one mouse was shown to produce specific antibodies detectable by the immunoprecipitation technique. Hybridoma cell lines were obtained by fusing splenocytes from the immune mouse with x68/Ag8 myeloma cells. Culture supernatants were tested for the presence of specific antibodies by the immunoprecipitation assay for the protein kinase activity (Materials and Methods). Five hybridomas were found to produce specific antibodies that could be used in an immunoprecipitation assay for protein kinase activity-i.e., by the phosphorylation of p68 and added histones. These hybridomas were further subcloned and screened for antibody production. The culture media from these clones (total of 255 clones) were first tested by the immunoprecipitation assay for the protein kinase activity. The selected clones were further studied by electrophoretic transfer immunoblot analysis and by immunoprecipitation of [35S]methionine-labeled proteins from interferon-treated Daudi cells (see below). Fig. 1 shows a typical test for protein kinase activity after immunoprecipitation of Daudi cell extracts with culture medium from a group of subclones. In all of these assays, the p68 phosphorylation was correlated with the phosphorylation of histones. Several subclones (71/5, 71/6, 71/8, 71/10, 71/12, 21/9, and 21/10) were selected and the antibody produced by each clone was characterized by polyacrylamide gel electrophoresis in one and two dimensions (21). All of the selected clones were producers of IgG1 class antibodies. The clone referred to as HL 71/10 was used in the experiments discussed here. Immune complexes prepared with the monoclonal antibody and extracts from control and interferon-treated human Daudi, HeLa, and W138 simian virus 40 cells have p68 ki-
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FIG. 1. Phosphorylation of p68 and added histones in immune complex preparations. Extracts (1 mg of protein) from interferontreated Daudi cells were incubated with 100 01 of culture medium from different subclones (samples 1-19) and the immune complexes recovered by protein A-Sepharose were assayed for the phosphorylation of p68 and added histones. The 32P-labeled proteins (indicated by the arrows on the right) were analyzed by polyacrylamide gel (12.5%) electrophoresis. An autoradiograph of a stained and dried gel is shown. The numbers on the left indicate protein molecular weight markers (shown as Mr x 10-3): bovine serum albumin, Mr 69,000; ovalbumin, Mr 46,000; carbonic anhydrase, Mr 30,000. nase activity. These monoclonal antibodies do not precipitate a similar protein kinase activity in interferon-treated mouse cells. Thus, they are specific of the antigen from hu-
man cells. The phosphorylation of p68 in immune complexes from control and interferon-treated Daudi cells is dependent on ds RNA. In addition to the ds RNA, heparin at 5 units/ml was found to be an efficient activator of the protein kinase activity. Therefore, for convenience, all of the phosphorylation reactions were carried out in buffer A (which contains heparin). Electropnoretic Transfer Immunoblot Analysis. Electrophoretic transfer immunoblot hybridization was performed to identify the protein that is recognized by the monoclonal antibody HL 71/10. In these experiments, we used extracts from both control and interferon-treated cells since a specific binding of the monoclonal antibody to its denatured antigen would be increased in extracts from cells treated with interferon. For this reason, poly(A)-poly(U)-Sepharose-bound enzyme fractions from control and interferon-treated Daudi cells were analyzed by electrophoresis in polyacrylamide gels and the antigen was then identified by the electrophoretic transfer blotting technique (Fig. 2). The monoclonal antibody was bound specifically to a Mr 68,000 protein in samples from control and interferon-treated cells. A higher level of antibody was bound to the protein from interferon-treated cells than that from control cells. The level of this antigen therefore was enhanced by interferon. A similar result was obtained by using crude NP-40 extracts from control and interferon-treated cells (Fig. 3). The p68 that was present in control cells (Fig. 3, lane 1) was enhanced after treatment with interferon (Fig. 3, lanes 2-6). It should be noted that when crude extracts were used for the electrophoretic transfer blot analysis, then several proteins were labeled unspecifically. These proteins were also labeled when control culture medium was used (data not shown). This is probably due to the fact that all of these proteins are present at concentrations at least 1000-fold higher than p68, which cannot be detected in these samples even after silver nitrate stain-
ing.
Immunoprecipitation of [35S]Methionine-Labeled p68. [35S]Methionine-labeled extracts from control and interferon-treated Daudi cells were immunoprecipitated by the monoclonal antibody HL 71/10 in the presence of protein ASepharose, under experimental conditions similar to those for the protein kinase assay described in the legend to Fig. 1. A Mr 68,000 protein was specifically recovered in immune
FIG. 2. Electrophoretic transfer immunoblot analysis of p68. Extracts from control (lanes C) and interferon-treated (IFN) Daudi cells (2 x 106 cells) were purified in poly(A)-poly(U)-Sepharose (17). The samples then were electrophoresed in NaDodSO4/polyacrylamide slab gels (10%) and processed for electrophoretic transfer blot hybridization. The immobilized antigens were visualized by incubation with culture medium from clone HL 71/10 (Ig+ section) and the subsequent binding of 1251I-labeled goat anti-mouse IgG. The Igsection shows the results obtained by using medium from hybridoma cell cultures that were negative for the p68 kinase activity in an immunoprecipitation assay. An autoradiograph is shown. The arrow on the right indicates the position of p68. On the left, the positions of molecular weight markers are indicated (shown as Mr x 10-3).
complex preparations from both control and interferon-treated cells. The level of this [35S]methionine-labeled p68 was enhanced in cells treated with interferon (Fig. 4A, lanes 2 and 4). In addition to p68, several other proteins were found in immune complex preparations. However, the precipitation of these proteins was not specific since in the absence of the monoclonal antibody they were also bound to protein ASepharose (Fig. 4A, lanes 1 and 3). Such preparations (in the absence of antibody) showed no protein kinase activity on endogenous or exogenous substrates. Unspecifically bound proteins in immune complex preparations were eliminated by washing in RIPA buffer (Fig. 4B, lanes 2 and 4). By this procedure the protein kinase activity was lost-i.e., addition of [y-32P]ATP no longer resulted in the phosphorylation of p68 or phosphorylation of added histones. These results suggest that p68 might be the substrate of the ds RNA-dependent protein kinase. The identity of this protein kinase remains to be studied. In the experiment illustrated in Fig. 4, the protein concentration of the kinase might have been too 1 2 3 4 5 6
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FIG. 3. Electrophoretic transfer blot analysis of p68 in crude cell extracts. NP-40 extracts from control (lane 1) and interferon-treated (lanes 2-6) Daudi cells (106 cells) were analyzed by polyacrylamide gel (10%o) electrophoresis and then were processed by electrophoretic transfer blot hybridization using HL 71/10 monoclonal antibodies. Treatment of cells with 1 (lane 2), 10 (lane 3), 102 (lane 4), 103 (lane 5), and 104 (lane 6) NIH units of a interferon per ml was for 18 hr. An autoradiograph is shown. The arrow on the right indicates the position of p68. On the left are the molecular weight markers (shown as Mr X 10 3). The labeling of proteins other than p68 was unspecific since they were also observed in the absence of the monoclonal
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FIG. 4. Immunoprecipitation of [35S]methionine-labeled p68. [35S]Methionine-labeled extracts from control (lanes 1 and 2) and interferon-treated (lanes 3 and 4) Daudi cells (2.5 x 10O cells) were incubated with 20 Ag of control mouse immunoglobulins (lanes 1 and 3) or 20 pg of HL 71/10 monoclonal antibody (lanes 2 and 4) in the presence of protein A-Sepharose (100 /4). (A) Immune complex preparations were washed with buffer A. (B) Immune complex preparations were washed with RIPA buffer. All of the samples were analyzed by polyacrylainide gel (10%) electrophoresis. A fluorograph is shown. Lanes m show the electrophoretic profile of 14Clabeled protein markers (Amersham) (shown as Mr 10-3): myosin, Mr 200,000; phosphorylase b, Mr 92,500; bovine serum albumin, Mr 69,000; ovalbumin, Mr 46,600; carbonic anhydrase, Mr 30,000. The arrow on the right indicates the position of [35S]methionine-labeled p68. X
low to be detectable by labeling of cells with [35S]methionine. The protein kinase probably exists in a complexed form bound to its substrate (p68) and thus it coprecipitates with p68 during the immunoprecipitation assay. Addition of detergent-containing buffer leads to the separation of the protein kinase from its substrate that remains attached to the monoclonal antibody. The RIPA-washed p68 can become phosphorylated by a heterologous ds RNA-dependent protein kinase preparation from interferon-treated mouse L-929 cells. These observations are in favor of p68 being the substrate.
Other possibilities for the loss of kinase activity might include inactivation of the protein kinase or denaturation of the catalytic subunit in p68 (in the case of autophosphorylation). However, this is most unlikely since the protein kinase activity was not modified in partially purified enzyme fractions (3) dialyzed against RIPA buffer. Furthermore, similar results were obtained by using another buffer that contains high concentrations of salts [10 mM Tris HCl, pH 7.6/1 M KCI/0.5 M magnesium acetate/10%o glycerol (vol/vol)]. Immune complex preparations washed with high salt buffer retained p68 but the protein kinase activity became undetectable (data not shown, similar to Fig. 4). To confirm that [35S]methionine-labeled p68 is the protein that becomes phosphorylated, we investigated its molecular weight and pI after saturation with phosphate (8). Immune complexes were prepared by using the monoclonal antibody and [35S]methionine-labeled extracts from interferon-treated Daudi cells. These samples were washed under experimental conditions similar to those for the protein kinase assay and then were incubated in the absence or presence of ATP (0.1 mM). After saturation with phosphate, the samples were washed with RIPA buffer before analysis by polyacrylamide gel electrophoresis. It is apparent in Fig. SA that the molecular weight of p68 is increased in the immune complex preparation incubated with ATP. This is in accord with previous results that indicated that the molecular weight of a highly phosphate-saturated protein is increased by 1500-2000 compared to a partially saturated protein (8). Further evidence to confirm the identity of [35S]methionine-labeled p68 was provided by two-dimensional gel electrophoretic analysis. In the
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FIG. 5. Molecular weight and pI of p68. (A) [35S]Methionine-labeled extracts from interferon-treated Daudi cells (10 x 106 cells) were immunoprecipitated by the HL 71/10 monoclonal antibody (25 ,ug) in the presence of protein A-Sepharose. The immune complex preparations were washed with buffer A and incubated (45 min, 30°C) in the absence (lane 0) or presence (lane 0.1) of 0.1 mM ATP. Both preparations then were washed with RIPA buffer and analyzed by polyacrylamide gel (7.5%) electrophoresis. A section of the fluorograph in the region of bovine serum albumin is shown. Lanes M show the band of 14C-labeled bovine serum albumin. The phosphatesaturated p68 is revealed as a wider band compared to the unsaturated protein. (B) Two-dimensional gel electrophoretic analysis of [35S]methionine-labeled p68 after phosphorylation with unlabeled ATP ([35S]Met.p68.PO4) or [y_32P]ATP ([35S]Met.p68.32P04). The immune complex preparations containing [35S]methionine-labeled p68 were washed in buffer A and were incubated (45 min, 30°C) with 0.1 mM ATP in the absence or presence of 10 jA of [y_32P]ATP (1 ,uCi/A; 3000 Ci/mmol). Both samples were analyzed by two-dimensional gel electrophoresis as described (8). The pH gradient obtained by isoelectric focusing (first dimension) was between 8.5 and 7.2. Sections of the fluorograph and the autoradiograph are shown.
absence of incubation with ATP, [35S]methionine-labeled p68 was observed as a very faint band showing the existence of several subspecies with p1 values ranging between 8.2 and 7.4 (data not shown). However, after saturation with phosphate, the subspecies at the acidic pI values (pH between 7.8 and 7.4) became enriched (Fig. SB). These pI values coincided with the pI values of the 12P-labeled protein. Induction and Synthesis of p68 in Daudi Cells. Fig. 6 shows that the level of [35S]methionine-labeled p68 is highly correlated with its degree of phosphorylation. A significant enhancement of the protein kinase activity and [35S]methionine-labeled p68 was observed in cells treated with different doses of a interferon, with a maximal enhancement at 100 units/ml. Higher doses of interferon resulted in a lower level of enhancement, which is probably due to the anticellular action of interferon in these cells (23). Therefore, higher levels of the substrate were correlated with enhanced levels of protein kinase activity. These results suggest that immunoprecipitation of p68 leads to coprecipitation of the protein kinase, which might bind specifically to its substrate. Therefore, the level of protein kinase activity in immune complex preparations would depend on the presence of p68. Synthesis of p68 occurs within a few hours after addition of interferon, with a maximal rate of synthesis between 6 and 9 hr (Fig. 7; Pulse). Addition of actinomycin D at different times (0.5, 5, 8, 12 hr) after interferon inhibits induction of p68 (Fig. 7; Kinetics). These results suggest that although maximal synthesis of this protein occurs 6-9 hr after addition
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FIG. 6. Levels of [355]methionine-labeled p68 and p68 kinase in cells treated with different concentrations of interferon (IFN). The level of [355]methionine-labeled p68 (ordinate on the left; *) and the level of p68 kinase (ordinate on the right; u) were measured in immune complex preparations from control (no IFN) and a interferontreated (IFN: 1, 10, 102, 103, and 104 NIH units/ml) Daudi cells. Immune complex preparations containing [35S]methionine-labeled proteins were washed with RIPA buffer. The different samples supplemented with bovine serum albumin (10 ,ug) were analyzed by polyacrylamide gel (10%) electrophoresis. The gels were stained and pieces of gels containing [35S]methionine or 32P04-labeled p68 were cut out according to the position of albumin and were assayed for radioactivity by liquid scintillation spectroscopy (ordinates). Extracts from 2.5 x 106 and 5 x 106 Daudi cells were used for the assay of [35S]methionine-labeled p68 and 32PO4-labeled p68, respectively.
of interferon, its induction continues even at 12 hr. The Use of Monoclonal Antibodies Specific for p68. The mechanisms involved in the ds RNA-dependent protein kinase system are still not well defined. This protein kinase system in crude extracts from human cells is manifested by Pulse 10
Kinetics
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Time, hr FIG. 7. Synthesis of p68 in Daudi cells treated with interferon. The level of [355]methionine-labeled p68 (ordinates) was measured in immune complex preparations as described in the legend to Fig. 6. The abscissa shows the time after addition of a interferon (300 NIH units/ml). Either these interferon-treated cells were pulse labeled at 3-hr intervals (Pulse) or the kinetics of the synthesis of p68 was studied (Kinetics) in the absence (*-o) or presence (o---o) of actinomycin D (2.5 ,ug/ml) added at times 0.5, 5, 8, and 12 hr (indicated by the arrows). Extracts from 2.5 x 106 Daudi cells were used for each individual point.
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the phosphorylation of p68 and also in some extracts by the phosphorylation of Mr 35,000 protein (p35), the smallest subunit of protein initiation factor eIF2. The precise relation existing between the ds RNA activation process and phosphorylation of both p68 and p35 remains to be clarified. Previously, we have suggested that p68 is the substrate of ds RNA-dependent protein kinase (8). The results presented here confirm this suggestion. The protein kinase and p68 might exist in a complexed form that could be dissociated by nonionic and ionic detergents or by high concentrations of salt. Therefore, it will be possible to separate the protein kinase from p68 and to isolate each component. The availability of monoclonal antibodies specific for human p68 should prove to be of great use for the purification and identification of this protein. Once purified preparations of p68 are obtained, then the identification of the protein kinase and the characterization of the ds RNA dependence might be defined. This work was supported in part by a grant from the Association pour la Recherche sur le Cancer, Villejuif, France. 1. Lebleu, B. & Content, J. (1982) in Interferon 1982, ed. Gresser, I. (Academic, London), pp. 47-94. 2. Galabru, J. & Hovanessian, A. G. (1984) Bull. Inst. Pasteur (Paris) 82, 283-334. 3. Hovanessian, A. G. & Kerr, I. M. (1979) Eur. J. Biochem. 93, 515-526. 4. Sen, G. C., Taira, H. & Lengyel, P. (1978) J. Biol. Chem. 253, 5915-5921. 5. Kimchi, A., Zilberstein, A., Schmidt, A., Shulman, L. & Revel, M. (1979) J. Biol. Chem. 254, 9846-9853. 6. Lasky, S. R., Jacobs, B. L. & Samuel, C. E. (1982) J. Biol. Chem. 257, 11087-11093. 7. Samuel, C. (1979) Proc. Natl. Acad. Sci. USA 76, 515-519. 8. Krust, B., Galabru, J. & Hovanessian, A. G. (1984) J. Biol. Chem. 259, 8494-8498. 9. Farrel, P. J., Balkow, K., Hunt, T., Jackson, J. & Trachsel, H. (1977) Cell 11, 187-200. 10. Zilberstein, A., Kimchi, A., Schmidt, A. & Revel, M. (1978) Proc. Natl. Acad. Sci. USA 75, 4734-4738. 11. De Benedetti, A. & Baglioni, C. (1984) Nature (London) 311, 79-81. 12. Gupta, S. L., Holmes, S. L. & Mehra, L. L. (1982) Virology 120, 495-499. 13. Nilsen, T. W., Maroney, P. A. & Baglioni, C. (1982) J. Biol. Chem. 257, 14593-145%. 14. Revel, M. (1979) in Interferon 1979, ed. Gresser, I. (Academic, London), pp. 102-163. 15. Ohtsuki, K. & Baron, S. (1979) J. Biochem. 85, 1495-1502. 16. Ohtsuki, K., Nakamura, M., Koike, T., Ishida, N. & Baron, S. (1980) Nature (London) 287, 65-67. 17. Hovanessian, A. G., Riviere, Y. & Krust, B. (1983) Anal. Biochem. 129, 349-356. 18. Meurs, E., Rougeot, C., Svab, J., Laurent, A. G., Hovanessian, A. G., Robert, N., Gruest, J., Montagnier, L. & Dray, F. (1982) Infect. Immun. 47, 919-926. 19. Galabru, J., Krust, B. & Hovanessian, A. G. (1984) J. Interferon Res. 4, 469-480. 20. Laurent, A. G., Krust, B., Svab, J. & Hovanessian, A. G. (1984) Biochem. Biophys. Res. Commun. 125, 1-7. 21. Laurent, A. G., Gruest, J., Krust, B. & Montagnier, L. (1982)
Hybridoma 1, 313-321. 22. Burnette, W. N. (1981) Anal. Biochem. 112, 195-230. 23. Stewart, W. E., II, Gresser, I., Tovey, M. G., Bandu, M. T. & Legoff, S. (1976) Nature (London) 262, 300-303.
