Immunology Letters
30 (1991)297-300
Elsevier IMLET 01674
A protein kinase C inhibitory activity is present in the human T lymphoblast cell line Jurkat S i m o n e Columelli, A n n e - M a r i e H o u l l i e r a n d J e a n - L o u i s P 6 r i g n o n Laboratoire de Biochimie, INSERM U75, Facult~ de M~decine Necker-Enfants Malades, Paris, France
(Received2 May 1991; revision received28 June 1991;accepted 1 July 1991)
1. Summary
The human lymphoblast cell line Jurkat is widely used as a model system for studying signal transduction pathways during lymphocyte activation. We report the presence of a potent endogenous inhibitor of protein kinase C (PKC) in the cytosolic fraction of Jurkat cells. This inhibitor is not diffusible and is thermolabile; it is assumed to be a protein. It was separated from PKC by ion-exchange chromatography on DEAE-cellulose. The inhibitory activity was partially reversed by increasing the concentration of the PKC substrate; increasing that of PKC activators (calcium and phospholipids) was without effect. PKC activity was inhibited by more than 90% in the crude cytosolic fraction but the inhibition could be completely reversed by diluting the cell extract. This inhibitory activity could not be detected in the cytosol from normal lymphocytes or from lymphoblasts from leukemic patients. 2. Introduction
The fact that the activation of protein kinase C (PKC) is involved in T lymphocyte activation is well established, although its precise role with respect to that of tyrosine kinases, and the nature of the specific target proteins ~phosPhorylated, are largely undetermined [1]. As part of a study of T lymphocyte Key words." Jurkat cell line; Protein kinase C inhibition; T
lym-
phoblast Correspondence to." Jean-Louis P6rignon, Laboratoire de Biochimie, INSERM U75, Facult6 de M6decine Necker-Enfants Malades, 156, rue de Vaugirard, 75730 Paris Cedex 15, France.
activation, we assayed PKC activity in the human T lymphoblast cell line Jurkat. Jurkat is a T helper (CD4 +) cell line derived from a patient with lymphoblastic leukemia, which can be activated through the T cell receptor/CD3 complex, and is widely used as a model system for the study of T cell activation. Here we report the presence of a PKC inhibitory activity in the cytosolic fraction of Jurkat cell and briefly describe some of its properties. 3. Materials and Methods
The Jurkat cell line was kept in continuous culture in RPMI-1640 medium as previously described [2]. Peripheral blood mononuclear cells from normal controls and lymphoblasts from patients with acute lymphoblastic leukemia (peripheral blood or bone marrow aspirates obtained before chemotherapy) were isolated by gradient centrifugation on FicollHypaque (Pharmacia, Uppsala, Sweden). Because of the large number of cells ( > 10s) needed for the detection of the PKC inhibitor, 2 pools of normal peripheral blood mononuclear cells and one pool of lymphoblasts were made. Lysine-rich histone I (IIIS), 1,2-diolein, brain extract type III (containing 8 0 - 8 5 % phosphatidylserine), aprotinin, pepstatin A, phenylmethylsulfonyl fluoride (PMSF), soybean trypsin inhibitor, 12-O-tetradecanoylphorbo! 13acetate (TPA), EDTA and EGTA were from Sigma (Saint-Louis, MO). Leupeptin and DTT were from Boehringer (Mannheim, FRG). [7-32p]ATP (110 TBq mmo1-1) was from the Radionuclear Center (Amersham Int., Amersham, GB). DE-52 cellulose and Sephacryl $300 were from Pharmacia (Uppsala, Sweden), and P81 chromatography paper from
0165 - 2478 / 91 / $ 3.50 © 1991 ElsevierSciencePublishers B.V.All rights reserved
297
Whatman (Maidstone, U.K.). Buffer A consisted of 20 mM Hepes/2 mM EDTA/10 mM EGTA/0.3 M sucrose/2 mM DTT, pH 7.5, supplemented with proteinase inhibitors: 2 mM PMSF/10 ~g/ml soybean trypsin inhibitor/10/~g/ml pepsatin A/10 ~g/ml leupeptin/25 #g/ml aprotinin. Buffer B consisted of 20 mM Hepes/2 mM EDTA/2 mM EGTA/1 mM DTT, pH 7.5/supplemented with 2 mM P M S E Cells were washed twice in cold RPMI-1640 medium. Pellets of about 20x106 cells were frozen and stored in liquid nitrogen until use. The pellets were then rapidly lysed in 50/zl distilled water aspirated through a Hamilton syringe for 20 s. Immediately, 1.0 ml of ice-cold buffer A was added. Cytosol and particulate fractions were prepared by centrifugation at 100000xg for 60 min. The centrifuged membrane pellets were sonicated in 1 ml of buffer A supplemented with 0.5°70 Triton X100, were gently rotated for 30 rain at 4 °C, and were centrifuged at 100000×g for 15 min. PKC and the inhibitory activity were partially purified by ion-exchange chromatography on a DEAEcellulose column (1×2 cm, flow rate 1 ml/min) equilibrated with buffer B. The column was washed with 35 ml of buffer B, then eluted with a linear gradient of NaC1 ( 0 - 0 . 3 M) in 2×10 ml buffer B. For the purification of the inhibitory activity, this linear gradient elution was immediately followed by 20 ml of 0.3 M NaC1 in buffer B. 1-ml fractions were collected. The activity of PKC was determined by measuring the incorporation of 32p from ['y-32p]ATP into histone, by a modification of the method of Castagna et al. [3]. The reaction mixture consisted of 20 mM Hepes, pH 7.5/10 mM MgC12/0.2 mg/ml histone III-S/0.5 mM EGTA/1 mM DTT/10/zM [3'32p]ATP with or without 1.5 mM Ca 2+, phosphatidylserine (20/~g/ml) and 1,2-diolein (2/zg/ml). The reaction was initiated by the addition of 10 #l of enzyme preparation, in a final volume of 60 ~l. Following a 6-min incubation at 30°C, 50-#1 aliquots of the reaction mixture were spotted onto 2 x 2 cm squares of P81 cation-exchange paper. The filters were washed five times in 1% phosphoric acid then counted in 5 ml of Opti-fluor O (Packard, Groningen, The Netherlands) using a Packard-300 liquid scintillation counter. PKC activity was calculated as the difference in histone phosphorylation 298
with or without Ca 2+ and phospholipids. For the assay of the inhibitory activity, the I0/zl of enzyme preparation consisted in a 50/50 mixture of partially purified PKC and of the fraction to be tested. 4. Results
4.1. Effect of dilution on PKC activity Many authors have assayed PKC activity by simplified methods avoiding the time-consuming partial purification of the enzyme by ion exchange chromatography [4, 5]. However, there is a major drawback with the particulate fraction, since the 0.5°70 concentration of Triton X-100 used to extract the enzyme from the membranes completely inhibits the enzyme activity. However, we found in a preliminary study that it was possible to by-pass this difficulty by diluting 1/10 in buffer B the particulate fraction after this step of extraction: indeed, the 0.05% final concentration of Triton X-100 thus obtained does not affect PKC activity. In order to similarly treat both the cytosolic and particulate fractions, we also diluted the cytosolic extract to 1/I0 in buffer B. Surprisingly, we found that the specific activity of PKC in the diluted extract was much higher than that measured in the crude extract; a more detailed study revealed that, in fact, PKC activity markedly increased with increasing dilutions (Fig. 1). We first suspected that this phenomenon might be due to an unknown inhibitor present in the extraction buffer A, inactivated by dilution in buffer B. Since a major difference between buffers A and B is the presence of a cocktail of proteinase inhibitors in buffer A, we repeated the experiment with diluting the cytosolic extract in a modified buffer B, containing all the proteinase inhibitors: the same results were obtained (Fig. 1, inset). The PKC activity was enhanced by dilution even after dialysis of the cytosolic fraction, indicating that the putative inhibitor was not diffusible (Fig. 1). We finally studied a preparation of PKC partially purified by DEAEchromatography of the cytosolic fraction: as shown in Fig. l, the specific activity of this preparation was not enhanced by dilution, which is a further indication of the presence of an inhibitor in the crude cytosolic fraction. The total cytosolic PKC activity recovered from the DEAE-cellulose column was
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30 (1991)297-300
Elsevier IMLET 01674
A protein kinase C inhibitory activity is present in the human T lymphoblast cell line Jurkat S i m o n e Columelli, A n n e - M a r i e H o u l l i e r a n d J e a n - L o u i s P 6 r i g n o n Laboratoire de Biochimie, INSERM U75, Facult~ de M~decine Necker-Enfants Malades, Paris, France
(Received2 May 1991; revision received28 June 1991;accepted 1 July 1991)
1. Summary
The human lymphoblast cell line Jurkat is widely used as a model system for studying signal transduction pathways during lymphocyte activation. We report the presence of a potent endogenous inhibitor of protein kinase C (PKC) in the cytosolic fraction of Jurkat cells. This inhibitor is not diffusible and is thermolabile; it is assumed to be a protein. It was separated from PKC by ion-exchange chromatography on DEAE-cellulose. The inhibitory activity was partially reversed by increasing the concentration of the PKC substrate; increasing that of PKC activators (calcium and phospholipids) was without effect. PKC activity was inhibited by more than 90% in the crude cytosolic fraction but the inhibition could be completely reversed by diluting the cell extract. This inhibitory activity could not be detected in the cytosol from normal lymphocytes or from lymphoblasts from leukemic patients. 2. Introduction
The fact that the activation of protein kinase C (PKC) is involved in T lymphocyte activation is well established, although its precise role with respect to that of tyrosine kinases, and the nature of the specific target proteins ~phosPhorylated, are largely undetermined [1]. As part of a study of T lymphocyte Key words." Jurkat cell line; Protein kinase C inhibition; T
lym-
phoblast Correspondence to." Jean-Louis P6rignon, Laboratoire de Biochimie, INSERM U75, Facult6 de M6decine Necker-Enfants Malades, 156, rue de Vaugirard, 75730 Paris Cedex 15, France.
activation, we assayed PKC activity in the human T lymphoblast cell line Jurkat. Jurkat is a T helper (CD4 +) cell line derived from a patient with lymphoblastic leukemia, which can be activated through the T cell receptor/CD3 complex, and is widely used as a model system for the study of T cell activation. Here we report the presence of a PKC inhibitory activity in the cytosolic fraction of Jurkat cell and briefly describe some of its properties. 3. Materials and Methods
The Jurkat cell line was kept in continuous culture in RPMI-1640 medium as previously described [2]. Peripheral blood mononuclear cells from normal controls and lymphoblasts from patients with acute lymphoblastic leukemia (peripheral blood or bone marrow aspirates obtained before chemotherapy) were isolated by gradient centrifugation on FicollHypaque (Pharmacia, Uppsala, Sweden). Because of the large number of cells ( > 10s) needed for the detection of the PKC inhibitor, 2 pools of normal peripheral blood mononuclear cells and one pool of lymphoblasts were made. Lysine-rich histone I (IIIS), 1,2-diolein, brain extract type III (containing 8 0 - 8 5 % phosphatidylserine), aprotinin, pepstatin A, phenylmethylsulfonyl fluoride (PMSF), soybean trypsin inhibitor, 12-O-tetradecanoylphorbo! 13acetate (TPA), EDTA and EGTA were from Sigma (Saint-Louis, MO). Leupeptin and DTT were from Boehringer (Mannheim, FRG). [7-32p]ATP (110 TBq mmo1-1) was from the Radionuclear Center (Amersham Int., Amersham, GB). DE-52 cellulose and Sephacryl $300 were from Pharmacia (Uppsala, Sweden), and P81 chromatography paper from
0165 - 2478 / 91 / $ 3.50 © 1991 ElsevierSciencePublishers B.V.All rights reserved
297
Whatman (Maidstone, U.K.). Buffer A consisted of 20 mM Hepes/2 mM EDTA/10 mM EGTA/0.3 M sucrose/2 mM DTT, pH 7.5, supplemented with proteinase inhibitors: 2 mM PMSF/10 ~g/ml soybean trypsin inhibitor/10/~g/ml pepsatin A/10 ~g/ml leupeptin/25 #g/ml aprotinin. Buffer B consisted of 20 mM Hepes/2 mM EDTA/2 mM EGTA/1 mM DTT, pH 7.5/supplemented with 2 mM P M S E Cells were washed twice in cold RPMI-1640 medium. Pellets of about 20x106 cells were frozen and stored in liquid nitrogen until use. The pellets were then rapidly lysed in 50/zl distilled water aspirated through a Hamilton syringe for 20 s. Immediately, 1.0 ml of ice-cold buffer A was added. Cytosol and particulate fractions were prepared by centrifugation at 100000xg for 60 min. The centrifuged membrane pellets were sonicated in 1 ml of buffer A supplemented with 0.5°70 Triton X100, were gently rotated for 30 rain at 4 °C, and were centrifuged at 100000×g for 15 min. PKC and the inhibitory activity were partially purified by ion-exchange chromatography on a DEAEcellulose column (1×2 cm, flow rate 1 ml/min) equilibrated with buffer B. The column was washed with 35 ml of buffer B, then eluted with a linear gradient of NaC1 ( 0 - 0 . 3 M) in 2×10 ml buffer B. For the purification of the inhibitory activity, this linear gradient elution was immediately followed by 20 ml of 0.3 M NaC1 in buffer B. 1-ml fractions were collected. The activity of PKC was determined by measuring the incorporation of 32p from ['y-32p]ATP into histone, by a modification of the method of Castagna et al. [3]. The reaction mixture consisted of 20 mM Hepes, pH 7.5/10 mM MgC12/0.2 mg/ml histone III-S/0.5 mM EGTA/1 mM DTT/10/zM [3'32p]ATP with or without 1.5 mM Ca 2+, phosphatidylserine (20/~g/ml) and 1,2-diolein (2/zg/ml). The reaction was initiated by the addition of 10 #l of enzyme preparation, in a final volume of 60 ~l. Following a 6-min incubation at 30°C, 50-#1 aliquots of the reaction mixture were spotted onto 2 x 2 cm squares of P81 cation-exchange paper. The filters were washed five times in 1% phosphoric acid then counted in 5 ml of Opti-fluor O (Packard, Groningen, The Netherlands) using a Packard-300 liquid scintillation counter. PKC activity was calculated as the difference in histone phosphorylation 298
with or without Ca 2+ and phospholipids. For the assay of the inhibitory activity, the I0/zl of enzyme preparation consisted in a 50/50 mixture of partially purified PKC and of the fraction to be tested. 4. Results
4.1. Effect of dilution on PKC activity Many authors have assayed PKC activity by simplified methods avoiding the time-consuming partial purification of the enzyme by ion exchange chromatography [4, 5]. However, there is a major drawback with the particulate fraction, since the 0.5°70 concentration of Triton X-100 used to extract the enzyme from the membranes completely inhibits the enzyme activity. However, we found in a preliminary study that it was possible to by-pass this difficulty by diluting 1/10 in buffer B the particulate fraction after this step of extraction: indeed, the 0.05% final concentration of Triton X-100 thus obtained does not affect PKC activity. In order to similarly treat both the cytosolic and particulate fractions, we also diluted the cytosolic extract to 1/I0 in buffer B. Surprisingly, we found that the specific activity of PKC in the diluted extract was much higher than that measured in the crude extract; a more detailed study revealed that, in fact, PKC activity markedly increased with increasing dilutions (Fig. 1). We first suspected that this phenomenon might be due to an unknown inhibitor present in the extraction buffer A, inactivated by dilution in buffer B. Since a major difference between buffers A and B is the presence of a cocktail of proteinase inhibitors in buffer A, we repeated the experiment with diluting the cytosolic extract in a modified buffer B, containing all the proteinase inhibitors: the same results were obtained (Fig. 1, inset). The PKC activity was enhanced by dilution even after dialysis of the cytosolic fraction, indicating that the putative inhibitor was not diffusible (Fig. 1). We finally studied a preparation of PKC partially purified by DEAEchromatography of the cytosolic fraction: as shown in Fig. l, the specific activity of this preparation was not enhanced by dilution, which is a further indication of the presence of an inhibitor in the crude cytosolic fraction. The total cytosolic PKC activity recovered from the DEAE-cellulose column was
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