A sensitive, non- radioactive and fast method for detection of JNK/SAPK activity in leukemic T cells

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TO THE EDITOR

Stimuli that stress cells, such as chemotherapeutic drugs stimulate a cytoplasmic signaling system consisting of a cascade of protein kinases including the c-Jun kinase (JNK)/stress-activated protein kinase (SAPK).123 Activation of JNK/SAPKs has been linked to the induction of apoptosis45678910111213 and may be a prognostic parameter for the apoptosis sensitivity of leukemia cells.

In conventional detection methods JNK/SAPKs are immunoprecipitated from cell lysates and an in vitro kinase assay is performed by incubating purified JNK/SAPKs together with GST-cJun protein and 32P-γ-ATP. Since JNK/SAPKs phosphorylate and thereby activate transcription factors such as cJun114 the amount of phosphorylated GST-cJun protein reflects activity of JNK/SAPKs which is detected by SDS-PAGE and autoradiography.15

We developed a non-radioactive, sensitive JNK/SAPK assay which offers a fast and cheap alternative to the classical radioactive JNK/SAPK-assay. 1 × 107 leukemic T cells (JURKAT) are washed in ice-cold PBS and lysed in 400 μl of Flag-buffer (20 mM Tris pH 7.5, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 25 mM β-glycerophosphate, 2 mM Na-pyrophosphate, 1 mM Na3VO4 pH 10.0, 1 mM PMSF, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 10 μg/ml leupeptin). Lysates are cleared by centrifugation for 20 min at 2°C and 13 000 r.p.m., and nuclei-free supernatant is normalized for protein content. Then 1.5 μl of polyclonal IgG rabbit antibodies raised against JNK1 and JNK3 (C-17, sc-474, raised against AA 369 to 384 at the C-terminus; Santa Cruz, Heidelberg, Germany) or JNK1, JNK2 and JNK3 (FL, sc-571, AA 1 to 384; Santa Cruz) are added and the mixture is rotated for 45 min at room temperature. Twenty-five μl of a 10% suspension of protein A sepharose beads (Sigma, Deisenhofen, Germany) are added and the mixture is rotated for 1 h at room temperature. Beads are washed twice with Flag-buffer and once with kinase buffer to remove nonspecifically bound proteins. The kinase reaction is carried out in the presence of non-labeled ATP and GST-cJun fusion protein which contains the two N-terminal phosphorylation sites, Ser63 and Ser73 essential for phosphorylation by JNK/SAPKs (Figure 1). In brief, protein A sepharose beads complexed with anti-JNK antibodies bound to JNK proteins are suspended in 50 μl kinase buffer consisting of 25 mM Hepes, pH 7.4, 25 mM MgCl2, 25 mM β-glycerophosphate, 0.1 mM Na3VO4 supplemented with 100 μM ATP (Roche, Mannheim, Germany) and 2 μg GST-cJun 1–166 prepared as described16 or, alternatively, 2 μg GST-cJun 1–79 (Santa Cruz). After 25 min at 30°C, the reaction is terminated by addition of 30 μl 3× SDS sample buffer (187.5 mM Tris-HCl, pH 6.8, 6% SDS, 30% glycerol, 10% β-mercaptoethanol, 0.3% bromphenol blue). The products are resolved by 12% SDS-PAGE under reducing conditions and transferred on to ECL membranes (Amersham Pharmacia, Freiburg, Germany). Phosphorylated GST-cJun protein is stained with a rabbit polyclonal IgG phospho-specific cJun (Ser 73) antibody (New England BioLabs, Schwalbach, Germany) which is used in a dilution of 1:10000. This antibody specifically measures JNK/SAPK-induced phosphorylation of c-Jun at Ser 73, a site important for c-Jun-dependent transcriptional activity. Bound antibodies are detected with anti-rabbit-horseradish peroxidase conjugate (Santa Cruz) in a dilution of 1:5000 and enhanced chemiluminescence (Amersham Pharmacia, Freiburg, Germany). Specificity of the assay is ensured by performing the kinase reaction in the absence or presence of either GST-cJun, immunoprecipitate or ATP. Only the kinase reaction which contained all reagents together gives a strong signal (Figure 2). A slight band visible in the reaction without GST-cJun protein might result from phosphorylation of endogenous Jun protein which is co-precipitated with JNK/SAPKs. The doubleband corresponds to mono- or biphosphorylated Jun protein. This assay has exposure times of seconds and a very low background binding. Thus, this assay is highly sensitive and can, at least in principle, detect ratio over conventional 32P assays since the 32P-γ-ATP label is highly diluted (usually to 100 μM ATP).

Figure 1
figure1

 Non-radioactive JNK/SAPK assay overview. Immunoprecipitated JNK/SAPKs, in complex with specific antibodies (FL, C-17) and bound to protein A sepharose are incubated with non-radioactive ATP and GST-cJun protein. Activity of JNK/SAPKs results in phosporylation of GST-cJun protein on Ser-63 and 73. Phosporylation of GST-cJun is detected by Western blot analysis using a phospho-cJun- specific antibody.

Figure 2
figure2

 Specific phosphorylation of GST-cJun 1/166 in a non-radioactive JNK/SAPK assay. Lysates of JURKAT cells, which have been treated for 4 h with cisplatin (1 μg/ml), were used to immunoprecipitate JNK/SAPKs with the rabbit polyclonal IgG antibodies FL and C17 (Santa Cruz) and protein A sepharose. The kinase reaction was performed by incubating the immunoprecipitate with kinase buffer and cold ATP (a), or with kinase buffer, cold ATP and GST-cJun 1/166 as substrate (d). Alternatively, kinase buffer was incubated with either GST-cJun 1/166 and cold ATP in the absence of immunmoprecipitate (b). The different reaction mixtures or kinase buffer alone (c) were separated by 12% SDS-PAGE and analyzed by Western blot. Phosphorylation of the GST-cJun protein was detected by a rabbit polyclonal phospho-cJun-Ser73-specific IgG antibody (New England BioLabs) and enhanced chemiluminescence. The molecular weight in kDa is shown on the left.

Recent data obtained with cultured leukemic T cells1718 and childhood ALL strongly suggest that induction of JNK/SAPKs within 24 h following therapy constitutes a critical parameter for therapeutic effectiveness. Taking this into consideration, a rapid and reproducible method is required for detection of JNK/SAPK activity. We think our non-radioactive JNK/SAPK assay fulfils these criteria.

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Acknowledgements

We thank Dr P Angel for the GST-cJun expression plasmid. This work was supported by grants from the Deutsche Forschungsgemeinschaft.

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Correspondence to K-M Debatin.

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Herr, I., Krilleke, D. & Debatin, K. A sensitive, non- radioactive and fast method for detection of JNK/SAPK activity in leukemic T cells. Leukemia 14, 1859–1860 (2000) doi:10.1038/sj.leu.2401911

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