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).
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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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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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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