Identification of MAP kinase domains by redirecting stress signals into growth factor responses

Science

Volumn 272, Issue 5268, 1996, Pages 1652-1655

  Brunet, Anne ^a   Pouysségur, Jacques ^a  

^a CNRS   (France)

Author keywords

[No Author keywords available]

Indexed keywords

GROWTH FACTOR; MITOGEN ACTIVATED PROTEIN KINASE;

AMINO ACID SEQUENCE; AMINO TERMINAL SEQUENCE; ANIMAL CELL; ARTICLE; CARBOXY TERMINAL SEQUENCE; CHIMERA; CONTROLLED STUDY; ENZYME SPECIFICITY; MITOGENESIS; NONHUMAN; PRIORITY JOURNAL; PROTEIN DOMAIN; SIGNAL TRANSDUCTION; STRESS;

EID: 0029893708     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5268.1652     Document Type: Article

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29. 5 cells per 35-mm well) were transfected with 6 μg of DNA by the calcium phosphate technique. Two days after transfection, cells were serum-deprived for 5 hours and stimulated with various agonists. Kinase assays were done as described (18). Briefly, cells were lysed in 400 μl of Triton lysis buffer. Equal amounts of proteins (300 μg) were immunoprecipitated on protein A-Sepharose beads coupled with the antibody to HA (anti-HA) (Babco, Emeryville). Activity of the kinases were assayed in 40 μl of kinase buffer with various substrates: glutathione-S-transferase (GST)-ATF2 (1-109), GST-Myc (1-143), GST-Elk1 (307-428), and MBP at a final concentration of 10 μg and 3 μCi of (γ-32P], 50 μM adenosine 5′-triphosphate (ATP) (ICN) for 30 min at 30°C. Reactions were stopped by addition of 25 μl of Laemmli buffer and heated for 5 min at 95°C. Samples were resolved on SDS-polyacrylamide gel electrophoresis (PAGE); the gels were autoradiographed, stained with Coomassie brilliant blue, and the bands corresponding to the substrates were excised and counted.
30. All chimerae were constructed from p44 and p38 MAPK vectors by introduction of polymerase chain reaction (PCR) fragments through use of restriction sites in p38MAPK {Ava I [93 base pairs (bp), domain I], PpuM I (200 bp, domain III), BstE II (312 bp, domain V), Sac I (491 bp, domain VII), and Kpn I (564 bp, domain VIII)} or in p44MAPK [Afl II (508 bp, domain VII) and Kpn I (600 bp, domain VIII)]. All the PCR fragments and the junctions were verified by sequencing.
31. Protein immunoblot experiments were done with anti-HA after resolution of proteins on SDS-PAGE (18).
32. rsk (Santa Cruz).
33. 32P], 50 μM ATP for 15 min at 30°C. Reactions were stopped by addition of 25 μl of Laemmli buffer and heated for 5 min at 95°C. Samples were resolved on SDS-PAGE and the gel was autoradiographed.
34. 5 cells per well in 24-well culture plates) were transfected with 0.25 μg of the c-fos-luciferase reporter gene and 0.75 μg of the relevant constructs. One day after transfection, cells were incubated in serum-free medium for 24 hours, then stimulated with fetal calf serum (20%) or IL-1β (10 ng/ml; Boehringer) for 16 hours. Luciferase activity was measured according to the Promega protocol.
35. rsk and the antibody directed against it, R. Davis for the GST-ATF2 construct, D. Czernilofsky for the c-fos promoter-luciferase construct, R. Ulevitch for the p38MAPK cDNA, G. Thomas for the 40S ribosomal S6 protein and R. Treisman for the GST-elk1 construct, D. Grall and M. Valetti for technical assistance, B. Chabanne for assistance with photography, V. Dulić and F. R. McKenzie for helpful comments on the manuscript, and J.-C. Chambard, B. Dérijard, and G. Pagès for stimulating discussions. We are especially indebted to V. Dulić for help and encouragement. Supported by the CNRS, the Institut National de la Santé et de la Recherche Médicale/MSD 91AN13, the Ligue Nationale contre le Cancer, and the Association pour la Recherche contre le Cancer.