Requirement of Rsk-2 for epidermal growth factor-activated phosphorylation of histone H3

Science

Volumn 285, Issue 5429, 1999, Pages 886-891

  Sassone Corsi, Paolo ^a   Mizzen, Craig A ^b   Cheung, Peter ^b   Crosio, Claudia ^a   Monaco, Lucia ^a   Jacquot, Sylvie ^a   Hanauer, André ^a   Allis, C David ^b  

^a CNRS   (France)

^b UNIVERSITY OF VIRGINIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EPIDERMAL GROWTH FACTOR; HISTONE H3; MITOGEN ACTIVATED PROTEIN KINASE; PROTEIN RSK2; UNCLASSIFIED DRUG;

ANIMAL CELL; ARTICLE; CONTROLLED STUDY; EARLY RESPONSE GENE; GENE DISRUPTION; GENE EXPRESSION REGULATION; GROWTH REGULATION; HUMAN; HUMAN CELL; MOLECULAR DYNAMICS; MOUSE; NONHUMAN; PRIORITY JOURNAL; PROTEIN DETERMINATION; PROTEIN FAMILY; PROTEIN PHOSPHORYLATION; PROTEIN PROTEIN INTERACTION; PROTEIN TARGETING; STEM CELL;

3T3 CELLS; ABNORMALITIES, MULTIPLE; ANIMALS; CA(2+)-CALMODULIN DEPENDENT PROTEIN KINASE; CELL LINE, TRANSFORMED; CELL NUCLEUS; CELLS, CULTURED; EPIDERMAL GROWTH FACTOR; GENE EXPRESSION REGULATION; GENE TARGETING; HISTONES; HUMANS; MICE; MITOSIS; MUTATION; PHOSPHORYLATION; RIBOSOMAL PROTEIN S6 KINASES; SIGNAL TRANSDUCTION; STEM CELLS; SYNDROME;

ANIMALIA; MAMMALIA; MURINAE;

EID: 0033529706     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5429.886     Document Type: Article

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28. Fibroblasts were maintained in serum-free medium for 1 to 2 days before stimulation. After treatment, cells were washed twice in cold phosphate-buffered saline (PBS) and lysed directly in boiling SDS sample buffer. Cell lysates were resolved by SDS-PAGE (8 or 10% gels); equal loading was confirmed by staining parallel gels with Coomassie blue. Resolved samples were electroblotted onto PVDF membranes, and equal transfer was confirmed by staining blots with Ponceau S. Membranes were blocked in 5% low-fat dry milk in PBS for 2 hours at room temperature and then incubated with primary antibody for 12 hours at 4°C. Membranes were washed five times in PBS before incubation with peroxidase-conjugated secondary antiserum for 2 hours at room temperature. After five washes in PBS, immunocomplexes were detected by enhanced chemiluminescence.
29. Equivalent numbers of normal and CLS fibroblasts were seeded on separate cover slips within 60-mm culture dishes and grown for 24 hours. Cells were then serum-starved for 24 hours, mock-treated or treated with EGF (50 ng/ml) for 30 min, and fixed in PBS (pH7.5) containing 4% paraformaldehyde. Fixed cells were permeabilized with PBS containing 0.2% Triton X-100, blocked with 2% goat serum in PBS, and sequentially incubated with the pS10 antiserum and rhodamine- or fluorescein-conjugated goat anti-rabbit secondary antisera. Immunostained cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) before microscopy. Cells stained with rhodamine-conjugated secondary antiserum were photographed and color prints were scanned in RGB mode to generate electronic image files. Cells stained with fluorescein-conjugated secondary antiserum were captured as grayscale images by using a charge-coupled device camera. Both types of files were converted to CMYK images and colorized with Adobe Photoshop.
30. NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS, 10%). When cells were ∼80% confluent, growth medium was replaced with DMEM without serum. Typically, cells were serum-starved for 24 hours before mitogen stimulation. Normal human and CLS (Rsk-2 deficient) fibroblasts, immortalized with Epstein-Barr virus (14), were cultured as above. The CLS cells used in this study are derived from a patient in which a splice site mutation leads to the use of a cryptic acceptor site and premature termination at amino acid 422. Consequently, Rsk-2 protein in these cells lacks the second kinase domain and is inactive (14, 15). Stimulation with EGF (50 ng/ml, Sigma) was performed as described (5, 15).
31. 4.
32. 32P]ATP (adenosine 5′-triphosphate) (3000 Ci/mmol, NEN), and incubated on a rocking platform at 30°C for 4 hours. After extensive washing in 1% (w/v) tetrasodium pyrophosphate, gels were stained with Coomassie blue R-250 and destained before autoradiography.
33. Histone substrates were prepared by acid extraction of isolated chicken erythrocyte nuclei. Individual histone fractions were purified by rpHPLC Chromatin fractions were prepared by sucrose density centrifugation of chromatin solubilized by micrococcal nuclease digestion of isolated chicken erythrocyte nuclei. Conventional kinase assays used the protocol supplied with an S6 kinase assay kit (Upstate Biotechnology). Where appropriate, portions of assay mixtures were recovered by trichloroacetic acid (TCA) precipitation (20% final) or supplemented with 5× sample buffer, resolved by SDS-PAGE (12% gels), and processed for autoradiography or protein immunoblotting.
34. 32P]ATP for 90 min at 30°C. The sample was recovered by TCA precipitation and H3 was purified by rpHPLC before microsequence analysis. Sequencing was performed as described [Y. Wei, C. A. Mizzen, R. G. Cook, M. A. Gorovsky, C D. Allis, Proc. Natl. Acad. Sci. U.S.A. 95, 7480 (1998)]. The repetitive yield for the analysis shown in Fig. 2B was 92%.
35. 2/M boundary by exposure to nocodazole (0.05 μg/ml) for 12 hours. The cultures were then washed and incubated with fresh DMEM without serum for 6 or 24 hours before stimulation with EGF (100 ng/ml) for 30 min. Lysates were prepared and analyzed by immunoblotting (22).
36. Parallel cultures of CLS cells (in 60-mm culture dishes) at 80% confluency were transfected either with 1.0 μg of vector DNA (pSG5) [S. Green, I. Issemann, E. Sheer, Nucleic Acids Res. 16, 369 (1988)] or with 1.0 μg of the human Rsk-2 expression plasmid pTHL-Rsk2 (human Rsk-2 cDNA cloned into the pSG5 vector) by using Lipofectamine Plus transfection reagent (Life Sciences). Transfected cells were cultured in medium containing 10% FBS for 24 hours and then in serum-free medium for another 24 hours. Cells were then mock-treated or treated with EGF (50 ng/ml) for 30 min before harvesting. Lysates were prepared and analyzed by immunoblotting (22). For immunofluorescence analyses, CLS cells were grown on cover slips in 60-mm dishes and transfected as described above. Indirect immunoftuorescence was performed as described (23).
37. Protein microsequencing and peptide synthesis were done at Baylor College of Medicine (Houston, TX) by R. G. Cook. We thank J. Davie, D. De Cesare, M. J. Hendzel, L Mahadevan, K. Merienne, M. Montminy, and A. Vertegal for helpful discussions; E. Heitz and J. Zhou for technical assistance; and T. Parsons and T. Sturgill for comments on this manuscript. Supported by grants to C.D.A (NIH-GM40922) and P.S.-C (Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire Régional, Fondation de la Recherche Médicale, and Association pour Recherche sur le Cancer).