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Fig. 5. H₂O₂-induced DAP kinase (DAPK) activation is downstream of ERK. (A) HEK293 cells were transfected with plasmids expressing the dominant-negative FLAG-tagged DAPK (DAPK K42A). The next day, cells were incubated in phosphate-free culture medium in the presence of H₃[³²P]O₄ for 90 minutes. Cells were pre-treated with vehicle only (DMSO, 0.25%) or with the MEK inhibitor PD098059 (50 µM, 60 minutes; P) and treated or not with H₂O₂ for 10 minutes (250 µM). The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against FLAG. Immunoprecipitated proteins (IP) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Incorporation of ³²PO₄ into the corresponding bands was visualized using Phosphor Imager. A loading control of the immunoprecipitated FLAG-DAPK is shown (second panel). Total extracts (TE) were kept before the immunoprecipitation to monitor ERK activation (third panel) by immunodetection of phospho-ERK. Total ERK is shown in the lower panel. (B) HEK293 cells were transfected with plasmids expressing the dominant-negative FLAG-tagged DAPK K42A or a double-mutant DAPK K42A S735A. The next day, cells were incubated in phosphate-free culture medium in the presence of H₃[³²P]O₄ for 90 minutes. Cells were processed as in A. Incorporation of ³²PO₄ into the corresponding FLAG-DAPK bands was visualized using PhosphorImager. A loading control of the immunoprecipitated FLAG-DAPK is shown (lower panel). (C) HEK293 cells were transfected with plasmids expressing the dominant-negative FLAG-tagged DAPK K42A. The next day, cells pre-treated with vehicle only (DMSO, 0.25%) or with the ERK inhibitor UO126 (50 µM, 60 minutes; U) were treated or not with H₂O₂ (250 µM) for 10 minutes. The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against FLAG. Immunoprecipitated proteins (IP) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was processed for immunodetection of ERK (upper panel) and DAPK (second panel). Total extracts (TE) were kept before the immunoprecipitation and processed as in A. (D) Exponentially growing HUVECs were pre-treated for 60 minutes with vehicle only (0.25% DMSO) or with the MEK inhibitor UO126 (50 µM; U) and were treated or not with H₂O₂ (250 µM, 10 minutes). Next, cells were extracted and processed for endogenous DAPK immunoprecipitation. Immunoprecipitated proteins were used for an in vitro kinase assay using purified MLC as a substrate, as described in Materials and Methods. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was processed for immunodetection of phospho-Ser19 within MLC (upper panel), the total MLC (second panel), the immunoprecipitated DAPK (third panel) or phospho-ERK (lower panel) using specific antibodies.

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