Figure 8.

DNA-PK_cs interacts with stress kinases and is subject to phosphorylation by SAPK/JNK. (A) SAPK/JNK was immunoprecipitated (IP) from untreated (Con) or MMS-treated (MMS, 1 mM, 6 h) DNA-PK_cs-proficient (WT) cells. Coprecipitation of DNA-PK_cs and Ku-86 was analyzed by Western blot analysis. As controls, immunoprecipitation was performed with heat-inactivated (95°C, 5 min) JNK antibody and antibody directed against p38 kinase. (B) p-JNK, p-SEK, and MKP-1 protein was immunoprecipitated from untreated (Con) and MMS-treated (1 mM, 6 h) DNA-PK_cs-proficient (WT) cells by use of the corresponding antibody. Coprecipitation of DNA-PK_cs, Ku-86, and the mismatch repair protein MSH2 was analyzed by Western blot analysis. (C) SAPK/JNK was immunoprecipitated from untreated (Con) DNA-PK_cs-proficient (BK4) and -deficient (SCID) cells. As a control, immunoprecipitation was performed using heat-inactivated JNK antibody as described in A. Coprecipitation of DNA-PKcs and Ku-86 was analyzed by Western blot analysis. (D) SAPK/JNK was immunoprecipitated from untreated (Con) or MMS (1 mM, 4 h) treated mouse fibroblasts. Afterward, purified DNA-PK_cs protein or recombinant GST-Jun protein was added, and SAPK/JNK-mediated ³²P-phosphorylation of DNA-PK_cs and GST-Jun was assayed in vitro as described in Materials and Methods. Shown is the autoradiography of a representative experiment.