Immunofluorescence studies have revealed that H2AX is phosphorylated at the sites of DNA double-strand breaks induced by ionizing radiation and is required for recruitment of repair factors into nuclear foci after DNA damage. Therefore, the function of H2AX is believed to be associated primarily with repair of DNA damage. Here, we report a function of H2AX in cellular apoptosis. Our data showed that H2AX is phosphorylated by UVA-activated JNK. We also provided evidence showing that UVA induces caspase-3 and caspase-activated DNase (CAD) activity in both H2AX wild-type and H2AX knockout mouse embryonic fibroblasts (MEFs). However, DNA fragmentation occurred only in H2AX wild-type MEFs. Furthermore, H2AX phosphorylation was critical for DNA degradation triggered by CAD in vitro. Taken together, these data indicated that H2AX phosphorylation is required for DNA ladder formation, but not for the activation of caspase-3; and the JNK/H2AX pathway cooperates with the caspase-3/CAD pathway resulting in cellular apoptosis.
Bibliographical noteFunding Information:
We thank Dr. André Nussenzweig (Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD) for providing the immortalized H2AX-wt and H2AX −/− MEFs, Dr. Yang Xu (Department of Biology, University of California, San Diego, CA) for ATM-wt and ATM −/− MEFs, Dr. Roger J. Davis (Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, MA) for the JNK2 plasmid, Dr. Xiaodong Wang (Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX) for the pET-15b-DFF40His, Dr. Joseph S. Siino (Division of Biological Science, University of California, Davis, CA) for the H2AX cDNA, Dr. Craig H. Bassing (CBR Institute for Biomedical Research, Boston, MA) for helpful suggestions pertaining to this work, and Todd Schuster (Hormel Institute, University of Minnesota) for help performing the cell apoptosis assays. This work is supported by the Hormel Foundation and National Institutes of Health grants CA77646, CA81064, CA88961, CA27502, and CA11135.