Ectopic expression of histone H2AX mutants reveals a role for its post-translational modifications.
Recent evidence from a wide variety of biological systems has indicated important regulatory roles for post-translation histone modifications in cellular processes such as regulation of gene expression, DNA damage response and recombination. Phosphorylation of histone H2AX at serine 139 is a critical event in the response to DNA damage, but the functional implications of this modification are not yet clear. To investigate the role of H2AX phosphorylation we ectopically expressed epitope-tagged H2AX or mutants at the phosphorylation site. GFP-tagged wild type H2AX, H2AX Ser139Ala or H2AX Ser139Glu proteins were efficiently expressed, localizing exclusively to the interphase nucleus and to condensed chromosomes during mitosis. Biochemical fractionation indicated that epitope-tagged H2AX proteins are incorporated into nucleosomes. Expression of H2AX Ser139Ala, which disrupts the phosphorylation site partially suppressed early G(2)/M arrest following ionizing radiation, and cells expressing this mutant were more sensitive to DNA damage. Conversely, expression of H2AX Ser139Glu, designed as phosphorylation mimic, induced a decrease in the number of cells in mitosis in the absence of DNA damage. Interestingly, this decrease induced by H2AX Ser139Glu was independent of the formation of 53BP1-containing foci and was partially suppressed in CHK2-deficient cells, suggesting a role for CHK2 in this process. Further analyses revealed that expression of either mutant lead to apoptosis and induced higher caspase-3/7 activity compared to expression of wild type H2AX. In addition, we also identified Lys119 as a site for ubiquitination that controls H2AX half-life. Phosphorylation of Ser139 and ubiquitination of K119 are not interdependent. Taken together these results demonstrate a role for H2AX Serine 139 phosphorylation in cell cycle regulation and apoptosis, and for Lysine 119 in the control of H2AX turnover.
Rios-Doria
Jonathan
J
Risk Assessment, Detection and Intervention Program, H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida 33612 , USA.
Velkova
Aneliya
A
Dapic
Virna
V
Galán-Caridad
José M
JM
Dapic
Vesna
V
Carvalho
Marcelo A
MA
Melendez
Johana
J
Monteiro
Alvaro N A
AN
D019943:Amino Acid Substitution; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002869:Chromosome Aberrations; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D045325:HCT116 Cells; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D054875:Ubiquitination
C468783:H2AX protein, human; D006657:Histones; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012694:Serine; D046988:Proteasome Endopeptidase Complex; D008239:Lysine