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The consensus sequence at the phosphorylation site implicates the phosphatidylinositol 3\u2010like family of protein kinases in H2AX phosphorylation. It remains open whether ATM (ataxia telangiectasia mutated) is the major H2AX kinase, or whether other members of the family, such as DNA\u2010PK (DNA dependent protein kinase) or ATR (ATM and Rad3 related), contribute in a functionally complementary manner. To address this question, we measured global H2AX phosphorylation in cell lysates and foci formation in individual cells of either wild type or mutant (ATM or DNA\u2010PK) genetic background. Normal global phosphorylation kinetics is observed after irradiation in cells defective either in ATM or DNA\u2010PK alone, suggesting a complementary contribution to H2AX phosphorylation. This is further supported by the observation that initial H2AX phosphorylation is delayed when both kinases are inhibited by wortmannin, as well as when ATM is inhibited by caffeine in DNA\u2010PK deficient cells. However, robust residual global phosphorylation is detectable under all conditions of genetic or chemical inhibition suggesting the function of additional kinases, such as ATR. Treatment with wortmannin, caffeine, or UCN\u201001 produces a strong DNA\u2010PK dependent late global hyperphosphorylation of H2AX, uncoupled from DNA DSB rejoining and compatible with an inhibition of late steps in DNA DSB processing. Evaluation of \u03b3\u2010H2AX foci formation confirms the major conclusions made on the basis of global H2AX phosphorylation, but also points to differences particularly several hours after exposure to IR. The results in aggregate implicate DNA\u2010PK, ATM and possibly other kinases in H2AX phosphorylation. The functional significance and the mechanisms of coordination in space and time of these multiple inputs require further investigation. \u00a9 2004 Wiley\u2010Liss, Inc.<\/jats:p>","DOI":"10.1002\/jcp.20141","type":"journal-article","created":{"date-parts":[[2004,8,9]],"date-time":"2004-08-09T22:29:31Z","timestamp":1092090571000},"page":"492-502","source":"Crossref","is-referenced-by-count":175,"title":["Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA\u2010PK in human cells exposed to ionizing radiation and treated with kinase inhibitors"],"prefix":"10.1002","volume":"202","author":[{"given":"Hongyan","family":"Wang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minli","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huichen","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wilfried","family":"B\u00f6cker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"George","family":"Iliakis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2004,8,9]]},"reference":[{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.2307\/3578948"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/0968-0004(93)90144-C"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-642-50140-1_7"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1667\/0033-7587(2001)156[0002:FMIPTG]2.0.CO;2"},{"key":"e_1_2_6_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.onc.1203953"},{"key":"e_1_2_6_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(99)80486-2"},{"key":"e_1_2_6_8_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.C100466200"},{"key":"e_1_2_6_9_1","first-page":"2108","article-title":"The 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