We demonstrate that AML1 (RUNX1) is phosphorylated on S276, S293, T300, S303 and S462 after phorbol ester treatment in K562 cells, and these phosphorylations control the transcriptional activity of this protein and regulate varied promoters including TCRb, GM-CSF, M-CSF receptor, and myeloperoxidase gene (

JBC 279: 53116–53125, 2004
). In addition, we find that phosphorylation of the AML1 protein controls the binding of AML1 to the nuclear matrix, and regulates its degradation (
MCR 3: 391–401, 2005
). We have now derived rabbit phosphopeptide antisera to S276, S303, and S462 in the AML1 protein. Using these antibodies, we find that in unsynchronized Jurkat T cells AML1 contain low levels of phosphorylation at all three sites. To examine whether this phosphoryaltion is cell cycle regulated, we blocked cells in G1/S with aphidicolin or in G2/M with nocodazole. In contrast to G1/S block that had no effect on phosphorylation, nocodazole treatment induces marked increases in phosphorylation of S276, S303, and S462. The nocodazole increase in phosphorylation was markedly inhibited by staurosporine, a PKC inhibitor, and to a lesser extent by the JNK inhibitor SP600125, and the p38 inhibitor, SB202190. Using elutriation to examine untreated Jurkat cells, we demonstrate increases in the level of AML1 protein as cells progress through the cell cycle to G2/M. However, the actual level of phosphorylated protein as judged by phospho303 and 462 antibodies decreases. Nocodazole treatment of Jurkat cells not only induces G2/M arrest, but also induces apoptosis. We are currently investigating the hypothesis that DNA damage causes phosphorylation of AML1 that regulates both its activity and location.

Topics:
dna damage, phosphorylation, nocodazole, antibodies, aphidicolin, granulocyte-macrophage colony-stimulating factor, immune sera, macrophage colony-stimulating factor receptor, peroxidase, phorbol esters

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2005, The American Society of Hematology
2005
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