AML1/Runx1 recruits calcineurin to regulate granulocyte macrophage colony-stimulating factor by Ets1 activation.
Acute myeloid leukemia 1 (AML1), also denoted Runx1, is a transcription factor essential for hematopoiesis, and the AML1 gene is the most common target of chromosomal translocations in human leukemias. AML1 binds to sequences present in the regulatory regions of a number of hematopoiesis-specific genes, including certain cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) up-regulated after T cell receptor stimulation. Here we show that both subunits of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN), which is activated upon T cell receptor stimulation, interact directly with the N-terminal runt homology domain-containing part of AML1. The regulatory CN subunit binds AML1 with a higher affinity and in addition also interacts with the isolated runt homology domain. The related Runx2 transcription factor, which is essential for bone formation, also interacts with CN. A constitutively active derivative of CN is shown to activate synergistically the GM-CSF promoter/enhancer together with AML1 or Runx2. We also provide evidence that relief of the negative effect of the AML1 sites is important for Ca(2+) activation of the GM-CSF promoter/enhancer and that AML1 overexpression increases this Ca(2+) activation. Both subunits of CN interact with AML1 in coimmunoprecipitation analyses, and confocal microscopy analysis of cells expressing fluorescence-tagged protein derivatives shows that CN can be recruited to the nucleus by AML1 in vivo. Mutant analysis of the GM-CSF promoter shows that the Ets1 binding site of the promoter is essential for the synergy between AML1 and CN in Jurkat T cells. Analysis of the effects of inhibitors of the protein kinase glycogen synthase kinase-3beta and in vitro phosphorylation/dephosphorylation analysis of Ets1 suggest that glycogen synthase kinase-3beta-phosphorylated Ets1 is a target of AML1-recruited CN phosphatase at the GM-CSF promoter.
Liu
Hebin
H
Department of Molecular Biology, Umeå University, Umeå S-901 87, Sweden.
Holm
Magnus
M
Xie
Xiao-Qi
XQ
Wolf-Watz
Magnus
M
Grundström
Thomas
T
D000818:Animals; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit; D050676:Core Binding Factor Alpha 2 Subunit; D016572:Cyclosporine / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D019169:Jurkat Cells; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D050659:Core Binding Factor Alpha 1 Subunit; D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; D004791:Enzyme Inhibitors; C494042:Ets1 protein, mouse; D007476:Ionophores; D009363:Neoplasm Proteins; D021122:Protein Subunits; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; C493728:RUNX1 protein, human; C493726:Runx1 protein, mouse; D014157:Transcription Factors; D015759:Ionomycin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D016572:Cyclosporine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3; D019703:Calcineurin; D002118:Calcium