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.

Nyckelord

Animals

Calcineurin/genetics/*metabolism

Calcium/metabolism

Core Binding Factor Alpha 1 Subunit

Core Binding Factor Alpha 2 Subunit

Cyclosporine/pharmacology

DNA-Binding Proteins/genetics/*metabolism

Enhancer Elements (Genetics)

Enzyme Inhibitors/pharmacology

Gene Expression Regulation/drug effects

Genes; Reporter

Glycogen Synthase Kinase 3/antagonists & inhibitors/metabolism

Granulocyte-Macrophage Colony-Stimulating Factor/genetics/*metabolism

Humans

Ionomycin/pharmacology

Ionophores/pharmacology

Jurkat Cells

Mice

Neoplasm Proteins/genetics/metabolism

Phosphorylation

Promoter Regions (Genetics)

Protein Structure; Tertiary

Protein Subunits/genetics/*metabolism

Protein Transport/physiology

Proto-Oncogene Protein c-ets-1

Proto-Oncogene Proteins/genetics/*metabolism

Proto-Oncogene Proteins c-ets

Transcription Factors/genetics/*metabolism

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