Calcium regulation of GM-CSF by calmodulin-dependent kinase II phosphorylation of Ets1

The multipotent cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is involved in particular in the physiological response to infection and in inflammatory responses. GM-CSF is produced by many cell types, including T lymphocytes responding to T-cell receptor activation and mantle zone B lymphocytes. B-cell receptor and T-cell receptor activation generates two major signals: an increase in intracellular Ca2+ concentration and a protein kinase cascade. Previous studies have shown that the Ca2+/calmodulin-dependent phosphatase calcineurin mediates stimulation of GM-CSF transcription in response to Ca2+. In this study, we show that Ca2+ signaling also regulates GM-CSF transcription negatively through Ca2+ /calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Wild-type Ets1 negatively affects GM-CSF transcription on Ca2+ stimulation in the presence of cyclosporin A, which inhibits calcineurin. Conversely, Ets1 with mutated CaMK II target serines showed an increase in transactivation of the GM-CSF promoter/enhancer. Moreover, constitutively active CaMK II inhibited transactivation of GM-CSF by wild-type Ets1 but not by Ets1 with mutated CaMK II sites. Mutation of CaMK II target serines in Ets1 also relieves inhibition of cooperative transactivation of GM-CSF with the Runx1/AML1 transcription factor. In addition, the Ca2+-dependent phosphorylation of Ets1 reduces the binding of Ets1 to the GM-CSF promoter in vivo.