Article Title: Calcium Regulation of GM-CSF by Calmodulin-Dependent Kinase II Phosphorylation of Ets1
Figure Lengend Snippet: Schematic illustrating the discussed models for regulation of GM-CSF transcription. The binding of NF-AT, NF-κB, Ets1, AP-1, and Runx1 transcription factors to the promoter is shown. For simplicity, only one NF-AT site and one Ets1 site are drawn, although the GM-CSF promoter/enhancer contains multiple binding sites for these factors (Thomas et al., 1995 ; Shannon et al., 1997 ; Thomas et al., 1997 ; McKinlay et al., 1998 ; Shang et al., 1999 ). The autoinhibitory domain of Ets1 (gray) and the Ets domain are also shown. Nonphosphorylated serines at positions 251, 257, 282, and 285 in the autoinhibitory domain are indicated with S and phosphorylation of these serines with P. Ca2+ signaling, which can be induced by TCR or BCR activation or by ionomycin treatment, positively regulates transcription from the GM-CSF promoter/enhancer through calcineurin activation of the transcription factors NF-AT, NF-κB, and AP-1 (Shannon et al., 1997 ). In the present study, we show that Ca2+ signaling can also negatively regulate the GM-CSF promoter/enhancer through CaMK II phosphorylation of serines in the autoinhibitory domain for DNA binding of Ets1. Serine phosphorylation in the autoinhibitory domain stabilizes the conformation that inhibits the DNA binding of the Ets domain (Cowley and Graves, 2000 ). The negative effect of wild-type Ets1 suggests that it functions as a dominant inhibitory protein that decreases transactivation by a functionally related protein(s), such as the p42 splice form of Ets1, that lacks the autoinhibitory domain or another Ets family member(s) that is less inhibited by CaMK II. The finding that the mutations also increased the Ets1 activity in the absence of Ca2+/CaMK II–activating treatment may, as illustrated, indicate that another kinase can mediate a partial phosphorylation in the absence of Ca2+ signaling. The circular arrow indicates a possible dynamic balance between Ca2+-dependent CaMK II and autophosphorylation that makes CaMK II independent of Ca2+ (Lukas et al., 1998 ). The intermediate nucleotides between the Runx1 and Ets1 sites in the GM-CSF promoter constitute a binding site for AP-1. The results suggest that when Ets1 functions in cooperation with nearby AP-1, at least a large part of the autoinhibition through phosphorylation of the autoinhibitory domain remains and is not relieved by interaction between Ets1 and Runx1.
Article Snippet: Bars represent average ratio of luciferase/β-galactosidase activity in arbitrary units from three independent transfections ± SD, using β-galactosidase expression from an hCMV-β-gal plasmid for normalization. (C) Quantification of the expression level of Ets1 wild-type and the mutants in the transfected DG75 cells by Western blot using the C-275 anti-Ets1 antibody (Santa Cruz).
Techniques: Binding Assay, Activation Assay, Activity Assay