Threonine188 of C/EBPβ is important for the interaction of ERK2 with C/EBPβ that activates G-CSF promoter.

Abstract

<p>(<b>A</b>) RAW264.7 cells were co-transfected with a mixture of two reporter plasmids (pG-CSF(-283/+35)-Luc and phRLTK) and the CA-ERK2, p50, Oct-2, or C/EBPβ plasmid alone or the C/EBPβ plasmid plus the CA-ERK2, p50, or Oct-2 plasmid, then luciferase activity was measured at 24 h after transfection using the Dual-luciferase reporter assay system. (<b>B</b>) RAW264.7 cells were pretreated for 30 min with DMSO (D) or U0126 (U) (10 μM), then were incubated with LPS (100 ng/ml) (L) for another 4 h, after which a nuclear extract (N.E.) and cytosolic extract (C.E.) were isolated and levels of total and phosphorylated C/EBPβ, phosphorylated ERK1/2, β-actin (cytosol internal control), and laminin A/C (nuclear internal control) analyzed by Western blotting. The data shown are typical of those obtained in three experiments. (<b>C</b>) Cells were cotransfected with reporter plasmids (0.5 μg of pG-CSF (-283/+35)-Luc mixed with 0.1 μg of phRLTK), 0.15 μg of CA-ERK expression plasmid, and 0.15 μg of the WT, T188A, or S64A C/EBP expression plasmid or 0.15 μg of pcDNA3.1 (vector control), then the reporter luciferase activities were analyzed 24 h after transfection. (<b>D</b>) Cells were co-transfected with pG-CSF(-283/+35)-Luc carrying the wild type or mutant C/EBPβ binding site, 0, 0.15, 0.3, 0.6 μg of CA-ERK2 expression plasmid, and the internal control phRLTK, then luciferase activities were measured 24 h after transfection. All luciferase activities are shown relative to the vector control (relative value = 1). In A, C, and D, values are the mean ± SD for three independent experiments. *<i>p</i><0.05 compared to wild-type cells.</p