FIG. 1.

Phosphorylation and activation of MEF2A or MEF2C by different p38 MAP kinases. (A) Diagram illustrating the domain structure of full-length MEF2C and truncated MEF2A and MEF2C proteins fused to either GST or the GAL4 DNA binding domain (open boxes). The location of the DNA binding domain (DBD), minimal TAD, and p38α (T293 and T300) and ERK5 (S387) phosphorylation sites in MEF2C are indicated. The black box represents the putative kinase docking domain (D-domain) of MEF2A/C, and the numbers of the N- and C-terminal amino acids in the MEF2A or MEF2C moiety are indicated. The sequences of MEF2A and MEF2C around the major phosphoacceptor motifs for p38α MAP kinase are shown. (B) The phosphorylation of GST-MEF2A, GST-MEF2C, and GST–Elk-310 by the p38 MAP kinase subtypes p38α (lane 1), p38β₂ (lane 2), p38γ (lane 3), and p38δ (lane 4) was examined by a protein kinase assay. The activity of each protein kinase was standardized towards the substrate GST–Elk-310 (bottom panel). Kinase assays were performed for 15 min at 30°C with 5 pmol of GST-MEF2A and GST-MEF2C as substrates. (C) COS-7 cells were cotransfected with either GAL4-MEF2A or GAL4-MEF2C expression vectors, a constitutively activated form of MKK6 (MKK6[E]), the indicated p38 MAP kinases, and a GAL4-driven luciferase reporter plasmid. (D) COS-7 cells were cotransfected with expression vectors encoding GAL4-MEF2A, an overexpressed (MKK7β) or constitutively activated form of MKK (MEK and MKK6), a MAP kinase (ERK2, JNK2, and p38β₂), and a GAL4-driven luciferase reporter plasmid. (E) HeLa cells were cotransfected with vectors encoding GAL4-MEF2A and a GAL4-driven luciferase reporter plasmid. Cells either were left unstimulated or were stimulated with IL-1 for 18 h in the absence or presence of the indicated JNK pathway (cotransfected dominant negative form of MKK4 [DN-MKK4]) or p38 pathway (SB202190) inhibitors. (F) COS-7 (for EGF stimulation of ERKs), CHO (for IL-1 stimulation of JNKs), and HeLa (for IL-1 stimulation of p38s) cells were cotransfected with vectors encoding GAL4-MEF2A or GAL4–Elk-1 and a GAL4-driven luciferase reporter plasmid. The cells either were left unstimulated (white bars) or were stimulated (black bars for Elk-1 and grey bars for MEF2A) with EGF or IL-1 as in panel E. Transfection efficiencies were monitored by using the β-galactosidase expression vector pCH110. The normalized luciferase activities (means ± standard errors; n, 2 or 3) are presented.