FIG. 6.
The p38-binding domain of MEF2A (D-domain) functions in a heterologous context. The phosphorylation and activation of GST and GAL4 fusions to MEF2A–Elk-1 (A to D) and MEF2A-cJun (E to H) chimeras by MAP kinases were analyzed. (A and E) Diagrams illustrating fusions of GST to Elk-1ΔD, MEF2A–Elk-1, cJun, and MEF2A-cJun. The numbers of the N- and C-terminal Elk-1/cJun (roman type) and MEF2A (italics) amino acids included in these constructs are indicated above and below each construct, respectively. The MEF2A D-domain and cJun δ-domain are indicated by solid and open boxes, respectively. The Elk-1 and cJun transcriptional activation domains are shown by open and black boxes, respectively. (B and F) Kinase assays of GST fusion proteins (5 pmol of each substrate) for the indicated MAP kinases were carried out for 15 min as described in the legend to Fig. 1. (C and G) Phosphorylation of the GST–MEF2A–Elk-1 and GST-MEF2A-cJun chimeras by MAP kinases was analyzed in the presence of competitor peptides as described in the legend to Fig. 4. Five picomoles of each chimeric protein was used in the reactions. The indicated p38 MAP kinases were preincubated in the absence (lane 1) or presence of 500 pmol of the following peptides: MEF2A WT D-domain (lane 2; MEFD[WT]), MEF2A mutant D-domain peptide (lane 3; MEFD[M2]), SAP-1 D-domain peptide (lane 4; SAPD), and Elk-1 D-domain peptide (lane 5; ElkD). (D and H) COS-7 cells were cotransfected with vectors encoding GAL4–Elk-1ΔD, GAL4–MEF2A–Elk-1, GAL4-cJun, and GAL4-MEF2A-cJun fusions, a GAL4-driven luciferase reporter plasmid, and vectors encoding MAP kinases (ERK2, JNK2, and p38β2) and an overexpressed (MKK7β) or constitutively activated form (MEKΔN and MKK6[E]) of the upstream MAP kinase kinases. The expression levels of the GAL4 fusion proteins in unstimulated cells were examined by Western blotting with an anti-GAL4 antibody (bottom panel).