Figure 1.

Mutations of critical residues in the NFAT1 dimer interface disrupt dimer formation without affecting DNA binding affinity or cooperation with AP-1. (A) EMSAs were performed using a probe containing a κB-like site and increasing amounts of recombinant NFAT1 RHR. Two complexes are identified: NFAT1 monomers (N) and dimers (N–N). Graph shows the relative percentage of dimer complexes compared with the total RHR bound to DNA. FP, free probe. (B) EMSAs were performed using recombinant NFAT1 RHR and either a probe containing a consensus κB-like site (left) or a probe in which the two core NFAT binding sites in a κB-like site were separated by 6 nt. (C) Representation of the crystal structure of RHR-C domains in the NFAT1 dimer. Residues involved in the dimer interface are highlighted. (D) Binding of recombinant WT NFAT1 RHR or the dimerization mutants ΔCC′ and ΔCC′QQ-AA was assayed using a probe containing a κB-like dimer site incubated with increasing concentration of these proteins. (E) Increasing amounts of recombinant c-Fos and c-Jun proteins were incubated with constant amounts of recombinant WT NFAT1 RHR or the ΔCC′QQ-AA mutant to analyze their ability to form ternary complexes on a probe containing a NFAT–AP-1 composite site. N, RHR monomer; N-A, RHR–cFos–cJun complex. (F–H). Nuclear extracts prepared from HEK293 cells transfected with plasmids expressing GFP (-), WT NFAT1, or the ΔCC′QQ-EE mutant were analyzed by EMSA to determine the ability of these proteins to form dimers (F), using a probe containing a κB-like site; to bind DNA (G); and to form complexes with AP-1 (H), using a probe containing an NFAT–AP-1 composite site. N-A, NFAT1–cFos–cJun complex. *, unspecific bands.