Analysis of the interaction between c-Jun and c-Jun N-terminal kinase in vivo

Regulation of c-Jun transcriptional activity is believed to depend on a phy sical interaction with c-Jun N-terminal kinase (JNK) that facilitates signa l-regulated phosphorylation of multiple regulatory phosphoacceptor sites wi thin the activation domain. Here we have investigated the structural requir ements and consequences of regulatory phosphorylation for the interaction b etween c-Jun and JNK in vivo. We show that binding of JNK to c-Jun in vivo does not require JNK catalytic activity or the presence of the potential ph osphoacceptor sites within c-Jun. and that JNK retains the capacity to bind to a pseudo-phosphorylated mutant of c-Jun where these sites are replaced by phospho-mimetic aspartic acid residues. The c-Jun delta region docking s ite is essential for interaction with JNK in vivo but is not sufficient, be cause a c-Jun mutant that retains this region but that lacks the C-terminal DNA-binding domain fails to interact. Experiments using purified recombina nt c-Jun and JNK proteins show that the c-Jun DNA-binding domain harbors an auxiliary interaction domain that has the potential to bind to JNK indepen dently. Our results suggest that JNK can be tethered passively to c-Jun in situ through multiple interacting regions and, when activated, can stimulat e c-Jun phosphorylation without necessarily dissociating from its substrate . Auxiliary interactions mediated by the DNA-binding domain could play a ro le in targeting JNK preferentially to c-Jun in specific homo- or heterodime ric complexes.