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 physical interaction with c-Jun N-terminal kinase (JNK) that facilitates signal-regulated phosphorylation of multiple regulatory phosphoacceptor sites within the activation domain. Here we have investigated the structural requirements and consequences of regulatory phosphorylation for the interaction between 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 phosphoacceptor 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 site is essential for interaction with JNK in vivo but is not sufficient, because a c-Jun mutant that retains this region but that lacks the C-terminal DNA-binding domain fails to interact. Experiments using purified recombinant 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 independently. Our results suggest that JNK can be tethered passively to c-Jun in situ through multiple interacting regions and, when activated, can stimulate c-Jun phosphorylation without necessarily dissociating from its substrate. Auxiliary interactions mediated by the DNA-binding domain could play a role in targeting JNK preferentially to c-Jun in specific homo- or heterodimeric complexes.
May
G H
GH
Beatson Institute for Cancer Research, Cancer Research Campaign Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, United Kingdom. g.may@beatson.gla.ac.uk
Allen
K E
KE
Clark
W
W
Funk
M
M
Gillespie
D A
DA
D019943:Amino Acid Substitution; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002642:Chick Embryo; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D016755:Proto-Oncogene Proteins c-jun; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases