Interacting JNK-docking sites in MKK7 promote binding and activation of JNK mitogen-activated protein kinases.
D-sites are a class of MAPK-docking sites that have been found in many MAPK regulators and substrates. A single functional, high affinity D-site has been identified near the N terminus of each of the MAPK kinases (MKKs or MEKs) MEK1, MEK2, MKK3, MKK4, and MKK6. Here we demonstrated that MKK7 recognizes its target JNK by a novel mechanism involving a partially cooperative interaction of three low affinity D-sites in the N-terminal domain of MKK7. Mutations of the conserved residues within any one of the three docking sites (D1, D2, and D3) disrupted the ability of the N-terminal domain of MKK7beta to bind JNK1 by about 50-70%. Moreover, mutation of any two of the three D-sites reduced binding by about 80-90%, and mutation of all three reduced binding by 95%. Full-length MKK7 containing combined D1/D2 mutations was compromised for binding to JNK1 and exhibited reduced JNK1 kinase activity when compared with wild-type MKK7. Peptide versions of the D-sites from MKK4 or the JIP-1 scaffold protein inhibited MKK7-JNK binding, suggesting that all three JNK regulators bind to the same region of JNK. Moreover, peptide versions of any of the three D-sites of MKK7 inhibited the ability of JNK1 and JNK2 to phosphorylate their transcription factor substrates c-Jun and ATF2, suggesting that D-site-containing substrates also compete with MKK7 for docking to JNK. Finally, MKK7-derived D-site peptides exhibited selective inhibition of JNK1 versus ERK2. We conclude that MKK7 contains three JNK-docking sites that interact to selectively bind JNK and contribute to JNK signal transmission and specificity.
Department of Developmental and Cell Biology, University of California, Irvine, California 92697.
David T
DT
Ho
Department of Developmental and Cell Biology, University of California, Irvine, California 92697.
A Jane
AJ
Bardwell
Department of Developmental and Cell Biology, University of California, Irvine, California 92697.
Seema
S
Grewal
Department of Developmental and Cell Biology, University of California, Irvine, California 92697.
Corey
C
Iverson
Department of Developmental and Cell Biology, University of California, Irvine, California 92697. Electronic address: bardwell@uci.edu.
Lee
L
Bardwell
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D048688:MAP Kinase Kinase 7 / Q000737:chemistry* / Q000378:metabolism*; D048728:MAP Kinase Kinase Kinase 1 / Q000378:metabolism; D048730:MAP Kinase Kinase Kinase 2 / Q000378:metabolism; D011485:Protein Binding
D048031:JNK Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; D048730:MAP Kinase Kinase Kinase 2; D048688:MAP Kinase Kinase 7; C482569:MAP2K7 protein, human