A docking site in MKK4 mediates high affinity binding to JNK MAPKs and competes with similar docking sites in JNK substrates.
Specific docking interactions between MAPKs and their activating MAPK kinases (MKKs or MEKs) are crucial for efficient and accurate signal transmission. Here, we report the identification of a MAPK-docking site, or "D-site," in the N terminus of human MKK4/JNKK1. This docking site conforms to the consensus sequence for known D-sites in other MKKs and contains the first of the two cleavage sites for anthrax lethal factor protease that have been found in the N terminus of MKK4. This docking site was both necessary and sufficient for the high affinity binding of the MAPKs JNK1, JNK2, JNK3, p38 alpha, and p38 beta to MKK4. Mutations that altered conserved residues in this docking site reduced JNK/p38 binding. In addition, a peptide version of this docking site, as well as a peptide version of the JNK-binding site of the JIP-1 scaffold protein, inhibited both MKK4/JNK binding and MKK4-mediated phosphorylation of JNK1. These same peptides also inhibited JNK2-mediated phosphorylation of c-Jun and ATF2, suggesting that transcription factors, MKK4, and the JIP scaffold compete for docking to JNK. Finally, the selectivity of the MKK4, MEK1, and MEK2 D-sites for JNK versus ERK was quantified. The MEK1 and MEK2 D-sites displayed a strong selectivity for their cognate MAPK (ERK2) versus a non-cognate MAPK (JNK). In contrast, the MKK4 D-site exhibited only limited selectivity for JNK versus ERK.
Ho
David T
DT
Department of Developmental and Cell Biology, University of California, Irvine, California 92697, USA.
Bardwell
A Jane
AJ
Abdollahi
Mahsa
M
Bardwell
Lee
L
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D003062:Codon; D017124:Conserved Sequence; D017931:DNA Primers / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D048670:MAP Kinase Kinase 4; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048057:Mitogen-Activated Protein Kinase 10; D048290:Mitogen-Activated Protein Kinase 11; D048055:Mitogen-Activated Protein Kinase 8; D048056:Mitogen-Activated Protein Kinase 9; D020929:Mitogen-Activated Protein Kinase Kinases / Q000737:chemistry* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D048051:p38 Mitogen-Activated Protein Kinases
D003062:Codon; D017931:DNA Primers; D010455:Peptides; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D048057:Mitogen-Activated Protein Kinase 10; D048056:Mitogen-Activated Protein Kinase 9; D011505:Protein-Tyrosine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048290:Mitogen-Activated Protein Kinase 11; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; C482559:MAP2K4 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases