Interacting JNK-docking Sites in MKK7 Promote Binding and Activation of JNK Mitogen-activated Protein Kinases

Seema Grewal,David T Ho,Lee Bardwell,Corey Iverson,A Jane Bardwell

doi:10.1074/jbc.m601010200

Seema Grewal, David T Ho + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m601010200

Copy DOI

Abstract

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.

Highlights

Coupling of signal to cellular response is an important and unresolved issue that is currently the subject of intense investigation [3,4,5,6,7]
The negative control MKK4EAG peptide did not inhibit. These results indicate that MKK7 makes contacts with regions of Jun N-terminal kinase (JNK) that at least partially overlap with the regions that are contacted by the MKK4 and JIP-1 D-sites
D-site Peptides from MKK7 Inhibit JNK1/2 Phosphorylation of c-Jun and ATF2—Previously, we demonstrated that the D-site peptide derived from MKK4 strongly inhibited the ability of JNK2 to phosphorylate the downstream transcription factors c-Jun and ATF2, providing evidence that the D-site of MKK4 was competing with the known c-Jun and ATF2 D-sites for binding to JNK [40]

Summary

Introduction

Coupling of signal to cellular response is an important and unresolved issue that is currently the subject of intense investigation [3,4,5,6,7]. MAPK-docking sites are found in the N-terminal regulatory domains of many MKKs, where they contribute to accurate and efficient enzymesubstrate recognition by promoting the formation of relatively stable, high affinity MKK1⁄7MAPK complexes [2]. This paradigm of MAPK recognition was first established for the yeast MEK Ste (36 –38) and has since been extended to mammalian MEK1 [38, 39], MEK2 [38], MKK3 and MKK6 [2], MKK4 [40], and MEK5 [41]. We shall refer to this class of MAPK-docking sites as “D-sites.” D-sites have been found in MAPK scaffolds, phosphatases, and substrates [2, 42, 43]

Results

Discussion

Conclusion