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. 1997 Jul 8;94(14):7337-42.
doi: 10.1073/pnas.94.14.7337.

Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase

C Tournier  1 , A J Whitmarsh, J Cavanagh, T Barrett, R J Davis
Affiliations

Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase

C Tournier et al. Proc Natl Acad Sci U S A. .

Abstract

The c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases is activated by phosphorylation on Thr and Tyr. Here we report the molecular cloning of a new member of the mammalian MAP kinase kinase group (MKK7) that functions as an activator of JNK. In vitro protein kinase assays demonstrate that MKK7 phosphorylates and activates JNK, but not the p38 or extracellular signal-regulated kinase groups of MAP kinase. Expression of MKK7 in cultured cells causes activation of the JNK signal transduction pathway. MKK7 is therefore established to be a novel component of the JNK signal transduction pathway.

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Figures

Figure 1
Figure 1
MKK7 is related to the Drosophila MAP kinase kinase hep. (A) The primary structure of MKK7 and HEP was deduced from the sequence of cDNA clones. These sequences were compared with the MAP kinase kinases MKK1, MKK2, MKK3, MKK4, MKK5, and MKK6 using the pile-up program (version 7.2; Wisconsin Genetics Computer Group, Madison). Gaps introduced into the sequences to optimize the alignment are illustrated with a dash (-). The sites of activating phosphorylation of MAP kinase kinases (, –29) are indicated with asterisks (∗). The sequences of the MKK7, MKK7b, MKK7c, and hep cDNAs have been deposited in GenBank with accession numbers U93030, U93031, AF003199, and U93032, respectively. (B) The relationship between members of the MAP kinase kinase group (kinase subdomains I–XI) is presented as a dendrogram created by the unweighted pair-group method using arithmetic averages (pile-up program). The MAP kinase kinases MKK1, MKK2, MKK3, MKK4, MKK5, MKK6, and MKK7 and the Drosophila MAP kinase kinase hep are presented.
Figure 2
Figure 2
MKK7 is widely expressed in mammalian tissues. The expression of MKK4 and MKK7 was examined by Northern blot analysis of poly(A)+ mRNA isolated from various murine tissues. Autoradiographs of the dried blots are presented.
Figure 3
Figure 3
MKK7 is an activator of the JNK protein kinase in vitro. (A) Recombinant MAP kinases were incubated with GST (Control) or with GST–MKK7 in an in vitro protein kinase assay using the substrate ATP[γ-32P]. MKK7 phosphorylates JNK1, but not GST, ERK2, or p38α. MKK7 is phosphorylated by p38α and JNK1. (B) GST-MKK7 was incubated in a protein kinase assay with recombinant JNK1 or JNK1(APF). The mutated JNK1 protein (APF) was constructed by replacing the dual phosphorylation motif Thr-Pro-Tyr with Ala-Pro-Phe. The JNK activity was measured by including the JNK substrate ATF2 in each assay.
Figure 4
Figure 4
MKK7 is an activator of the JNK protein kinase in vivo. (A) CHO cells were cotransfected with epitope-tagged JNK1 together with an empty expression vector (Control) or an expression vector encoding MKK4 or MKK7. The JNK1 was isolated by immunoprecipitation using an HA monoclonal antibody, and the protein kinase activity was measured in the immunecomplex with [γ-32P]ATP and c-Jun as substrates. The product of the phosphorylation reaction was visualized after SDS/PAGE by autoradiography. (B) The cells were cotransfected with epitope-tagged p38 MAP kinase together with an empty expression vector (Control) or an expression vector encoding MKK6 or MKK7. The activity of p38α MAP kinase was examined in an immunecomplex kinase assay using ATF2 as the substrate. (C) The cells were cotransfected with epitope-tagged ERK2 together with an empty expression vector (Control) or an expression vector encoding MKK1 or MKK7. The activity of ERK2 was examined in an immunecomplex kinase assay using c-Myc as the substrate.
Figure 5
Figure 5
MKK7 is an activator of the JNK signal transduction pathway. (A) CHO cells were cotransfected with the AP-1 reporter plasmid pTRE-luciferase together with an empty expression vector, or expression vectors for MKK4, MKK7, JNK1, and JNK1 (APF). The mutated JNK1 protein (APF) was constructed by replacing the dual phosphorylation motif Thr-Pro-Tyr with Ala-Pro-Phe. Transfection efficiency was monitored by cotransfection with a β-galactosidase expression vector. The relative luciferase/β-galactosidase activity detected in the cell lysates is presented. The data shown are the mean ± SD (n = 3). (B) The transcriptional activity of a GAL4–ATF2 fusion protein was measured in a cotransfection assay in CHO cells using the reporter plasmid pG5E1bLuc. The effect of cotransfection with an empty expression vector or expression vectors for MKK4, MKK7, JNK1, and JNK1 (APF) was examined. Control experiments were performed using a mutated GAL4–ATF2 vector (mATF-2) in which the sites of ATF2 phosphorylation by JNK (Thr-69 and Thr-71) are replaced with Ala. Transfection efficiency was monitored by cotransfection with a β-galactosidase expression vector. The relative luciferase/β-galactosidase activity detected in the cell lysates is presented. The data shown are the mean ± SD (n = 3).
Figure 6
Figure 6
MKK7 is an activator of the JNK signal transduction pathway. The ERK, BMK1/ERK5, p38, and JNK signal transduction pathways are illustrated schematically. MKK1 and MKK2 are activators of the ERK subgroup of MAP kinase. MKK3, MKK4, and MKK6 are activators of p38 MAP kinase. MKK5 is an activator of BMK1/ERK5. MKK7 is a specific activator of the JNK group of MAP kinases, whereas MKK4 activates both the p38 and JNK subgroups of MAP kinase.
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References

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